Genetic diversity in envelope genes of contemporary U.S. porcine reproductive and respiratory syndrome virus strains influences viral antigenicity.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases in swine caused by porcine reproductive and respiratory syndrome virus (PRRSV). Genome sequences of sixty-six PRRSV strains were obtained using metagenomic sequencing of serum samples collected in the U.S. in 2014 to explore contemporary genetic diversity. Phylogenetic analysis of the genes encoding the envelope proteins identified four to eight distinct lineages with >87% intraclade identity. To explore the effect of the observed genetic diversity on antigenicity, the genome regions encoding either GP2a-GP3-GP4 or GP5-M in strain SD95-21 were replaced with alleles from each of eight distinct PRRSV strains using reverse genetics. The GP2a-GP3-GP4 region from only four of the eight strains yielded viable recombinant virus. When viable, both GP2a-GP3-GP4 and GP5-M variably affected antigenicity. A strain-dependent significant loss in cross reactivity was variably observed by indirect immunofluorescence assays using antisera from pigs vaccinated with commercial modified-live vaccines following replacement of GP2a-GP3-GP4 or GP5-M. Significantly reduced neutralization titers were similarly measured using antisera from naturally PRRSV-exposed pigs. These results illustrate the need to consider genomic regions besides GP5 for PRRSV epidemiology and vaccination.

An Amino Acid in the Stalk Domain of N1 Neuraminidase Is Critical for Enzymatic Activity.

Neuraminidase (NA) is a sialidase expressed on the surface of influenza A viruses that releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. It is a homotetramer, with each monomer consisting of a transmembrane region, a stalk, and a globular head with sialidase activity. We recently characterized two swine viruses of the pandemic H1N1 lineage, A/swine/Virginia/1814-1/2012 (pH1N1low-1) and A/swine/Virginia/1814-2/2012 (pH1N1low-2), with almost undetectable NA enzymatic activity compared to that of the highly homologous A/swine/Pennsylvania/2436/2012 (pH1N1-1) and A/swine/Minnesota/2499/2012 (pH1N1-2) viruses. pH1N1-1 transmitted to aerosol contact ferrets, but pH1N1low-1 did not. The aim of this study was to identify the molecular determinants associated with low NA activity as potential markers of aerosol transmission. We identified the shared unique substitutions M19V, A232V, D248N, and I436V (N1 numbering) in pH1N1low-1 and pH1N1low-2. pH1N1low-1 also had the unique Y66D substitution in the stalk domain, where 66Y was highly conserved in N1 NAs. Restoration of 66Y was critical for the NA activity of pH1N1low-1 NA, although 19M or 248D in conjunction with 66Y was required to recover the level of activity to that of pH1N1 viruses. Studies of NA stability and molecular modeling revealed that 66Y likely stabilized the NA homotetramer. Therefore, 66Y in the stalk domain of N1 NA was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity. IMPORTANCE: Neuraminidase (NA) is a sialidase that is one of the major surface glycoproteins of influenza A viruses and the target for the influenza drugs oseltamivir and zanamivir. NA is important as it releases progeny viruses from the surface of infected cells and prevents viruses becoming trapped in mucus. Mutations in the globular head domain that decrease enzymatic activity but confer resistance to NA inhibitors have been characterized; however, the importance of specific mutations in the stalk domain is unknown. We identified 66Y (N1 numbering), a highly conserved amino acid that was critical for the stability of the NA tetramer and, subsequently, for NA enzymatic activity.

Replication and Transmission of the Novel Bovine Influenza D Virus in a Guinea Pig Model.

UNLABELLED: Influenza D virus (FLUDV) is a novel influenza virus that infects cattle and swine. The goal of this study was to investigate the replication and transmission of bovine FLUDV in guinea pigs. Following direct intranasal inoculation of animals, the virus was detected in nasal washes of infected animals during the first 7 days postinfection. High viral titers were obtained from nasal turbinates and lung tissues of directly inoculated animals. Further, bovine FLUDV was able to transmit from the infected guinea pigs to sentinel animals by means of contact and not by aerosol dissemination under the experimental conditions tested in this study. Despite exhibiting no clinical signs, infected guinea pigs developed seroconversion and the viral antigen was detected in lungs of animals by immunohistochemistry. The observation that bovine FLUDV replicated in the respiratory tract of guinea pigs was similar to observations described previously in studies of gnotobiotic calves and pigs experimentally infected with bovine FLUDV but different from those described previously in experimental infections in ferrets and swine with a swine FLUDV, which supported virus replication only in the upper respiratory tract and not in the lower respiratory tract, including lung. Our study established that guinea pigs could be used as an animal model for studying this newly emerging influenza virus. IMPORTANCE: Influenza D virus (FLUDV) is a novel emerging pathogen with bovine as its primary host. The epidemiology and pathogenicity of the virus are not yet known. FLUDV also spreads to swine, and the presence of FLUDV-specific antibodies in humans could indicate that there is a potential for zoonosis. Our results showed that bovine FLUDV replicated in the nasal turbinate and lungs of guinea pigs at high titers and was also able to transmit from an infected animal to sentinel animals by contact. The fact that bovine FLUDV replicated productively in both the upper and lower respiratory tracts of guinea pigs, similarly to virus infection in its native host, demonstrates that guinea pigs would be a suitable model host to study the replication and transmission potential of bovine FLUDV.

Discovery of a novel putative atypical porcine pestivirus in pigs in the USA.

Pestiviruses are some of the most significant pathogens affecting ruminants and swine. Here, we assembled a 11 276 bp contig encoding a predicted 3635 aa polyprotein from porcine serum with 68 % pairwise identity to that of a recently partially characterized Rhinolophus affinis pestivirus (RaPV) and approximately 25-28 % pairwise identity to those of other pestiviruses. The virus was provisionally named atypical porcine pestivirus (APPV). Metagenomic sequencing of 182 serum samples identified four additional APPV-positive samples. Positive samples originated from five states and ELISAs using recombinant APPV Erns found cross-reactive antibodies in 94 % of a collection of porcine serum samples, suggesting widespread distribution of APPV in the US swine herd. The molecular and serological results suggest that APPV is a novel, highly divergent porcine pestivirus widely distributed in US pigs.

An inactivated vaccine made from a U.S. field isolate of porcine epidemic disease virus is immunogenic in pigs as demonstrated by a dose-titration.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV), a highly pathogenic and transmissible virus in swine, was first detected in the U.S. in May, 2013, and has caused tremendous losses to the swine industry. Due to the difficulty in isolating and growing this virus in cell culture, few vaccine studies using cell culture propagated PEDV have been performed on U.S. strains in pigs. Therefore, the objective of this study was to evaluate the humoral immune response to the selected inactivated PEDV vaccine candidate in a dose-titration manner. RESULTS: PEDV was isolated from a pig with diarrhea and complete genome sequencing found >99% nucleotide identity to other U.S. PEDV. Inactivated adjuvanted monovalent vaccines were administered intramuscularly to five week old pigs in a dose titration experimental design, ranging from 6.0-8.0 log10 tissue culture infective dose (TCID50/mL), to evaluate immunogenicity using a fluorescent foci neutralization assay (FFN), fluorescent microsphere immunoassay (FMIA), and enzyme-linked immunosorbent assay (ELISA) on sera. Pigs vaccinated with 8.0 log10 TCID50/mL inactivated virus showed significantly higher FFN titers as well as FMIA and ELISA values than 6.0 log10 TCID50/mL vaccinates and the negative controls. CONCLUSIONS: These results demonstrate the immunogenicity of a PEDV inactivated viral vaccine with a U.S. strain via dose-titration. A future vaccination-challenge study would illustrate the efficacy of an inactivated vaccine and help evaluate protective FFN titers and ELISA and FMIA responses.

Pandemic Swine H1N1 Influenza Viruses with Almost Undetectable Neuraminidase Activity Are Not Transmitted via Aerosols in Ferrets and Are Inhibited by Human Mucus but Not Swine Mucus.

UNLABELLED: A balance between the functions of the influenza virus surface proteins hemagglutinin (HA) and neuraminidase (NA) is thought to be important for the transmission of viruses between humans. Here we describe two pandemic H1N1 viruses, A/swine/Virginia/1814-1/2012 and A/swine/Virginia/1814-2/2012 (pH1N1low-1 and -2, respectively), that were isolated from swine symptomatic for influenza. The enzymatic activity of the NA of these viruses was almost undetectable, while the HA binding affinity for α2,6 sialic acids was greater than that of the highly homologous pH1N1 viruses A/swine/Pennsylvania/2436/2012 and A/swine/Minnesota/2499/2012 (pH1N1-1 and -2), which exhibited better-balanced HA and NA activities. The in vitro growth kinetics of pH1N1low and pH1N1 viruses were similar, but aerosol transmission of pH1N1low-1 was abrogated and transmission via direct contact in ferrets was significantly impaired compared to pH1N1-1, which transmitted by direct and aerosol contact. In normal human bronchial epithelial cells, pH1N1low-1 was significantly inhibited by mucus but pH1N1-1 was not. In Madin-Darby canine kidney cell cultures overlaid with human or swine mucus, human mucus inhibited pH1N1low-1 but swine mucus did not. These data show that the interaction between viruses and mucus may be an important factor in viral transmissibility and could be a barrier for interspecies transmission between humans and swine for influenza viruses. IMPORTANCE: A balance between the functions of the influenza virus surface proteins hemagglutinin (HA) and neuraminidase (NA) is thought to be important for transmission of viruses from swine to humans. Here we show that a swine virus with extremely functionally mismatched HA and NAs (pH1N1low-1) cannot transmit via aerosol in ferrets, while another highly homologous virus with HA and NAs that are better matched functionally (pH1N1-1) can transmit via aerosol. These viruses show similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, but pH1N1low-1 is significantly inhibited by mucus in normal human bronchial epithelial cells whereas pH1N1-1 is not. Further, human mucus could inhibit these viruses, but swine mucus could not. These data show that the interaction between viruses and mucus may be an important factor in viral transmissibility and could be a species barrier between humans and swine for influenza viruses.

Bovine rhinitis viruses are common in U.S. cattle with bovine respiratory disease.

Bovine rhinitis viruses (BRV) are established etiological agents of bovine respiratory disease complex however little research into their epidemiology and ecology has been published for several decades. In the U.S., only bovine rhinitis A virus 1 (BRAV1) has been identified while bovine rhinitis A virus 2 (BRAV2) and bovine rhinitis B virus (BRBV) were previously only identified in England and Japan, respectively. Metagenomic sequencing of a nasal swab from a bovine respiratory disease (BRD) diagnostic submission from Kansas identified contigs with approximately 90% nucleotide similarity to BRAV2 and BRBV. A combination of de novo and templated assemblies using reference genomes yielded near complete BRAV2 and BRBV genomes. The near complete genome of bovine rhinitis A virus 1 (BRAV1) was also determined from a historical isolate to enable further molecular epidemiological studies. A 5'-nuclease reverse transcription PCR assay targeting the 3D polymerase gene was designed and used to screen 204 archived BRD clinical specimens. Thirteen (6.4%) were positive. Metagenomic sequencing of six positive samples identified mixed BRAV1/BRAV2, BRAV1/BRBV and BRAV2/BRBV infections for five samples. One sample showed infection only with BRAV1. Seroprevalence studies using a cell culture adapted BRBV found immunofluorescence assay-reactive antibodies were common in the herds analyzed. Altogether, these results demonstrate that BRV infections are common in cattle with respiratory disease and that BRAV1, BRAV2 and BRBV co-circulate in U.S. cattle and have high similarity to viruses isolated more than 30 years ago from diverse locations.

Cocirculation of two distinct genetic and antigenic lineages of proposed influenza D virus in cattle.

UNLABELLED: Viruses with approximately 50% homology to human influenza C virus (ICV) have recently been isolated from swine and cattle. The overall low homology to ICV, lack of antibody cross-reactivity to ICV in hemagglutination inhibition (HI) and agar gel immunodiffusion assays, and inability to productively reassort with ICV led to the proposal that these viruses represented a new genus of influenza virus, influenzavirus D (IDV). To further our understanding of the epidemiology of IDV, real-time reverse transcription-PCR was performed on a set of 208 samples from bovines with respiratory disease. Ten samples (4.8%) were positive and six viruses were successfully isolated in vitro. Phylogenetic analysis of full-genome sequences of these six new viruses and four previously reported viruses revealed two distinct cocirculating lineages represented by D/swine/Oklahoma/1334/2011 (D/OK) and D/bovine/Oklahoma/660/2013 (D/660), which frequently reassorted with one another. Antigenic analysis using the HI assay and lineage-representative D/OK and D/660 antiserum found up to an approximate 10-fold loss in cross-reactivity against heterologous clade antiserum. One isolate, D/bovine/Texas/3-13/2011 (D/3-13), clustered with the D/660 lineage, but also had high HI titers to heterologous (D/OK) clade antiserum. Molecular modeling of the hemagglutinin esterase fusion protein of D/3-13 identified a mutation at position 212 as a possible antigenic determinant responsible for the discrepant HI results. These results suggest that IDV is common in bovines with respiratory disease and that at least two genetic and antigenically distinct clades cocirculate. IMPORTANCE: A novel bovine influenza virus was recently identified. Detailed genetic and antigenic studies led to the proposal that this virus represents a new genus of influenza, influenzavirus D (IDV). Here, we show that IDV is common in clinical samples of bovine respiratory disease complex (BRDC), with a prevalence similar to that of other established BRDC etiological agents. These results are in good agreement with the near-ubiquitous seroprevalence of IDV previously found. Phylogenetic analysis of complete genome sequences found evidence for two distinct cocirculating lineages of IDV which freely reassort. Significant antigenic differences, which generally agreed with the surface glycoprotein hemagglutinin esterase phylogeny, were observed between the two lineages. Based on these results, and on the ability of IDV to infect and transmit in multiple mammalian species, additional studies to determine the pathogenic potential of IDV are warranted.

Characterization of a novel influenza virus in cattle and Swine: proposal for a new genus in the Orthomyxoviridae family.

We have recently reported the isolation of a novel virus, provisionally designated C/swine/Oklahoma/1334/2011 (C/OK), with 50% overall homology to human influenza C viruses (ICV), from a pig in Oklahoma. Deep RNA sequencing of C/OK virus found a matrix 1 (M1) protein expression strategy that differed from that of ICV. The novelty of C/OK virus prompted us to investigate whether C/OK virus could exist in a nonswine species. Significantly, we found that C/OK virus was widespread in U.S. bovine herds, as demonstrated by reverse transcription (RT)-PCR and serological assays. Genome sequencing of three bovine viruses isolated from two herds in different states further confirmed these findings. To determine whether swine/bovine C/OK viruses can undergo reassortment with human ICV, and to clarify the taxonomic status of C/OK, in vitro reassortment and serological typing by agar gel immunodiffusion (AGID) were conducted. In vitro reassortment using two human ICV and two swine and bovine C/OK viruses demonstrated that human ICV and C/OK viruses were unable to reassort and produce viable progeny. Antigenically, no cross-recognition of detergent split virions was observed in AGID between human and nonhuman viruses by using polyclonal antibodies that were reactive to cognate antigens. Taken together, these results demonstrate that C/OK virus is genetically and antigenically distinct from ICV. The classification of the new virus in a separate genus of the Orthomyxoviridae family is proposed. The finding of C/OK virus in swine and bovine indicates that this new virus may spread and establish infection in other mammals, including humans. IMPORTANCE Influenza C viruses (ICV) are common human pathogens, infecting most people during childhood and adolescence, and typically cause mild respiratory symptoms. While ICV have been isolated from both pigs and dogs, humans are thought to be the natural viral reservoir. Previously, we characterized an ICV-like virus isolated from pigs exhibiting symptoms of influenza virus-like illness. Here, we show molecular and serological data demonstrating widespread circulation of similar viruses in bovines. Deep RNA sequencing, phylogenetic analysis, and in vitro reassortment experiments demonstrate that animal ICV-like viruses are genetically distinct from human ICV. Antigenically, we show that ICV-like viruses are not recognized by ICV antibodies. En masse, these results suggest that bovine influenza virus warrants classification as a new genus of influenza virus. The finding of this novel virus that can infect multiple mammalian species warrants further research into its role in human health.

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.

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.