Ducks: The “Trojan Horses” of H5N1 influenza

Abstract Wild ducks are the main reservoir of influenza A viruses that can be transmitted to domestic poultry and mammals, including humans. Of the 16 hemagglutinin (HA) subtypes of influenza A viruses, only the H5 and H7 subtypes cause highly pathogenic (HP) influenza in the natural hosts. Several duck species are naturally resistant to HP Asian H5N1 influenza viruses. These duck species can shed and spread virus from both the respiratory and intestinal tracts while showing few or no disease signs. While the HP Asian H5N1 viruses are 100% lethal for chickens and other gallinaceous poultry, the absence of disease signs in some duck species has led to the concept that ducks are the “Trojan horses” of H5N1 in their surreptitious spread of virus. An important unresolved issue is whether the HP H5N1 viruses are maintained in the wild duck population of the world. Here, we review the ecology and pathobiology of ducks infected with influenza A viruses and ducks’ role in the maintenance and spread of HP H5N1 viruses. We also identify the key questions about the role of ducks that must be resolved in order to understand the emergence and control of pandemic influenza. It is generally accepted that wild duck species can spread HP H5N1 viruses, but there is insufficient evidence to show that ducks maintain these viruses and transfer them from one generation to the next.     

The ecology and adaptive evolution of influenza A interspecies transmission

Since 2013, there have been several alarming influenza‐related events; the spread of highly pathogenic avian influenza H5 viruses into North America, the detection of H10N8 and H5N6 zoonotic infections, the ongoing H7N9 infections in China and the continued zoonosis of H5N1 viruses in parts of Asia and the Middle East. The risk of a new influenza pandemic increases with the repeated interspecies transmission events that facilitate reassortment between animal influenza strains; thus, it is of utmost importance to understand the factors involved that promote or become a barrier to cross‐species transmission of Influenza A viruses (IAVs). Here, we provide an overview of the ecology and evolutionary adaptations of IAVs, with a focus on a review of the molecular factors that enable interspecies transmission of the various virus gene segments.     

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。     

2009 Pandemic Influenza A (H1N1): Diagnosis,Management, and Prevention— Lessons Learned

The 2009 pandemic influenza A (H1N1) was responsible for the first influenza pandemic of the 21st century. The virus— a previously unknown triple-reassortant virus containing segments of avian, human, and swine origins— generally caused mild disease. Unlike seasonal influenza, 2009 pandemic influenza A (H1N1) primarily affected adults 18 to 64 years of age. During the course of the pandemic, public health officials tried to facilitate diagnostic procedures and share information about treatment modalities globally. Efforts to contain the spread of 2009 pandemic influenza A (H1N1) included personal protective mechanisms and the 2009 H1N1 vaccine, which was not produced quickly enough or in large enough quantities. The lessons learned from this pandemic should be applied to ensure better preparedness in case of future pandemics.     

Results of the influenza virus surveillance in wild birds in Western part of Mongolia

ObjectiveTo present results of virological study of wild birds inhabiting Western Mongolia.MethodsOver a period of 2003–2008, we isolated 13 influenza A viruses: H1N1, H3N6, H13N8 and H4N6 subtypes. We did not isolate any H5N1 subtype, that still cause epizooty in wild birds and poultry.ResultsWe revealed taxonomic and ecological heterogeneity of the birds involved in maintenance of circulation of influenza viruses in the given territory. Influenza viruses were isolated from birds of 6 orders; among them there are species preferring water and semi-aquatic biotopes, one species preferring dry plain region, and also one species which can inhabit both dry and water biotopes.ConclusionsRepresentatives of all main orders of Western Mongolia avifauna are involved in support of influenza A virus circulation, highly pathogenic H5N1 influenza viruses were registered in Mongolia thus it's necessary to continue permanent influenza virus surveillance in wild birds' populations.     

Viral and Host Factors Required for Avian H5N1 Influenza A Virus Replication in Mammalian Cells

Following the initial and sporadic emergence into humans of highly pathogenic avian H5N1 influenza A viruses in Hong Kong in 1997, we have come to realize the potential for avian influenza A viruses to be transmitted directly from birds to humans. Understanding the basic viral and cellular mechanisms that contribute to infection of mammalian species with avian influenza viruses is essential for developing prevention and control measures against possible future human pandemics. Multiple physical and functional cellular barriers can restrict influenza A virus infection in a new host species, including the cell membrane, the nuclear envelope, the nuclear environment, and innate antiviral responses. In this review, we summarize current knowledge on viral and host factors required for avian H5N1 influenza A viruses to successfully establish infections in mammalian cells. We focus on the molecular mechanisms underpinning mammalian host restrictions, as well as the adaptive mutations that are necessary for an avian influenza virus to overcome them. It is likely that many more viral and host determinants remain to be discovered, and future research in this area should provide novel and translational insights into the biology of influenza virus-host interactions.     

Temporal Dynamics of Influenza A(H5N1) Subtype before and after the Emergence of H5N8

Highly pathogenic avian influenza (HPAI) viruses continue to circulate worldwide, causing numerous outbreaks among bird species and severe public health concerns. H5N1 and H5N8 are the two most fundamental HPAI subtypes detected in birds in the last two decades. The two viruses may compete with each other while sharing the same host population and, thus, suppress the spread of one of the viruses. In this study, we performed a statistical analysis to investigate the temporal correlation of the HPAI H5N1 and HPAI H5N8 subtypes using globally reported data in 2015–2020. This was joined with an in-depth analysis using data generated via our national surveillance program in Egypt. A total of 6412 outbreaks were reported worldwide during this period, with 39% (2529) as H5N1 and 61% (3883) as H5N8. In Egypt, 65% of positive cases were found in backyards, while only 12% were found in farms and 23% in live bird markets. Overall, our findings depict a trade-off between the number of positive H5N1 and H5N8 samples around early 2017, which is suggestive of the potential replacement between the two subtypes. Further research is still required to elucidate the underpinning mechanisms of this competitive dynamic. This, in turn, will implicate the design of effective strategies for disease control.     

A veterinarian's experience of the spring 2004 avian influenza outbreak in Laos

Since it was first reported in December 2003, the outbreak of avian influenza A/H5N1 has spread to at least nine countries in Asia, affected multiple species of animals, and caused at least 42 human deaths. The magnitude and extent of this zoonotic outbreak are unprecedented, continue to grow, and threaten the start of a global human influenza pandemic. Control of the H5N1 outbreak has required the implementation of integrated human and veterinary health surveillance and response efforts. These efforts have also necessitated an unprecedented level of bilateral and multilateral international communication and cooperation. This report describes the contribution of one public-health veterinarian to the H5N1 outbreak response effort in Laos, and emphasises the value of multidisciplinary approaches to addressing this and future emerging infectious disease outbreaks.     

Establishment of multiple sublineages of H5N1 influenza virus in Asia: implications for pandemic control

Preparedness for a possible influenza pandemic caused by highly pathogenic avian influenza A subtype H5N1 has become a global priority. The spread of the virus to Europe and continued human infection in Southeast Asia have heightened pandemic concern. It remains unknown from where the pandemic strain may emerge; current attention is directed at Vietnam, Thailand, and, more recently, Indonesia and China. Here, we report that genetically and antigenically distinct sublineages of H5N1 virus have become established in poultry in different geographical regions of Southeast Asia, indicating the long-term endemicity of the virus, and the isolation of H5N1 virus from apparently healthy migratory birds in southern China. Our data show that H5N1 influenza virus, has continued to spread from its established source in southern China to other regions through transport of poultry and bird migration. The identification of regionally distinct sublineages contributes to the understanding of the mechanism for the perpetuation and spread of H5N1, providing information that is directly relevant to control of the source of infection in poultry. It points to the necessity of surveillance that is geographically broader than previously supposed and that includes H5N1 viruses of greater genetic and antigenic diversity.     

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.     

Highly Pathogenic Avian Influenza Clade 2.3.4.4b Subtype H5N8 Virus Isolated from Mandarin Duck in South Korea, 2020

In October 2020, a highly pathogenic avian influenza (HPAI) subtype H5N8 virus was identified from a fecal sample of a wild mandarin duck (Aix galericulata) in South Korea. We sequenced all eight genome segments of the virus, designated as A/Mandarin duck/Korea/K20-551-4/2020(H5N8), and conducted genetic characterization and comparative phylogenetic analysis to track its origin. Genome sequencing and phylogenetic analysis show that the hemagglutinin gene belongs to H5 clade 2.3.4.4 subgroup B. All genes share high levels of nucleotide identity with H5N8 HPAI viruses identified from Europe during early 2020. Enhanced active surveillance in wild and domestic birds is needed to monitor the introduction and spread of HPAI via wild birds and to inform the design of improved prevention and control strategies.     

Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the influenza A virus subtypes responsible for the 20th‐century pandemics

Please cite this paper as: Pasricha et al. (2012) Comprehensive global amino acid sequence analysis of PB1F2 protein of influenza A H5N1 viruses and the Influenza A virus subtypes responsible for the 20th‐century pandemics. Influenza and Other Respiratory Viruses 7(4), 497–505. Background PB1F2 is the 11th protein of influenza A virus translated from +1 alternate reading frame of PB1 gene. Since the discovery, varying sizes and functions of the PB1F2 protein of influenza A viruses have been reported. Selection of PB1 gene segment in the pandemics, variable size and pleiotropic effect of PB1F2 intrigued us to analyze amino acid sequences of this protein in various influenza A viruses. Methods Amino acid sequences for PB1F2 protein of influenza A H5N1, H1N1, H2N2, and H3N2 subtypes were obtained from Influenza Research Database. Multiple sequence alignments of the PB1F2 protein sequences of the aforementioned subtypes were used to determine the size, variable and conserved domains and to perform mutational analysis. Results Analysis showed that 96·4% of the H5N1 influenza viruses harbored full‐length PB1F2 protein. Except for the 2009 pandemic H1N1 virus, all the subtypes of the 20th‐century pandemic influenza viruses contained full‐length PB1F2 protein. Through the years, PB1F2 protein of the H1N1 and H3N2 viruses has undergone much variation. PB1F2 protein sequences of H5N1 viruses showed both human‐ and avian host‐specific conserved domains. Global database of PB1F2 protein revealed that N66S mutation was present only in 3·8% of the H5N1 strains. We found a novel mutation, N84S in the PB1F2 protein of 9·35% of the highly pathogenic avian influenza H5N1 influenza viruses. Conclusions Varying sizes and mutations of the PB1F2 protein in different influenza A virus subtypes with pandemic potential were obtained. There was genetic divergence of the protein in various hosts which highlighted the host‐specific evolution of the virus. However, studies are required to correlate this sequence variability with the virulence and pathogenicity.     

Isolation of Genetically Diverse H5N8 Avian Influenza Viruses in Poultry in Egypt, 2019–2021

The global spread of avian influenza virus (AIV) of clade 2.3.4.4b since 2016 has caused severe losses in wild birds and poultry and has posed a risk for the infection of mammals including humans. The vaccination of poultry has been used to limit the spread of the virus and mitigate its socioeconomic impact. Here, we describe H5N8 epidemics in chickens, turkeys and ducks from different localities in Egypt from 2019 to 2021. About 41.7% (n = 88/211) flocks were tested positive by RT-qPCR for H5N8 viruses with prevalence rates of 45.1% (n = 65/144) and 34.3% (n = 23/67) in vaccinated and non-vaccinated flocks, respectively. A sequence analysis of the hemagglutinin and neuraminidase genes indicated not only the multiple introduction events of H5N8 viruses in Egypt but also the establishment of endemic viruses in commercial poultry in 2020/2021. The recent H5N8 viruses in poultry in Egypt are genetically distinct from the majority of licensed vaccines used in the field. Together, our findings indicate that poultry in Egypt is an endemic center for clade 2.3.4.4b in the Middle East. The efficiency of current vaccines should be regularly evaluated and updated to fully protect poultry flocks in Egypt against H5N8 viruses.     

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.     

An influenza A H1N1 virus revival – pandemic H1N1/09 virus

In April 2009, a novel H1N1 influenza A virus, the so-called pandemic H1N1/09 virus (former designations include swine influenza, novel influenza, swine-origin influenza A [H1N1] virus [S-OIV], Mexican flu, North American Flu) was identified in Mexico. The virus has since spread throughout the world and caused an influenza pandemic as defined by the criteria of the World Health Organization. This represents the first influenza A virus pandemic since the emergence of H3N2 (‘‘Hong Kong’’ Flu) in 1968. Vaccine production has started, and vaccines are expected to become available during the course of 2009. Although the pandemic H1N1/09 virus originates from the triple-reassortant swine influenza (H1) virus circulating in North American pigs, it is not epidemic in pigs. Although the H1N1/09 virus pandemic is currently mild, concerns remain that it may become more aggressive during spreading. The distribution of proper information to the public on the status of the H1N1/09 virus pandemic will be important to achieve a broad awareness of the potential risks and the optimum code of behavior during the pandemic. Here, the features of pandemic H1N1/09 virus are discussed within the framework of knowledge gained from previous influenza A virus pandemics.     

Monoclonal antibodies to the hemagglutinin Sa antigenic site of a/pr/8/34 influenza virus distinguish biologic mutants of swine influenza virus.

The dimorphic L and H hemagglutinin mutants of A/NJ/11/76(H1N1) (swine) influenza virus differ pleiotropically in their replication and virulence characteristics and in their antigenicity. L mutants replicate less well in chicken embryos and Madin-Darby canine kidney cells and are more infective for swine than are H mutants. L and H mutants are not antigenically distinguishable in cross-neutralization tests with homotypic antisera, but they can be identified with certain heterotypic heterogeneous antisera. The present studies demonstrate that two monoclonal antibodies (Sa-5 and Sa-13) to the Sa antigenic site of the hemagglutinin of A/PR/8/34H1N1 influenza virus react with mutants and viral reassortants containing the H hemagglutinin in radioimmunoassay, neutralization, and hemagglutination-inhibition tests but to a lesser degree or not at all with L mutants and reassortants. Conversely, monoclonal antibody (9C8) to the L mutant does not react with H mutants. L to H and H to L revertants, whether or not selected with monoclonal antibody, demonstrate concomitant change in biological and antigenic phenotype. Reactivity of H mutants with Sa monoclonal antibodies localizes the mutational site to a position on the hemagglutinin near the receptor binding site--a position in which single amino acid changes could readily influence both antigenic and biologic activity.     

福建省人感染H5N6禽流感病毒与外环境禽流感病毒相关性分析

目的 分析福建省外环境H5亚型禽流感病毒分布情况及与人感染H5N6禽流感的关系,为人感染禽流感的科学防治提供依据。方法 收集2013-2017年福建省外环境常规监测标本信息,进行统计分析。从数据库下载相关禽流感病毒基因序列,进行基因同源性比对和系统进化分析。结果 外环境监测结果统计分析显示:2013-2017年共采集样本4 903份,H5亚型阳性率为4.24%,且不同的网络实验室(χ²=101.033)、监测场所(χ²=45.526)、标本类型(χ²=31.751)和季节(χ²=48.174)阳性率均存在统计学差异(P<0.05)。福建省首例人感染H5N6禽流感(A/Fujian-Sanyuan/21099/2017)病毒全基因序列与6株福建省外环境H5N6病毒的同源性在85.1%~98.5%,与病家院中采集的1株加强监测外环境标本的HA和NA基因同源性分别为100.0%和99.9%。系统进化分析显示:福建省人感染H5N6病毒HA基因与加强监测的外环境H5N6病毒的进化距离最近,与其余6株距离较远,处在不同的进化分枝中,其基因源于H5N8病毒;而NA基因则与7株外环境H5N6病毒的进化距离较近,源于H5N6病毒。结论 福建省冬春季外环境中禽流感活动较为活跃,应加强活禽交易市场的卫生管理工作。福建省发现的首例人感染H5N6禽流感与外环境禽流感病毒的重配可能存在一定的相关性。     

A consensus-hemagglutinin-based DNA vaccine that protects mice against divergent H5N1 influenza viruses

H5N1 influenza viruses have spread extensively among wild birds and domestic poultry. Cross-species transmission of these viruses to humans has been documented in over 380 cases, with a mortality rate of ≈60%. There is great concern that a H5N1 virus would acquire the ability to spread efficiently between humans, thereby becoming a pandemic threat. An H5N1 influenza vaccine must, therefore, be an integral part of any pandemic preparedness plan. However, traditional methods of making influenza vaccines have yet to produce a candidate that could induce potently neutralizing antibodies against divergent strains of H5N1 influenza viruses. To address this need, we generated a consensus H5N1 hemagglutinin (HA) sequence based on data available in early 2006. This sequence was then optimized for protein expression before being inserted into a DNA plasmid (pCHA5). Immunizing mice with pCHA5, delivered intramuscularly via electroporation, elicited antibodies that neutralized a panel of virions that have been pseudotyped with the HA from various H5N1 viruses (clades 1, 2.1, 2.2, 2.3.2, and 2.3.4). Moreover, immunization with pCHA5 in mice conferred complete (clades 1 and 2.2) or significant (clade 2.1) protection from H5N1 virus challenges. We conclude that this vaccine, based on a consensus HA, could induce broad protection against divergent H5N1 influenza viruses and thus warrants further study.     

Changes in H5N1 influenza virus hemagglutinin receptor binding domain affect systemic spread

The HA of influenza virus is a receptor-binding and fusion protein that is required to initiate infection. The HA receptor-binding domain determines the species of sialyl receptors recognized by influenza viruses. Here, we demonstrate that changes in the HA receptor-binding domain alter the ability of the H5N1 virus to spread systemically in mice. The A/Vietnam/1203/04 (VN1203) and A/Hong Kong/213/03 (HK213) viruses are consistently lethal to domestic chickens but differ in their pathogenicity to mammals. Insertion of the VN1203 HA and neuraminidase (NA) genes into recombinant HK213 virus expanded its tissue tropism and increased its lethality in mice; conversely, insertion of HK213 HA and NA genes into recombinant VN1203 virus decreased its systemic spread and lethality. The VN1203 and HK213 HAs differ by 10 aa, and HK213 HA has shown greater binding affinity for synthetic α2,6-linked sialyl receptor. Introduction of an S227N change and removal of N-linked glycosylation at residue 158 increased the α2,6-binding affinity of VN1203 HA. Recombinant VN1203 virus carrying the S227N change alone or with the residue-158 glycosylation site removed showed reduced lethality and systemic spread in mice but not in domestic chickens. Wild-type VN1203 virus exhibited the greatest efficiency in systemic spread after intramuscular inoculation and in infection of mouse bone marrow-derived dendritic cells and conventional pulmonary dendritic cells. These results show that VN1203 HA glycoprotein confers pathogenicity by facilitating systemic spread in mice; they also suggest that a minor change in receptor binding domain may modulate the virulence of H5N1 viruses.     

Isolation of clade 2.3.4.4b A(H5N8), a highly pathogenic avian influenza virus,from a worker during an outbreak on a poultry farm,Russia, December 2020

This study presents the isolation of influenza A(H5N8) virus clade 2.3.4.4b from a poultry worker during an outbreak of highly pathogenic avian influenza A(H5N8) among chickens at a poultry farm in Astrakhan, Russia in December 2020. Nasopharyngeal swabs collected from seven poultry workers were positive for influenza A(H5N8), as confirmed by RT-PCR and sequencing. The influenza A(H5N8) virus was isolated from one of the human specimens and characterised. Sporadic human influenza A(H5)2.3.4.4. infections represent a possible concern for public health.