Compatibility of H9N2 avian influenza surface genes and 2009 pandemic H1N1 internal genes for transmission in the ferret model

In 2009, a novel H1N1 influenza (pH1N1) virus caused the first influenza pandemic in 40 y. The virus was identified as a triple reassortant between avian, swine, and human influenza viruses, highlighting the importance of reassortment in the generation of viruses with pandemic potential. Previously, we showed that a reassortant virus composed of wild-type avian H9N2 surface genes in a seasonal human H3N2 backbone could gain efficient respiratory droplet transmission in the ferret model. Here we determine the ability of the H9N2 surface genes in the context of the internal genes of a pH1N1 virus to efficiently transmit via respiratory droplets in ferrets. We generated reassorted viruses carrying the HA gene alone or in combination with the NA gene of a prototypical H9N2 virus in the background of a pH1N1 virus. Four reassortant viruses were generated, with three of them showing efficient respiratory droplet transmission. Differences in replication efficiency were observed for these viruses; however, the results clearly indicate that H9N2 avian influenza viruses and pH1N1 viruses, both of which have occasionally infected pigs, have the potential to reassort and generate novel viruses with respiratory transmission potential in mammals.     

Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model

Avian influenza A H5N1 viruses continue to spread globally among birds, resulting in occasional transmission of virus from infected poultry to humans. Probable human-to-human transmission has been documented rarely, but H5N1 viruses have not yet acquired the ability to transmit efficiently among humans, an essential property of a pandemic virus. The pandemics of 1957 and 1968 were caused by avian-human reassortant influenza viruses that had acquired human virus-like receptor binding properties. However, the relative contribution of human internal protein genes or other molecular changes to the efficient transmission of influenza viruses among humans remains poorly understood. Here, we report on a comparative ferret model that parallels the efficient transmission of H3N2 human viruses and the poor transmission of H5N1 avian viruses in humans. In this model, an H3N2 reassortant virus with avian virus internal protein genes exhibited efficient replication but inefficient transmission, whereas H5N1 reassortant viruses with four or six human virus internal protein genes exhibited reduced replication and no transmission. These findings indicate that the human virus H3N2 surface protein genes alone did not confer efficient transmissibility and that acquisition of human virus internal protein genes alone was insufficient for this 1997 H5N1 virus to develop pandemic capabilities, even after serial passages in a mammalian host. These results highlight the complexity of the genetic basis of influenza virus transmissibility and suggest that H5N1 viruses may require further adaptation to acquire this essential pandemic trait.     

Avian influenza and human health

Natural infections with influenza A viruses have been reported in a variety of animal species including humans, pigs, horses, sea mammals, mustelids and birds. Occasionally devastating pandemics occur in humans. Although viruses of relatively few HA and NA subtype combinations have been isolated from mammalian species, all 15 HA subtypes and all 9 NA subtypes, in most combinations, have been isolated from birds.In the 20th century the sudden emergence of antigenically different strains transmissible in humans, termed antigenic shift, has occurred on four occasions, 1918 (H1N1), 1957 (H2N2), 1968 (H3N2) and 1977 (H1N1), each time resulting in a pandemic. Genetic analysis of the isolates demonstrated that 'new' strains most certainly emerged after reassortment of genes of viruses of avian and human origin in a permissive host. The leading theory is that the pig represents the 'mixing vessel' where this genetic reassortment may occur.In 1996, an H7N7 influenza virus of avian origin was isolated from a woman with a self-limiting conjunctivitis. During 1997 in Hong Kong, an H5N1 avian influenza virus was recognised as the cause of death of 6 of 18 infected patients. Genetic analysis revealed these human isolates of H5N1 subtype to be indistinguishable from a highly pathogenic avian influenza virus that was endemic in the local poultry population. More recently, in March 1999, two independent isolations of influenza virus subtype H9N2 were made from girls aged one to four who recovered from flu-like illnesses in Hong Kong. Subsequently, five isolations of H9N2 virus from humans on mainland China in August 1998 were reported. H9N2 viruses were known to be widespread in poultry in China and other Asian countries.In all these cases there was no evidence of human to human spread except with the H5N1 infections where there was evidence of very limited spread. This is in keeping with the finding that all these viruses possessed all eight genes of avian origin. It may well be that infection of humans with avian influenza viruses occurs much more frequently than originally assumed, but due to their limited effect go unrecognised.For the human population as a whole the main danger of direct infection with avian influenza viruses appears to be if people infected with an 'avian' virus are infected simultaneously with a 'human' influenza virus. In such circumstances reassortment could occur with the potential emergence of a virus fully capable of spread in the human population, but with antigenic characteristics for which the human population was immunologically naive. Presumably this represents a very rare coincidence, but one which could result in a true influenza pandemic.     

Critical role of segment-specific packaging signals in genetic reassortment of influenza A viruses

The fragmented nature of the influenza A genome allows the exchange of gene segments when two or more influenza viruses infect the same cell, but little is known about the rules underlying this process. Here, we studied genetic reassortment between the A/Moscow/10/99 (H3N2, MO) virus originally isolated from human and the avian A/Finch/England/2051/91 (H5N2, EN) virus and found that this process is strongly biased. Importantly, the avian HA segment never entered the MO genetic background alone but always was accompanied by the avian PA and M fragments. Introduction of the 5′ and 3′ packaging sequences of HA_MO into an otherwise HA_EN backbone allowed efficient incorporation of the chimerical viral RNA (vRNA) into the MO genetic background. Furthermore, forcing the incorporation of the avian M segment or introducing five silent mutations into the human M segment was sufficient to drive coincorporation of the avian HA segment into the MO genetic background. These silent mutations also strongly affected the genotype of reassortant viruses. Taken together, our results indicate that packaging signals are crucial for genetic reassortment and that suboptimal compatibility between the vRNA packaging signals, which are detected only when vRNAs compete for packaging, limit this process.The mechanisms by which animal viruses are introduced into and are disseminated through the human population remain to be addressed. In particular, emerging pathogenic influenza viruses, such as the highly pathogenic avian H5N1 virus and the 2009 “swine” H1N1 virus (H1N1pdm2009), pose major public health and scientific challenges (1, 2). Even though the natural reservoirs of influenza A viruses are wild aquatic birds, influenza A viruses exhibit a broad host range and a wide antigenic diversity, represented by combinations of 17 hemagglutinin (HA) and nine neuraminidase (NA) subtypes (3). Two subtypes of influenza A viruses, H1N1 and H3N2, currently are circulating in the human population.The genome of influenza A viruses is composed of eight single-stranded, negative-sense viral RNA (vRNA) segments. Each segment is associated with the heterotrimeric polymerase complex consisting of polymerase basic proteins 1 and 2 and polymeric acid (PB1/PB2/PA) and is covered by the viral nucleoprotein (NP) to form a viral ribonucleoparticle (vRNP). The fragmented nature of the genome allows the exchange of gene segments when two or more influenza viruses coinfect the same cell, in a process named “genetic reassortment” (4). Genetic reassortment is a major feature of influenza evolution and cross-species transmission and also is important for the generation of antigenically novel isolates by introducing novel HA segments in compatible genetic backgrounds (5–7). Future pandemic viruses most likely will carry different HA genes to which human populations are immunologically naive. The strains giving rise to the 1918 Spanish, 1957 Asian, and 1968 Hong Kong influenza pandemics all harbored an HA segment derived from an avian virus. The avian viruses circulating in the waterfowl are the source of the HA genes most likely to be introduced into the human population (8). Phylogenetic, epidemic, epizootic, and virology studies suggest that swine serve as “mixing vessels” for the generation of human–avian–swine reassortant viruses.When the reassortment process takes place between a human and an avian influenza virus, there are in theory 127 possible reassortant viruses harboring the avian HA segment. Two studies used forced reverse genetics (i.e., a minimal set of reverse genetic plasmids allowing no competition between segments) to generate all 127 reassortant viruses carrying the HA segment from an avian H5N1 virus in the genetic background of a human H3N2 or the HA segment from an avian H9N2 virus in the genetic background of the human 2009 pandemic H1N1 virus (9, 10). They showed that 49% (H5N1/H3N2) and 58% (H9N2/H1N1) of these reassortant viruses replicated efficiently in Madin–Darby canine kidney (MDCK) cells (9, 10). However, several reports indicated that the number of observed natural or experimental reassortant viruses is much smaller than 127, suggesting that reassortment is somehow restricted (4, 11, 12). When analyzing viruses from the nasal secretions of ferrets coinfected with human H3N2 and avian H5N1 viruses, Jackson et al. (13) observed that only 3.1% were reassortant viruses possessing the HA H5 gene, and they corresponded to only five distinct genotypes. Genetic reassortment between human H3N2 and an equine H7N7 virus has been studied using cotransfection (4). Only 1.6% of purified viruses, corresponding to two genotypes, were reassortant viruses possessing the HA H7 gene (4). In contrast, a high frequency of genetic reassortment was observed recently between swine-origin H1N1 and avian H5N1 viruses: 64% of purified viruses, corresponding to 20 different genotypes, were reassortant viruses possessing the HA H5 gene (14). In this case, the high reassortment rate was attributed to the triple reassortant internal gene cassette, consisting of the avian PA and PB2 genes, the nonstructural (NS), NP, and matrix (M) swine genes, and the human PB1 gene (15).The low number of reassortant genotypes usually generated from genetically diverse influenza viruses suggests incompatibilities at the protein and/or genomic level. Accumulating evidence indicates that protein incompatibility among the vRNP components is a limiting factor for reassortment between two viruses (4, 16–18), but little is known about genetic incompatibilities between the vRNA segments. Although incompatibility between proteins is expected to have similar effects in cotransfection or coinfection experiments and in forced reverse genetic experiments, genomic incompatibilities may have several possible effects, especially at the level of the vRNA-packaging signals. Some incompatibilities between packaging signals might reduce viral replication in the absence of competition (absolute incompatibility), whereas more subtle ones might be revealed only when vRNA segments from the two parental viruses compete for packaging (suboptimal compatibility). Reverse genetics-derived reassortant viruses (RGd-RV) that possess the H5N1 (H5) HA in an otherwise H3N2 genetic background show high replicative capacities in MDCK cells (10). Similarly, RGd-RV with the HA gene from H5N1 virus in the H1N1pdm2009 genetic background replicated efficiently in primary human respiratory epithelial cells and caused 100% mortality in mice (19). However, phylogenetic analyses of natural or experimental reassortant viruses have shown that the HA segment from avian, swine, or equine viruses was never incorporated alone in the genetic background of a human virus (13, 14, 20): The HA segment is packaged with additional groups of gene segments depending on the viral subtypes involved in the coinfection process (13, 14).The inability to obtain a virus containing a nonhuman HA gene in an otherwise human genetic background, in contrast with the ability to produce “7+1” RGd-RV with a high yield of replication, suggests that the reassortment process might be restricted by suboptimal compatibility between the vRNA-packaging signals (10).To predict how pandemic influenza viruses can emerge, the complex molecular mechanisms limiting or facilitating genetic reassortment must be deciphered. Using reverse genetics, cis-packaging signals of the human H1N1 WSN and PR8 strains were found to reside at both ends of each vRNA, including the UTRs, along with up to 80 bases of adjacent coding sequences (21–28). In this study, we generated reassortant viruses in vitro from avian H5N2 and human H3N2 viruses to identify incompatibilities between the two parental viruses arising at the vRNA level. Our experiments focusing on the generation of reassortant viruses containing the HA H5 gene segment in an H3N2 genetic background indicate that genomic suboptimal compatibility driven by the selective packaging mechanism limits the generation of HA H5 reassortant viruses in vitro.     

Virulence and transmissibility of H1N2 influenza virus in ferrets imply the continuing threat of triple-reassortant swine viruses

Efficient worldwide swine surveillance for influenza A viruses is urgently needed; the emergence of a novel reassortant pandemic H1N1 (pH1N1) virus in 2009 demonstrated that swine can be the direct source of pandemic influenza and that the pandemic potential of viruses prevalent in swine populations must be monitored. We used the ferret model to assess the pathogenicity and transmissibility of predominant Korean triple-reassortant swine (TRSw) H1N2 and H3N2 influenza viruses genetically related to North American strains. Although most of the TRSw viruses were moderately pathogenic, one [A/Swine/Korea/1204/2009; Sw/1204 (H1N2)] was virulent in ferrets, causing death within 10 d of inoculation, and was efficiently transmitted to naive contact ferrets via respiratory droplets. Although molecular analysis did not reveal known virulence markers, the Sw/1204 virus acquired mutations in hemagglutinin (HA) (Asp-225-Gly) and neuraminidase (NA) (Ser-315-Asn) proteins during the single ferret passage. The contact-Sw/1204 virus became more virulent in mice, replicated efficiently in vitro, extensively infected human lung tissues ex vivo, and maintained its ability to replicate and transmit in swine. Reverse-genetics studies further indicated that the HA_225G and NA_315N substitutions contributed substantially in altering virulence and transmissibility. These findings support the continuing threat of some field TRSw viruses to human and animal health, reviving concerns on the capacity of pigs to create future pandemic viruses. Apart from warranting continued and enhanced global surveillance, this study also provides evidence on the emerging roles of HA_225G and NA_315N as potential virulence markers in mammals.     

Diversity of influenza viruses in swine and the emergence of a novel human pandemic influenza A (H1N1)

Abstract The novel H1N1 influenza virus that emerged in humans in Mexico in early 2009 and transmitted efficiently in the human population with global spread has been declared a pandemic strain. Here we review influenza infections in swine since 1918 and the introduction of different avian and human influenza virus genes into swine influenza viruses of North America and Eurasia. These introductions often result in viruses of increased fitness for pigs that occasionally transmit to humans. The novel virus affecting humans is derived from a North American swine influenza virus that has acquired two gene segments [Neuraminidase (NA) and Matrix (M)] from the European swine lineages. This reassortant appears to have increased fitness in humans. The potential for increased virulence in humans and of further reassortment between the novel H1N1 influenza virus and oseltamivir resistant seasonal H1N1 or with highly pathogenic H5N1 influenza stresses the need for urgent pandemic planning.     

The Effects of Genetic Variation on H7N9 Avian Influenza Virus Pathogenicity

Since the H7N9 avian influenza virus emerged in China in 2013, there have been five seasonal waves which have shown human infections and caused high fatality rates in infected patients. A multibasic amino acid insertion seen in the HA of current H7N9 viruses occurred through natural evolution and reassortment, and created a high pathogenicity avian influenza (HPAI) virus from the low pathogenicity avian influenza (LPAI) in 2017, and significantly increased pathogenicity in poultry, resulting in widespread HPAI H7N9 in poultry, which along with LPAI H7N9, contributed to the severe fifth seasonal wave in China. H7N9 is a novel reassorted virus from three different subtypes of influenza A viruses (IAVs) which displays a great potential threat to public health and the poultry industry. To date, no sustained human-to-human transmission has been recorded by the WHO. However, the high ability of evolutionary adaptation of H7N9 and lack of pre-existing immunity in humans heightens the pandemic potential. Changes in IAVs proteins can affect the viral transmissibility, receptor binding specificity, pathogenicity, and virulence. The multibasic amino acid insertion, mutations in hemagglutinin, deletion and mutations in neuraminidase, and mutations in PB2 contribute to different virological characteristics. This review summarized the latest research evidence to describe the impacts of viral protein changes in viral adaptation and pathogenicity of H7N9, aiming to provide better insights for developing and enhancing early warning or intervention strategies with the goal of preventing highly pathogenic IAVs circulation in live poultry, and transmission to humans.     

Identification of H2N3 influenza A viruses from swine in the United States

Although viruses of each of the 16 influenza A HA subtypes are potential human pathogens, only viruses of the H1, H2, and H3 subtype are known to have been successfully established in humans. H2 influenza viruses have been absent from human circulation since 1968, and as such they pose a substantial human pandemic risk. In this report, we isolate and characterize genetically similar avian/swine virus reassortant H2N3 influenza A viruses isolated from diseased swine from two farms in the United States. These viruses contained leucine at position 226 of the H2 protein, which has been associated with increased binding affinity to the mammalian α2,6Gal-linked sialic acid virus receptor. Correspondingly, the H2N3 viruses were able to cause disease in experimentally infected swine and mice without prior adaptation. In addition, the swine H2N3 virus was infectious and highly transmissible in swine and ferrets. Taken together, these findings suggest that the H2N3 virus has undergone some adaptation to the mammalian host and that their spread should be very closely monitored.     

Genetic Characterization and Pathogenicity of H7N7 and H7N9 Avian Influenza Viruses Isolated from South Korea

H7 low pathogenic avian influenza viruses (LPAIVs) can mutate into highly pathogenic avian influenza viruses (HPAIVs). In addition to avian species, H7 avian influenza viruses (AIVs) also infect humans. In this study, two AIVs, H7N9 (20X-20) and H7N7 (34X-2), isolated from the feces of wild birds in South Korea in 2021, were genetically analyzed. The HA cleavage site of the two H7 Korean viruses was confirmed to be ELPKGR/GLF, indicating they are LPAIVs. There were no amino acid substitutions at the receptor-binding site of the HA gene of two H7 Korean viruses compared to that of A/Anhui/1/2013 (H7N9), which prefer human receptors. In the phylogenetic tree analysis, the HA gene of the two H7 Korean viruses shared the highest nucleotide similarity with the Korean H7 subtype AIVs. In addition, the HA gene of the two H7 Korean viruses showed high nucleotide similarity to that of the A/Jiangsu/1/2018(H7N4) virus, which is a human influenza virus originating from avian influenza virus. Most internal genes (PB2, PB1, PA, NP, NA, M, and NS) of the two H7 Korean viruses belonged to the Eurasian lineage, except for the M gene of 34X-2. This result suggests that active reassortment occurred among AIVs. In pathogenicity studies of mice, the two H7 Korean viruses replicated in the lungs of mice. In addition, the body weight of mice infected with 34X-2 decreased 7 days post-infection (dpi) and inflammation was observed in the peribronchiolar and perivascular regions of the lungs of mice. These results suggest that mammals can be infected with the two H7 Korean AIVs. Our data showed that even low pathogenic H7 AIVs may infect mammals, including humans, as confirmed by the A/Jiangsu/1/2018(H7N4) virus. Therefore, continuous monitoring and pathogenicity assessment of AIVs, even of LPAIVs, are required.     

鸭呼吸道和消化道流感病毒SA受体的分布特征

目的探讨鸭流感病毒受体分布特点及其作用。方法应用凝集素组织化学染色技术检测鸭呼吸道和消化道流感病毒SA受体的分布,用荧光素Alexa488标记禽流感病毒H9N1、人流感病毒H1N1,观察这两种病毒与鸭呼吸道、消化道各解剖部位结合特点。结果SAα-2,3Gal受体在鸭呼吸道气管、支气管、次级支气管、副支气管和消化道结肠呈高密度分布,而SAot-2,6Gal受体缺乏或仅极少量表达。禽流感病毒H9N1能与鸭呼吸道和消化道上皮细胞结合,而人流感病毒H1N1与副支气管和结肠未见结合反应,仅极少量与气管、支气管、次级支气管结合。结论水禽类鸭流感病毒SA受体的分布以SAα-2,3Gal受体为主,在呼吸道和消化道均呈高密度分布,有利于各亚型禽流感病毒在其复制、基因重配。     

猪流感病毒基因池中的新成员:新甲型H1N1流感病毒基因片段的存在及潜在威胁

新甲型H1N1流感病毒(2009 pandemic H1N1 virus,pdm/09)于2009年在人群中暴发以后,迅速在全球范围内传播,引起了21世纪的第一次流感大流行。pdm/09是由人的流感病毒、禽流感和猪流感病毒(swine influenza virus,SIV)经过重配后形成的病毒,它的基因片段已经进入了猪流感病毒当中并开始产生新的变异毒株,这些新的变异流感毒株在欧亚大陆、北美大陆及中国南部的各个地区被不断报道和发现,这表明猪源性pdm/09在人间流行后可返传给猪,成为猪流感病毒基因池中的固有组成,获得与SIV重组形成新的重配病毒的能力,并可能仍然具有感染人类的潜能。因此,必须关注新型重配病毒的进化:包括其在猪群中的生长适应、以及适应性感染人的进化过程。不仅如此,还必须加强对猪群及人群流感病毒的检测,了解重配病毒在人和猪两个种群中的进化过程。     

Insights into Genetic Characteristics and Virological Features of Endemic Avian Influenza A (H9N2) Viruses in Egypt from 2017–2021

From 2010 to 2013, genotype I avian influenza A(H9N2) viruses of the G1-lineage were isolated from several poultry species in Egypt. In 2014, novel reassortant H9N2 viruses were detected in pigeons designated as genotype II. To monitor the subsequent genetic evolution of Egyptian A(H9N2) viruses, we characterized the full genomes of 173 viruses isolated through active surveillance from 2017 to 2022. In addition, we compared the virological characteristics and pathogenicity of representative viruses. Phylogenetic analysis of the HA indicated that all studied sequences from 2017–2021 were grouped into G1-like H9N2 viruses previously detected in Egypt. Phylogenetic analysis indicated that the Egyptian A(H9N2) viruses had undergone further reassortment, inheriting four genes (PB2, PB1, PA, NS) from genotype II, with their remaining segments deriving from genotype I viruses (these viruses designated as genotype III). Studying the virological features of the two most dominant genotypes (I and III) of Egyptian H9N2 viruses in vitro and in vivo indicated that both replicated well in mammalian cells, but did not show any clinical signs in chickens, ducks, and mice. Monitoring avian influenza viruses through surveillance programs and understanding the genetic and antigenic characteristics of circulating H9N2 viruses are essential for risk assessment and influenza pandemic preparedness.     

Multiple Neuraminidase Containing Influenza Virus-like Particle Vaccines Protect Mice from Avian and Human Influenza Virus Infection

Avian influenza virus remains a threat for humans, and vaccines preventing both avian and human influenza virus infections are needed. Since virus-like particles (VLPs) expressing single neuraminidase (NA) subtype elicited limited heterosubtypic protection, VLPs expressing multiple NA subtypes would enhance the extent of heterosubtypic immunity. Here, we generated avian influenza VLP vaccines displaying H5 hemagglutinin (HA) antigen with or without avian NA subtypes (N1, N6, N8) in different combinations. BALB/c mice were intramuscularly immunized with the VLPs to evaluate the resulting homologous and heterosubtypic immunity upon challenge infections with the avian and human influenza viruses (A/H5N1, A/H3N2, A/H1N1). VLPs expressing H5 alone conferred homologous protection but not heterosubtypic protection, whereas VLPs co-expressing H5 and NA subtypes elicited both homologous and heterosubtypic protection against human influenza viruses in mice. We observed that VLP induced neuraminidase inhibitory activities (NAI), virus-neutralizing activity, and virus-specific antibody (IgG, IgA) responses were strongly correlated with the number of different NA subtype expressions on the VLPs. VLPs expressing all 3 NA subtypes resulted in the highest protection, indicated by the lowest lung titer, negligible body weight changes, and survival in immunized mice. These results suggest that expressing multiple neuraminidases in avian HA VLPs is a promising approach for developing a universal influenza A vaccine against avian and human influenza virus infections.     

The A/Mallard/6750/78 avian-human, but not the A/Ann Arbor/6/60 cold-adapted, influenza A/Kawasaki/86 (H1N1) reassortant virus vaccine retains partial virulence for infants and children

Characteristics of avian-human (ah) and cold-adapted (ca) influenza A/Kawasaki/9/86 (H1N1) reassortant vaccine viruses were compared in 37 seronegative adults and 122 seronegative infants and children. The 50% human infectious dose (HID50) in infants and children was 10(2.9) and 10(2.6) TCID50 for the ah and ca vaccine, respectively. The ah influenza A/Kawasaki/9/86 reassortant was reactogenic: 24% of infants and children infected with greater than or equal to 100 HID50 had fever greater than or equal to 39.4 degrees C. Since H3N2 ah vaccines were previously shown to be adequately attenuated, it is reasonable to suggest that the genes that code for hemagglutinin and neuraminidase of the H1N1 virus apparently influence the reactogenicity of reassortant viruses derived from the avian influenza A/Mallard/New York/6750/78 donor virus. Because this avian virus does not reproducibly confer a satisfactory level of attenuation to each subtype of influenza A virus, it is not a suitable donor virus for attenuation of wild-type influenza viruses. In contrast, the ca A/Ann Arbor/6/60 donor virus reliably confers attenuation characteristics to a variety of H1N1 and H3N2 influenza A viruses.     

Serologic evidence of human influenza virus infections in swine populations,Cambodia

Background This study was conducted from 2006 to 2010 and investigated the seroprevalence of influenza A viruses in Cambodian pigs, including human H1N1, H3N2, 2009 pandemic H1N1 (A(H1N1)pdm09), and highly pathogenic avian H5N1 influenza A viruses. Methods A total of 1147 sera obtained from pigs in Cambodia were tested by haemagglutination inhibition (HI) assays for antibody to human influenza A viruses along with both HI and microneutralization (MN) tests to assess immunological responses to H5N1 virus. The results were compared by year, age, and province. Results Antibodies against a human influenza A virus were detected in 14·9% of samples. A(H1N1)pdm09 virus were dominant over the study period (23·1%), followed by those to human H1N1 (17·3%) and H3N2 subtypes (9·9%). No pigs were serologically positive for avian H5 influenza viruses. The seroprevalence of human H1N1 and H3N2 influenza viruses peaked in 2008, while that of A(H1N1)pdm09 reached a peak in 2010. No significant differences in seroprevalence to human influenza subtypes were observed in different age groups. Conclusions Cambodian pigs were exposed to human strains of influenza A viruses either prior to or during this study. The implications of these high prevalence rates imply human‐to‐swine influenza virus transmission in Cambodia. Although pigs are mostly raised in small non‐commercial farms, our preliminary results provide evidence of sustained human influenza virus circulation in pig populations in Cambodia.     

Avian influenza virus infections in humans

Seroepidemiologic and virologic studies since 1889 suggested that human influenza pandemics were caused by H1, H2, and H3 subtypes of influenza A viruses. If not for the 1997 avian A/H5N1 outbreak in Hong Kong of China, subtype H2 is the likely candidate for the next pandemic. However, unlike previous poultry outbreaks of highly pathogenic avian influenza due to H5 that were controlled by depopulation with or without vaccination, the presently circulating A/H5N1 genotype Z virus has since been spreading from Southern China to other parts of the world. Migratory birds and, less likely, bird trafficking are believed to be globalizing the avian influenza A/H5N1 epidemic in poultry. More than 200 human cases of avian influenza virus infection due to A/H5, A/H7, and A/H9 subtypes mainly as a result of poultry-to-human transmission have been reported with a > 50% case fatality rate for A/H5N1 infections. A mutant or reassortant virus capable of efficient human-to-human transmission could trigger another influenza pandemic. The recent isolation of this virus in extrapulmonary sites of human diseases suggests that the high fatality of this infection may be more than just the result of a cytokine storm triggered by the pulmonary disease. The emergence of resistance to adamantanes (amantadine and rimantadine) and recently oseltamivir while H5N1 vaccines are still at the developmental stage of phase I clinical trial are causes for grave concern. Moreover, the to-be pandemic strain may have little cross immunogenicity to the presently tested vaccine strain. The relative importance and usefulness of airborne, droplet, or contact precautions in infection control are still uncertain. Laboratory-acquired avian influenza H7N7 has been reported, and the laboratory strains of human influenza H2N2 could also be the cause of another pandemic. The control of this impending disaster requires more research in addition to national and international preparedness at various levels. The epidemiology, virology, clinical features, laboratory diagnosis, management, and hospital infection control measures are reviewed from a clinical perspective.     

Genetic Characterization of Novel H7Nx Low Pathogenic Avian Influenza Viruses from Wild Birds in South Korea during the Winter of 2020–2021

Zoonotic infection with avian influenza viruses (AIVs) of subtype H7, such as H7N9 and H7N4, has raised concerns worldwide. During the winter of 2020–2021, five novel H7 low pathogenic AIVs (LPAIVs) containing different neuraminidase (NA) subtypes, including two H7N3, an H7N8, and two H7N9, were detected in wild bird feces in South Korea. Complete genome sequencing and phylogenetic analysis showed that the novel H7Nx AIVs were reassortants containing two gene segments (hemagglutinin (HA) and matrix) that were related to the zoonotic Jiangsu–Cambodian H7 viruses causing zoonotic infection and six gene segments originating from LPAIVs circulating in migratory birds in Eurasia. A genomic constellation analysis demonstrated that all H7 isolates contained a mix of gene segments from different viruses, indicating that multiple reassortment occurred. The well-known mammalian adaptive substitution (E627K and D701N) in PB2 was not detected in any of these isolates. The detection of multiple reassortant H7Nx AIVs in wild birds highlights the need for intensive surveillance in both wild birds and poultry in Eurasia.     

Cell-Culture Adaptation of H3N2 Influenza Virus Impacts Acid Stability and Reduces Airborne Transmission in Ferret Model

Airborne transmission of seasonal and pandemic influenza viruses is the reason for their epidemiological success and public health burden in humans. Efficient airborne transmission of the H1N1 influenza virus relies on the receptor specificity and pH of fusion of the surface glycoprotein hemagglutinin (HA). In this study, we examined the role of HA pH of fusion on transmissibility of a cell-culture-adapted H3N2 virus. Mutations in the HA head at positions 78 and 212 of A/Perth/16/2009 (H3N2), which were selected after cell culture adaptation, decreased the acid stability of the virus from pH 5.5 (WT) to pH 5.8 (mutant). In addition, the mutant H3N2 virus replicated to higher titers in cell culture but had reduced airborne transmission in the ferret model. These data demonstrate that, like H1N1 HA, the pH of fusion for H3N2 HA is a determinant of efficient airborne transmission. Surprisingly, noncoding regions of the NA segment can impact the pH of fusion of mutant viruses. Taken together, our data confirm that HA acid stability is an important characteristic of epidemiologically successful human influenza viruses and is influenced by HA/NA balance.     

A novel highly pathogenic H5N8 avian influenza virus isolated from a wild duck in China

Migrating wild birds are considered natural reservoirs of influenza viruses and serve as a potential source of novel influenza strains in humans and livestock. During routine avian influenza surveillance conducted in eastern China, a novel H5N8 (SH‐9) reassortant influenza virus was isolated from a mallard duck in China. blast analysis revealed that the HA, NA, PB1, PA, NP, and M segments of SH‐9 were most closely related to the corresponding segments of A/duck/Jiangsu/k1203/2010 (H5N8). The SH‐9 virus preferentially recognized avian‐like influenza virus receptors and was highly pathogenic in mice. Our results suggest that wild birds could acquire the H5N8 virus from breeding ducks and spread the virus via migratory bird flyways.