Evaluation of Screening Assays for the Detection of Influenza A Virus Serum Antibodies in Swine

Increased surveillance of influenza A virus (IAV) infections in human and swine populations is mandated by public health and animal health concerns. Antibody assays have proven useful in previous surveillance programmes because antibodies provide a record of prior exposure and the technology is inexpensive. The objective of this research was to compare the performance of influenza serum antibody assays using samples collected from pigs (vaccinated or unvaccinated) inoculated with either A/Swine/OH/511445/2007 γ H1N1 virus or A/Swine/Illinois/02907/2009 Cluster IV H3N2 virus and followed for 42 days. Weekly serum samples were tested for anti‐IAV antibodies using homologous and heterologous haemagglutination‐inhibition (HI) assays, commercial swine influenza H1N1 and H3N2 indirect ELISAs, and a commercial influenza nucleoprotein (NP)‐blocking ELISA. The homologous HIs showed 100% diagnostic sensitivity, but largely failed to detect infection with the heterologous virus. With diagnostic sensitivities of 1.4% and 4.9%, respectively, the H1N1 and H3N2 indirect ELISAs were ineffective at detecting IAV antibodies in swine infected with the contemporary influenza viruses used in the study. At a cut‐off of S/N ≤ 0.60, the sensitivity and specificity of the NP‐blocking ELISA were estimated at 95.5% and 99.6%, respectively. Statistically significant factors which affected S/N results include vaccination status, inoculum (virus subtype), day post‐inoculation and the interactions between those factors (P < 0.0001). Serum antibodies against NP provide an ideal universal diagnostic screening target and could provide a cost‐effective approach for the detection and surveillance of IAV infections in swine populations.     

Influenza A Virus in Backyard Pigs and Poultry in Rural Cambodia

Surveillance of influenza virus in humans and livestock is critical, given the worldwide public health threats and livestock production losses. Livestock farming involving close proximity between humans, pigs and poultry is often practised by smallholders in low‐income countries and is considered an important driver of influenza virus evolution. This study determined the prevalence and genetic characteristics of influenza A virus (IAV) in backyard pigs and poultry in Cambodia. A total of 751 animals were tested by matrix gene‐based rRT‐PCR, and influenza virus was detected in 1.5% of sampled pigs, 1.4% of chickens and 1.0% of ducks, but not in pigeons. Full‐length genome sequencing confirmed triple reassortant H3N2 in all IAV‐positive pigs and various low pathogenic avian influenza subtypes in poultry. Phylogenetic analysis of the swine influenza viruses revealed that these had haemagglutinin and neuraminidase genes originating from human H3N2 viruses previously isolated in South‐East Asia. Phylogenetic analysis also revealed that several of the avian influenza subtypes detected were closely related to internal viral genes from highly pathogenic H5N1 and H9N2 formerly sequenced in the region. High sequence homology was likewise found with influenza A viruses circulating in pigs, poultry and wild birds in China and Vietnam, suggesting transboundary introduction and cocirculation of the various influenza subtypes. In conclusion, highly pathogenic subtypes of influenza virus seem rare in backyard poultry, but virus reassortment, involving potentially zoonotic and pandemic subtypes, appears to occur frequently in smallholder pigs and poultry. Increased targeted surveillance and monitoring of influenza circulation on smallholdings would further improve understanding of the transmission dynamics and evolution of influenza viruses in humans, pigs and poultry in the Mekong subregion and could contribute to limit the influenza burden.     

Identification and genomic characterization of influenza viruses with different origin in Mexican pigs

Swine influenza is a worldwide disease, which causes damage to the respiratory system of pigs. The H1N1 and H3N2 subtypes circulate mainly in the swine population of Mexico. There is evidence that new subtypes of influenza virus have evolved genetically and have been rearranged with human viruses and from other species; therefore, the aim of our study was to identify and characterize the genetic changes that have been generated in the different subtypes of the swine influenza virus in Mexican pigs. By sequencing and analyzing phylogenetically the eight segments that form the virus genome, the following subtypes were identified: H1N1, H3N2, H1N2 and H5N2; of which, a H1N1 subtype had a high genetic relationship with the human influenza virus. In addition, a H1N2 subtype related to the porcine H1N2 virus reported in the United States was identified, as well as, two other viruses of avian origin from the H5N2 subtype. Particularly for the H5N2 subtype, this is the first time that its presence has been reported in Mexican pigs. The analysis of these sequences demonstrates that in the swine population of Mexico, circulate viruses that have suffered punctual‐specific mutations and rearrangements of their proteins with different subtypes, which have successfully adapted to the Mexican swine population.     

The PB2 and M genes are critical for the superiority of genotype S H9N2 virus to genotype H in optimizing viral fitness of H5Nx and H7N9 avian influenza viruses in mice

Genotype S H9N2 avian influenza virus, which has been predominant in China since 2010, contributed its entire internal gene cassette to the genesis of novel reassortant influenza viruses, including H5Nx, H7N9 and H10N8 viruses that pose great threat to poultry and humans. A key feature of the genotype S H9N2 virus is the substitution of G1‐like M and PB2 genes for the earlier F/98‐like M and PB2 of genotype H virus. However, how this gene substitution has influenced viral adaptability of emerging influenza viruses in mammals remains unclear. We report here that reassortant H5Nx and H7N9 viruses with the genotype S internal gene cassette displayed enhanced replication and virulence over those with genotype H internal gene cassette in cell cultures as well as in the mouse models. We showed that the G1‐like PB2 gene was associated with increased polymerase activity and improved nuclear accumulation compared with the F/98‐like counterpart, while the G1‐like M gene facilitated effective translocation of RNP to cytoplasm. Our findings suggest that the genotype S H9N2 internal gene cassette, which possesses G1‐like M and PB2 genes, is superior to that of genotype H, in optimizing viral fitness, and thus have implications for assessing the potential risk of these gene introductions to generate emerging influenza viruses.     

Characteristics of the first H16N3 subtype influenza A viruses isolated in western China

The first documented avian influenza virus subtype H16N3 was isolated in 1975 and is currently detectable in many countries worldwide. However, the prevalence, biological characteristics and threat to humans of the avian influenza virus H16N3 subtype in China remain poorly understood. We performed avian influenza surveillance in major wild bird gatherings across the country from 2017 to 2019, resulting in the isolation of two H16N3 subtype influenza viruses. Phylogenetic analysis showed these viruses belong to the Eurasian lineage, and both viruses presented the characteristics of inter‐species reassortment. In addition, the two viruses exhibited limited growth capacity in MDCK and A549 cells. Receptor‐binding assays indicated that the two H16N3 viruses presented dual receptor‐binding profiles, being able to bind to both human and avian‐type receptors, where GBHG/NX/2/2018(H16N3) preferentially bound the avian‐type receptor, while GBHG/NX/1/2018(H16N3) showed greater binding to the human‐type receptor, even the mice virulence data showed the negative results. Segments from other species have been introduced into the H16N3 avian influenza virus, which may alter its pathogenicity and host tropism, potentially posing a threat to animal and human health in the future. Consequently, it is necessary to increase monitoring of the emergence and spread of avian influenza subtype H16N3 in wild birds.     

Detection of Influenza A Virus Nucleoprotein Antibodies in Oral Fluid Specimens From Pigs Infected Under Experimental Conditions Using a Blocking ELISA

In commercial swine populations, influenza is an important component of the porcine respiratory disease complex (PRDC) and a pathogen with major economic impact. Previously, a commercial blocking ELISA (FlockChek^™ Avian Influenza Virus MultiS‐Screen^® Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME, USA) designed to detect influenza A nucleoprotein (NP) antibodies in avian serum was shown to accurately detect NP antibodies in swine serum. The purpose of this study was to determine whether this assay could detect NP antibodies in swine oral fluid samples. Initially, the procedure for performing the NP‐blocking ELISA on oral fluid was modified from the serum testing protocol by changing sample dilution, sample volume, incubation time and incubation temperature. The detection of NP antibody was then evaluated using pen‐based oral fluid samples (n = 182) from pigs inoculated with either influenza A virus subtype H1N1 or H3N2 under experimental conditions and followed for 42 days post inoculation (DPI). NP antibodies in oral fluid were detected from DPI 7 to 42 in all inoculated groups, that is, the mean sample‐to‐negative (S/N) ratio of influenza‐inoculated pigs was significantly different (P < 0.0001) from uninoculated controls (unvaccinated or vaccinated‐uninoculated groups) through this period. Oral fluid versus serum S/N ratios from the same pen showed a correlation of 0.796 (Pearson's correlation coefficient, P < 0.0001). The results showed that oral fluid samples from influenza virus‐infected pigs contained detectable levels of NP antibodies for ≥42 DPI. Future research will be required to determine whether this approach could be used to monitor the circulation of influenza virus in commercial pig populations.     

Isolation and characterization of avian influenza viruses from raw poultry products illegally imported to Japan by international flight passengers

The transportation of poultry and related products for international trade contributes to transboundary pathogen spread and disease outbreaks worldwide. To prevent pathogen incursion through poultry products, many countries have regulations about animal health and poultry product quarantine. However, in Japan, animal products have been illegally introduced into the country in baggage and confiscated at the airport. Lately, the number of illegally imported poultry and the incursion risk of transboundary pathogens through poultry products have been increasing. In this study, we isolated avian influenza viruses (AIV s) from raw poultry products illegally imported to Japan by international passengers. Highly (H5N1 and H5N6) and low (H9N2 and H1N2) pathogenic AIV s were isolated from raw chicken and duck products carried by flight passengers. H5 and H9 isolates were phylogenetically closely related to viruses isolated from poultry in China, and haemagglutinin genes of H5N1 and H5N6 isolates belonged to clades 2.3.2.1c and 2.3.4.4, respectively. Experimental infections of H5 and H9 isolates in chickens and ducks demonstrated pathogenicity and tissue tropism to skeletal muscles. To prevent virus incursion by poultry products, it is important to encourage the phased cleaning based on the disease control and eradication and promote the reduction in contamination risk in animal products.     

Infection Dynamics of Pandemic 2009 H1N1 Influenza Virus in a Two‐Site Swine Herd

Influenza A viruses are common causes of respiratory disease in pigs and can be transmitted among multiple host species, including humans. The current lack of published information on infection dynamics of influenza viruses within swine herds hinders the ability to make informed animal health, biosecurity and surveillance programme decisions. The objectives of this serial cross‐sectional study were to describe the infection dynamics of influenza virus in a two‐site swine system by estimating the prevalence of influenza virus in animal subpopulations at the swine breeding herd and describing the temporal pattern of infection in a selected cohort of growing pigs weaned from the breeding herd. Nasal swab and blood samples were collected at approximately 30‐day intervals from the swine breeding herd (Site 1) known to be infected with pandemic 2009 H1N1 influenza virus. Sows, gilts and neonatal pigs were sampled at each sampling event, and samples were tested for influenza virus genome using matrix gene RRT‐PCR. Influenza virus was detected in neonatal pigs, but was not detected in sow or gilt populations via RRT‐PCR. A virus genetically similar to that detected in the neonatal pig population at Site 1 was also detected at the wean‐to‐finish site (Site 2), presumably following transportation of infected weaned pigs. Longitudinal sampling of nasal swabs and oral fluids revealed that influenza virus persisted in the growing pigs at Site 2 for at least 69 days. The occurrence of influenza virus in neonatal pigs, but not breeding females, at Site 1 emphasizes the potential for virus maintenance in this dynamic subpopulation, the importance of including this subpopulation in surveillance programmes and the potential transport of influenza virus between sites via the movement of weaned pigs.     

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Influenza A (H1N1) viruses are distributed worldwide and pose a threat to public health. Swine, as a natural host and mixing vessel of influenza A (H1N1) virus, play a critical role in the transmission of this virus to humans. Furthermore, swine influenza A (H1N1) viruses have provided all eight genes or some genes to the genomes of influenza strains that historically have caused human pandemics. Hence, persistent surveillance of influenza A (H1N1) virus in swine herds could contribute to the prevention and control of this virus. Here, we report a novel reassortant influenza A (H1N1) virus generated by reassortment between 2009 pandemic H1N1 viruses and swine viruses. We also found that this virus is prevalent in swine herds in Shandong Province, eastern China. Our findings suggest that surveillance of the emergence of the novel reassortant influenza A (H1N1) virus in swine is imperative.     

Complete genome sequence of a novel influenza A H1N2 virus circulating in swine from Central Bajio region,Mexico

The aim of this study was to perform the complete genome sequence of a swine influenza A H1N2 virus strain isolated from a pig in Guanajuato, México (A/swine/Mexico/GtoDMZC01/2014) and to report its seroprevalence in 86 counties at the Central Bajio zone. To understand the evolutionary dynamics of the isolate, we undertook a phylogenetic analysis of the eight gene segments. These data revealed that the isolated virus is a reassortant H1N2 subtype, as its genes are derived from human (HA, NP, PA) and swine (M, NA, PB1, PB2 and NS) influenza viruses. Pig serum samples were analysed by the hemagglutination inhibition test, using wild H1N2 and H3N2 strains (A/swine/México/Mex51/2010 [H3N2]) as antigen sources. Positive samples to the H1N2 subtype were processed using the field‐isolated H1N1 subtype (A/swine/México/Ver37/2010 [H1N1]). Seroprevalence to the H1N2 subtype was 26.74% in the sampled counties, being Jalisco the state with highest seroprevalence to this subtype (35.30%). The results herein reported demonstrate that this new, previously unregistered influenza virus subtype in México that shows internal genes from other swine viral subtypes isolated in the past 5 years, along with human virus‐originated genes, is widely distributed in this area of the country.     

Poultry trading behaviours in Vietnamese live bird markets as risk factors for avian influenza infection in chickens

Vietnamese poultry are host to co‐circulating subtypes of avian influenza viruses, including H5N1 and H9N2, which pose a great risk to poultry productivity and to human health. AIVs circulate throughout the poultry trade network in Vietnam, with live bird markets being an integral component to this network. Traders at LBMs exhibit a variety of trading practices, which may influence the transmission of AIVs. We identified trading practices that impacted on AIV prevalence in chickens marketed in northern Vietnamese LBMs. We generated sequencing data for 31 H9N2 and two H5N6 viruses. Viruses isolated in the same LBM or from chickens sourced from the same province were genetically closer than viruses isolated in different LBMs or from chickens sourced in different provinces. The position of a vendor in the trading network impacted on their odds of having AIV‐infected chickens. Being a retailer and purchasing chickens from middlemen was associated with increased odds of infection, whereas odds decreased if vendors purchased chickens directly from large farms. Odds of infection were also higher for vendors having a greater volume of ducks unsold per day. These results indicate how the spread of AIVs is influenced by the structure of the live poultry trading network.     

Unusually High Mortality in Waterfowl Caused by Highly Pathogenic Avian Influenza A(H5N1) in Bangladesh

Mortality in ducks and geese caused by highly pathogenic avian influenza A(H5N1) infection had not been previously identified in Bangladesh. In June–July 2011, we investigated mortality in ducks, geese and chickens with suspected H5N1 infection in a north‐eastern district of the country to identify the aetiologic agent and extent of the outbreak and identify possible associated human infections. We surveyed households and farms with affected poultry flocks in six villages in Netrokona district and collected cloacal and oropharyngeal swabs from sick birds and tissue samples from dead poultry. We conducted a survey in three of these villages to identify suspected human influenza‐like illness cases and collected nasopharyngeal and throat swabs. We tested all swabs by real‐time RT‐PCR, sequenced cultured viruses, and examined tissue samples by histopathology and immunohistochemistry to detect and characterize influenza virus infection. In the six villages, among the 240 surveyed households and 11 small‐scale farms, 61% (1789/2930) of chickens, 47% (4816/10 184) of ducks and 73% (358/493) of geese died within 14 days preceding the investigation. Of 70 sick poultry swabbed, 80% (56/70) had detectable RNA for influenza A/H5, including 89% (49/55) of ducks, 40% (2/5) of geese and 50% (5/10) of chickens. We isolated virus from six of 25 samples; sequence analysis of the hemagglutinin and neuraminidase gene of these six isolates indicated clade 2.3.2.1a of H5N1 virus. Histopathological changes and immunohistochemistry staining of avian influenza viral antigens were recognized in the brain, pancreas and intestines of ducks and chickens. We identified ten human cases showing signs compatible with influenza‐like illness; four were positive for influenza A/H3; however, none were positive for influenza A/H5. The recently introduced H5N1 clade 2.3.2.1a virus caused unusually high mortality in ducks and geese. Heightened surveillance in poultry is warranted to guide appropriate diagnostic testing and detect novel influenza strains.     

Genetic characterization of an H13N2 low pathogenic avian influenza virus isolated from gulls in China

Low pathogenic avian influenza viruses circulate in wild birds but are occasionally transmitted to other species, including poultry, mammals and humans. To date, infections with low pathogenic avian influenza viruses of HA subtype 6, HA subtype 7, HA subtype 9 and HA subtype 10 among humans have been reported. However, the epidemiology, genetics and ecology of low pathogenic avian influenza viruses have not been fully understood thus far. Therefore, persistent surveillance of low pathogenic avian influenza virus infections in wild birds and other species is needed. Here, we found a low pathogenic avian influenza virus of the subtype H13N2 (abbreviated as WH42) in black‐tailed gulls in China. All gene sequences of this H13N2 virus were determined and used for subsequent analysis. Phylogenetic analysis of the HA gene and NA gene indicated that WH42 was derived from the Eurasian lineage. We analysed the timing of the reassortment events and found that WH42 was a reassortant whose genes were transferred from avian influenza viruses circulating in Asia, Europe and North America. Additionally, WH42 possessed several molecular markers associated with mammalian virulence and mammalian transmissibility. Interestingly, we also found low but detectable haemagglutination inhibition antibodies against H13N2 low pathogenic avian influenza virus in serum samples collected from chickens. Taken together, our findings show that the H13 virus may have been introduced into poultry and that sustainable surveillance in gulls and poultry is required.     

Molecular and Antigenic Characterization of Triple‐Reassortant H3N2 Swine Influenza Viruses Isolated from Pigs,Turkey and Quail in Canada

In 2005, triple‐reassortant H3N2 (trH3N2) influenza A viruses were isolated from swine and turkeys in Canada. Subsequently, these viruses were isolated from humans and mink in 2006 and 2007, respectively. Following full genome sequencing, H3N2 viruses isolated from turkeys (2005), quail (2008) and swine (2009) in Canada, were characterized as trH3N2. The 2005 turkey isolate was found to be almost identical to other viruses isolated in that year, with quail and pig isolates related very closely to the 2005 trH3N2. Minimal antigenic evolution of the swine isolates relative to the reference 2005 virus was observed. These results suggest the establishment of a stable lineage of trH3N2 in Canadian pigs, with evidence for interspecies transmission to turkeys and quails.     

Experimental infection of clade 1.1.2 (H5N1), clade 2.3.2.1c (H5N1) and clade 2.3.4.4 (H5N6) highly pathogenic avian influenza viruses in dogs

Since the emergence of highly pathogenic avian influenza (HPAI) H5N1 in Asia, the haemagglutinin (HA) gene of this virus lineage has continued to evolve in avian populations, and H5N1 lineage viruses now circulate concurrently worldwide. Dogs may act as an intermediate host, increasing the potential for zoonotic transmission of influenza viruses. Virus transmission and pathologic changes in HPAI clade 1.1.2 (H5N1)‐, 2.3.2.1c (H5N1)‐ and 2.3.4.4 (H5N6)‐infected dogs were investigated. Mild respiratory signs and antibody response were shown in dogs intranasally infected with the viruses. Lung histopathology showed lesions that were associated with moderate interstitial pneumonia in the infected dogs. In this study, HPAI H5N6 virus replication in dogs was demonstrated for the first time. Dogs have been suspected as a “mixing vessel” for reassortments between avian and human influenza viruses to occur. The replication of these three subtypes of the H5 lineage of HPAI viruses in dogs suggests that dogs could serve as intermediate hosts for avian–human influenza virus reassortment if they are also co‐infected with human influenza viruses.     

Epidemiological Analysis of Influenza A Infection in Cambodian Pigs and Recommendations for Surveillance Strategies

This study analysed the available data of seroprevalence to human influenza viruses in pigs in Cambodia using generalized linear mixed models in order to improve understanding of factors underlying the spread of human influenza viruses in Cambodian pigs. The associations between seroprevalence against seasonal H1N1 influenza virus in pigs and the population density of humans and pigs were not significant. However, a positive association between anti‐H3 antibodies in pigs and the human population density was identified. In contrast, there was a negative association between seroprevalence of H3N2 in pigs and the pig population density. Our study has highlighted the difficulty in identifying epidemiological risk factors when a limited data set is used for analyses. We therefore provide recommendations on data collection for future epidemiological analyses that could be improved by collecting metadata related to the animals sampled. In addition, serosurveillance for influenza A viruses in pigs in high‐risk areas or at slaughterhouses is recommended in resource‐limited countries.     

Appearance of US‐like porcine epidemic diarrhoea virus (PEDV) strains before US outbreaks and genetic heterogeneity of PEDVs collected in Northern Vietnam during 2012–2015

Porcine epidemic diarrhoea virus (PEDV ) is the aetiologic agent of porcine epidemic diarrhoea (PED ), a highly contagious enteric disease that is threatening the swine industry globally. Since PED was first reported in Southern Vietnam in 2009, the disease has spread throughout the country and caused substantial economic losses. To identify PEDV s responsible for the recent outbreaks, the full‐length spike (S) gene of 25 field PEDV strains collected from seven northern provinces of Vietnam was sequenced and analysed. The sequence analysis revealed that the S genes of Vietnamese PEDV s were heterogeneous and classified into four genotypes, namely North America and Asian non‐S INDEL , Asian non‐S INDEL , new S INDEL and classical S INDEL . This study reported the pre‐existence of US ‐like PEDV strains in Vietnam. Thirteen Vietnamese variants had a truncated S protein that was 261 amino acids shorter than the normal protein. We also detected one novel variant with an 8‐amino acid insertion located in the receptor‐binding region for porcine aminopeptidase N. Compared to the commercial vaccine strains, the emerging Vietnamese strains were genetically distant and had various amino acid differences in epitope regions and N‐glycosylation sites in the S protein. The development of novel vaccines based on the emerging Vietnamese strains may be contributive to the control of the current PED outbreaks.     

Evidence Obtained from anova to Reason Cross‐species Infection and Cross‐subtype Mutation in Neuraminidases of Influenza A Viruses

The current pandemic of A/H1N1 influenza raises a serious question on cross‐species infection and cross‐subtype mutation because our previous focus on possible influenza pandemic laid on H5N1 subtype and the cross‐species infection between avian and human. In this study, we analyse 3874 neuraminidases from influenza A viruses using anova to answer the question of if there is barrier between species and between subtypes. The results show that there is no cross‐species barrier in some species, and the intra‐species variation is larger than the inter‐species variation in some species hosting the viruses, and the cross‐subtype mutation is possible because there is no cross‐subtype barrier in some subtypes and the intra‐subtype variation is larger than the inter‐subtype variation in some subtypes. These results highlight the state of barrier of influenza A virus, which can help us understand the current pandemic and manufacture more effective vaccines.