Minimal molecular constraints for respiratory droplet transmission of an avian–human H9N2 influenza A virus

Pandemic influenza requires interspecies transmission of an influenza virus with a novel hemagglutinin (HA) subtytpe that can adapt to its new host through either reassortment or point mutations and transmit by aerosolized respiratory droplets. Two previous pandemics of 1957 and 1968 resulted from the reassortment of low pathogenic avian viruses and human subtypes of that period; however, conditions leading to a pandemic virus are still poorly understood. Given the endemic situation of avian H9N2 influenza with human-like receptor specificity in Eurasia and its occasional transmission to humans and pigs, we wanted to determine whether an avian–human H9N2 reassortant could gain respiratory transmission in a mammalian animal model, the ferret. Here we show that following adaptation in the ferret, a reassortant virus carrying the surface proteins of an avian H9N2 in a human H3N2 backbone can transmit efficiently via respiratory droplets, creating a clinical infection similar to human influenza infections. Minimal changes at the protein level were found in this virus capable of respiratory droplet transmission. A reassortant virus expressing only the HA and neuraminidase (NA) of the ferret-adapted virus was able to account for the transmissibility, suggesting that currently circulating avian H9N2 viruses require little adaptation in mammals following acquisition of all human virus internal genes through reassortment. Hemagglutinin inhibition (HI) analysis showed changes in the antigenic profile of the virus, which carries profound implications for vaccine seed stock preparation against avian H9N2 influenza. This report illustrates that aerosolized respiratory transmission is not exclusive to current human H1, H2, and H3 influenza subtypes.     

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.     

From the Cover: Dating the emergence of pandemic influenza viruses

Pandemic influenza viruses cause significant mortality in humans. In the 20th century, 3 influenza viruses caused major pandemics: the 1918 H1N1 virus, the 1957 H2N2 virus, and the 1968 H3N2 virus. These pandemics were initiated by the introduction and successful adaptation of a novel hemagglutinin subtype to humans from an animal source, resulting in antigenic shift. Despite global concern regarding a new pandemic influenza, the emergence pathway of pandemic strains remains unknown. Here we estimated the evolutionary history and inferred date of introduction to humans of each of the genes for all 20th century pandemic influenza strains. Our results indicate that genetic components of the 1918 H1N1 pandemic virus circulated in mammalian hosts, i.e., swine and humans, as early as 1911 and was not likely to be a recently introduced avian virus. Phylogenetic relationships suggest that the A/Brevig Mission/1/1918 virus (BM/1918) was generated by reassortment between mammalian viruses and a previously circulating human strain, either in swine or, possibly, in humans. Furthermore, seasonal and classic swine H1N1 viruses were not derived directly from BM/1918, but their precursors co-circulated during the pandemic. Mean estimates of the time of most recent common ancestor also suggest that the H2N2 and H3N2 pandemic strains may have been generated through reassortment events in unknown mammalian hosts and involved multiple avian viruses preceding pandemic recognition. The possible generation of pandemic strains through a series of reassortment events in mammals over a period of years before pandemic recognition suggests that appropriate surveillance strategies for detection of precursor viruses may abort future pandemics.     

H6N1亚型禽流感病毒跨种属传播研究进展

禽流感病毒H6N1亚型广泛存在于水禽和陆禽,是最常分离到的甲型流感病毒亚型,遗传分析表明该病毒可能是高致病禽流感病毒H5N1的前体。随着病毒基因的持续进化,H6N1可跨种间屏障传播至哺乳动物,其对于哺乳动物小鼠、猪和雪貂已经具有较强感染能力。血清流行病学调查结果显示少数人H6禽流感病毒抗体阳性,2013年5月,我国台湾出现全球首例人类感染H6N1亚型流感病毒。因此,H6N1病毒宿主范围不断扩大,在这些宿主内可发生病毒基因突变、基因重配,进而演变为具有感染人类潜能的新变异株的可能。本文从病原学、流行病学、病毒感染哺乳动物和人类等方面对H6N1亚型禽流感病毒的研究进展进行综述,以期为H6N1禽流感病毒的防控提供参考。     

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.     

Transmission of influenza A(H1N1) 2009 pandemic viruses in Australian swine

Please cite this paper as: Deng et al. (2012). Transmission of influenza A(H1N1) 2009 pandemic viruses in Australian swine. Influenza and Other Respiratory Viruses 6(3), e42–e47. Background Swine have receptors for both human and avian influenza viruses and are a natural host for influenza A viruses. The 2009 influenza A(H1N1) pandemic (H1N1pdm) virus that was derived from avian, human and swine influenza viruses has infected pigs in various countries. Objectives To investigate the relationship between the H1N1pdm viruses isolated from piggery outbreaks in Australia and human samples associated with one of the outbreaks by phylogenetic analysis, and to determine whether there was any reassortment event occurring during the human‐pig interspecies transmission. Methods Real‐time RT‐PCR and full genome sequencing were carried out on RNA isolated from nasal swabs and/or virus cultures. Phylogenetic analysis was performed using the Geneious package. Results The influenza H1N1pdm outbreaks were detected in three pig farms located in three different states in Australia. Further analysis of the Queensland outbreak led to the identification of two distinct virus strains in the pigs. Two staff working in the same piggery were also infected with the same two strains found in the pigs. Full genome sequence analysis on the viruses isolated from pigs and humans did not identify any reassortment of these H1N1pdm viruses with seasonal or avian influenza A viruses. Conclusions This is the first report of swine infected with influenza in Australia and marked the end of the influenza‐free era for the Australian swine industry. Although no reassortment was detected in these cases, the ability of these viruses to cross between pigs and humans highlights the importance of monitoring swine for novel influenza infections.     

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.     

上海地区2009年人群甲型流行性感冒病毒抗体水平检测分析

目的 了解上海地区人群季节性H1、H3亚型流感病毒抗体水平及职业人群中H5、H9亚型禽流感抗体的检出情况.方法 应用血凝抑制试验对上海地区2009年与禽类密切接触的职业人群356人以及一般人群332人各年龄组进行H1、H3、H5、H9亚型流感病毒抗体的血清学监测.结果 A/Brisbane/59/2007(H1N1)抗体阳性分布,一般人群有275人,占82.8%,职业人群有263人,占73.9%;A/Brisbane/10/2007(H3N2)抗体阳性,一般人群有168人,占50.6%,职业人群有195人,占54.8%;人群中A/Brisbane/59/2007(H1N1)抗体阳性率明显高于H3,与2008年上海地区流感病原学监测情况相吻合.职业人群与一般人群H5亚型抗体阳性率分别为4.2%(15/356)和0.3%(1/332);H9亚型抗体阳性率分别为34.6%(123/356)和2.4%(8/332).不同年龄组中,6个月～5岁年龄组和≥60岁的老年组A/Brisbane/59/2007(H1N1)、A/Brisbane/10/2007(H3N2)抗体阳性率较低.结论 上海地区人群对H1、H3季节性流感有较强的免疫保护,儿童和老年人群H1、H3季节性流感抗体水平较低.职业人群禽流感病毒H5、H9抗体有明显升高趋势,要加强对职业人群流感样患者病原学和血清流行病学监测.     

Influenza: prospect for prevention and control

Influenza is an emerging and re-emerging disease. Since the late 1930s influenza viruses have been isolated yearly from different parts of the world during epidemics and pandemics. The "epidemiologic success" of influenza is due largely to rapid and unpredictable antigenic changes (antigenic drift) among human influenza viruses, and the emergence of new subtypes (antigenic shift), mostly from reassortment between human and avian influenza viruses. Antigenic shifts were attributed to the global pandemic viruses of 1957 (H2N2 Asian flu) and 1968 (H3N2 Hong Kong flu). Concern over possible new pandemics has been heightened by recent reports of human infection in Asia in 1997 with avian viruses (H5N1) and in 1999 (H9N2) and isolation of human-avian reassorted viruses from pigs and humans in Europe. Influenza has a high rate of inapparent infection, short incubation and high infectivity; epidemics usually start abruptly and spread rapidly to neighboring communities and countries. Isolation and quarantine are often unsuccessful in preventing the spread of the infection. Although not perfect, immunization and chemoprophylaxis are highly effective at minimizing the spread of influenza and reducing morbidity and mortality, social disruption and economic loss. Plans for future influenza epidemics and pandemics require national and international programs to be in place for the monitoring of influenza activity, the dissemination and exchange of information and the provision and delivery of sufficient quantities of vaccines and antiviral agents. This paper reviews and discusses the antigenic variations of the influenza virus, potential influenza pandemics, protective efficacy of inactivated vaccines and antiviral agents and preparation for control of future epidemics and pandemics.     

H9N2 influenza A virus circulates in H5N1 endemically infected poultry population in Egypt

We describe the identification and characterization of the H9N2 influenza subtype reported in Egyptian broiler and broiler breeder farms for the first time. Circulation of this subtype in a highly pathogenic H5N1 influenza virus endemic population provides an opportunity for genetic reassortment and emergence of novel viruses.     

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.     

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。     

A Characterization and an Evolutionary and a Pathogenicity Analysis of Reassortment H3N2 Avian Influenza Virus in South China in 2019–2020

Seasonal H3N2 influenza virus has always been a potential threat to public health. The reassortment of the human and avian H3N2 influenza viruses has resulted in major influenza outbreaks, which have seriously damaged human life and health. To assess the possible threat of the H3N2 avian influenza virus to human health, we performed whole-genome sequencing and genetic evolution analyses on 10 H3N2 field strains isolated from different hosts and regions in 2019–2020 and selected representative strains for pathogenicity tests on mice. According to the results, the internal gene cassettes of nine strains had not only undergone reassortment with the H1, H2, H4, H6, and H7 subtypes, which circulate in poultry and mammals, but also with H10N8, which circulates in wild birds in the natural environment. Three reassorted strains were found to be pathogenic to mice, of these one strain harboring MP from H10N8 showed a stronger virulence in mice. This study indicates that reassorted H3N2 AIVs may cross the host barrier to infect mammals and humans, thereby, necessitating persistent surveillance of H3N2 AIVs.     

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.     

Genomic and Phylogenetic Characterization of Novel,Recombinant H5N2 Avian Influenza Virus Strains Isolated from Vaccinated Chickens with Clinical Symptoms in China

Infection of poultry with diverse lineages of H5N2 avian influenza viruses has been documented for over three decades in different parts of the world, with limited outbreaks caused by this highly pathogenic avian influenza virus. In the present study, three avian H5N2 influenza viruses, A/chicken/Shijiazhuang/1209/2013, A/chicken/Chiping/0321/2014, and A/chicken/Laiwu/0313/2014, were isolated from chickens with clinical symptoms of avian influenza. Complete genomic and phylogenetic analyses demonstrated that all three isolates are novel recombinant viruses with hemagglutinin (HA) and matrix (M) genes derived from H5N1, and remaining genes derived from H9N2-like viruses. The HA cleavage motif in all three strains (PQIEGRRRKR/GL) is characteristic of a highly pathogenic avian influenza virus strain. These results indicate the occurrence of H5N2 recombination and highlight the importance of continued surveillance of the H5N2 subtype virus and reformulation of vaccine strains.     

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.     

Avian influenza: Myth or mass murder?

The purpose of the present article was to determine whether avian influenza (AI) is capable of causing a pandemic. Using research from a variety of medical journals, books and texts, the present paper evaluates the probability of the AI virus becoming sufficiently virulent to pose a global threat.Previous influenza A pandemics from the past century are reviewed, focusing on the mortality rate and the qualities of the virus that distinguish it from other viruses. Each of the influenza A viruses reviewed were classified as pandemic because they met three key criteria: first, the viruses were highly pathogenic within the human population; second, the viruses were easily transmissible from person to person; and finally, the viruses were novel, such that a large proportion of the population was susceptible to infection. Information about the H5N1 subtype of AI has also been critically assessed. Evidence suggests that this AI subtype is both novel and highly pathogenic. The mortality rate from epidemics in Thailand in 2004 was as high as 66%. Clearly, this virus is aggressive. It causes a high death rate, proving that humans have a low immunity to the disease.To date, there has been little evidence to suggest that AI can spread among humans. There have been cases where the virus has transferred from birds to humans, in settings such as farms or open markets with live animal vending. If AI were to undergo a genetic reassortment that allowed itself to transmit easily from person to person, then a serious pandemic could ensue, resulting in high morbidity and mortality. Experts at the World Health Organization and the United States Centers for Disease Control and Prevention agree that AI has the potential to undergo an antigenic shift, thus triggering the next pandemic.     

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.     

Genetic Variations among Different Variants of G1-like Avian Influenza H9N2 Viruses and Their Pathogenicity in Chickens

Since it was first discovered, the low pathogenic avian influenza (LPAI) H9N2 subtype has established linages infecting the poultry population globally and has become one of the most prevalent influenza subtypes in domestic poultry. Several different variants and genotypes of LPAI H9N2 viruses have been reported in Egypt, but little is known about their pathogenicity and how they have evolved. In this study, four different Egyptian LPAI H9N2 viruses were genetically and antigenically characterized and compared to representative H9N2 viruses from G1 lineage. Furthermore, the pathogenicity of three genetically distinct Egyptian LPAI H9N2 viruses was assessed by experimental infection in chickens. Whole-genome sequencing revealed that the H9N2 virus of the Egy-2 G1-B lineage (pigeon-like) has become the dominant circulating H9N2 genotype in Egypt since 2016. Considerable variation in virus shedding at day 7 post-infections was detected in infected chickens, but no significant difference in pathogenicity was found between the infected groups. The rapid spread and emergence of new genotypes of the influenza viruses pinpoint the importance of continuous surveillance for the detection of novel reassortant viruses, as well as monitoring the viral evolution.