- Volume 93, Issue 4, 2012

Vesicular stomatitis virus (VSV) matrix protein (M) has a flexible amino-terminal part that recruits cellular partners. It contains a dynamin-binding site that is required for efficient virus assembly, and two motifs, 24PPPY27 and 37PSAP40, that constitute potential late domains. Late domains are present in proteins of several enveloped viruses and are involved in the ultimate step of the budding process (i.e. fission between viral and cellular membranes). In baby hamster kidney (BHK)-21 cells, it has been demonstrated that the 24PPPY27 motif binds the Nedd4 (neuronal precursor cell-expressed developmentally downregulated 4) E3 ubiquitin ligase for efficient virus budding and that the 37PSAP40 motif, although conserved among M proteins of vesiculoviruses, does not possess late-domain activity. In this study, we have re-examined the contribution of the PSAP motif to VSV budding. First, we demonstrate that VSV M indeed binds TSG101 [tumour susceptibility gene 101; a component of the ESCRT1 (endosomal sorting complex required for transport 1)] through its PSAP motif. Second, we analysed the phenotype of several recombinant mutants. We show that a double mutant with point mutations in both the PSAP and the PPPY motifs is impaired compared with a single mutant in the PPPY motif, indicating that the PSAP motif partially compensates for the lack of the PPPY motif. Mutants’ phenotypes depend on cell lines: in CERA (chicken embryo-related, Alger clone) cells, a recombinant virus with a single mutation in the PSAP motif was impaired compared with the wild type, and a mutant with a single mutation in the dynamin-binding motif was much less impaired in Vero cells than in BSR (clones of BHK-21) cells. These results have implications for the VSV budding pathway that will be discussed.

In this study, the complete genome sequences of seven equine group A rotavirus (RVA) strains (RVA/Horse-tc/GBR/L338/1991/G13P[18], RVA/Horse-wt/IRL/03V04954/2003/G3P[12] and RVA/Horse-wt/IRL/04V2024/2004/G14P[12] from Europe; RVA/Horse-wt/ARG/E30/1993/G3P[12], RVA/Horse-wt/ARG/E403/2006/G14P[12] and RVA/Horse-wt/ARG/E4040/2008/G14P[12] from Argentina; and RVA/Horse-wt/ZAF/EqRV-SA1/2006/G14P[12] from South Africa) were determined. Multiple novel genotypes were identified and genotype numbers were assigned by the Rotavirus Classification Working Group: R9 (VP1), C9 (VP2), N9 (NSP2), T12 (NSP3), E14 (NSP4), and H7 and H11 (NSP5). The genotype constellation of L338 was unique: G13-P[18]-I6-R9-C9-M6-A6-N9-T12-E14-H11. The six remaining equine RVA strains showed a largely conserved genotype constellation: G3/G14-P[12]-I2/I6-R2-C2-M3-A10-N2-T3-E2/E12-H7, which is highly divergent from other known non-equine RVA genotype constellations. Phylogenetic analyses revealed that the sequences of these equine RVA strains are related distantly to non-equine RVA strains, and that at least three lineages exist within equine RVA strains. A small number of reassortment events were observed. Interestingly, the three RVA strains from Argentina possessed the E12 genotype, whereas the three RVA strains from Ireland and South Africa possessed the E2 genotype. The unusual E12 genotype has until now only been described in Argentina among RVA strains collected from guanaco, cattle and horses, suggesting geographical isolation of this NSP4 genotype. This conserved genetic configuration of equine RVA strains could be useful for future vaccine development or improvement of currently used equine RVA vaccines.

Infectious bursal disease virus (IBDV) causes an important disease in young chickens. Chicken heat-shock protein 90 (cHsp90) has been shown to be a functional component of the cellular receptor complex for IBDV infection. This study demonstrates the inhibitory effect of vector-expressed anti-cHsp90α microRNA (miRNA) on IBDV infection. The reporter vectors pcHsp90α-EGFP and pcHsp90β-EGFP were constructed to facilitate effective miRNA selection. Two anti-cHsp90α and one anti-cHsp90β miRNA-expression vectors were constructed for a stable transfection study. Poly(A)-tailed RT-PCR detected sequence-specific miRNA transcription in transfected cells. Semiquantitative RT-PCR showed inhibition of cHsp90 transcription in transfected cells. A virus-titration assay showed that the anti-cHsp90α miRNA, but not the anti-cHsp90β miRNA, had inhibitory effects on IBDV infection. These results suggest that cHsp90α is a functional component of the cellular receptor complex for IBDV infection, and that anti-cHsp90α miRNA could be used as an anti-IBDV reagent.

Viral diseases are significant impediments to the sustainability of shrimp aquaculture. In addition to endemic disease, new viral diseases continue to emerge and cause significant impact on the shrimp industry. Disease caused by infectious myonecrosis virus (IMNV) has caused tremendous losses in farmed Pacific white shrimp (Litopenaeus vannamei) since it emerged in Brazil and translocated to Indonesia. There are no existing antiviral interventions, outside of pathogen exclusion, to mitigate disease in commercial shrimp operations. Here, we describe an iterative process of panning the genome of IMNV to discover RNA interference trigger sequences that initiate a robust and long-lasting protective response against IMNV in L. vannamei. Using this process, a single, low dose (0.02 µg) of an 81 or 153 bp fragment, with sequence corresponding to putative cleavage protein 1 in ORF1, protected 100 % of animals from disease and mortality caused by IMNV. Furthermore, animals that were treated with highly efficacious dsRNA survived an initial infection and were resistant to subsequent infections over 50 days later with a 100-fold greater dose of virus. This protection is probably sequence dependent, because targeting the coding regions for the polymerase or structural genes of IMNV conferred lesser or no protection. Interestingly, non-sequence specific dsRNA did not provide any degree of protection to animals as had been described for other shrimp viruses. Our data indicate that the targeted region for dsRNA is a crucial factor in maximizing the degree of protection and lowering the dose required to induce a protective effect against IMNV infection in shrimp.

Human immunodeficiency virus type 2 (HIV-2) emerged in West Africa and has spread further to countries that share socio-historical ties with this region. However, viral origins and dispersal patterns at a global scale remain poorly understood. Here, we adopt a Bayesian phylogeographic approach to investigate the spatial dynamics of HIV-2 group A (HIV-2A) using a collection of 320 partial pol and 248 partial env sequences sampled throughout 19 countries worldwide. We extend phylogenetic diffusion models that simultaneously draw information from multiple loci to estimate location states throughout distinct phylogenies and explicitly attempt to incorporate human migratory fluxes. Our study highlights that Guinea-Bissau, together with Côte d’Ivoire and Senegal, have acted as the main viral sources in the early stages of the epidemic. We show that convenience sampling can obfuscate the estimation of the spatial root of HIV-2A. We explicitly attempt to circumvent this by incorporating rate priors that reflect the ratio of human flow from and to West Africa. We recover four main routes of HIV-2A dispersal that are laid out along colonial ties: Guinea-Bissau and Cape Verde to Portugal, Côte d’Ivoire and Senegal to France. Within Europe, we find strong support for epidemiological linkage from Portugal to Luxembourg and to the UK. We demonstrate that probabilistic models can uncover global patterns of HIV-2A dispersal providing sampling bias is taken into account and we provide a scenario for the international spread of this virus.

Feline leukemia virus (FeLV) is a gammaretrovirus that is a significant cause of neoplastic-related disorders affecting cats worldwide. Treatment options for FeLV are limited, associated with serious side effects, and can be cost-prohibitive. The development of drugs used to treat a related retrovirus, human immunodeficiency virus type 1 (HIV-1), has been rapid, leading to the approval of five drug classes. Although structural differences affect the susceptibility of gammaretroviruses to anti-HIV drugs, the similarities in mechanism of replication suggest that some anti-HIV-1 drugs may also inhibit FeLV. This study demonstrates the anti-FeLV activity of four drugs approved by the US FDA (Food and Drug Administration) at non-toxic concentrations. Of these, tenofovir and raltegravir are anti-HIV-1 drugs, while decitabine and gemcitabine are approved to treat myelodysplastic syndromes and pancreatic cancer, respectively, but also have anti-HIV-1 activity in cell culture. Our results indicate that these drugs may be useful for FeLV treatment and should be investigated for mechanism of action and suitability for veterinary use.

Lettuce necrotic yellows virus (LNYV), Sonchus yellow net virus (SYNV) and Potato yellow dwarf virus (PYDV) are members of the family Rhabdoviridae that infect plants. LNYV is a cytorhabdovirus that replicates in the cytoplasm, while SYNV and PYDV are nucleorhabdoviruses that replicate in the nuclei of infected cells. LNYV and SYNV share a similar genome organization with a gene order of nucleoprotein (N), phosphoprotein (P), putative movement protein (Mv), matrix protein (M), glycoprotein (G) and polymerase (L). PYDV contains an additional predicted gene of unknown function located between N and P. In order to gain insight into the associations of viral structural and non-structural proteins and the mechanisms by which they may function, we constructed protein localization and interaction maps. Subcellular localization was determined by transiently expressing the viral proteins fused to green or red fluorescent protein in leaf epidermal cells of Nicotiana benthamiana. Protein interactions were tested in planta by using bimolecular fluorescence complementation. All three viruses showed Mv to be localized at the cell periphery and the G protein to be membrane associated. Comparing the interaction maps revealed that only the N–P and M–M interactions are common to all three viruses. Associations unique to only one virus include P–M for LNYV, G–Mv for SYNV and M–Mv, M–G and N–M for PYDV. The cognate N–P proteins of all three viruses interacted and exhibited characteristic changes in localization when co-expressed.