Migration of the Swine Influenza Virus -δ Cluster Hemagglutinin NLinked 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 \u3e8-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

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 nonhuman 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

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 log10 TCID50/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

No Reliable Association between Runs of Homozygosity and Schizophrenia in a Well-Powered Replication Study

It is well known that inbreeding increases the risk of recessive monogenic diseases, but it is less certain whether it contributes to the etiology of complex diseases such as schizophrenia. One way to estimate the effects of inbreeding is to examine the association between disease diagnosis and genome-wide autozygosity estimated using runs of homozygosity (ROH) in genome-wide single nucleotide polymorphism arrays. Using data for schizophrenia from the Psychiatric Genomics Consortium (n = 21,868), Keller et al. (2012) estimated that the odds of developing schizophrenia increased by approximately 17% for every additional percent of the genome that is autozygous (β = 16.1, CI(β) = [6.93, 25.7], Z = 3.44, p = 0.0006). Here we describe replication results from 22 independent schizophrenia case-control datasets from the Psychiatric Genomics Consortium (n = 39,830). Using the same ROH calling thresholds and procedures as Keller et al. (2012), we were unable to replicate the significant association between ROH burden and schizophrenia in the independent PGC phase II data, although the effect was in the predicted direction, and the combined (original + replication) dataset yielded an attenuated but significant relationship between Froh and schizophrenia (β = 4.86,CI(β) = [0.90,8.83],Z = 2.40,p = 0.02). Since Keller et al. (2012), several studies reported inconsistent association of ROH burden with complex traits, particularly in case-control data. These conflicting results might suggest that the effects of autozygosity are confounded by various factors, such as socioeconomic status, education, urbanicity, and religiosity, which may be associated with both real inbreeding and the outcome measures of interest

Four pigs were added to each of four rooms containing pigs challenged intranasally with 2 mL of 6.2 log10 TCID50/mL with H1N2, pH1N1, R1 or R2 at day one post infection.

<p>Nasal swabs were collected daily and analyzed by rt-RT-PCR for virus shedding. Data represent the mean Ct value for the four nasal swabs collected at each time point. Samples with different subscripts at a time point are statistically different (P<0.05).</p

Multicycle growth curves for pH1N1 and H1N2, both before (x+0) and after 10 passes (x+10) in A549 cells, as well as reassortant viruses R1 and R2.

<p>Data represent means of triplicate growth curves.</p

Single nucleotide polymorphisms identified in pH1N1 and H1N2 following 10 passages in ST cells as compared to genome sequences prior to cell passaging^a.

<p>For non-synonymous mutations, amino acid changes are shown in parentheses.</p>a<p>For reassortant viruses R1 and R2, single nucleotide polymorphisms are reported based on changes observed from the parent segment at x+0.</p

Lung samples were collected on day five post infection and analyzed for TCID50 by virus replication assay in ST cells.

<p>Data represent the mean log₁₀ TCID₅₀/mL for the four lung samples per treatment group. Samples with different subscripts within the same treatment group are statistically different (P<0.05).</p

Isolation of a Novel Swine Influenza Virus from Oklahoma in 2011 Which Is Distantly Related to Human Influenza C Viruses

<div><p>Of the <em>Orthomyxoviridae</em> 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.</p> </div

Ultrastructural analysis of C/OK virus isolate in cell culture as observed by negative stain (A) and thin-section (B) electron microscopy.

<p>(<b>A</b>) Negative stain shows features of an orthomyxovirus particle (Bar = 200 nm) (<b>B</b>) Infected cells visualized with uranyl acetate and lead contrast. Note assembly and budding of virions at the apical pole. Free spherical virions 70–90 nm in diameter present surface spikes and internal electron-dense dots. Bar = 500 nm.</p