Coxsackieviruses (CV) A1, CV-A19 and CV-A22 have historically comprised a distinct phylogenetic clade within enterovirus C. Several novel serotypes that are genetically similar to these three viruses have been recently discovered and characterized. Here we report the coding sequence analysis of two genotypes of a previously uncharacterized serotype EV-C113 from Bangladesh and demonstrate that it is most similar to CV-A22 and EV-C116 within the capsid region. We sequenced novel genotypes of CV-A1, CV-A19, CV-A22 from Bangladesh and observed a high rate of recombination within this group. We also report genomic analysis of the rarely reported EV-C104 circulating in the Gambia in 2009. All available EV-C104 sequences display a high degree of similarity within the structural genes but form two clusters within the nonstructural genes. One cluster includes the recently reported EV-C117, suggesting an ancestral recombination between these two serotypes. Phylogenetic analysis of all available complete genome sequences indicates the existence of two subgroups within this distinct enterovirus C clade: one has been exclusively recovered from gastrointestinal samples, while the other cluster has been implicated in respiratory disease.

Bats host a broad diversity of coronaviruses (CoVs), including close relatives of human pathogens. There is only limited data on neotropical bat CoVs. We analyzed fecal, blood and intestine specimens from 1,562 bats sampled in Costa Rica, Panama, Ecuador and Brazil for CoVs by broad-range PCR. CoV RNA was detected in 50 bats representing nine different species, both frugivorous and insectivorous. These bat CoVs were unrelated to known human or animal pathogens, indicating an absence of recent zoonotic spill-over events. Based on RNA-dependent RNA Polymerase (RdRp)-based grouping units (RGUs) as a surrogate for CoV species identification, the 50 viruses represented five different alphacoronavirus RGUs and two betacoronavirus RGUs. Closely related alphacoronaviruses were detected in Carollia perspicillata and C. brevicauda across a geographic distance exceeding 5,600 km. Our study expands the knowledge on CoV diversity in neotropical bats and emphasises the association of distinct CoVs and bat host genera.

Vaccinia virus (VACV) expresses many proteins that are non-essential for virus replication but promote virulence by inhibiting components of the host immune response to infection. These immunomodulators include a family of proteins that have, or are predicted to have, a structure related to the B-cell lymphoma (Bcl)-2 protein. Five members of the VACV Bcl-2 family (N1, B14, A52, F1 and K7) have had their crystal structure solved, others have been characterised and a function assigned (C6, A46), and others are predicted to be Bcl-2 proteins but are uncharacterised hitherto (N2, B22, C1). Data presented here show that N2 is a nuclear protein that is expressed early during infection and inhibits the activation of interferon regulatory factor (IRF)3. Consistent with its nuclear localisation, N2 inhibits IRF3 downstream of the TANK-binding kinase (TBK)-1 and after IRF3 translocation into the nucleus. A mutant VACV strain Western Reserve lacking the N2L gene showed normal replication and spread in cultured cells compared to wild type parental (vN2) and revertant (vN2-rev) viruses, but was attenuated in two murine models of infection. After intranasal infection, the vdeltaN2 mutant induced lower weight loss and signs of illness, and virus was cleared more rapidly from the infected tissue. In the intradermal model of infection, vdeltaN2 induced smaller lesions that were resolved more rapidly. In summary, the N2 protein is an intracellular virulence factor that inhibits IRF3 activity in the nucleus.

Nonstructural protein 1beta (nsp1beta) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a papain-like cysteine protease (PLP-beta) domain and was identified to be the main viral protein antagonizing host innate immune response. In this study, nsp1beta was determined to suppress the expression of reporter genes as well as suppress 'self-expression' in transfected cells, and this activity appeared to associate with its interferon (IFN) antagonist function. To knock down the effect of nsp1beta on IFN activity, a panel of site-specific mutations in nsp1beta was analyzed. Double mutations K130A/R134A (Type 1 PRRSV) or K124A/R128A (Type 2 PRRSV), targeting on a highly conserved motif of nsp1beta, GKYLQRRLQ, impaired the nsp1beta's ability to suppress IFN-beta and reporter gene expression, as well as to suppress 'self-expression' in vitro. Subsequently, viable recombinant viruses, vSD01-08-K130A/R134A and vSD95-21-K124A/R128A, containing double mutations in GKYLQRRLQ motif were generated using reverse genetics. In comparison to wild-type viruses, these nsp1beta mutants showed impaired growth ability in infected cells, but the PLP-beta cleavage function was not directly affected. The expression of selected innate immune genes was determined in vSD95-21-K124A/R128A mutant-infected cells. The results consistently showed that gene expression levels of IFN-alpha, IFN-beta and ISG15 were up-regulated in cells that were infected with the vSD95-21-K124A/R128A compared with that of wild-type virus. These data suggest that PRRSV nsp1beta may selectively suppress cellular gene expression, including those involved in host innate immune function. Modifying the key residues on the highly conserved GKYLQRRLQ motif could attenuate virus growth and improve cellular innate immune responses.

Emaravirus is a recently described genus of negative-strand RNA plant viruses. Emaravirus P4 protein localizes to plasmodesmata, suggesting that it could be a viral movement protein (MP). In the current study, we showed that the P4 protein of Raspberry leaf blotch emaravirus (RLBV) rescues the cell-to-cell movement of a defective Potato virus X (PVX) that has a deletion mutation in the Triple Gene Block 1 (TGB1) movement-associated protein. This demonstrates that RLBV P4 is a functional MP. Sequence analyses revealed that P4 is a distant member of the 30K superfamily of MPs. All MPs of this family contain two highly conserved regions predicted to form β-strands, namely β1 and β2. We explored by alanine mutagenesis the role of two residues of P4 (Ile106 and Asp127) located in each of these strands. We also made the equivalent substitutions in the 29K MP of Tobacco rattle virus (TRV), another member of the 30K superfamily. All substitutions abolished the ability to complement PVX movement, except the I106A substitution in the β1 region of P4. This region has been shown to mediate membrane association of 30K MPs; our results show that it is possible to make non-conservative substitutions of a well conserved aliphatic residue within β1 without disrupting the membrane association or movement function of P4.

Development of transgenic mouse models expressing heterologous prion proteins (PrP) has facilitated and advanced in vivo studies of prion diseases affecting humans and animals. Here, novel transgenic mouse lines expressing a chimeric murine/ovine (Mu/Ov) PrP transgene, including amino acid residues alanine, histidine and glutamine at ovine polymorphic codons 136, 154 and 171 (A136H154Q171), were generated to provide a means of assessing the susceptibility of the OvPrPAHQ allele to ruminant prion diseases in an in vivo model. Transmission studies showed that the highest level of transgene overexpression, in Tg(Mu/OvPrPAHQ)EM16 mice, conferred high susceptibility to ruminant prions. Highly efficient primary transmission of atypical scrapie from sheep was shown, irrespective of donor sheep PrP genotype, with mean incubation periods (IPs) of 154-178 days post-inoculation (dpi), 100% disease penetrance, and early Western blot detection of protease-resistant fragments (PrPres) of the disease-associated isoform, PrPSc, in Tg(Mu/OvPrPAHQ)EM16 brain from 110 dpi onwards. Tg(Mu/OvPrPAHQ)EM16 mice were also highly susceptible to classical scrapie and BSE, with mean IPs 320 and 246 days faster, respectively, than wild-type mice. Primary passage of atypical scrapie, classical scrapie and BSE showed that the PrPres profiles associated with disease in the natural host were faithfully maintained in Tg(Mu/OvPrPAHQ)EM16 mice and were distinguishable based on molecular masses, antibody reactivities and glycoform percentages. Immunohistochemistry was used to confirm PrPSc deposition in brain sections from terminal phase TSE-challenged Tg(Mu/OvPrPAHQ)EM16 mice. The findings indicate that Tg(Mu/OvPrPAHQ)EM16 mice represent a suitable bioassay model for detection of atypical scrapie infectivity and offer the prospect of differentiation of ruminant prions.

The nucleocytoplasmic export of cytomegaloviral capsids is regulated by formation of a multicomponent nuclear egress complex (NEC), essentially based on viral proteins pUL50 and pUL53. In this study, the generation of BACmid-derived recombinant human cytomegaloviruses, expressing tagged versions of pUL50 and pUL53, enabled the investigation of NEC formation in infected primary fibroblasts. For these recombinant viruses, a wild-type-like mode of pUL50-pUL53 interaction and recruitment of both proteins to the nuclear envelope could be demonstrated. Importantly, pUL50 was translocated from initial cytoplasmic distribution to the nuclear rim, whereas pUL53 accumulated in the nucleus before attaining overall rim colocalization with pUL50. Specified experimental settings illustrated that pUL50 and pUL53 were subject to different pathways of intracellular trafficking. Importantly, a novel NLS could be identified and functionally verified for pUL53 (amino acids 18-27), whereas no NLS proved to be present in pUL50. Analysis of amino acid replacement mutants further illustrated the differential modes of nuclear import of the two essential viral egress proteins. Taken together, our findings suggest a combination of classical nuclear import (pUL53) and interaction-mediated recruitment (pUL50) as the driving forces for core NEC formation and viral nuclear egress.

Porcine sialoadhesin (pSn; a sialic acid-binding lectin) and porcine CD163 (pCD163) are molecules that facilitate infectious entry of porcine reproductive and respiratory syndrome virus (PRRSV) into alveolar macrophages. In this study, it was shown that murine (mSn) and human (hSn) sialoadhesin, like pSn, can promote PRRSV infection of pCD163-expressing cells. Intact sialic acid-binding domains are crucial, since non-sialic acid-binding mutants of pSn, mSn and hSn did not promote infection. Endodomain-deletion mutants of pSn, mSn and hSn promoted PRRSV infection less efficiently, but also showed markedly reduced expression levels, making further research into the potential role of the sialoadhesin endodomain in PRRSV receptor activity necessary. These data further complement our knowledge on sialoadhesin as an important PRRSV receptor, and suggest - in combination with other published data - that species differences in the main PRRSV entry mediators sialoadhesin and CD163 do not account for the strict host species specificity displayed by the virus.

The flavivirus nonstructural protein 5 (NS5) is a large protein that is structurally conserved among members of the genus, making it an attractive target for antiviral drug development. The protein contains a methyltransferase (MTase) domain and an RNA dependent RNA polymerase (POL) domain. Previous studies with dengue viruses have identified a genetic interaction between residues 46-49 in the αA3-motif in the MTase and residue 512 in POL. These genetic interactions are consistent with structural modeling of these domains in WNV NS5 that predict close proximity of these regions of the two domains, and potentially a functional interaction mediated via the αA3-motif. To demonstrate an interaction between the MTase and POL domains of the WNV NS5 protein, we co-expressed affinity-tagged recombinant MTase and POL proteins in COS293T cells and performed pulldown assays using an antibody to the Flag tag on POL. Western blot analysis with an anti-MTase mAb revealed that the MTase protein was specifically co-immunoprecipitated with POL, providing the first evidence of a specific interaction between these domains. To further assess the role of the αA3 helix in this interaction, selected residues in this motif were mutated in the recombinant MTase and the effect on POL interaction determined by the pulldown assay. These mutations were also introduced into a WNV infectious clone (FLSDX) and the replication properties of these mutant viruses assessed. While none of the αA3 mutations had a significant effect on the MTase-POL association in pulldown assays, suggesting that these residues were not specific to the interaction, an E46L mutation completely abolished virus viability indicating a critical requirement of this residue in replication. Failure to generate compensatory mutations in POL to rescue replication, even after several passages of the transfection supernatant in Vero cells, precluded further conclusion of the role of this residue in the context of MTase-POL interactions.

The foot-and-mouth disease virus (FMDV) structural protein precursor, P1-2A, is cleaved by the virus-encoded 3C protease (3Cpro) into the capsid proteins VP0, VP1 and VP3 (and 2A). In some systems, it is difficult to produce large amounts of these processed capsid proteins since 3Cpro can be toxic for cells. The expression level of 3Cpro activity has now been reduced relative to the P1-2A, and the effect on the yield of processed capsid proteins and their assembly into empty capsid particles within mammalian cells has been determined. Using a vaccinia virus based transient expression system, P1-2A (from serotypes O and A) and 3Cpro were expressed from monocistronic cDNA cassettes as P1-2A-3C, or from dicistronic cassettes with the 3Cpro expression dependent on a mutant FMDV IRES (P1-2A-mIRES-3C). The effects of using a mutant 3Cpro with reduced catalytic activity or using two different mutant IRES elements (the wild-type GNRA tetraloop sequence GCGA converted, in the cDNA, to GAGA or GTTA) were analysed. For both serotypes, the P1-2A-mIRES-3C construct containing the inefficient GTTA mutant IRES produced the highest amount of processed capsid proteins. These products self-assembled to form FMDV empty capsid particles, which have a related, but distinct, morphology (as determined by electron microscopy and reconstruction) from that determined previously by X-ray crystallography. The assembled empty capsids bind, in a divalent cation-dependent manner, to the RGD-dependent integrin αvβ6, a cellular receptor for FMDV, and are recognized appropriately in serotype-specific antigen ELISAs.