Isolation of a novel swine influenza virus from Oklahoma in 2011 which is distantly related to human influenza C viruses.

Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution.

Whole genome analysis of epizootic hemorrhagic disease virus identified limited genome constellations and preferential reassortment.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides transmitted orbivirus that causes haemorrhagic disease in wild and domestic ruminants. A collection of 44 EHDV isolated from 2008 to 2012 was fully sequenced and analysed phylogenetically. Serotype 2 viruses were the dominant serotype all years except 2012 when serotype 6 viruses represented 63 % of the isolates. High genetic similarity (>94 % identity) between serotype 1 and 2 virus VP1, VP3, VP4, VP6, NS1, NS2 and NS3 segments prevented identification of reassortment events for these segments. Additionally, there was little genetic diversity (>96 % identity) within serotypes for VP2, VP5 and VP7. Preferential reassortment within the homologous serotype was observed for VP2, VP5 and VP7 segments for type 1 and type 2 viruses. In contrast, type 6 viruses were all reassortants containing VP2 and VP5 derived from an exotic type 6 with the remaining segments most similar to type 2 viruses. These results suggest that reassortment between type 1 and type 2 viruses requires conservation of the VP2, VP5 and VP7 segment constellation while type 6 viruses only require VP2 and VP5 and are restricted to type 2-lineage VP7. As type 6 VP2 and VP5 segments were exclusively identified in viruses with type 2-derived VP7, these results suggest functional complementation between type 2 and type 6 VP7 proteins.

Mobuck virus genome sequence and phylogenetic analysis: identification of a novel Orbivirus isolated from a white-tailed deer in Missouri, USA.

The genus Orbivirus includes a diverse group of segmented dsRNA viruses that are transmitted via arthropods, have a global distribution and affect a wide range of hosts. A novel orbivirus was co-isolated with epizootic haemorrhagic disease virus (EHDV) from a white-tailed deer (Odocoileus virginianus) exhibiting clinical signs characteristic of EHDV. Using antiserum generated against EHDV, a pure isolate of the novel non-cytopathic orbivirus was obtained in Aedes albopictus cell culture. Genomic sequencing and phylogenetic analysis of predicted ORFs showed that eight of the ten ORFs were most homologous to Peruvian horse sickness virus (PHSV), with amino acid identities of 44.3-73.7 %. The remaining two ORFs, VP3 and VP5, were most similar to Middle Point orbivirus (35.9 %) and Yunnan orbivirus (59.8 %), respectively. Taxonomic classification of orbiviruses is largely based on homology of the major subcore structural protein VP2(T2), encoded by segment 2 for mobuck virus. With only 69.1 % amino acid identity to PHSV, we propose mobuck virus as the prototype of a new species of Orbivirus.

Antigenic categorization of contemporary H3N2 Swine influenza virus isolates using a high-throughput serum neutralization assay.

In vivo, neutralizing antibodies are critical for viral clearance. A high-throughput serum neutralization (HTSN) assay was developed to antigenically categorize Swine influenza virus (SIV) isolates. Uncategorized viruses were tested using a panel of antisera representing the H3N2 SIV subtypes and the results expressed as a serum neutralization ratio. Antisera were generated against contemporary isolates representing circulating H3N2 SIV subtypes (clusters I, III, IV). Reference viruses and the corresponding antisera were evaluated using traditional hemagglutination inhibition (HI) and the HTSN assays and good correlation (r = 0.84) was observed between the 2 tests. Categorical clustering of 40 recent (2008-2009) SIV isolates was assessed using the HTSN assay. The H3N2 SIV isolates with amino acid similarity >97% to the commonly used H3N2 cluster IV reference strain A/Swine/Ontario/33853/2005 (ON05) showed strong reactivity with cluster IV antisera. Isolates with <97% amino acid similarity to ON05 sporadically or completely failed to react with any antiserum. A cluster of 3 isolates with weak reaction with cluster III antiserum may be a potential emerging cluster of H3N2 with moderate genetic similarity to cluster II H3N2 (93% similarity). Potential uses of the HTSN assay include identification of broadly cross-reactive or antigenically distinct SIV isolates for use in vaccine virus selection or as part of surveillance efforts monitoring antigenic drift.

Migration of the swine influenza virus δ-cluster hemagglutinin N-linked glycosylation site from N142 to N144 results in loss of antibody cross-reactivity.

Routine antigenic characterization of swine influenza virus isolates in a high-throughput serum neutralization (HTSN) assay found that approximately 20% of isolates were not neutralized by a panel of reference antisera. Genetic analysis revealed that nearly all of the neutralization-resistant isolates possessed a seasonal human-lineage hemagglutinin (HA; δ cluster). Subsequent sequencing analysis of full-length HA identified a conserved N144 residue present only in neutralization-resistant strains. N144 lies in a predicted N-linked glycosylation consensus sequence, i.e., N-X-S/T (where X is any amino acid except for proline). Interestingly, neutralization-sensitive viruses all had predicted N-linked glycosylation sites at N137 or N142, with threonine (T) occupying position 144 of HA. Consistent with the HTSN assay, hemagglutination inhibition (HI) and serum neutralization (SN) assays demonstrated that migration of the potential N-linked glycosylation site from N137 or N142 to N144 resulted in a >8-fold decrease in titers. These results were further confirmed in a reverse genetics system where syngeneic viruses varying only by predicted N-glycosylation sites at either N142 or N144 exhibited distinct antigenic characteristics like those observed in field isolates. Molecular modeling of the hemagglutinin protein containing N142 or N144 in complex with a neutralizing antibody suggested that N144-induced potential glycosylation may sterically hinder access of antibodies to the hemagglutinin head domain, allowing viruses to escape neutralization. Since N-linked glycosylation at these sites has been implicated in genetic and antigenic evolution of human influenza A viruses, we conclude that the relocation of the hemagglutinin N-linked glycosylation site from N142 to N144 renders swine influenza virus δ-cluster viruses resistant to antibody-mediated neutralization.

In vitro reassortment between endemic H1N2 and 2009 H1N1 pandemic swine influenza viruses generates attenuated viruses.

The pandemic H1N1 (pH1N1) influenza virus was first reported in humans in the spring of 2009 and soon thereafter was identified in numerous species, including swine. Reassortant viruses, presumably arising from the co-infection of pH1N1 and endemic swine influenza virus (SIV), were subsequently identified from diagnostic samples collected from swine. In this study, co-infection of swine testicle (ST) cells with swine-derived endemic H1N2 (MN745) and pH1N1 (MN432) yielded two reassortant H1N2 viruses (R1 and R2), both possessing a matrix gene derived from pH1N1. In ST cells, the reassortant viruses had growth kinetics similar to the parental H1N2 virus and reached titers approximately 2 log(10) TCID(50)/mL higher than the pH1N1 virus, while in A549 cells these viruses had similar growth kinetics. Intranasal challenge of pigs with H1N2, pH1N1, R1 or R2 found that all viruses were capable of infecting and transmitting between direct contact pigs as measured by real time reverse transcription PCR of nasal swabs. Lung samples were also PCR-positive for all challenge groups and influenza-associated microscopic lesions were detected by histology. Interestingly, infectious virus was detected in lung samples for pigs challenged with the parental H1N2 and pH1N1 at levels significantly higher than either reassortant virus despite similar levels of viral RNA. Results of our experiment suggested that the reassortant viruses generated through in vitro cell culture system were attenuated without gaining any selective growth advantage in pigs over the parental lineages. Thus, reassortant influenza viruses described in this study may provide a good system to study genetic basis of the attenuation and its mechanism.