Appearance of reassortant European avian‐origin H1 influenza A viruses of swine in Vietnam

Three subtypes—H1N1, H1N2 and H3N2—of influenza A viruses of swine (IAV s‐S) are currently endemic in swine worldwide, but there is considerable genotypic diversity among each subtype and limited geographical distribution. Through IAV s‐S monitoring in Vietnam, two H1N2 influenza A viruses were isolated from healthy pigs in Ba Ria‐Vung Tau Province, Southern Vietnam, on 2 December 2016. BLAST and phylogenetic analyses revealed that their HA and NA genes were derived from those of European avian‐like H1N2 IAV s‐S that contained avian‐origin H1 and human‐like N2 genes, and were particularly closely related to those of IAV s‐S circulating in the Netherlands, Germany or Denmark. In addition, the internal genes of these Vietnamese isolates were derived from human A(H1N1)pdm09 viruses, suggesting that the Vietnamese H1N2 IAV s‐S are reassortants between European H1N2 IAV s‐S and human A(H1N1)pdm09v. The appearance of European avian‐like H1N2 IAV s‐S in Vietnam marks their first transmission outside Europe. Our results and statistical analyses of the number of live pigs imported into Vietnam suggest that the European avian‐like H1N2 IAV s‐S may have been introduced into Vietnam with their hosts through international trade. These findings highlight the importance of quarantining imported pigs to impede the introduction of new IAV s‐S.     

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.     

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.     

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.     

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.     

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Five novel H5N6 influenza viruses, including four highly pathogenic avian influenza viruses and one low pathogenic avian influenza virus, were isolated from migratory birds in Ningxia, China, in November 2017. To understand the genetic origination of the novel H5N6 virus, and the infectivity and pathogenicity of the four highly pathogenic avian influenza viruses in mammals, phylogeographic analyses and infection studies in mice were performed. The phylogenetic and phylogeographic analyses showed that the H5N6 isolates, which are closely related to the viruses from Korea, Japan and the Netherlands, originated from reassortant virus between H5N8 and HxN6 viruses from western Russia. The animal study revealed that the SBD‐87 isolate presented moderate virulence in mice, suggesting a potential public risk to humans and a potential threat to public health.     

Seroprevalence of highly pathogenic avian influenza (H5N1) virus infection among humans in mainland China: A systematic review and meta‐analysis

Although the effective transmission of the H5N1 virus from humans to humans has yet to be further observed, humans are at increased risk of a pandemic caused by H5N1. In order to fully evaluate the seroprevalence and risk factor of highly pathogenic avian influenza A (H5N1) virus infection among in mainland China, we performed a systematic review and meta‐analysis. In this review, we searched literature on the seroprevalence of H5N1 infection among humans in mainland China from 1 January 1997 to 20 October 2018 in English and Chinese databases, including PubMed, Google scholar, Cochrane library, Clinical Trial, VIP, CNKI and WanFang database. We made a selection according to the title and the abstract of paper, and then, we excluded duplicated literature, and data incomplete literature according to the exclusion criteria we formulated. Finally, we extracted how many humans have H5N1 infection from the obtained studies to establish the seroprevalence of H5N1 infection among humans in mainland China. A total of 56 studies (including data of 35,159 humans) were compliant with our criteria. In China, the overall seroprevalence of H5N1 infection among humans was 2.45% (862/35,159), while the seroprevalence of H5N1 infection among humans from central China was 7.32% (213/2,911), higher than those in other regions of China. The seroprevalence of H5N1 infection was associated with test method, sampling time and demographic characteristics of humans. However, there was no significant difference in the effect of gender on the seroprevalence of H5N1 among humans in China. The purpose of this review was to better understand the real infection rate of H5N1 virus among humans and evaluate the potential risk factors for the zoonotic spread of H5N1 virus to humans. Sufficient epidemiological data are important to explore and understand the prevalent status of AIVs throughout the country and to disease control.     

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.     

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.     

Evolutionary and Ecological Dynamics of Transboundary Disease Caused by H5N1 Virus in Southeast Asia

Southeast Asia has been the breeding ground for many emerging diseases in the past decade, and it is in this region that new genetic variants of HPAI H5N1 viruses have been emerging. Cross‐border movement of animals accelerates the spread of H5N1, and the changing environmental conditions also exert strong selective pressure on the viruses. The transboundary zoonotic diseases caused by H5N1 pose a serious and continual threat to global economy and public health. Here, we divided the H5N1 viruses isolated in Southeast Asia during 2003–2009 into four groups according to their phylogenetic relationships among HA gene sequences. Molecular evolution analysis suggests populations in expansion rather than a positive selection for group 2 and group 3, yet group 4 is under strong positive selection. Site 193 was found to be a potential glycosylation site and located in receptor‐binding domain. Note that site 193 tends to appear in avian isolates instead of human strains. Population dynamics analysis reveals that the effective population size of infections in Southeast Asia has undergone three obvious increases, and the results are consistent with the epidemiological analysis. Ecological and phylogeographical analyses show that agro‐ecological environments, migratory birds, domestic waterfowl, especially free‐ranging ducks, are crucial in the occurrence, maintenance and spread of H5N1 virus. The epidemiological links between Indonesia and Suphanburi observed suggest that viruses in Indonesia were originated from multiple introductions.     

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.     

Genetic relationship between poultry and wild bird viruses during the highly pathogenic avian influenza H5N6 epidemic in the Netherlands, 2017–2018

In the Netherlands, three commercial poultry farms and two hobby holdings were infected with highly pathogenic avian influenza (HPAI) H5N6 virus in the winter of 2017–2018. This H5N6 virus is a reassortant of HPAI H5N8 clade 2.3.4.4 group B viruses detected in Eurasia in 2016. H5N6 viruses were also detected in several dead wild birds during the winter. However, wild bird mortality was limited compared to the caused by the H5N8 group B virus in 2016–2017. H5N6 virus was not detected in wild birds after March, but in late summer infected wild birds were found again. In this study, the complete genome sequences of poultry and wild bird viruses were determined to study their genetic relationship. Genetic analysis showed that the outbreaks in poultry were not the result of farm‐to‐farm transmissions, but rather resulted from separate introductions from wild birds. Wild birds infected with viruses related to the first outbreak in poultry were found at short distances from the farm, within a short time frame. However, no wild bird viruses related to outbreaks 2 and 3 were detected. The H5N6 virus isolated in summer shares a common ancestor with the virus detected in outbreak 1. This suggests long‐term circulation of H5N6 virus in the local wild bird population. In addition, the pathogenicity of H5N6 virus in ducks was determined, and compared to that of H5N8 viruses detected in 2014 and 2016. A similar high pathogenicity was measured for H5N6 and H5N8 group B viruses, suggesting that biological or ecological factors in the wild bird population may have affected the mortality rates during the H5N6 epidemic. These observations suggest different infection dynamics for the H5N6 and H5N8 group B viruses in the wild bird population.     

Seasonal risk of low pathogenic avian influenza virus introductions into free‐range layer farms in the Netherlands

Poultry can become infected with avian influenza viruses (AIV) via (in) direct contact with infected wild birds. Free‐range chicken farms in the Netherlands were shown to have a higher risk for introduction of low pathogenic avian influenza (LPAI) virus than indoor chicken farms. Therefore, during outbreaks of highly pathogenic avian influenza (HPAI), free‐range layers are confined indoors as a risk mitigation measure. In this study, we characterized the seasonal patterns of AIV introductions into free‐range layer farms, to determine the high‐risk period. Data from the LPAI serological surveillance programme for the period 2013–2016 were used to first estimate the time of virus introduction into affected farms and then assess seasonal patterns in the risk of introduction. Time of introduction was estimated by fitting a mathematical model to seroprevalence data collected longitudinally from infected farms. For the period 2015–2016, longitudinal follow‐up included monthly collections of eggs for serological testing from a cohort of 261 farms. Information on the time of introduction was then used to estimate the monthly incidence and seasonality by fitting harmonic and Poisson regression models. A significant yearly seasonal risk of introduction that lasted around 4 months (November to February) was identified with the highest risk observed in January. The risk for introduction of LPAI viruses in this period was on average four times significantly higher than the period of low risk around the summer months. Although the data for HPAI infections were limited in the period 2014–2018, a similar risk period for introduction of HPAI viruses was observed. The results of this study can be used to optimize risk‐based surveillance and inform decisions on timing and duration of indoor confinement when HPAI viruses are known to circulate in the wild bird population.     

Characterization of H7N9 avian influenza viruses isolated from duck meat products

Avian influenza H7N9 viruses have caused five epidemic waves of human infections since the first human cases were reported in 2013. In 2016, the initial low pathogenic avian influenza (LPAI) H7N9 viruses became highly pathogenic, acquiring multi‐basic amino acids at the haemagglutinin cleavage site. These highly pathogenic avian influenza (HPAI) H7N9 viruses have been detected in poultry and humans in China, causing concerns of a serious threat to global public health. In Japan, both HPAI and LPAI H7N9 viruses were isolated from duck meat products carried illegally and relinquished voluntarily at the border by passengers on flights from China to Japan between 2016 and 2017. Some of the LPAI and HPAI H7N9 viruses detected at the border in Japan were characterized previously in chickens and ducks; however, their pathogenicity and replicative ability in mammals remain unknown. In this study, we assessed the biological features of two HPAI H7N9 virus isolates [A/duck/Japan/AQ‐HE29‐22/2017 (HE29‐22) and A/duck/Japan/AQ‐HE29‐52/2017 (HE29‐52); both of these viruses were isolated from duck meat at the border)] and an LPAI H7N9 virus isolate [A/duck/Japan/AQ‐HE28‐3/2016 (HE28‐3)] in mice and ferrets. In mice, HE29‐52 was more pathogenic than HE29‐22 and HE28‐3. In ferrets, the two HPAI virus isolates replicated more efficiently in the lower respiratory tract of the animals than did the LPAI virus isolate. Our results indicate that HPAI H7N9 viruses with the potential to cause severe diseases in mammals have been illegally introduced to Japan.     

Co‐circulation and characterization of HPAI‐H5N1 and LPAI‐H9N2 recovered from a duck farm,Yogyakarta, Indonesia

In July 2016, an avian influenza outbreak in duck farms in Yogyakarta province was reported to Disease Investigation Center (DIC), Wates, Indonesia, with approximately 1,000 ducks died or culled. In this study, two avian influenza (AI) virus subtypes, A/duck/Bantul/04161291‐OR/2016 (H5N1) and A/duck/Bantul/04161291‐OP/2016 (H9N2) isolated from ducks in the same farm during an AI outbreak in Bantul district, Yogyakarta province, were sequenced and characterized. Our results showed that H5N1 virus was closely related to the highly pathogenic AI (HPAI) H5N1 of clade 2.3.2.1c, while the H9N2 virus was clustered with LPAI viruses from China, Vietnam and Indonesia H9N2 (CVI lineage). Genetic analysis revealed virulence characteristics for both in avian and in mammalian species. In summary, co‐circulation of HPAI‐H5N1 of clade 2.3.2.1c and LPAI‐H9N2 was identified in a duck farm during an AI outbreak in Yogyakarta province, Indonesia. Our findings raise a concern of the potential risk of the viruses, which could increase viral transmission and/or threat to human health. Routine surveillance of avian influenza viruses should be continuously conducted to understand the dynamic and diversity of the viruses for influenza prevention and control in Indonesia and SEA region.     

One‐Health Simulation Modelling: A Case Study of Influenza Spread between Human and Swine Populations using NAADSM

The circulation of zoonotic influenza A viruses including pH1N1 2009 and H5N1 continue to present a constant threat to animal and human populations. Recently, an H3N2 variant spread from pigs to humans and between humans in limited numbers. Accordingly, this research investigated a range of scenarios of the transmission dynamics of pH1N1 2009 virus at the swine–human interface while accounting for different percentages of swine workers initially immune. Furthermore, the feasibility of using NAADSM (North American Animal Disease Spread Model) applied as a one‐health simulation model was assessed. The study population included 488 swine herds and 29, 707 households of people within a county in Ontario, Canada. Households were categorized as follows: (i) rural households with swine workers, (ii) rural households without swine workers, and (iii) urban households without swine workers. Forty‐eight scenarios were investigated, based on the combination of six scenarios around the transmissibility of the virus at the interface and four vaccination coverage levels of swine workers (0–60%), all under two settings of either swine or human origin of the virus. Outcomes were assessed in terms of stochastic ‘die‐out’ fraction, size and time to peak epidemic day, overall size and duration of the outbreaks. The modelled outcomes indicated that minimizing influenza transmissibility at the interface and targeted vaccination of swine workers had significant beneficial effects. Our results indicate that NAADSM can be used as a framework to model the spread and control of contagious zoonotic diseases among animal and human populations, under certain simplifying assumptions. Further evaluation of the model is required. In addition to these specific findings, this study serves as a benchmark that can provide useful input to a future one‐health influenza modelling studies. Some pertinent information gaps were also identified. Enhanced surveillance and the collection of high‐quality information for more accurate parameterization of such models are encouraged.     

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.     

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.