Viruses

18 pages, 2582 KiB

Open AccessArticle

The CARD9-Associated C-Type Lectin, Mincle, Recognizes La Crosse Virus (LACV) but Plays a Limited Role in Early Antiviral Responses against LACV

by João T. Monteiro, Kathleen Schön, Tim Ebbecke, Ralph Goethe, Jürgen Ruland, Wolfgang Baumgärtner, Stefanie C. Becker and Bernd Lepenies

Viruses 2019, 11(3), 303; https://doi.org/10.3390/v11030303 - 26 Mar 2019

Cited by 27 | Viewed by 4513

Abstract

La Crosse virus (LACV) is a mosquito-transmitted arbovirus and the main cause of virus-mediated neurological diseases in children. To date, little is known about the role of C-type lectin receptors (CLRs)—an important class of pattern recognition receptors—in LACV recognition. DC-SIGN remains the only [...] Read more.

La Crosse virus (LACV) is a mosquito-transmitted arbovirus and the main cause of virus-mediated neurological diseases in children. To date, little is known about the role of C-type lectin receptors (CLRs)—an important class of pattern recognition receptors—in LACV recognition. DC-SIGN remains the only well-described CLR that recognizes LACV. In this study, we investigated the role of additional CLR/LACV interactions. To this end, we applied a flow-through chromatography method for the purification of LACV to perform an unbiased high-throughput screening of LACV with a CLR-hFc fusion protein library. Interestingly, the CARD9-associated CLRs Mincle, Dectin-1, and Dectin-2 were identified to strongly interact with LACV. Since CARD9 is a common adaptor protein for signaling via Mincle, Dectin-1, and Dectin-2, we performed LACV infection of Mincle^−/− and CARD9^−/− DCs. Mincle^−/− and CARD9^−/− DCs produced less amounts of proinflammatory cytokines, namely IL-6 and TNF-α, albeit no reduction of the LACV titer was observed. Together, novel CLR/LACV interactions were identified; however, the Mincle/CARD9 axis plays a limited role in early antiviral responses against LACV. Full article

(This article belongs to the Special Issue The Glycobiology of Viral Infections)

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Figure 1
Schematic representation of the flow-through chromatography process used for La Crosse virus (LACV) purification. Mock- and LACV-infected supernatants of VeroE6 cells were purified by sequential steps of ultrafiltration and liquid chromatographic processes to remove host-cell derived proteins (HCPs). At the end of the purification process, a virus enrichment in the LACV sample is attained by efficient removal of HCPs. Full article ">Figure 2
ELISA-based screening of LACV with a C-type lectin receptor (CLR)-hFc fusion protein library. DC-SIGN was reported to recognize Gc/Gn of LACV [<a href="#B23-viruses-11-00303" class="html-bibr">23</a>] and is considered a positive control. To discard possible false-positives, a 3-fold margin in the absorbance value relative to hFc (negative control) was given (dotted line), based on previous screenings with the CLR-hFc library [<a href="#B27-viruses-11-00303" class="html-bibr">27</a>]. Data depicted are the mean ± SEM of four independent experiments (duplicates each). Full article ">Figure 3
LACV internalization, replication in Mincle−/−, CARD9−/−, or WT dendritic cells (DCs), and LACV-dependent induction of Mincle expression. (A) Transmission electron microscopy (TEM) picture showing a LACV-infected WT DC at time 2 h (magnification 6300×). (B) Close-up TEM picture (magnification 25,000×) of highlighted region in (A). The black arrowheads show LACV particles inside vesicles in the phagolysosome. (C) Expression levels of LACV N mRNA at different time points in DCs. The time point 2 h was used as baseline (internalized LACV). In C, two distinct LACV MOI were used—MOI 5 and MOI 20. (D) Expression levels of Mincle mRNA at different time points in LACV-infected WT DCs. The mock-infected DCs were used as baseline. Data shown in (C) and (D) are mean ± SEM and three independent experiments were performed. A two-way ANOVA with a Tukey’s honest significance test was used to compare differences between the different groups and p < 0.05 was considered significant (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Full article ">Figure 4
Effector functions of Mincle−/−, CARD9−/−, and WT DCs stimulated with LACV. (A) Surface expression of MHC-I. (B) Surface expression of MHC-II. (C) Surface expression of CD80 in Mincle−/−, CARD9−/− or WT DCs after 24 h of stimulation. Data represented are mean ± SEM of three independent experiments. Data are presented as MFI (median fluorescence intensity) values. A two-way ANOVA with a Tukey’s honest significance test was performed and p < 0.05 was considered significant (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Full article ">Figure 5
Mincle−/− and CARD9−/− DCs display impaired proinflammatory cytokine production upon LACV challenge in the presence of Poly(I:C). IL-6 and TNF-α production after 8 h (A,B, respectively) and 24 h of stimulation (C,D, respectively). Data represented are mean ± SEM of three independent experiments. A two-way ANOVA with a Tukey’s honest significance test was performed and p < 0.05 was considered significant (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Full article ">Figure 6
Viral loads in Mincle−/−, CARD9−/−, and WT DCs challenged with LACV. Virus titer in the supernatant of Mincle−/−, CARD9−/− and WT DCs infected with LACV at a MOI 5 (A) and a MOI 20 (B) for 8 h and 24 h. Data shown are mean ± SEM of three independent experiments. A two-way ANOVA with a Tukey’s honest significance test was employed and p < 0.05 was considered significant (* p < 0.05, ** p < 0.01, *** p < 0.001). Full article ">

17 pages, 1741 KiB

Open AccessReview

IFN Signaling in Inflammation and Viral Infections: New Insights from Fish Models

by Christelle Langevin, Pierre Boudinot and Bertrand Collet

Viruses 2019, 11(3), 302; https://doi.org/10.3390/v11030302 - 26 Mar 2019

Cited by 26 | Viewed by 4981

Abstract

The overarching structure of the type I interferon (IFN) system is conserved across vertebrates. However, the variable numbers of whole genome duplication events during fish evolution offer opportunities for the expansion, diversification, and new functionalization of the genes that are involved in antiviral [...] Read more.

The overarching structure of the type I interferon (IFN) system is conserved across vertebrates. However, the variable numbers of whole genome duplication events during fish evolution offer opportunities for the expansion, diversification, and new functionalization of the genes that are involved in antiviral immunity. In this review, we examine how fish models provide new insights about the implication of virus-driven inflammation in immunity and hematopoiesis. Mechanisms that have been discovered in fish, such as the strong adjuvant effect of type I IFN that is used with DNA vaccination, constitute good models to understand how virus-induced inflammatory mechanisms can interfere with adaptive responses. We also comment on new discoveries regarding the role of pathogen-induced inflammation in the development and guidance of hematopoietic stem cells in zebrafish. These findings raise issues about the potential interferences of viral infections with the establishment of the immune system. Finally, the recent development of genome editing provides new opportunities to dissect the roles of the key players involved in the antiviral response in fish, hence enhancing the power of comparative approaches. Full article

(This article belongs to the Special Issue Viruses Ten-Year Anniversary)

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15 pages, 2663 KiB

Open AccessArticle

Zika Virus-Specific IgY Results Are Therapeutic Following a Lethal Zika Virus Challenge without Inducing Antibody-Dependent Enhancement

by Kyle L. O’Donnell, Bernadette Meberg, James Schiltz, Matthew L. Nilles and David S. Bradley

Viruses 2019, 11(3), 301; https://doi.org/10.3390/v11030301 - 26 Mar 2019

Cited by 19 | Viewed by 4154

Abstract

The Zika virus (ZIKV) is a newly emerged pathogen in the Western hemisphere. It was declared a global health emergency by the World Health Organization in 2016. There have been 223,477 confirmed cases, including 3720 congenital syndrome cases since 2015. ZIKV infection symptoms [...] Read more.

The Zika virus (ZIKV) is a newly emerged pathogen in the Western hemisphere. It was declared a global health emergency by the World Health Organization in 2016. There have been 223,477 confirmed cases, including 3720 congenital syndrome cases since 2015. ZIKV infection symptoms range from asymptomatic to Gullain–Barré syndrome and extensive neuropathology in infected fetuses. Passive and active vaccines have been unsuccessful in the protection from or the treatment of flaviviral infections due to antibody-dependent enhancement (ADE). ADE causes an increased viral load due to an increased monocyte opsonization by non-neutralizing, low-avidity antibodies from a previous dengue virus (DENV) infection or from a previous exposure to ZIKV. We have previously demonstrated that polyclonal avian IgY generated against whole-killed DENV-2 ameliorates DENV infection in mice while not inducing ADE. This is likely due to the inability of the Fc portion of IgY to bind to mammalian Fc receptors. We have shown here that ZIKV oligoclonal IgY is able to neutralize the virus in vitro and in IFNAR^−/− mice. The concentration of ZIKV-specific IgY yielding 50% neutralization (NT₅₀) was 25 µg/mL. The exposure of the ZIKV, prior to culture with ZIKV-specific IgY or 4G2 flavivirus-enveloped IgG, demonstrated that the ZIKV-specific IgY does not induce ADE. ZIKV IgY was protective in vivo when administered following a lethal ZIKV challenge in 3-week-old IFNAR^−/− mice. We propose polyclonal ZIKV-specific IgY may provide a viable passive immunotherapy for a ZIKV infection without inducing ADE. Full article

(This article belongs to the Section Viral Immunology, Vaccines, and Antivirals)

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Anti-ZIKV (Zika virus) IgY purification: The purity of anti-ZIKV IgY after the second polyethylene glycol (PEG) precipitation. Lane 1, supernatant from the PEG precipitation; lane 2, resuspended PEG pellet; Lanes 3 and 4, molecular weight concentration and diafiltration; Lane 5, molecular weight marker; and Lane 6, control naïve IgY that was previously purified via mercapto-ethyl pyridine HyperCel Hydrophobic Charged Induced Chromatograph for comparison. Full article ">Figure 2
Anti-ZIKV IgY neutralizes ZIKV in vitro without antibody-dependent enhancement. (A) Anti-ZIKV IgY neutralized the ZIKV infection in vitro with a NT50 of 25 µg/mL. (B) Purified anti-ZIKV IgY did not enhance the ZIKV infection in vitro at 100 µg/mL. (C) Purified anti-ZIKV IgY did not enhance the DENV2 infection in vitro at 100 µg/mL, whereas 4G2 flavivirus IgG significantly enhanced the viral load. (D) Purified anti-ZIKV IgY did not enhance the ZIKV infection in vitro at any level tested, whereas 4G2 flavivirus IgG significantly enhanced the viral load at 1, 10, and 100 µg. **** p < 0.0001, ns = not significant. Full article ">Figure 2 Cont.
Anti-ZIKV IgY neutralizes ZIKV in vitro without antibody-dependent enhancement. (A) Anti-ZIKV IgY neutralized the ZIKV infection in vitro with a NT50 of 25 µg/mL. (B) Purified anti-ZIKV IgY did not enhance the ZIKV infection in vitro at 100 µg/mL. (C) Purified anti-ZIKV IgY did not enhance the DENV2 infection in vitro at 100 µg/mL, whereas 4G2 flavivirus IgG significantly enhanced the viral load. (D) Purified anti-ZIKV IgY did not enhance the ZIKV infection in vitro at any level tested, whereas 4G2 flavivirus IgG significantly enhanced the viral load at 1, 10, and 100 µg. **** p < 0.0001, ns = not significant. Full article ">Figure 3
The therapeutic efficacy of anti-ZIKV IgY in vivo: Three-week-old IFNAR−/− mice were administered a lethal dose of ZIKV intravenously (i.v.) in 100 µL total volume. At 24 and 48 h postinfection, the mice were immunized i.p. with the indicated amount of antibody in a volume of 100 µL. The control mice were treated with 100 uL of PBS. (A,B) The survival outcome of the treatments and (C,D) the weights were monitored daily until day 14 or until morbid. The weights were reported as a percent of the starting weight; the p-values are denoted when comparing the groups to the viral control. ** p < 0.01, *** p < 0.005, and **** p < 0.0001. Full article ">Figure 3 Cont.
The therapeutic efficacy of anti-ZIKV IgY in vivo: Three-week-old IFNAR−/− mice were administered a lethal dose of ZIKV intravenously (i.v.) in 100 µL total volume. At 24 and 48 h postinfection, the mice were immunized i.p. with the indicated amount of antibody in a volume of 100 µL. The control mice were treated with 100 uL of PBS. (A,B) The survival outcome of the treatments and (C,D) the weights were monitored daily until day 14 or until morbid. The weights were reported as a percent of the starting weight; the p-values are denoted when comparing the groups to the viral control. ** p < 0.01, *** p < 0.005, and **** p < 0.0001. Full article ">Figure 4
The viral load reduction upon treatment with anti-ZIKV IgY: (A–C) The infectious viral load was quantified in the brain (A), spleen (B), and liver (C) via a plaque assay. (D–F) The viral genomic copy number was quantified in the brain (D), spleen (E), and liver (F) via RT-qPCR. The viral load of the treated mice was compared to the viral control group. * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.0001, and $ is the comparison to the matching ZIKV-specific IgY group. Full article ">Figure 5
An epitope map of the structural and nonstructural genes: (A) A heat map of the structural epitopes of the Zika virus recognized by Zika-specific IgY and naïve IgY and (B) a heat map of the non-structural epitopes of the Zika virus recognized by Zika-specific IgY and naïve IgY. The strength of binding is indicated on a colorimetric scale with red being a strong binding affinity and green being no binding. Full article ">

12 pages, 637 KiB

Open AccessReview

Innovation in Newcastle Disease Virus Vectored Avian Influenza Vaccines

by Shin-Hee Kim and Siba K. Samal

Viruses 2019, 11(3), 300; https://doi.org/10.3390/v11030300 - 26 Mar 2019

Cited by 30 | Viewed by 6720

Abstract

Highly pathogenic avian influenza (HPAI) and Newcastle disease are economically important avian diseases worldwide. Effective vaccination is critical to control these diseases in poultry. Live attenuated Newcastle disease virus (NDV) vectored vaccines have been developed for bivalent vaccination against HPAI viruses and NDV. [...] Read more.

Highly pathogenic avian influenza (HPAI) and Newcastle disease are economically important avian diseases worldwide. Effective vaccination is critical to control these diseases in poultry. Live attenuated Newcastle disease virus (NDV) vectored vaccines have been developed for bivalent vaccination against HPAI viruses and NDV. These vaccines have been generated by inserting the hemagglutinin (HA) gene of avian influenza virus into NDV genomes. In laboratory settings, several experimental NDV-vectored vaccines have protected specific pathogen-free chickens from mortality, clinical signs, and virus shedding against H5 and H7 HPAI viruses and NDV challenges. NDV-vectored H5 vaccines have been licensed for poultry vaccination in China and Mexico. Recently, an antigenically chimeric NDV vector has been generated to overcome pre-existing immunity to NDV in poultry and to provide early protection of poultry in the field. Prime immunization of one-day-old poults with a chimeric NDV vector followed by boosting with a conventional NDV vector has shown to protect broiler chickens against H5 HPAI viruses and a highly virulent NDV. This novel vaccination approach can provide efficient control of HPAI viruses in the field and facilitate poultry vaccination. Full article

(This article belongs to the Special Issue Avian Respiratory Viruses)

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16 pages, 3592 KiB

Open AccessArticle

Metagenomes of a Freshwater Charavirus from British Columbia Provide a Window into Ancient Lineages of Viruses

by Marli Vlok, Adrian J. Gibbs and Curtis A. Suttle

Viruses 2019, 11(3), 299; https://doi.org/10.3390/v11030299 - 25 Mar 2019

Cited by 16 | Viewed by 5017

Abstract

Charophyte algae, not chlorophyte algae, are the ancestors of ‘higher plants’; hence, viruses infecting charophytes may be related to those that first infected higher plants. Streamwaters from British Columbia, Canada, yielded single-stranded RNA metagenomes of Charavirus canadensis (CV-Can), that are similar in genomic [...] Read more.

Charophyte algae, not chlorophyte algae, are the ancestors of ‘higher plants’; hence, viruses infecting charophytes may be related to those that first infected higher plants. Streamwaters from British Columbia, Canada, yielded single-stranded RNA metagenomes of Charavirus canadensis (CV-Can), that are similar in genomic architecture, length (9593 nt), nucleotide identity (63.4%), and encoded amino-acid sequence identity (53.0%) to those of Charavirus australis (CV-Aus). The sequences of their RNA-dependent RNA-polymerases (RdRp) resemble those found in benyviruses, their helicases those of hepaciviruses and hepegiviruses, and their coat-proteins (CP) those of tobamoviruses; all from the alphavirus/flavivirus branch of the ‘global RNA virome’. The 5’-terminus of the CV-Can genome, but not that of CV-Aus, is complete and encodes a methyltransferase domain. Comparisons of CP sequences suggests that Canadian and Australian charaviruses diverged 29–46 million years ago (mya); whereas, the CPs of charaviruses and tobamoviruses last shared a common ancestor 212 mya, and the RdRps of charaviruses and benyviruses 396 mya. CV-Can is sporadically abundant in low-nutrient freshwater rivers in British Columbia, where Chara braunii, a close relative of C. australis, occurs, and which may be its natural host. Charaviruses, like their hosts, are ancient and widely distributed, and thus provide a window to the viromes of early eukaryotes and, even, Archaea. Full article

(This article belongs to the Special Issue Plant Virus Ecology and Biodiversity)

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Comparison of two Charavirus genomes; gene map and similarities of nucleotide sequences and predicted amino acids. The nucleotide similarity axis positively correlated with the similarity score. Predicted weight of putative proteins are indicated in kilo Daltons (kDa); identical amino acids in black, similar amino acids in grey and in line with the open reading frame (ORF) of Charavirus canadensis (CV-Can). Full article ">Figure 2
Maximum-likelihood phylogeny of the amino acid sequences of the RdRp-2 regions of the replicase proteins of the charaviruses, benyviruses, and selected tobamoviruses and relatives. Acronyms: ABV13, Agaricus bisporus virus 13 (AQM49942); BastlV-VN, Bastrovirus-like_virus-VietNam Bat (YP_009333174); BastV-Braz, Bastrovirus Brazil/sewage (ASM79505); BMoV, Burdock mottle virus (YP_008219063); BNYVV, Beet necrotic yellow vein virus (NP_612615); BSbMV, Beet soil-borne mosaic virus (NP_612601); CGMMV, Cucumber green mottle mosaic virus (NP_044577); CMMtV, Cactus mild mottle virus (YP_002455590); CTV, Cutthroat trout piscihepevirus (YP_004464917); CuMtV, Cucumber mottle virus (YP_908760); CV-Aus, Charavirus australis (AEJ33768); CV-Can, Charavirus canadensis (MK521928); HBlV1, Hubei Beny-like virus 1 (APG77690); HHlV1, Hubei hepe-like virus 1 (YP_009336840); HLSV, Hibiscus latent Singapore virus (YP_719997); HVlV16, Hubei virga-like virus 16 (YP_009336677); KGMMV, Kyuri green mottle mosaic virus (YP_908760); MILV, Mangifera indica latent virus (AMQ23297); OHV-A, Orthohepevirus A(ABB88699, AGE83293, AGE83340, AGT38396, ANW09725, BAE86910); OHV-B, Orthohepevirus B (AEX93357, CAQ16023, YP_009001465; OHV-C, Orthohepevirus C (ADB96199,AFL69932, ANJ02843, BAO47898, BAT70058); OHV-D, Orthohepevirus D (AIF74285, YP_006576507); RCNaV, Rattail cactus necrosis-associated virus (YP_0044936166); RMV, Ribgrass mosaic virus (YP_005476600); RStNV, Rice stripe necrosis virus (ABU94739); SanBV, San Bernardo virus (AQM55436); TbTlV, Tick borne tetravirus-like virus (AII01815); TMV, Tobacco mosaic virus (NP597746). The green discs mark nodes with >0.9 SH support; two thirds of the nodes in the Orthohepevirus cluster have >0.9 SH support, but, for clarity, are not marked. Full article ">Figure 3
Maximum-likelihood phylogeny of the amino acid sequences of the CPs, and CP-like proteins, of the charaviruses and relatives. These include: AV, Abisko virus (NC_035470); ASV, Adelphocoris suturalis virus (NC_032728); BCCV1, Beihai Charybdis crab virus 1 (NC_032449); BNYVV, Beet necrotic yellow vein virus (NC_003515); BSbMV, Beet soil-borne mosaic virus, (NC_003503); BSMV. Barley stripe mosaic virus (NC_003481); CGMtMV, Cucumber green mottle mosaic virus (NC_001801); CuMtV, Cucumber mottle virus (NC_008614); CV-Aus, Charavirus australis (JF824737); CV-Can, Charavirus canadensis (MK521928); HLSV, Hibiscus latent Singapore virus (NC_008310); HVlV2; Hubei virga-like virus 2 (NC_033158); HVlV9, Hubei virga-like virus 9 (NC_032765); HVlV11, Hubei virga-like virus 11 (NC_033082); HVlV12, Hubei virga-like virus 12 (NC_033269); KGMtMV, Kyuri green mottle mosaic virus (NC_003610); MMV, Maracuja mosaic virus (NC_008716); PCV, Peanut clump virus (NC_003668); PEBV, Pea early browning virus (NC_001368); RCNaV, Rattail cactus necrosis-associated virus (NC_016442); RMV, Ribgrass mosaic virus (NC_002792); SbWMV, Soil-borne wheat mosaic virus (NC_002042); TMV, Tobacco mosaic virus (NC_001367); TRV, Tobacco rattle virus (NC_003811); VTMtV, Velvet tobacco mottle virus (NC_014509); XM_014378609, a gene from Apis mellifera; XM_016912277, a gene from Trichogramma pretiosum. The circle marks the midpoint of the phylogeny, and the broken line is the branch to the outgroup of HVlV9 and VTMtV drawn only at 50% of its true length. The green discs mark nodes with >0.9 SH support and the yellow discs those with >0.8 < 0.9 SH support. Full article ">Figure 4
Correspondence analysis of tetra-nucleotide patterns of charaviruses and other sequence related viruses. Patterns were obtained for both RdRp sequences (A,B) used in <a href="#viruses-11-00299-f001" class="html-fig">Figure 1</a>, and capsid gene sequences (C,D) from <a href="#viruses-11-00299-f002" class="html-fig">Figure 2</a>. The separated helicase sequences of the two charaviruses (CV-Aus_H and CV-Can_H) were included to compare with the helicase sequences reported by [<a href="#B7-viruses-11-00299" class="html-bibr">7</a>], but can be seen to be distinct. Full article ">Figure 5
Molecular phylogeny of charophytes as determined by Perez et al. [<a href="#B49-viruses-11-00299" class="html-bibr">49</a>], with four additional species (red branches) extrapolated from the data of Sakayama et al. [<a href="#B48-viruses-11-00299" class="html-bibr">48</a>]. Full article ">Figure 6
CV-Can contigs were abundant and diverse in freshwater streams. Relative abundances calculated from rarefied read counts and normalized by contig size (A) suggest that CV-Can was most abundant at the first protected site (Prist1) compared to the urban and agricultural (Agri) sites. Single nucleotide variant (SNV) analysis (B) of the replicase gene at the two protected and one urban site indicate much genetic variation within the CV-Can population. Full article ">Figure 7
Summary of the estimates of gene divergence dates (million years before present) for the charavirus, tobamovirus, and benyvirus RdRp and CP proteins. The coloured triangles represent monophyletic clusters (i.e., two or more homologs) of the proteins found in extant viruses, and their left hand tip represents the earliest likely age of each extant virus cluster. Full article ">

19 pages, 745 KiB

Open AccessArticle

Functional RNA Structures in the 3′UTR of Tick-Borne, Insect-Specific and No-Known-Vector Flaviviruses

by Roman Ochsenreiter, Ivo L. Hofacker and Michael T. Wolfinger

Viruses 2019, 11(3), 298; https://doi.org/10.3390/v11030298 - 24 Mar 2019

Cited by 37 | Viewed by 6555

Abstract

Untranslated regions (UTRs) of flaviviruses contain a large number of RNA structural elements involved in mediating the viral life cycle, including cyclisation, replication, and encapsidation. Here we report on a comparative genomics approach to characterize evolutionarily conserved RNAs in the 3

^{'}

UTR [...] Read more.

Untranslated regions (UTRs) of flaviviruses contain a large number of RNA structural elements involved in mediating the viral life cycle, including cyclisation, replication, and encapsidation. Here we report on a comparative genomics approach to characterize evolutionarily conserved RNAs in the 3

^{'}

UTR of tick-borne, insect-specific and no-known-vector flaviviruses in silico. Our data support the wide distribution of previously experimentally characterized exoribonuclease resistant RNAs (xrRNAs) within tick-borne and no-known-vector flaviviruses and provide evidence for the existence of a cascade of duplicated RNA structures within insect-specific flaviviruses. On a broader scale, our findings indicate that viral 3

^{'}

UTRs represent a flexible scaffold for evolution to come up with novel xrRNAs. Full article

(This article belongs to the Special Issue Virus Bioinformatics)

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7 pages, 1446 KiB

Open AccessCommunication

A Novel Hepacivirus in Wild Rodents from South America

by William Marciel de Souza, Marcílio Jorge Fumagalli, Gilberto Sabino-Santos, Jr., Felipe Gonçalves Motta Maia, Sejal Modha, Márcio Roberto Teixeira Nunes, Pablo Ramiro Murcia and Luiz Tadeu Moraes Figueiredo

Viruses 2019, 11(3), 297; https://doi.org/10.3390/v11030297 - 24 Mar 2019

Cited by 12 | Viewed by 4844

Abstract

The Hepacivirus genus comprises single-stranded positive-sense RNA viruses within the family Flaviviridae. Several hepaciviruses have been identified in different mammals, including multiple rodent species in Africa, Asia, Europe, and North America. To date, no rodent hepacivirus has been identified in the South [...] Read more.

The Hepacivirus genus comprises single-stranded positive-sense RNA viruses within the family Flaviviridae. Several hepaciviruses have been identified in different mammals, including multiple rodent species in Africa, Asia, Europe, and North America. To date, no rodent hepacivirus has been identified in the South American continent. Here, we describe an unknown hepacivirus discovered during a metagenomic screen in Akodon montensis, Calomys tener, Oligoryzomys nigripes, Necromys lasiurus, and Mus musculus from São Paulo State, Brazil. Molecular detection of this novel hepacivirus by RT-PCR showed a frequency of 11.11% (2/18) in Oligoryzomys nigripes. This is the first identification of hepavivirus in sigmondonine rodents and in rodents from South America. In sum, our results expand the host range, viral diversity, and geographical distribution of the Hepacivirus genus. Full article

(This article belongs to the Section Animal Viruses)

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13 pages, 2412 KiB

Open AccessArticle

A Field Recombinant Strain Derived from Two Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV-1) Modified Live Vaccines Shows Increased Viremia and Transmission in SPF Pigs

by Julie Eclercy, Patricia Renson, Arnaud Lebret, Edouard Hirchaud, Valérie Normand, Mathieu Andraud, Frédéric Paboeuf, Yannick Blanchard, Nicolas Rose and Olivier Bourry

Viruses 2019, 11(3), 296; https://doi.org/10.3390/v11030296 - 23 Mar 2019

Cited by 50 | Viewed by 5122

Abstract

In Europe, modified live vaccines (MLV) are commonly used to control porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, they have been associated with safety issues such as reversion to virulence induced by mutation and/or recombination. On a French pig farm, we [...] Read more.

In Europe, modified live vaccines (MLV) are commonly used to control porcine reproductive and respiratory syndrome virus (PRRSV) infection. However, they have been associated with safety issues such as reversion to virulence induced by mutation and/or recombination. On a French pig farm, we identified a field recombinant strain derived from two PRRSV-1 MLV (MLV1). As a result, we aimed to evaluate its clinical, virological, and transmission parameters in comparison with both parental strains. Three groups with six pigs in each were inoculated with either one of the two MLV1s or with the recombinant strain; six contact pigs were then added into each inoculated group. The animals were monitored daily for 35 days post-inoculation (dpi) for clinical symptoms; blood samples and nasal swabs were collected twice a week. PRRS viral load in inoculated pigs of recombinant group was higher in serum, nasal swabs, and tonsils in comparison with both vaccine groups. The first viremic contact pig was detected as soon as 2 dpi in the recombinant group compared to 10 and 17 dpi for vaccine groups. Estimation of transmission parameters revealed fastest transmission and longest duration of infectiousness for recombinant group. Our in vivo study showed that the field recombinant strain derived from two MLV1s demonstrated high viremia, shedding and transmission capacities. Full article

(This article belongs to the Special Issue Porcine Viruses 2019)

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13 pages, 253 KiB

Open AccessReview

Towards Inhaled Phage Therapy in Western Europe

by Sandra-Maria Wienhold, Jasmin Lienau and Martin Witzenrath

Viruses 2019, 11(3), 295; https://doi.org/10.3390/v11030295 - 23 Mar 2019

Cited by 33 | Viewed by 6155

Abstract

The emergence of multidrug-resistant bacteria constitutes a great challenge for modern medicine, recognized by leading medical experts and politicians worldwide. Rediscovery and implementation of bacteriophage therapy by Western medicine might be one solution to the problem of increasing antibiotic failure. In some Eastern [...] Read more.

The emergence of multidrug-resistant bacteria constitutes a great challenge for modern medicine, recognized by leading medical experts and politicians worldwide. Rediscovery and implementation of bacteriophage therapy by Western medicine might be one solution to the problem of increasing antibiotic failure. In some Eastern European countries phage therapy is used for treating infectious diseases. However, while the European Medicines Agency (EMA) advised that the development of bacteriophage-based therapies should be expedited due to its significant potential, EMA emphasized that phages cannot be recommended for approval before efficacy and safety have been proven by appropriately designed preclinical and clinical trials. More evidence-based data is required, particularly in the areas of pharmacokinetics, repeat applications, immunological reactions to the application of phages as well as the interactions and effects on bacterial biofilms and organ-specific environments. In this brief review we summarize advantages and disadvantages of phage therapy and discuss challenges to the establishment of phage therapy as approved treatment for multidrug-resistant bacteria. Full article

(This article belongs to the Special Issue Hurdles for Phage Therapy (PT) to Become a Reality)

20 pages, 557 KiB

Open AccessReview

Chikungunya in Infants and Children: Is Pathogenesis Increasing?

by Kelli L. Barr and Vedana Vaidhyanathan

Viruses 2019, 11(3), 294; https://doi.org/10.3390/v11030294 - 23 Mar 2019

Cited by 29 | Viewed by 5240

Abstract

Chikungunya virus (CHIKV) was first extensively described in children during outbreaks in India and South Asia during the mid-1960s. Prior to the 2005 emergence of CHIKV on Reunion Island, CHIKV infection was usually described as a dengue-like illness with arthralgia in Africa and [...] Read more.

Chikungunya virus (CHIKV) was first extensively described in children during outbreaks in India and South Asia during the mid-1960s. Prior to the 2005 emergence of CHIKV on Reunion Island, CHIKV infection was usually described as a dengue-like illness with arthralgia in Africa and febrile hemorrhagic disease in Asia. Soon after the 2005 emergence, severe CNS consequences from vertical and perinatal transmission were described and as CHIKV continued to emerge in new areas over the next 10 years, severe manifestation of infection and sequelae were increasingly reported in infants and neonates. The following review describes the global reemergence and the syndromes of Chikungunya fever (CHIKF) in infants and children. The various manifestations of CHIKF are described and connected to the viral lineage that was documented in the area at the time the disease was described. The data show that certain manifestations of CHIKF occur with specific viral lineages and genetic motifs, which suggests that severe manifestations of CHIKF in the very young may be associated with the emergence of new viral lineages. Full article

(This article belongs to the Special Issue Chikungunya Virus and (Re-) Emerging Alphaviruses)

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18 pages, 4458 KiB

Open AccessArticle

Autophagy Promotes Infectious Particle Production of Mopeia and Lassa Viruses

by Nicolas Baillet, Sophie Krieger, Alexandra Journeaux, Valérie Caro, Frédéric Tangy, Pierre-Olivier Vidalain and Sylvain Baize

Viruses 2019, 11(3), 293; https://doi.org/10.3390/v11030293 - 23 Mar 2019

Cited by 11 | Viewed by 4228

Abstract

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related Old-World mammarenaviruses. LASV causes severe hemorrhagic fever with high mortality in humans, whereas no case of MOPV infection has been reported. Comparing MOPV and LASV is a powerful strategy to unravel pathogenic [...] Read more.

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related Old-World mammarenaviruses. LASV causes severe hemorrhagic fever with high mortality in humans, whereas no case of MOPV infection has been reported. Comparing MOPV and LASV is a powerful strategy to unravel pathogenic mechanisms that occur during the course of pathogenic arenavirus infection. We used a yeast two-hybrid approach to identify cell partners of MOPV and LASV Z matrix protein in which two autophagy adaptors were identified, NDP52 and TAX1BP1. Autophagy has emerged as an important cellular defense mechanism against viral infections but its role during arenavirus infection has not been shown. Here, we demonstrate that autophagy is transiently induced by MOPV, but not LASV, in infected cells two days after infection. Impairment of the early steps of autophagy significantly decreased the production of MOPV and LASV infectious particles, whereas a blockade of the degradative steps impaired only MOPV infectious particle production. Our study provides insights into the role played by autophagy during MOPV and LASV infection and suggests that this process could partially explain their different pathogenicity. Full article

(This article belongs to the Special Issue Medical Advances in Viral Hemorrhagic Fever Research)

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19 pages, 3385 KiB

Open AccessArticle

The PB2 Polymerase Host Adaptation Substitutions Prime Avian Indonesia Sub Clade 2.1 H5N1 Viruses for Infecting Humans

by Pui Wang, Wenjun Song, Bobo Wing-Yee Mok, Min Zheng, Siu-Ying Lau, Siwen Liu, Pin Chen, Xiaofeng Huang, Honglian Liu, Conor J. Cremin and Honglin Chen

Viruses 2019, 11(3), 292; https://doi.org/10.3390/v11030292 - 22 Mar 2019

Cited by 7 | Viewed by 3733

Abstract

Significantly higher numbers of human infections with H5N1 virus have occurred in Indonesia and Egypt, compared with other affected areas, and it is speculated that there are specific viral factors for human infection with avian H5N1 viruses in these locations. We previously showed [...] Read more.

Significantly higher numbers of human infections with H5N1 virus have occurred in Indonesia and Egypt, compared with other affected areas, and it is speculated that there are specific viral factors for human infection with avian H5N1 viruses in these locations. We previously showed PB2-K526R is present in 80% of Indonesian H5N1 human isolates, which lack the more common PB2-E627K substitution. Testing the hypothesis that this mutation may prime avian H5N1 virus for human infection, we showed that: (1) K526R is rarely found in avian influenza viruses but was identified in H5N1 viruses 2–3 years after the virus emerged in Indonesia, coincident with the emergence of H5N1 human infections in Indonesia; (2) K526R is required for efficient replication of Indonesia H5N1 virus in mammalian cells in vitro and in vivo and reverse substitution to 526K in human isolates abolishes this ability; (3) Indonesian H5N1 virus, which contains K526R-PB2, is stable and does not further acquire E627K following replication in infected mice; and (4) virus containing K526R-PB2 shows no fitness deficit in avian species. These findings illustrate an important mechanism in which a host adaptive mutation that predisposes avian H5N1 virus towards infecting humans has arisen with the virus becoming prevalent in avian species prior to human infections occurring. A similar mechanism is observed in the Qinghai-lineage H5N1 viruses that have caused many human cases in Egypt; here, E627K predisposes towards human infections. Surveillance should focus on the detection of adaptation markers in avian strains that prime for human infection. Full article

(This article belongs to the Special Issue Avian Respiratory Viruses)

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Phylogenetic tree of the PB2 gene of Indonesian H5N1 virus. The tree was constructed using the neighbor-joining method with the Tajima-Nei model of nucleotide substitution using MEGA X. Blue indicates avian strains carrying Arg at position 526 of the PB2 gene (526R). Human strains are highlighted in boldface. Numbers represent the bootstrap values (percentages) from 1000 replicates: only bootstrap values greater than 50 are shown. Strains in red are the avian isolate, A/Chicken/Indonesia/2A/2003 (CK2A), and the human isolate, A/Indonesia/5/2005 (IND5), used as backbones for constructing reverse genetic versions of viruses in this study. Full article ">Figure 2
RNP polymerase activity in HEK293T and DF-1 cells. RNP expression plasmids containing NP, PB1, PA and wild type PB2, or the corresponding PB2 526 mutant derived from CK2A or IND5, along with a firefly luciferase RNP reporter plasmid and the Renilla luciferase expressing plasmid, pRL-TK (as an internal control), were transfected into HEK293T cells. The transfected cells were incubated for 24 h at: 37 °C (A); or 33 °C (B). DF-1 cells were transfected with the same sets of RNP complex plasmids but with an avian specific RNP firefly reporter and Renilla luciferase control (C). DF-1 cells were incubated at 39 °C. Luciferase activities were measured at 24 h post transfection using a Dual-Luciferase Reporter Assay System (Promega). Firefly RNP activity was normalized against Renilla activity. RNP without PB2 was used as a negative control. Data represent mean normalized luciferase activity from three independent experiments. Error bars represent standard deviation calculated from three separate experiments. Statistical significance was analyzed by Student’s t-test. *** p < 0.001, ** p < 0.01. Full article ">Figure 3
Growth kinetics of H5N1 viruses in mammalian and avian cells. Reverse genetic (RG) versions of the human H5N1 isolate, A/Indonesia/5/05 (IND5), and a mutant virus containing 526K-PB2 (IND5-526K) were used to infect MDCK and DF-1 cells at an MOI of 0.001, or A549 cells at an MOI of 0.01 (A). Similarly, RG versions of the avian H5N1 isolate, A/Chicken/Indonesia/2A/03 (CK2A), and a mutant virus containing 526R-PB2 (CK2A-526R) were used to infect MDCK and DF-1 cells at an MOI of 0.001, or A549 cells at an MOI of 0.01 (B). Growth of IND5 (C) and CK2A (D) viruses at 33 °C was estimated in MDCK and A549 cells. After adsorption for 1 h, the cells were washed and overlaid with infection medium containing 1 µg/mL of TPCK-trypsin. At the indicated time points, culture supernatants were collected for virus titration by plaque assay in MDCK cells. Data represent mean viral titers from three independent experiments. Error bars represent standard deviation calculated from three separate experiments. Statistical significance was analyzed by one-way ANOVA, corrected by the Bonferroni post test: *** p < 0.001., h.p.i., h post infection. Full article ">Figure 4
Effect of PB2 526R on H5N1 viral RNA and protein synthesis. (A) A549 cells were infected with reverse genetic wild type IND5 or CK2A H5N1 viruses or mutant versions of these viruses containing either 526K or 526R PB2, respectively, at an MOI of 1. Total RNA was extracted at 4 or 8 h post infection and reverse transcription (RT) performed using uni-12 or oligo dT primers, to detect vRNA or mRNA, respectively. Expression of these two RNA species of NP was quantified by relative quantitative real time RT-PCR and normalized against the β-actin gene. Data represent mean relative NP expression levels from three independent experiments. Statistical significance was analyzed by one-way ANOVA, corrected by the Bonferroni post test: *** p < 0.001and * p < 0.05. (B) A549 cells were infected as above. Cells were lysed at 8 h post infection and expression levels of NP and PB2 estimated by Western blot using anti-NP (1:5000) and anti-PB2 (1:1000) antibodies. Expression levels of β-tubulin were detected using anti-β-tubulin antibody, and served as a loading control. Full article ">Figure 5
Effect of 526R PB2 on RNP activity in the presence of NEP and NP-PB2 interaction. Increasing amounts of NEP expression vector, or empty vector, were co-transfected with expression plasmids for the RNP complexes of IND5 and IND5-526K PB2 (A), or CK2A and CK2A-526R PB2 (B), together with RNP luciferase reporter and pRL-TK control plasmids, into HEK293T cells, followed by culture at 37 °C. Similarly, RNP polymerase activity with the RNP complexes of CK2A and CK2A-526R and increasing amounts of NEP expression was examined in DF-1 cells, cultured at 39 °C (C). Luciferase activities were measured at 24 h post transfection. Firefly RNP polymerase activity was normalized against Renilla activity, and RNP without PB2 was used as a negative control. Data represent mean normalized luciferase activity from three independent experiments. Error bars represent standard deviation from three separate experiments. Statistical significance was analyzed by Student’s t-test. *** p < 0.001. (D) Interaction between NP and PB2 in RNP complexes. Different sets of plasmids expressing PB1, PA, NP and Flag-tagged PB2 from IND5-526K or CK2A-526R, were transfected into HEK293T cells. At 48 h post transfection, cell lysates were prepared and rabbit polyclonal NP antibody used to co-precipitate PB2. Proteins from co-precipitated complexes were resolved using SDS-PAGE and detected by Western blot using anti-NP (1:5000) and anti-Flag (Sigma) (1:5000) antibodies. Full article ">Figure 6
Evaluation of H5N1 virus infection and replication in mice. Groups of four or six BALB/c mice, aged 4–6 weeks, were intranasally inoculated with 103 (A) or 102 (B) PFU of virus containing wild type IND5 or CK2A, or the 526K (IND5-526K) or 526R (CK2A-526R) mutant viruses, in 25ul PBS. Body weight and survival were monitored daily for 14 days post infection. Results of infection with high doses of virus (104, 105 and 106 PFU) are shown in the <a href="#app1-viruses-11-00292" class="html-app">Supplementary Materials (Figure S1)</a>. The MLD50 was calculated by the method of Reed and Muench [<a href="#B30-viruses-11-00292" class="html-bibr">30</a>]. (C) Replication efficiency of viruses in lung tissues of infected mice. To determine virus titers in mouse lung tissues, groups of three mice were infected with 103 PFU of the respective viruses described above and then euthanized at 72 h post infection, with lung tissues from each mouse being collected and homogenized for virus titration by plaque assay using MDCK cells. Error bars represent standard deviation from virus-infected mice mouse in the group. Statistical significance was analyzed by one-way ANOVA or Student’s t-test *** p < 0.001, ** p < 0.01 and * p < 0.05. Full article ">Figure 7
Comparison of effects of PB2 526R and 627K on Indonesian H5N1 virus replication. (A) Growth kinetics of reverse genetic versions of CK2A virus containing PB2 526R or 627K in avian cells. DF-1 cells were infected separately with CK2A, CK2A-526R or CK2A-627K RG virus and cultured at 39 °C. Culture supernatant was collected at the indicated time points and virus titrated by plaque assay in MDCK cells. (B) Growth competition assay: CK2A wild type versus CK2A-526R virus in avian cells. DF-1 cells were infected at an MOI of 0.001 with CK2A and CK2A-526R viruses, mixed at a ratio of 1:1. Continuous cultures were performed for four passages. Viral RNA was isolated from culture supernatants at 48 h post infection, and the PB2 gene amplified by RT-PCR and sequenced. Representative sequencing chromatograms from each passage are displayed; the genetic code for PB2 526R is AGA, whereas 526K is AAA. (C) Growth competition assay: CK2A wild type versus CK2A-627K virus in avian cells. The assay was conducted similarly to (B). Representative sequencing chromatograms are shown: the genetic code for PB2 627E is GAG, whereas 627K is AAG. (D) Growth kinetics of reverse genetic versions of CK2A virus containing 526R or 627K. A549 cells were infected separately with RG CK2A-526R or CK2A-627K viruses and cultured at 37 °C. Culture supernatant was collected at the indicated time points and virus titrated by plaque assay in MDCK cells. (E) Growth competition assay: CK2A-526R versus CK2A-627K virus in A549 cells. The procedure was performed as in (B). Sequencing chromatograms representing mixed populations of 526R/K and 627E/K are shown. h.p.i., hours post infection. Error bars represent standard deviation from three separate experiments. Statistical significance was analyzed by one-way ANOVA or Student’s t-test *** p < 0.001. Full article ">Figure 8
Effect of PB2 R288Q on viral replication and RNP activity in Indonesian H5N1 viruses. R288Q, alone and in combination with K526R, was introduced into the PB2 segment of the H5N1 avian isolate, A/Chicken/Indonesia/2A/2003, and alternatively, the back mutations Q288R and R526K, separately and in combination, were introduced into the PB2 of the H5N1 human isolate, A/Indonesia/5/2005. Plasmids of the minigenome system, consisting of NP, PB1, PA and wild type PB2, or the corresponding PB2 mutants derived from CK2A or IND5, together with a firefly RNP luciferase reporter plasmid and the Renilla luciferase expressing plasmid pRL-TK (as an internal control) were transfected into HEK293T cells. (A) Effect of R288Q, alone or in conjunction with K526R, on RNP polymerase activity in the background of other RNP proteins from A/Chicken/Indonesia/2A/2003. (B) Effect of Q288R, alone or in conjunction with R526K, on RNP polymerase activity, with other RNP proteins derived from A/Indonesia/5/2005. Luciferase activities were measured at 24 h post transfection using a Dual Luciferase Reporter Assay System (Promega). Firefly RNP activity was normalized against Renilla activity. RNP without PB2 was used as a negative control. Data represent mean normalized luciferase activity from three independent experiments. Statistical significance was analyzed by Student’s t-test. *** p < 0.001. (C) Growth kinetics of wild type CK2A virus and mutant viruses containing R288Q PB2 or R288Q/K526R PB2. A549 cells were infected with CK2A, CK2A-R288Q or CK2A-K526R/R288Q viruses at an MOI of 0.01 and cultured at 37 °C. Culture supernatants were collected at the indicated time points and virus titrated by plaque assay in MDCK cells. Error bars represent standard deviation from three separate experiments. Full article ">

11 pages, 1889 KiB

Open AccessArticle

Differential Innate Immune Responses Elicited by Nipah Virus and Cedar Virus Correlate with Disparate In Vivo Pathogenesis in Hamsters

by Tony Schountz, Corey Campbell, Kaitlyn Wagner, Joel Rovnak, Cynthia Martellaro, Blair L DeBuysscher, Heinz Feldmann and Joseph Prescott

Viruses 2019, 11(3), 291; https://doi.org/10.3390/v11030291 - 22 Mar 2019

Cited by 32 | Viewed by 5814

Abstract

Syrian hamsters (Mesocricetus auratus) are a pathogenesis model for the Nipah virus (NiV), and we sought to determine if they are also susceptible to the Cedar virus (CedPV). Following intranasal inoculation with CedPV, virus replication occurred in the lungs and spleens [...] Read more.

Syrian hamsters (Mesocricetus auratus) are a pathogenesis model for the Nipah virus (NiV), and we sought to determine if they are also susceptible to the Cedar virus (CedPV). Following intranasal inoculation with CedPV, virus replication occurred in the lungs and spleens of infected hamsters, a neutralizing antibody was produced in some hamsters within 8 days post-challenge, and no conspicuous signs of disease occurred. CedPV replicated to a similar magnitude as NiV-Bangladesh in type I IFN-deficient BHK-21 Syrian hamster fibroblasts but replicated 4 logs lower in type I IFN-competent primary Syrian hamster and human pulmonary endothelial cells, a principal target of henipaviruses. The coinfection of these cells with CedPV and NiV failed to rescue CedPV titers and did not diminish NiV titers, suggesting the replication machinery is virus-specific. Type I IFN response transcripts Ifna7, Ddx58, Stat1, Stat2, Ccl5, Cxcl10, Isg20, Irf7, and Iigp1 were all significantly elevated in CedPV-infected hamster endothelial cells, whereas Ifna7 and Iigp1 expression were significantly repressed during NiV infection. These results are consistent with the hypothesis that CedPV’s inability to counter the host type I IFN response may, in part, contribute to its lack of pathogenicity. Because NiV causes a fatal disease in Syrian hamsters with similarities to human disease, this model will provide valuable information about the pathogenic mechanisms of henipaviruses. Full article

(This article belongs to the Special Issue Viruses and Bats 2019)

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The cedar virus replication in hamsters is more robust via the intranasal route. The hamsters were challenged with 105 TCID50 CedPV via the intranasal (A) or intraperitoneal (B) routes. The tissues were homogenized, and the total RNA was extracted and used to quantitate CedPV viral RNA by qRT-PCR. At each time point, intranasal inoculation resulted in a more robust replication in the lungs, with the exception of day 14. No viral RNA was detected at day 28 postinoculation. The geometric means are plotted and error bars represent the 95% CI. Full article ">Figure 2
Intranasal inoculation leads to a more frequent seroconversion in hamsters. The hamsters intranasally challenged with CedPV as described in <a href="#viruses-11-00291-f001" class="html-fig">Figure 1</a> all seroconverted as measured by CedPV neutralization by day 14, whereas only 2 of 4 of those intraperitoneally challenged seroconverted, which had similar titers to intranasally inoculated hamsters. Full article ">Figure 3
CedPV and NiV replicate similarly in hamster BHK-21 cells. Regardless of MOI, CedPV and NiV replicate with similar kinetics and to similar titers in type I IFN-deficient BHK-21 cells. The data are represented as the geometric mean titers and 95% CI from the titration of the supernatant of infected monolayers of BHK-21 cells. A 2-way ANOVA with Bonferroni’s posttest was used to compare the conditions and showed no significant differences. Full article ">Figure 4
CedPV replicates poorly in primary hamster (A) and human (B) pulmonary endothelial cells. Monolayers of the cells were inoculated with CedPV or NiV at 0.1 MOI, and the supernatants were sampled for 3 days, at which time NiV replicated to more than 4 logs more than CedPV. The data are represented as the geometric mean titers and 95% CI. A 2-way ANOVA with Bonferroni’s posttest was used to compare the viruses (* p < 0.05, ** p < 0.01, and *** p < 0.001). Full article ">Figure 5
The coinfection of IFN-competent primary hamster endothelial cells does not alter virus replication kinetics. CedPV (A) or NiV (B) replication was measured by virus-specific qRT-PCR. A similar viral RNA abundance was measured regardless of MOI. The data are represented as the geometric mean titers and 95% CI. Full article ">Figure 6
The robust innate gene expression in primary hamster endothelial cells infected with CedPV: Hamster endothelial cells were infected with 0.1 MOI of CedPV or NiV, and the total RNA was extracted at 24 and 48 h. Real-time PCR was performed to determine the fold-change in gene expression. At 24 h, the expression of Ifna7, Isg20, and Iigp1 were not detected. At 48 h, Ccl5 expression was not detected in the NiV-infected cells. Full article ">

18 pages, 1424 KiB

Open AccessReview

Arthritogenic Alphavirus-Induced Immunopathology and Targeting Host Inflammation as A Therapeutic Strategy for Alphaviral Disease

by Helen Mostafavi, Eranga Abeyratne, Ali Zaid and Adam Taylor

Viruses 2019, 11(3), 290; https://doi.org/10.3390/v11030290 - 22 Mar 2019

Cited by 26 | Viewed by 6141

Abstract

Arthritogenic alphaviruses are a group of medically important arboviruses that cause inflammatory musculoskeletal disease in humans with debilitating symptoms, such as arthralgia, arthritis, and myalgia. The arthritogenic, or Old World, alphaviruses are capable of causing explosive outbreaks, with some viruses of major global [...] Read more.

Arthritogenic alphaviruses are a group of medically important arboviruses that cause inflammatory musculoskeletal disease in humans with debilitating symptoms, such as arthralgia, arthritis, and myalgia. The arthritogenic, or Old World, alphaviruses are capable of causing explosive outbreaks, with some viruses of major global concern. At present, there are no specific therapeutics or commercially available vaccines available to prevent alphaviral disease. Infected patients are typically treated with analgesics and non-steroidal anti-inflammatory drugs to provide often inadequate symptomatic relief. Studies to determine the mechanisms of arthritogenic alphaviral disease have highlighted the role of the host immune system in disease pathogenesis. This review discusses the current knowledge of the innate immune response to acute alphavirus infection and alphavirus-induced immunopathology. Therapeutic strategies to treat arthritogenic alphavirus disease by targeting the host immune response are also examined. Full article

(This article belongs to the Special Issue Viruses and Inflammation)

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12 pages, 1625 KiB

Open AccessReview

The Clinical Features, Pathogenesis and Methotrexate Therapy of Chronic Chikungunya Arthritis

by J. Kennedy Amaral, Peter C. Taylor, Mauro Martins Teixeira, Thomas E. “Tem” Morrison and Robert T. Schoen

Viruses 2019, 11(3), 289; https://doi.org/10.3390/v11030289 - 22 Mar 2019

Cited by 24 | Viewed by 6513

Abstract

Chikungunya fever (CHIKF) is an emerging viral infection that has spread widely, along with its Aedes vectors, throughout the tropics and beyond, causing explosive epidemics of acute illness and persistent disabling arthritis. The rheumatic symptoms associated with chikungunya virus (CHIKV) infection include polyarthralgia, [...] Read more.

Chikungunya fever (CHIKF) is an emerging viral infection that has spread widely, along with its Aedes vectors, throughout the tropics and beyond, causing explosive epidemics of acute illness and persistent disabling arthritis. The rheumatic symptoms associated with chikungunya virus (CHIKV) infection include polyarthralgia, polyarthritis, morning stiffness, joint edema, and erythema. Chronic CHIK arthritis (CCA) often causes severe pain and associated disability. The pathogenesis of CCA is not well understood. Proposed hypotheses include the persistence of a low level of replicating virus in the joints, the persistence of viral RNA in the synovium, and the induction of autoimmunity. In this review, we describe the main hypotheses of CCA pathogenesis, some of which support methotrexate (MTX) treatment which has been shown to be effective in preliminary studies in CCA. Full article

(This article belongs to the Special Issue Chikungunya Virus and (Re-) Emerging Alphaviruses)

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16 pages, 3482 KiB

Open AccessReview

Function, Architecture, and Biogenesis of Reovirus Replication Neoorganelles

by Raquel Tenorio, Isabel Fernández de Castro, Jonathan J. Knowlton, Paula F. Zamora, Danica M. Sutherland, Cristina Risco and Terence S. Dermody

Viruses 2019, 11(3), 288; https://doi.org/10.3390/v11030288 - 21 Mar 2019

Cited by 28 | Viewed by 11329

Abstract

Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release [...] Read more.

Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Reoviruses are common pathogens of mammals that have been linked to celiac disease and show promise for oncolytic applications. These viruses form nonenveloped, double-shelled virions that contain ten segments of double-stranded RNA. Replication organelles in reovirus-infected cells are nucleated by viral nonstructural proteins µNS and σNS. Both proteins partition the endoplasmic reticulum to form the matrix of these structures. The resultant membranous webs likely serve to anchor viral RNA–protein complexes for the replication of the reovirus genome and the assembly of progeny virions. Ongoing studies of reovirus replication organelles will advance our knowledge about the strategies used by viruses to commandeer host biosynthetic pathways and may expose new targets for therapeutic intervention against diverse families of pathogenic viruses. Full article

(This article belongs to the Special Issue Viruses Ten-Year Anniversary)

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The reovirus replication cycle. VI—viral inclusions; ER—endoplasmic reticulum. Full article ">Figure 2
Reovirus inclusions imaged by light and electron microscopy. (A–C) Human brain microvascular endothelial cells were infected with reovirus strain T1L M1-P208S for 24 h, and were fixed, permeabilized, and processed for immunofluorescence staining with a chicken anti-μNS polyclonal serum and a secondary antibody conjugated with Alexa 594 (green). This strain forms large, globular VIs. (A) The phase-contrast microscopy shows dark, dense, globular structures (asterisks) in the cytosol of reovirus-infected cells. (B) The localization of μNS by fluorescence microscopy confirms that the dense structures seen by phase-contrast microscopy are viral inclusions (asterisks). (C) The merging of phase-contrast and fluorescence microscopy images. (D,E) HeLa cells were infected with T1L M1-P208S and fixed at 24 h. (D) Ultrathin sections (~70 nm) of infected cells were imaged by transmission electron microscopy (TEM). A characteristic viral inclusion (VI) is shown. The VI contains mature virions (black arrowheads) and empty viral particles (white arrowheads). Mitochondria (mi), endoplasmic reticulum (ER) cisternae, and microtubules (arrows) surround the VI. N—nucleus. (E) VI as visualized by TEM of serial sections, 3D reconstruction, and image processing. The mitochondria (red) and ER cisternae (gold) surround a network of smooth membranes (light yellow) with mature virions (black) and empty viral particles (white). The nucleus is colored in blue and the microtubules in green. Scale bars are 10 μM in (A–C) and 250 nm in (D,E). Full article ">Figure 3
Reovirus inclusions contain ER membranes. (A,B) HeLa cells were infected with reovirus T1L M1-P208S for 14 h, frozen in liquid nitrogen, and sectioned at −120 °C. The thawed cryosections were processed for immunogold labeling with primary antibodies specific for two ER proteins—protein disulfide isomerase (PDI) (A) and calreticulin (CLT) (B)—and for secondary antibodies conjugated with 10 nm colloidal gold particles. The rough ER (RER) cisternae around the VIs and membranes inside the VIs are labeled with antibodies specific for ER proteins (white arrows in A and B). These membranes are in close contact with the viral particles (black arrows in B). V—viral particle. (C) Electron tomography (ET) of a single VI. A thawed cryosection was processed by single-tilt-axis ET, 3D reconstruction, and image processing. The 3D model shows that the VI is a collection of vesicles and tubules with viral particles attached to membranes (white arrows). RER—yellow; viral particles—light blue; mitochondria—red; nuclear membrane—dark blue; tubules and membrane fragments inside the inclusion—brown; vesicles inside the inclusion—orange. Scale bars are 500 nm in (A) and 200 nm in (B,C). Modified from Tenorio et al., 2018 [<a href="#B31-viruses-11-00288" class="html-bibr">31</a>]. Full article ">Figure 4
Model of ER remodeling and VI biogenesis. The ER in uninfected cells is composed of sheets and tubules. (A) In reovirus-infected cells, σNS binds to the ER tubules and transforms them into thin structures. (B) µNS binds to these thin tubules and triggers their fragmentation. Small tubules and vesicles coalesce to form the VI. The schematics at the bottom demonstrate how σNS and µNS might remodel the ER. NUC—nucleus. Modified from Tenorio et al., 2018 [<a href="#B31-viruses-11-00288" class="html-bibr">31</a>]. Full article ">

9 pages, 2566 KiB

Open AccessArticle

Non-Pathogenic Mopeia Virus Induces More Robust Activation of Plasmacytoid Dendritic Cells than Lassa Virus

by Justine Schaeffer, Stéphanie Reynard, Xavier Carnec, Natalia Pietrosemoli, Marie-Agnès Dillies and Sylvain Baize

Viruses 2019, 11(3), 287; https://doi.org/10.3390/v11030287 - 21 Mar 2019

Cited by 7 | Viewed by 3419

Abstract

Lassa virus (LASV) causes a viral haemorrhagic fever in humans and is a major public health concern in West Africa. An efficient immune response to LASV appears to rely on type I interferon (IFN-I) production and T-cell activation. We evaluated the response of [...] Read more.

Lassa virus (LASV) causes a viral haemorrhagic fever in humans and is a major public health concern in West Africa. An efficient immune response to LASV appears to rely on type I interferon (IFN-I) production and T-cell activation. We evaluated the response of plasmacytoid dendritic cells (pDC) to LASV, as they are an important and early source of IFN-I. We compared the response of primary human pDCs to LASV and Mopeia virus (MOPV), which is very closely related to LASV, but non-pathogenic. We showed that pDCs are not productively infected by either MOPV or LASV, but produce IFN-I. However, the activation of pDCs was more robust in response to MOPV than LASV. In vivo, pDC activation may support the control of viral replication through IFN-I production, but also improve the induction of a global immune response. Therefore, pDC activation could play a role in the control of LASV infection. Full article

(This article belongs to the Special Issue Medical Advances in Viral Hemorrhagic Fever Research)

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pDC infection by MOPV and LASV. (a) pDCs were infected with MOPV or LASV (MOI = 0.1) and infectious particles in the culture medium quantified. (b,c) pDCs were infected with MOPV or LASV (MOI = 0.1) for 1 h (day 0), 1 day, or 2 days. Viral RNA in the culture medium (b) or inside the cells (c) was quantified by RT-qPCR. (d–g) VeroE6 cells were infected with MOPV-Zflag or LASV-Zflag (MOI = 0.3). After 24 h, pDCs were added to the cells, or infected with MOPV-Zflag or LASV-Zflag (MOI = 0.1). 24 h later, cells were stained for phenotypic markers and the Z protein. Conditions were: infected VeroE6 ("veroE6"), VeroE6 cultured with pDCs ("coC"), and infected pDCs (“pDC”). Z-positive pDCs (d–f) and VeroE6 cells (e–g) were quantified by flow cytometry. All data are presented as the mean and standard error of mean (SEM) of three independent experiments. ANOVA on Ranks followed by pairwise comparisons (Tukey test) were performed. Differences are significant for p < 0.05. When significant, P values of the ANOVA are indicated on the graph. Significant pairwise comparisons are indicated by a star (*). Full article ">Figure 2
IFN-I production in LASV-infected pDCs is less long-lasting than that of MOPV-infected pDCs. (a) pDCs were infected with MOPV (MOI = 2). Every 6 h, from 0 to 24 hpi, IFN-I mRNA was quantified by RT-qPCR. Data are presented as the fold change in the mRNA/GAPDH ratio in MOPV-infected pDCs relative to uninfected pDCs. (b–d) pDCs were cultured for 7 h or 16 h in culture medium (mock), R848 (1 µg/mL), MOPV, or LASV (MOI = 2). IFNα1 (b), IFNα2 (c) and IFNβ (d) mRNAs were quantified by RT-qPCR. Data shown are the means and SEM of three (a), four (b–d – 7 hpi), or seven (b–d – 16 hpi) independent experiments. ANOVA on Ranks followed by pairwise comparisons (Tukey test) were performed. Differences are significant for p < 0.05. Significant pairwise comparisons are indicated by a star (*). Full article ">Figure 3
MOPV- and LASV-infected pDCs show different patterns of activation. (a) pDCs were cultured for 16 h with culture medium (mock), R848 (1 µg/mL), MOPV, or LASV (MOI = 2). Protein levels were quantified using the Luminex assay. Data are presented as the means and SEM of five independent experiments. Wilcoxon tests were performed, and differences are significant for p < 0.05 (*) or p < 0.01 (**). (b,c) pDCs were cultured for 12 h in culture medium (mock), MOPV, or LASV (MOI = 1). Cellular mRNA from three independent experiments was quantified by poly-A amplification and next-generation sequencing. (b) Data show the differential expression of genes in MOPV relative to LASV (MOPV/LASV) infected cells or in MOPV or LASV infected cells relative to mock (1, 2, 3, and mean). Genes shown in this figure displayed significant differences of expression (adjusted p < 0.05). (c) MA plots for all pairwise comparison of data sets (MOPV/mock, LASV/mock and MOPV/LASV). Red dots indicate significantly different genes between the two conditions. Triangles correspond to features having a too low/high fold change to be displayed on the plot. Full article ">

15 pages, 1285 KiB

Open AccessReview

Caliciviridae Other Than Noroviruses

by Ulrich Desselberger

Viruses 2019, 11(3), 286; https://doi.org/10.3390/v11030286 - 21 Mar 2019

Cited by 44 | Viewed by 7702

Abstract

Besides noroviruses, the Caliciviridae family comprises four other accepted genera: Sapovirus, Lagovirus, Vesivirus, and Nebovirus. There are six new genera proposed: Recovirus, Valovirus, Bavovirus, Nacovirus, Minovirus, and Salovirus. All Caliciviridae have closely related genome structures, but are genetically and antigenically [...] Read more.

Besides noroviruses, the Caliciviridae family comprises four other accepted genera: Sapovirus, Lagovirus, Vesivirus, and Nebovirus. There are six new genera proposed: Recovirus, Valovirus, Bavovirus, Nacovirus, Minovirus, and Salovirus. All Caliciviridae have closely related genome structures, but are genetically and antigenically highly diverse and infect a wide range of mammalian host species including humans. Recombination in nature is not infrequent for most of the Caliciviridae, contributing to their diversity. Sapovirus infections cause diarrhoea in pigs, humans and other mammalian hosts. Lagovirus infections cause systemic haemorrhagic disease in rabbits and hares, and vesivirus infections lead to lung disease in cats, vesicular disease in swine, and exanthema and diseases of the reproductive system in large sea mammals. Neboviruses are an enteric pathogen of cattle, differing from bovine norovirus. At present, only a few selected caliciviruses can be propagated in cell culture (permanent cell lines or enteroids), and for most of the cultivatable caliciviruses helper virus-free, plasmid only-based reverse genetics systems have been established. The replication cycles of the caliciviruses are similar as far as they have been explored: viruses interact with a multitude of cell surface attachment factors (glycans) and co-receptors (proteins) for adsorption and penetration, use cellular membranes for the formation of replication complexes and have developed mechanisms to circumvent innate immune responses. Vaccines have been developed against lagoviruses and vesiviruses, and are under development against human noroviruses. Full article

(This article belongs to the Special Issue Noroviruses)

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17 pages, 3495 KiB

Open AccessArticle

Initial Characterization of the Epstein–Barr Virus BSRF1 Gene Product

by Yusuke Yanagi, H. M. Abdullah Al Masud, Takahiro Watanabe, Yoshitaka Sato, Fumi Goshima, Hiroshi Kimura and Takayuki Murata

Viruses 2019, 11(3), 285; https://doi.org/10.3390/v11030285 - 21 Mar 2019

Cited by 13 | Viewed by 4301

Abstract

Epstein–Barr virus (EBV) is a ubiquitous virus that causes infectious mononucleosis and several types of cancer, such as Burkitt lymphoma, T/NK-cell lymphoma, and nasopharyngeal carcinoma. As a herpesvirus, it encodes more than 80 genes, many of which have not been characterized. EBV Bam [...] Read more.

Epstein–Barr virus (EBV) is a ubiquitous virus that causes infectious mononucleosis and several types of cancer, such as Burkitt lymphoma, T/NK-cell lymphoma, and nasopharyngeal carcinoma. As a herpesvirus, it encodes more than 80 genes, many of which have not been characterized. EBV BamHI S rightward reading frame 1 (BSRF1) encodes a tegument protein that, unlike its homologs herpes simplex virus unique long 51 (UL51) and human cytomegalovirus UL71, has not been extensively investigated. To examine the role of BSRF1, we prepared knockout and revertant strains using the bacterial artificial chromosome system. Unexpectedly, the disruption of the gene had little or no effect on EBV lytic replication and the transformation of primary B cells. However, the knockdown of BSRF1 in B95-8 cells decreased progeny production. An immunofluorescence assay revealed that BSRF1 localized to the Golgi apparatus in the cytoplasm, as did its homologs. BSRF1 also associated with BamHI G leftward reading frame 3.5 (BGLF3.5), BamHI B rightward reading frame 2 (BBRF2), and BamHI A leftward reading frame 1 (BALF1), and BALF1 was incorporated into the tegument fraction with BSRF1. Taken together, our results indicate that BSRF1 plays a role in secondary envelopment or virion egress in the cytoplasm, as do its homolog genes. Full article

(This article belongs to the Section Animal Viruses)

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16 pages, 2344 KiB

Open AccessArticle

Broad Bactericidal Activity of the Myoviridae Bacteriophage Lysins LysAm24, LysECD7, and LysSi3 against Gram-Negative ESKAPE Pathogens

by Nataliia P. Antonova, Daria V. Vasina, Anastasiya M. Lendel, Evgeny V. Usachev, Valentine V. Makarov, Alexander L. Gintsburg, Artem P. Tkachuk and Vladimir A. Gushchin

Viruses 2019, 11(3), 284; https://doi.org/10.3390/v11030284 - 21 Mar 2019

Cited by 60 | Viewed by 5634

Abstract

The extremely rapid spread of multiple-antibiotic resistance among Gram-negative pathogens threatens to move humankind into the so-called “post-antibiotic era” in which the most efficient and safe antibiotics will not work. Bacteriophage lysins represent promising alternatives to antibiotics, as they are capable of digesting [...] Read more.

The extremely rapid spread of multiple-antibiotic resistance among Gram-negative pathogens threatens to move humankind into the so-called “post-antibiotic era” in which the most efficient and safe antibiotics will not work. Bacteriophage lysins represent promising alternatives to antibiotics, as they are capable of digesting bacterial cell wall peptidoglycans to promote their osmotic lysis. However, relatively little is known regarding the spectrum of lysin bactericidal activity against Gram-negative bacteria. In this study, we present the results of in vitro activity assays of three putative and newly cloned Myoviridae bacteriophage endolysins (LysAm24, LysECD7, and LysSi3). The chosen proteins represent lysins with diverse domain organization (single-domain vs. two-domain) and different predicted mechanisms of action (lysozyme vs. peptidase). The enzymes were purified, and their properties were characterized. The enzymes were tested against a panel of Gram-negative clinical bacterial isolates comprising all Gram-negative representatives of the ESKAPE group. Despite exhibiting different structural organizations, all of the assayed lysins were shown to be capable of lysing Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Salmonella typhi strains. Less than 50 μg/mL was enough to eradicate growing cells over more than five orders of magnitude. Thus, LysAm24, LysECD7, and LysSi3 represent promising therapeutic agents for drug development. Full article

(This article belongs to the Special Issue Biotechnological Applications of Phage and Phage-Derived Proteins)

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Figure 1

Figure 1
Domain organization, purification, and physicochemical properties of LysAm24, LysECD7, and LysSi3. (a) Putative domain organization of LysAm24, LysECD7, and LysSi3 predicted from the deduced amino acid sequences. The prediction was done with the protein BLAST search (<a href="https://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">https://blast.ncbi.nlm.nih.gov/Blast.cgi</a>). (b) SDS-PAGE gel analysis of purified endolysins. The PageRuler Broad range Unstained Protein Ladder (Thermo Scientific, Vilnius, Lithuania) was used (c) Evaluation of the hydrodynamic diameter of E. coli-produced endolysins by DLS analysis. Statistical distribution of particle size by number. The data are presented as the mean values of three measurements ± SD. (d) Temperature dependence of the total light scattering intensity (upper panel) and the hydrodynamic diameters of the particles (lower panel). The data are presented as the mean values of three measurements made with an interval of 15 s ± SD. Full article ">Figure 2
Bactericidal activity and biochemical properties of LysAm24, LysECD7, and LysSi3. (a) Bactericidal activity of different concentrations of LysAm24 against A. baumannii Ts 50-16, LysECD7 against E. coli M15 and LysSi3 against the A. baumannii Ts 50-16 in exponential phase. Cell cultures without incubation with endolysins were used as controls. The number of surviving cells after 18 h is expressed as reduction in log10 CFU/mL compared to the control. NS, no statistical significance of the data compared to the untreated culture is observed (p > 0.05, Mann–Whitney test). (b) Activity of lysins against both exponentially growing cells (Exp) and stationary-phase cells (Stat) of A. baumannii Ts 50-16 without and in the presence of EDTA. The residual activity after 18 h of growth compared to the untreated culture is shown. NS, no statistical significance of the data compared to the exponential growth phase is observed (p > 0.05, Mann–Whitney test). (c) Effects of salts on the bactericidal activity against A. baumannii Ts 50-16. The residual activity after 18 h of growth compared to the untreated culture is shown. NS, no statistical significance of the data compared to the untreated culture is observed (p > 0.05, Mann–Whitney test). For all experiments, the mean values are shown (± standard error of the mean (SEM)) from three independent experiments. Asterisk (*) indicates significant effect on bactericidal activity. Full article ">Figure 3
Effects of pH and EDTA on the antibacterial activity of LysAm24, LysECD7, and LysSi3 against exponentially growing cells. The residual activity after 18 h of growth compared to the untreated culture is shown. For all experiments, the mean values are shown (±SEM) from three independent experiments. NS, no statistical significance of the data compared to the untreated control culture (p > 0.05, Mann–Whitney test). All other data points were statistically significant compared to the untreated control culture. Full article ">Figure 4
Endolysin activity against different strains of Gram-negative bacteria. Bacterial count (cfu/mL) was used as the method of choice for all strains: (a) LysAm24, (b) LysECD7, (c) LysSi3. For all experiments, the mean values are shown (±SEM) from three independent experiments. NA, no bactericidal activity was detected. All other data points were statistically significant compared to the untreated control culture (p < 0.05, Mann–Whitney test). Full article ">

17 pages, 6675 KiB

Open AccessHypothesis

RNA Back and Forth: Looking through Ribozyme and Viroid Motifs

by Marie-Christine Maurel, Fabrice Leclerc, Jacques Vergne and Giuseppe Zaccai

Viruses 2019, 11(3), 283; https://doi.org/10.3390/v11030283 - 21 Mar 2019

Cited by 9 | Viewed by 4203

Abstract

Current cellular facts allow us to follow the link from chemical to biochemical metabolites, from the ancient to the modern world. In this context, the “RNA world” hypothesis proposes that early in the evolution of life, the ribozyme was responsible for the storage [...] Read more.

Current cellular facts allow us to follow the link from chemical to biochemical metabolites, from the ancient to the modern world. In this context, the “RNA world” hypothesis proposes that early in the evolution of life, the ribozyme was responsible for the storage and transfer of genetic information and for the catalysis of biochemical reactions. Accordingly, the hammerhead ribozyme (HHR) and the hairpin ribozyme belong to a family of endonucleolytic RNAs performing self-cleavage that might occur during replication. Furthermore, regarding the widespread occurrence of HHRs in several genomes of modern organisms (from mammals to small parasites and elsewhere), these small ribozymes have been regarded as living fossils of a primitive RNA world. They fold into 3D structures that generally require long-range intramolecular interactions to adopt the catalytically active conformation under specific physicochemical conditions. By studying viroids as plausible remains of ancient RNA, we recently demonstrated that they replicate in non-specific hosts, emphasizing their adaptability to different environments, which enhanced their survival probability over the ages. All these results exemplify ubiquitous features of life. Those are the structural and functional versatility of small RNAs, ribozymes, and viroids, as well as their diversity and adaptability to various extreme conditions. All these traits must have originated in early life to generate novel RNA populations. Full article

(This article belongs to the Special Issue Viroid-2018: International Conference on Viroids and Viroid-Like RNAs)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
RNA 2D structures of Avocado Sun Blotch Viroid (ABSVd) (−) and ASBVd (+) (top and bottom, respectively). The full length genome of ASBVd can fold into 2D structures that preserve the hammerhead ribozyme (HHR) motif (regions in black) in both the (−) and (+) strands; the HHR motif of ASBVd (−) is more stable, with 3 base-pairs in stem III but only two base-pairs in ASBVd (+). Full article ">Figure 2
TGGE (Thermal Gradient Gel Electrophoresis) and melting curves of the monomeric forms of ASBVd: mASBVd (+) and mASBVd (−) transcripts. (A) TGGE analysis performed by native gel electrophoresis 8% PAGE in 0.2 TBE Buffer. (B) TGGE analysis performed by native gel electrophoresis 8% PAGE in 0.2 TBE Buffer containing 20 mM magnesium acetate. Migration was monitored between 20 °C and 65 °C. The arrows denote the transition temperatures. Derivative absorbance melting profile were determined either in 150 mM KCL and 10mM sodium cacodylate (pH 7.2). (C) or with in addition, 100 mM MgCl<math display="inline"><semantics> <msub> <mrow/> <mn>2</mn> </msub> </semantics></math> (D) or 1M NaCl. (E) The first derivative profiles are shown with a thick curve for mASBVd (+) and a thin curve for mASBVd (−). The apparatus fixes the temperature gradient, which is transferred from left to right on the figure; the arrows indicate the transition temperatures that correspond to the conformational changes. From Delan-Forino et al. [<a href="#B70-viruses-11-00283" class="html-bibr">70</a>]. Full article ">Figure 3
(A) Normalized forward scattered small-angle neutron scattering (SANS) intensity for HHR (−) in the presence of Mg++ at the corresponding temperature on the plot below. The values at 1.00 and 2.00 correspond to the intensity expected for the monomer (M) and dimer (D), respectively. (B) Normalized forward scattered SANS intensity for ASBVd (−) in presence (filled circles) and absence (open circles) of Mg++, at the corresponding temperature on the plot below. The data indicate a small amount of dimer formation at the lower temperature. (C) Arrhenius plots of HHR (−)/Mg rate of dimer dissociation (open circles, calculated from (A), and the ribozyme’s catalytic activity (filled circles), revealing parallel behaviour. Modified from Leclerc et al. [<a href="#B33-viruses-11-00283" class="html-bibr">33</a>]. Full article ">Figure 4
3D Model of self-association for the hammerhead ribozyme (HHR) from ASBVd(−). (A) 2D structures of the two monomers as they are in the HHR dimer. (B) 3D structures of the two monomers as they are in the HHR dimer. (C) Zoom in on the regions involved in the monomer-monomer interactions and in the 3D contacts within the first monomer (top). (D) 3D structure of the HHR dimer. The long-range intramolecular contacts are indicated in magenta and the cleavage site in green. The paired regions are blue (first monomer) and red (second monomer). Modified from Leclerc et al. [<a href="#B33-viruses-11-00283" class="html-bibr">33</a>]. Full article ">Figure 5
Northern blot analyses (by hybridization using riboprobes corresponding to the replicated ASBVd (−) (A) or (+) (B)) of the persistence of mASBVd in S. cerevisiae. (A) mASBVd (+) evidenced by riboprobe (−) and (B) mASBVd (−) evidenced by riboprobe (+) (cm: circular monomer; lm: linear monomer). (A,B) Total RNAs were extracted from the YCDF7P, YCDF8P(+), and YCDF8P(−) strains. From Delan-Forino et al. [<a href="#B71-viruses-11-00283" class="html-bibr">71</a>]. Full article ">Figure 6
Analysis of ASBVd replication in the filamentous cyanobacteria Anabaena. RNAs extracted from Anabaena harboring either the empty pRL25 plasmid (3,7), pRLASBVd (−) which is the plasmid recombinant ASBVd (−) derived from the pRL25 plasmid (5, 9) or pRLASBVd (+) (4, 8). Northern blot analysis by hybridization using riboprobes corresponding to the replicated ASBVd (−) (A) or (+) (B). Lane 5: linear replicate (+) detected by riboprobe (−) when RNAs/ pRLASBVd(−) was loaded. Lane 8: linear replicate (−) detected by riboprobe (+) when RNAs /pRLASBVd(+) was loaded. Modified from Latifi et al. [<a href="#B73-viruses-11-00283" class="html-bibr">73</a>]. Full article ">Figure 7
A model for the evolutionary path of the transition from proviroids (RNA World) to viroids (DNA World). In a clay mineral environment (RNA World), the proviroids are suboptimal catalysts that retain an optimal template activity. The catalytic activity can be increased by “bonded” dimerization (double-hammerhead structures). In a host (DNA World), “virulent” viroids are efficient as catalysts and suboptimal templates, while “latent” viroids (ASBVd) have a regulatory control of their catalytic activity by “non-bonded” dimerization (non-bonded dimeric structures). The bubbles indicate the enzymes required for the replication cycle. Full article ">

15 pages, 10360 KiB

Open AccessArticle

A Single V672F Substitution in the Spike Protein of Field-Isolated PEDV Promotes Cell–Cell Fusion and Replication in VeroE6 Cells

by Asawin Wanitchang, Janya Saenboonrueng, Challika Kaewborisuth, Kanjana Srisutthisamphan and Anan Jongkaewwattana

Viruses 2019, 11(3), 282; https://doi.org/10.3390/v11030282 - 20 Mar 2019

Cited by 9 | Viewed by 5382

Abstract

While porcine epidemic diarrhea virus (PEDV) infects and replicates in enterocytes lining villi of neonatal piglets with high efficiency, naturally isolated variants typically grow poorly in established cell lines, unless adapted by multiple passages. Cells infected with most cell-adapted PEDVs usually displayed large [...] Read more.

While porcine epidemic diarrhea virus (PEDV) infects and replicates in enterocytes lining villi of neonatal piglets with high efficiency, naturally isolated variants typically grow poorly in established cell lines, unless adapted by multiple passages. Cells infected with most cell-adapted PEDVs usually displayed large syncytia, a process triggered by the spike protein (S). To identify amino acids responsible for S-mediated syncytium formation, we constructed and characterized chimeric S proteins of the cell-adapted variant, YN144, in which the receptor binding domain (RBD) and S1/S2 cleavage site were replaced with those of a poorly culturable field isolate (G2). We demonstrated that the RBD, not the S1/S2 cleavage site, is critical for syncytium formation mediated by chimeric S proteins. Further mutational analyses revealed that a single mutation at the amino acid residue position 672 (V672F) could enable the chimeric S with the entire RBD derived from the G2 strain to trigger large syncytia. Moreover, recombinant PEDV viruses bearing S of the G2 strain with the single V672F substitution could induce extensive syncytium formation and replicate efficiently in VeroE6 cells stably expressing porcine aminopeptidase N (VeroE6-APN). Interestingly, we also demonstrated that while the V672F mutation is critical for the syncytium formation in VeroE6-APN cells, it exerts a minimal effect in Huh-7 cells, thereby suggesting the difference in receptor preference of PEDV among host cells. Full article

(This article belongs to the Special Issue Porcine Viruses 2019)

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7 pages, 591 KiB

Open AccessCommunication

Hepatitis A Strain Linked to the European Outbreaks During Gay Events between 2016 and 2017, Identified in a Brazilian Homosexual Couple in 2017

by Vinicius M. Mello, Barbara V. Lago, Paulo S. F. Sousa, Francisco C. A. Mello, Caroline B. Souza, Laura C. M. Pinto, Cleber F. Ginuino, Carlos A. S. Fernandes, Shirlei F. Aguiar, Lívia M. Villar, Elisabeth Lampe, Juliana G. Melgaço and Lia L. Lewis-Ximenez

Viruses 2019, 11(3), 281; https://doi.org/10.3390/v11030281 - 20 Mar 2019

Cited by 11 | Viewed by 3386

Abstract

Hepatitis A virus (HAV) outbreaks among men who have sex with men (MSM) have been reported worldwide and associated primarily with sexual transmission through oral-anal sex. Here, we provide the molecular and evolutionary description of a European strain, linked to HAV outbreaks among [...] Read more.

Hepatitis A virus (HAV) outbreaks among men who have sex with men (MSM) have been reported worldwide and associated primarily with sexual transmission through oral-anal sex. Here, we provide the molecular and evolutionary description of a European strain, linked to HAV outbreaks among MSM, detected in a Brazilian homosexual couple. Bayesian analysis provided evidence that the viral isolates were introduced in Brazil from Spain between the end of 2016 and the beginning of 2017. Full article

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14 pages, 1525 KiB

Open AccessReview

Host Determinants of MERS-CoV Transmission and Pathogenesis

by W. Widagdo, Syriam Sooksawasdi Na Ayudhya, Gadissa B. Hundie and Bart L. Haagmans

Viruses 2019, 11(3), 280; https://doi.org/10.3390/v11030280 - 19 Mar 2019

Cited by 56 | Viewed by 11701

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic pathogen that causes respiratory infection in humans, ranging from asymptomatic to severe pneumonia. In dromedary camels, the virus only causes a mild infection but it spreads efficiently between animals. Differences in the behavior of [...] Read more.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic pathogen that causes respiratory infection in humans, ranging from asymptomatic to severe pneumonia. In dromedary camels, the virus only causes a mild infection but it spreads efficiently between animals. Differences in the behavior of the virus observed between individuals, as well as between humans and dromedary camels, highlight the role of host factors in MERS-CoV pathogenesis and transmission. One of these host factors, the MERS-CoV receptor dipeptidyl peptidase-4 (DPP4), may be a critical determinant because it is variably expressed in MERS-CoV-susceptible species as well as in humans. This could partially explain inter- and intraspecies differences in the tropism, pathogenesis, and transmissibility of MERS-CoV. In this review, we explore the role of DPP4 and other host factors in MERS-CoV transmission and pathogenesis—such as sialic acids, host proteases, and interferons. Further characterization of these host determinants may potentially offer novel insights to develop intervention strategies to tackle ongoing outbreaks. Full article

(This article belongs to the Special Issue MERS-CoV)

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Schematic figure depicting four structural proteins of Middle East respiratory syndrome coronavirus (MERS-CoV), i.e., S, E, M, and N proteins (A); a cartoon representation of MERS-CoV S1 protein binding to DPP4 (PDB code 4L72) (B). The S protein consists of the S1 and S2 subunits. The α/β hydrolase domain of DPP4 is indicated in red, β-propeller domain in green, while part of the MERS-CoV S1 protein is shown in blue. Full article ">Figure 2
Schematic overview of viral RNA and infectious virus shedding of MERS-CoV-inoculated dromedary camels, pigs, and rabbits. Each data point represents the average data from previous experiments [<a href="#B17-viruses-11-00280" class="html-bibr">17</a>,<a href="#B33-viruses-11-00280" class="html-bibr">33</a>,<a href="#B84-viruses-11-00280" class="html-bibr">84</a>]. Viral RNA is measured in TCID50/mL genome equivalents, while infectious virus is expressed in TCID50/mL. Full article ">Figure 3
Schematic representation of DPP4 expression and MERS-CoV-recognized α2,3-sialic acid glycotopes in the respiratory tract of dromedary camel, pig, rabbit, human, and sheep. Full article ">Figure 4
MERS-CoV infection in the lungs of asymptomatic-to-mild (left panel) and severe-to-fatal cases (right panel). Shown is a hypothetical model with two critical host determinants, DPP4 and interferon, differentially expressed in asymptomatic-to-mild and severe-to-fatal MERS-CoV infection. Full article ">

10 pages, 934 KiB

Open AccessCommunication

A Novel Orthohepadnavirus Identified in a Dead Maxwell’s Duiker (Philantomba maxwellii) in Taï National Park, Côte d’Ivoire

by Jan F. Gogarten, Markus Ulrich, Nishit Bhuva, Joel Garcia, Komal Jain, Bohyun Lee, Therese Löhrich, Alexandra Oleynik, Emmanuel Couacy-Hymann, Terence Fuh Neba, Nischay Mishra, Thomas Briese, Sébastien Calvignac-Spencer, W. Ian Lipkin and Fabian H. Leendertz

Viruses 2019, 11(3), 279; https://doi.org/10.3390/v11030279 - 19 Mar 2019

Cited by 6 | Viewed by 3939

Abstract

New technologies enable viral discovery in a diversity of hosts, providing insights into viral evolution. We used one such approach, the virome capture sequencing for vertebrate viruses (VirCapSeq-VERT) platform, on 21 samples originating from six dead Maxwell’s duikers (Philantomba maxwellii) from [...] Read more.

New technologies enable viral discovery in a diversity of hosts, providing insights into viral evolution. We used one such approach, the virome capture sequencing for vertebrate viruses (VirCapSeq-VERT) platform, on 21 samples originating from six dead Maxwell’s duikers (Philantomba maxwellii) from Taï National Park, Côte d’Ivoire. We detected the presence of an orthohepadnavirus in one animal and characterized its 3128 bp genome. The highest viral copy numbers were detected in the spleen, followed by the lung, blood, and liver, with the lowest copy numbers in the kidney and heart; the virus was not detected in the jejunum. Viral copy numbers in the blood were in the range known from humans with active chronic infections leading to liver histolytic damage, suggesting this virus could be pathogenic in duikers, though many orthohepadnaviruses appear to be apathogenic in other hosts, precluding a formal test of this hypothesis. The virus was not detected in 29 other dead duiker samples from the Côte d’Ivoire and Central African Republic, suggesting either a spillover event or a low prevalence in these populations. Phylogenetic analysis placed the virus as a divergent member of the mammalian clade of orthohepadnaviruses, though its relationship to other orthohepadnaviruses remains uncertain. This represents the first orthohepadnavirus described in an artiodactyl. We have tentatively named this new member of the genus Orthohepadnavirus (family Hepadnaviridae), Taï Forest hepadnavirus. Further studies are needed to determine whether it, or some close relatives, are present in a broader range of artiodactyls, including livestock. Full article

(This article belongs to the Section Animal Viruses)

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(A) The genome organization of Taï Forest hepadnavirus. The innermost circles represent the GC (blue) and AT (green) content along a 50 nt sliding window. (B) Phylogenetic relationship of Taï Forest hepadnavirus to other viruses of the family Hepadnaviridae. Maximum likelihood phylogeny constructed using the amino acid sequence of the polymerase ORF from a range of vertebrate hepadnaviruses; and (C) using the core and (D) the surface protein ORFs in comparison to the mammalian orthohepadnaviruses; the common name of the host is indicated at the branch labels, along with each virus’s accession number. GenBank sequences were aligned to Taï Forest hepadnavirus ORF sequences using MAFFT (v7.307) and we selected conserved blocks using Gblocks, as implemented in SeaView V4 [<a href="#B28-viruses-11-00279" class="html-bibr">28</a>]. PhyML with smart model selection [<a href="#B29-viruses-11-00279" class="html-bibr">29</a>], and the Bayesian Information Criterion and subtree pruning and regrafting (SPR) was applied as the tree improvement approach, with otherwise default settings for tree building (selected models: polymerase ORF = LG + G + I + F; core ORF = JTT + G; surface ORF = JTT + G + F). We estimated the best-fitting root of these phylogenies using the heuristic residual mean squared function in the program TempEst, which minimizes the variance of root-to-tip distances [<a href="#B30-viruses-11-00279" class="html-bibr">30</a>]. To further assess the confidence in our phylogenetic trees, BMCMC analyses were run on each amino acid alignment using BEAST v1.10.4 under the assumption of a relaxed log-normal molecular clock and with tree shape modeled according to a birth-death speciation model and the amino acids substitution model supported by PhyML’s smart model selection [<a href="#B31-viruses-11-00279" class="html-bibr">31</a>]. We examined the output of three runs for convergence and appropriate sampling of the posterior using Tracer v1.7.1 [<a href="#B32-viruses-11-00279" class="html-bibr">32</a>] before merging runs using LogCombiner v1.10.4 [<a href="#B33-viruses-11-00279" class="html-bibr">33</a>]. The best representative tree was then identified from the posterior set of trees and annotated with TreeAnnotator v1.10.4 (distributed with BEAST). Branch support was assessed using Shimodaira-Hasegawa-like approximate likelihood ratio tests (SH-like aLRT), with branches supported by SH-like aLRT values < 0.95 and/or posterior probabilities <0.95 in the Bayesian Markov chain Monte Carlo tree indicated in gray. Branch lengths are representative of substitutions per site. SH-like aLRT values are indicated at each node. Full article ">

13 pages, 2239 KiB

Open AccessArticle

Early Transcriptional Response to DNA Virus Infection in Sclerotinia sclerotiorum

by Feng Ding, Jiasen Cheng, Yanping Fu, Tao Chen, Bo Li, Daohong Jiang and Jiatao Xie

Viruses 2019, 11(3), 278; https://doi.org/10.3390/v11030278 - 19 Mar 2019

Cited by 14 | Viewed by 3923

Abstract

We previously determined that virions of Sclerotinia sclerotiorum hypovirulence associated DNA virus 1 (SsHADV-1) could directly infect hyphae of Sclerotinia sclerotiorum, resulting in hypovirulence of the fungal host. However, the molecular mechanisms of SsHADV-1 virions disruption of the fungal cell wall barrier [...] Read more.

We previously determined that virions of Sclerotinia sclerotiorum hypovirulence associated DNA virus 1 (SsHADV-1) could directly infect hyphae of Sclerotinia sclerotiorum, resulting in hypovirulence of the fungal host. However, the molecular mechanisms of SsHADV-1 virions disruption of the fungal cell wall barrier and entrance into the host cell are still unclear. To investigate the early response of S. sclerotiorum to SsHADV-1 infection, S. sclerotiorum hyphae were inoculated with purified SsHADV-1 virions. The pre- and post-infection hyphae were collected at one–three hours post-inoculation for transcriptome analysis. Further, bioinformatic analysis showed that differentially expressed genes (DEGs) regulated by SsHADV-1 infection were identified in S. sclerotiorum. In total, 187 genes were differentially expressed, consisting of more up-regulated (114) than down-regulated (73) genes. The identified DEGs were involved in several important pathways. Metabolic processes, biosynthesis of antibiotics, and secondary metabolites were the most affected categories in S. sclerotiorum upon SsHADV-1 infection. Cell structure analysis suggested that 26% of the total DEGs were related to membrane tissues. Furthermore, 10 and 27 DEGs were predicted to be located in the cell membrane and mitochondria, respectively. Gene ontology enrichment analyses of the DEGs were performed, followed by functional annotation of the genes. Interestingly, one third of the annotated functional DEGs could be involved in the Ras-small G protein signal transduction pathway. These results revealed that SsHADV-1 virions may be able to bind host membrane proteins and influence signal transduction through Ras-small G protein-coupled receptors during early infection, providing new insight towards the molecular mechanisms of virions infection in S. sclerotiorum. Full article

(This article belongs to the Section Viruses of Plants, Fungi and Protozoa)

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Figure 1
(A) Schematic diagram of SsHADV-1 virion inoculation on an angled plate. White, sandy brown, and blue colors represent the mycelium, PDA, and SsHADV-1 virions, respectively. After the mycelium grew on cellophane-covered PDA for 36 h, the virions were added to the lower mycelial tips. Subsequently, the upper mycelia were collected at different time points. Briefly, 15 µL of SsHADV-1 virion (2 mg/mL) were added, and strain Ep-1PNA367 was cultured under 20 °C conditions. (B) SsHADV-1 detection in healthy tissue after mycelia inoculation with virions at different time points (0, 3, 6, 9, 12, 24, and 48 h). SsHADV-1 was detected with specific primers (<a href="#app1-viruses-11-00278" class="html-app">Table S6</a>) for five biological replicates. The PCR production size was about 1.2 kb. Full article ">Figure 2
Differentially expressed genes between virus-infected and virus-free samples. (A) Volcano plot displaying probability on the Y-axis, index of gene differences from NOIseq, and Log2 (fold-change (FC)-value) on the X-axis. The red dots represent up-regulated differentially expressed transcripts between virus-infected and virus-free samples, green dots represent down-regulated transcripts, and gray dots indicate genes not showing statistical significance. (B) Stacked column showing statistics of differential genes, and red represents significantly expression genes. (C) The expression of eight DEGs (four up-regulated and four down-regulated DEGs) were examined via RT-qPCR. Three technical replicates were performed for each RT-qPCR. The ubiquitin gene was used as a reference gene. Full article ">Figure 3
Subcellular localization of differential expression gene involved in SsHADV-1 virions infection. (A) Heat map enrichment analysis of all differentially expressed genes. Row clustering was carried out based on subcellular localization. The column represents the individual treatment sample. Down-regulated DEGs are displayed in green color, and up-regulated DEGs in red. The brightness of each color corresponds to the magnitude of the difference when compared against the average value. (B) Histogram of differentially expressed genes in various organelles, which respectively shows the number of up-regulated (red) and down-regulated (green) genes in each cell structure. (C) RT-qPCR validated the expression of cell membrane-related DEGs (seven up-regulated and three down-regulated DEGs). Three technical replicates were performed for each RT-qPCR. The ubiquitin gene was used as a reference gene. Full article ">Figure 4
Small GTPase mediated signal transduction involved in SsHADV-1 virion infection. (A) Functional gene map from EMBL-EBI (The European Bioinformatics Institute). The box contains the GO number and functions, and different colored lines represent different relationships. The label in bottom-right corner is shown for details. The red box indicates the biological process by which DEGs are enriched. (B) DEGs were re-confirmed by RT-qPCR. Briefly, 11 DEGs (six up-regulated and five down-regulated DEGs) enriched in small G protein signaling pathways were selected. Three technical replicates were performed for each RT-qPCR. The ubiquitin gene was used as a reference gene. Full article ">Figure 5
KEGG enrichment analysis of DEGs. The Y-axis of the bubble graph represents the enriched signal pathway. For each item, characters in square brackets are serial numbers related to the KEGG pathway. The X-axis is −log10 (p-value). The bubble size represents the number of genes, and the color is corrected p-value. The brightness of the blue color corresponds to the magnitude of reliability in the enrichment. Full article ">

16 pages, 997 KiB

Open AccessArticle

Protection of Phage Applications in Crop Production: A Patent Landscape

by Dominique Holtappels, Rob Lavigne, Isabelle Huys and Jeroen Wagemans

Viruses 2019, 11(3), 277; https://doi.org/10.3390/v11030277 - 19 Mar 2019

Cited by 16 | Viewed by 5761

Abstract

In agriculture, the prevention and treatment of bacterial infections represents an increasing challenge. Traditional (chemical) methods have been restricted to ensure public health and to limit the occurrence of resistant strains. Bacteriophages could be a sustainable alternative. A major hurdle towards the commercial [...] Read more.

In agriculture, the prevention and treatment of bacterial infections represents an increasing challenge. Traditional (chemical) methods have been restricted to ensure public health and to limit the occurrence of resistant strains. Bacteriophages could be a sustainable alternative. A major hurdle towards the commercial implementation of phage-based biocontrol strategies concerns aspects of regulation and intellectual property protection. Within this study, two datasets have been composed to analyze both scientific publications and patent documents and to get an idea on the focus of research and development (R&D) by means of an abstract and claim analysis. A total of 137 papers and 49 patent families were found from searching public databases, with their numbers increasing over time. Within this dataset, the majority of the patent documents were filed by non-profit organizations in Asia. There seems to be a good correlation between the papers and patent documents in terms of targeted bacterial genera. Furthermore, granted patents seem to claim rather broad and cover methods of treatment. This review shows that there is indeed growing publishing and patenting activity concerning phage biocontrol. Targeted research is needed to further stimulate the exploration of phages within integrated pest management strategies and to deal with bacterial infections in crop production. Full article

(This article belongs to the Special Issue Hurdles for Phage Therapy (PT) to Become a Reality)

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An overview of the number of scientific publications, patent families, patents, and patent applications in the field of phage biocontrol in crop production organized by year. The light blue area chart represents the number of scientific publications and the dark blue area chart the number of patent families per priority year. The bars represent the number of patent applications per priority year: green (“Granted patents”) corresponds to the number of granted patents, yellow (“Pending applications”) to pending applications, and grey (“Dead applications and patents”) to dead patents and applications. This last group consists of patents and applications that are abandoned, refused, withdrawn, deemed to be withdrawn, or lapsed. Full article ">Figure 2
Percentage of patents and applications organized per continent. The total height of the bars indicate the percentage of patents and applications per continent. Africa (ZA), Asia (CN, IN, JP, KR), Oceania (AU, NZ), Europe (DE, EA, EP, ES, GB, IT), North America (CA, US), South America (AR, BR, CL, CR, GT, MX, PE), and world applications. In green, the percentage of granted patents; in yellow, the percentage of pending applications; and in grey, the percentage of dead applications and dead patents. Full article ">Figure 3
Distribution of patent families and scientific publications classified according to the bacterial genera that is tackled by the phage product. On the basis of Mansfield et al., 2012, seven categories (Agrobacterium, Dickeya/Pectobacterium, Erwinia, Pseudomonas, Ralstonia, Xanthomonas/Xylella, and Other) were made to classify patent families and publications. Groupings of bacterial genera (Dickeya/Pectobacterium and Xanthomonas/Xylella) were created as phage cocktails were created to tackle both bacterial genera. The last category “Other” consists of patent families and publications that do not specify the bacterial pathogen that is being targeted or that tackle an alternative bacterial genera. Full article ">Figure 4
Claim and abstract analysis of the active, granted patents and scientific publications. In total, 79 independent claims from 21 patents and 137 abstracts from scientific papers were categorized among four different categories: (1) Phage—here the phage was described as the active ingredient or the isolation of a phage was described, (2) Cocktail—this category contains claims that protect the combination of phages and publications that describe a phage cocktail, (3) Production—ways of how the phage is produced, and (4) Treatment—claims that protect the use of phages to fight a specific bacterial infection or methods and application strategies for using the phage (e.g., bioassays, field trials). In blue, the percentages of publications are shown; in yellow, the percentage of claims are shown. Note: one publication and patent can be categorized in multiple categories. Full article ">

8 pages, 1821 KiB

Open AccessBrief Report

Localization of Frog Virus 3 Conserved Viral Proteins 88R, 91R, and 94L

by Emily Penny and Craig R. Brunetti

Viruses 2019, 11(3), 276; https://doi.org/10.3390/v11030276 - 19 Mar 2019

Cited by 2 | Viewed by 2852

Abstract

The characterization of the function of conserved viral genes is central to developing a greater understanding of important aspects of viral replication or pathogenesis. A comparative genomic analysis of the iridoviral genomes identified 26 core genes conserved across the family Iridoviridae. Three [...] Read more.

The characterization of the function of conserved viral genes is central to developing a greater understanding of important aspects of viral replication or pathogenesis. A comparative genomic analysis of the iridoviral genomes identified 26 core genes conserved across the family Iridoviridae. Three of those conserved genes have no defined function; these include the homologs of frog virus 3 (FV3) open reading frames (ORFs) 88R, 91R, and 94L. Conserved viral genes that have been previously identified are known to participate in a number of viral activities including: transcriptional regulation, DNA replication/repair/modification/processing, protein modification, and viral structural proteins. To begin to characterize the conserved FV3 ORFs 88R, 91R, and 94L, we cloned the genes and determined their intracellular localization. We demonstrated that 88R localizes to the cytoplasm of the cell while 91R localizes to the nucleus and 94L localizes to the endoplasmic reticulum (ER). Full article

(This article belongs to the Special Issue Family Iridoviridae: Molecular and Ecological Studies of a Family Infecting Invertebrates and Ectothermic Vertebrates)

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26 pages, 4113 KiB

Open AccessArticle

Characterization of the Filovirus-Resistant Cell Line SH-SY5Y Reveals Redundant Role of Cell Surface Entry Factors

by Francisco J. Zapatero-Belinchón, Erik Dietzel, Olga Dolnik, Katinka Döhner, Rui Costa, Barbara Hertel, Barbora Veselkova, Jared Kirui, Anneke Klintworth, Michael P. Manns, Stefan Pöhlmann, Thomas Pietschmann, Thomas Krey, Sandra Ciesek, Gisa Gerold, Beate Sodeik, Stephan Becker and Thomas von Hahn

Viruses 2019, 11(3), 275; https://doi.org/10.3390/v11030275 - 19 Mar 2019

Cited by 6 | Viewed by 6917

Abstract

Filoviruses infect a wide range of cell types with the exception of lymphocytes. The intracellular proteins cathepsin B and L, two-pore channel 1 and 2, and bona fide receptor Niemann–Pick Disease C1 (NPC1) are essential for the endosomal phase of cell entry. However, [...] Read more.

Filoviruses infect a wide range of cell types with the exception of lymphocytes. The intracellular proteins cathepsin B and L, two-pore channel 1 and 2, and bona fide receptor Niemann–Pick Disease C1 (NPC1) are essential for the endosomal phase of cell entry. However, earlier steps of filoviral infection remain poorly characterized. Numerous plasma membrane proteins have been implicated in attachment but it is still unclear which ones are sufficient for productive entry. To define a minimal set of host factors required for filoviral glycoprotein-driven cell entry, we screened twelve cell lines and identified the nonlymphocytic cell line SH-SY5Y to be specifically resistant to filovirus infection. Heterokaryons of SH-SY5Y cells fused to susceptible cells were susceptible to filoviruses, indicating that SH-SY5Y cells do not express a restriction factor but lack an enabling factor critical for filovirus entry. However, all tested cell lines expressed functional intracellular factors. Global gene expression profiling of known cell surface entry factors and protein expression levels of analyzed attachment factors did not reveal any correlation between susceptibility and expression of a specific host factor. Using binding assays with recombinant filovirus glycoprotein, we identified cell attachment as the step impaired in filovirus entry in SH-SY5Y cells. Individual overexpression of attachment factors T-cell immunoglobulin and mucin domain 1 (TIM-1), Axl, Mer, or dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) rendered SH-SY5Y cells susceptible to filovirus glycoprotein-driven transduction. Our study reveals that a lack of attachment factors limits filovirus entry and provides direct experimental support for a model of filoviral cell attachment where host factor usage at the cell surface is highly promiscuous. Full article

(This article belongs to the Collection Advances in Ebolavirus, Marburgvirus, and Cuevavirus Research)

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Figure 1
Unbiased cell screening for susceptibility to EBOV (EBOVpp) and MARV (MARVpp) GP-driven transduction. A panel of twelve cell lines were transduced with EBOV (A) or MARV (B) firefly luciferase pseudoparticles. Pseudoparticles with no envelope proteins (NoEnvpp) or VSV-G (VSV-Gpp) were used as negative and positive control, respectively. Seventy-two hours post transduction, cells were lysed and luciferase activity was measured. Data were normalized to VSV-Gpp activity as indicated in material and methods. Graphs plot the mean values of three independent experiments performed in triplicate (n = 9) with error bars representing the standard deviation (SD). Individual values are represented as dots, squares or triangles. Full article ">Figure 2
SH-SY5Y is resistant to EBOV and VSVΔG-EBOVGP infection. (A,B) Authentic filovirus infection. Susceptible HEK293T, SK-N-BE(2)-C as well as resistant SK-N-MC and SH-SY5Y cells were infected with EBOV or MARV or mock-infected for 1 h at a MOI of 0.1. Seventy-two hpi, cells were fixed and stained for EBOV and MARV NP (A) using DAPI as counterstaining. Images acquired with a 40× objective (B) In parallel, remaining cells were lysed, and analyzed by immune-blotting for virus NP. α-tubulin was used as internal control. Images and blots are representative of three independent infections. (C,D) rVSVΔG-EBOV infection. HEK293T and SH-SY5Y cells were infected with rVSV bearing EBOV Mayinga GP for 1 h at 37 ºC. Fresh media was added and cells further incubated for 48 h. A 10× objective was used for image acquisition (C) 48 hpi. GP-specific cell rounding and detachment. (D) Cell supernatant was collected 24 hpi and viral titers determined by TCID50. Full article ">Figure 3
SH-SY5Y cells are resistant to pseudoparticles bearing either Ebolavirus species GPs but susceptible to pseudoparticles bearing glycoproteins of other virus families. (A) SH-SY5Y cell susceptibility to Ebolavirus species and entry-enhancing variant GPs. HEK293T and SH-SY5Y cells were transduced for 6 h with denoted pseudoparticles encoding for a firefly luciferase reporter gene. After 72 h, 100 µL of cell lysates were measured for luciferase activity. Unspecific entry was determined by NoEnvpp RLU values. Data are the log10 RLU mean values of 3 independent transductions with 9 individual values. Error bars represent SD. (B) Cell–virus specificity analysis. Huh-7.5, HEK293T, SH-SY5Y, and Jurkat cells were transduced with GFP-encoding lentiviral particles pseudotyped with different GPs or No GP (NoEnvpp) for 6 h at a MOI of 0.1 (titers determined in Huh-7.5). Seventy-two hours later, cells were analyzed for GFP expression by flow cytometry. The graph is the representation of the mean percentage of transduced cells plus individual values of the three independent experiments ± SD. Full article ">Figure 4
SH-SY5Y cells do not express a dominant entry restriction factor. Huh-7.5 or SH-SY5Y cells stably expressing the tetracycline-inducible Tet On 3G transactivator protein were cocultured with HEK293T-H6 cells stably expressing transactivator-inducible ZsGreen1 green fluorescent protein for 24 h. Cells were chemically fused with PEG. One hour after fusion, cells were transduced with NoEnv, EBOV, MARV, or VSV-G pseudoparticles encoding mCherry for 6 h at 37 °C. Seventy-two hours post transduction, cells were fixed with 3% PFA and analyzed for heterokaryon formation (ZsGreen1 protein expression) and susceptibility to pseudoparticle infection (mCherry protein expression). (A) Confocal microscopy images of one representative experiment with SH-SY5Y and HEK293T-H6 cells. Scale bars = 50 µm (B) Quantification of transduced cells by flow cytometry. Left-hand side of graph represents transduction percentage of single cell line controls and right-hand side from heterokaryons (discriminated previously by gating on ZsGreen1 positive cells). Mean ± SD. of three independent cell fusions and subsequent transduction experiments (n = 3). Full article ">Figure 5
Intracellular entry factors are expressed and functional in SH-SY5Y cells. Endogenous protein expression of (A) cathepsin B and L, (C) NPC1, and (E) TPC1 and 2. Cell lysates were analyzed for protein expression by western blotting with protein specific abs. β-tubulin (55 kDa) was used as internal control. (B) Cathepsin B and L activity assay. Cathepsins substrate-specific proteolytic cleavage was measured with a commercially available kit as described in methods. Assay limit of detection (LOD) represented as a dotted line. Experiments were conducted thrice with graph bars representing mean experimental value, individual values and SD. (D) Intracellular cholesterol accumulation. HEK293T (upper panels) or SH-SY5Y cells (bottom panels) were treated either with vehicle (left) or with 10 µg/mL of the NPC1 inhibitor U18666A (right) for 24 h. Cells were fixed with 0.1% TX100 and stained for unesterified cholesterol using filipin. Images are representative of two independent experiments. Scale bar 200 µm. (F) EGF intracellular accumulation. HEK293T (upper panels) or SH-SY5Y cells (bottom panels) were treated either with vehicle alone (left) or with the TPC1/2 inhibitor tetrandrine (2 µg/mL) (right) for 24 h followed by incubation with EGF-Alexa Fluor 555 (red) for 30 min. Cells were fixed with 3% PFA, permeabilized with 0.1% TX100, stained with DAPI (blue) and analyzed by confocal microscopy. Scale bar 20 µm. Full article ">Figure 6
Hierarchical clustering analysis (HCA) does not correlate gene expression to susceptibility to filovirus infection. Microarray data of cell lines tested for susceptibility to filovirus infection as well as primary human hepatocytes (PHH) were clustered based on (A) their global gene expression or (B) gene expression of attachment factors implicated in filovirus entry using the heatmaps.2 of the R library “glplots” package. Transcript probes that yielded no detectable signal were removed prior to analysis. Heatmaps were generated by plotting cell lines as columns and genes as rows using the “complete” method for clustering and “Euclidean” method for distance calculation. In the bar above the heatmaps dark blue represents susceptible cell lines and light blue resistant cell lines. Full article ">Figure 7
Surface expression does not explain susceptibility to filovirus infection. (A–D) Axl and TIM-1 cell surface expression. Cells were surface stained with saturating concentrations of specific abs and their correspondent isotype controls and fluorescent signal quantified by flow cytometry. Protein expression profiles of (A) Axl and (C) TIM-1 in different cell lines are shown as histograms of a representative experiment. (B–D) Cell surface expression of Axl and TIM-1 in terms of average delta mean fluorescence intensity (ΔMFI) from three independent stainings. ΔMFI was calculated by subtracting the geometric mean intensity values of the isotype control from the specific staining values. SD is shown as error bars. Full article ">Figure 8
Impaired filovirus attachment on SH-SY5Y cells can be overcome by surface factor overexpression. (A) Binding of EBOV GP-Fc fusion protein to cell surface. EBOV-GP1 and human Fc (100 nM) were incubated for 1.5 h with the indicated cell lines and detected with a secondary ab against the human Fc fragment. MFI signal was recorded for Fc or EBOV GP1-Fc and subtracted from secondary ab MFI. Bars represent mean of two biological replicates performed in duplicate (n = 4) and symbols represent each individual value. Error bars represent the SD. (B) Overexpression of several cell surface factors and their role in filoviral GP-dependent entry. Susceptible HEK293T, resistant SH-SY5Y WT or SH-SY5Y cells genetically engineered to individually express different entry host factors were transduced with NoEnv, EBOV, MARV, and VSV-G luciferase-encoding pseudoparticles. Mock infection was performed to control background signal. Enhanced susceptibility to filovirus infection was calculated as the fold change difference over SH-SY5Y WT cells by dividing the RLU values of 100 µl lysed cells of each engineered cell line and HEK293T cells with the SH-SY5Y WT RLU values. Fold change differences between SH-SY5Y WT and HEK293T or engineered SH-SY5Y cell lines were used for statistical analysis. Graph is the representation of 3 independent transductions done in triplicate (n = 9). Error bars depicts SD. For the analysis of significance in all 3 graphs a multiple t test with a Holm–Sidak multiple comparison correction method was conducted. P-value significance is shown as: n.s. P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Full article ">

20 pages, 1865 KiB

Open AccessReview

Ebola Virus Entry: From Molecular Characterization to Drug Discovery

by Cristiano Salata, Arianna Calistri, Gualtiero Alvisi, Michele Celestino, Cristina Parolin and Giorgio Palù

Viruses 2019, 11(3), 274; https://doi.org/10.3390/v11030274 - 19 Mar 2019

Cited by 55 | Viewed by 16924

Abstract

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the Filoviridae family. The recent large outbreak of EVD in Western Africa (2013–2016) highlighted the worldwide threat represented by [...] Read more.

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the Filoviridae family. The recent large outbreak of EVD in Western Africa (2013–2016) highlighted the worldwide threat represented by the disease and its impact on global public health and the economy. The development of highly needed anti-Ebola virus antivirals has been so far hampered by the shortage of tools to study their life cycle in vitro, allowing to screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to study Ebola virus entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus-like particles, tremendously boosted both our knowledge of the viral life cycle and the identification of promising antiviral compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD. Full article

(This article belongs to the Collection Advances in Ebolavirus, Marburgvirus, and Cuevavirus Research)

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Schematic representation of EBOV entry. Following interaction with attachment factors (1), the virion is internalized by the macropinocytosis (2). Inside the membrane-bound vesicle, GP is cleaved by cysteine proteases to activate its fusogenic potential (3). Cleaved GP is then able to interact with the specific NPC1 viral receptor (4). Such event, in addition to the activity of the TPC2 calcium channel (5), helps triggering the fusion between the viral envelope and the endosomal/lysosomal membrane (6), leading to viral genome release followed by transcription and replication (7). Full article ">Figure 2
Recovery, growth and pseudotyping of rVSV-ΔG-GFP. The system is based on a plasmid encoding the viral genome, containing a reporter gene (GFP) instead of the native gene coding for the glycoprotein G, and four plasmids providing the packaging system (matrix M, polymerase L, phosphoprotein P and G). At the beginning, cells are cotransfected with the pVSV-ΔG-GFP plasmid along with the four packaging plasmids to recover the G-complemented rVSV-ΔG-GFP. To express the viral genome for the first viral rescue, a plasmid encoding the T7 RNA polymerase is also required (not shown). This virus can be used for the generation of a pseudotyped rVSV-ΔG-GFP by transducing cells preventively transfected with a plasmid encoding for the heterologous glycoproteins. Then, the pseudotyped virus can be used to transduce target cells. Full article ">Figure 3
Schematic representation of the production of a pseudotyped retroviral vector. This system is based on a plasmid encoding for the retroviral vector (cis-acting sequences, reporter gene), and constructs expressing the packaging system factors and the heterologous envelope glycoprotein. Packaging cells are cotransfected with the different plasmids to recover pseudotyped retroviral particles in the supernatant. Pseudotyped particles can be used to transduce target cells. Full article ">Figure 4
Transcription- and replication-competent eVLP (tr-eVLP). This system is based on a minigenome, encoding for a reporter gene, the viral proteins VP40, GP, and in some cases p24, co-transfected with the constructs expressing RNP proteins (N, VP35, VP30, and L). Inside the producer cells, VP40 drives the formation of eVLPs that harbor minigenome-containing nucleocapsids. These tr-eVLPs can transduce target cells and deliver the minigenome that undergoes primary transcription mediated by RNP proteins brought into the target cells within the tr-eVLPs (in the form of nucleocapsids), resulting into the expression of the reporter gene. If target cells are pre-transfected with plasmids encoding for RNPs, the minigenome is replicated and undergoes a secondary transcription (with the expression of the reporter gene) mediated by RNP proteins provided in trans from expression constructs. Furthermore, a new progeny of infectious tr-eVLPs is produced and can be used to transduce new target cells. Full article ">

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