Novel Reassortant Highly Pathogenic H5N2 Avian Influenza Viruses in Poultry in China

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.     

Spatiotemporal Structure of Molecular Evolution of H5N1 Highly Pathogenic Avian Influenza Viruses in Vietnam

Background

Vietnam is one of the countries most affected by outbreaks of H5N1 highly pathogenic avian influenza viruses. First identified in Vietnam in poultry in 2001 and in humans in 2004, the virus has since caused 111 cases and 56 deaths in humans. In 2003/2004 H5N1 outbreaks, nearly the entire poultry population of Vietnam was culled. Our earlier study (Wan et al., 2008, PLoS ONE, 3(10): e3462) demonstrated that there have been at least six independent H5N1 introductions into Vietnam and there were nine newly emerged reassortants from 2001 to 2007 in Vietnam. H5N1 viruses in Vietnam cluster distinctly around Hanoi and Ho Chi Minh City. However, the nature of the relationship between genetic divergence and geographic patterns is still unclear.

Methodology/Principal Findings

In this study, we hypothesized that genetic distances between H5N1 viruses in Vietnam are correlated with geographic distances, as the result of distinct population and environment patterns along Vietnam''s long north to south longitudinal extent. Based on this hypothesis, we combined spatial statistical methods with genetic analytic techniques and explicitly used geographic space to explore genetic evolution of H5N1 highly pathogenic avian influenza viruses at the sub-national scale in Vietnam. Our dataset consisted of 125 influenza viruses (with whole genome sets) isolated in Vietnam from 2003 to 2007. Our results document the significant effect of space and time on genetic evolution and the rise of two regional centers of genetic mixing by 2007. These findings give insight into processes underlying viral evolution and suggest that genetic differentiation is associated with the distance between concentrations of human and poultry populations around Hanoi and Ho Chi Minh City.

Conclusions/Significance

The results show that genetic evolution of H5N1 viruses in Vietnamese domestic poultry is highly correlated with the location and spread of those viruses in geographic space. This correlation varies by scale, time, and gene, though a classic isolation by distance pattern is observed. This study is the first to characterize the geographic structure of influenza viral evolution at the sub-national scale in Vietnam and can shed light on how H5N1 HPAIVs evolve in certain geographic settings.     

Phylogeography of Highly Pathogenic H5 Avian Influenza Viruses in China

Virologica Sinica - The spread of H5 highly pathogenic avian influenza viruses poses serious threats to the poultry industry, wild bird ecology and human health. Circulation of H5 viruses between...     

Live Attenuated Influenza Viruses Containing NS1 Truncations as Vaccine Candidates against H5N1 Highly Pathogenic Avian Influenza

Due to the high mortality associated with recent, widely circulating strains of H5N1 influenza virus in poultry, the recurring introduction of H5N1 viruses from birds to humans, and the difficulties in H5N1 eradication by elimination of affected flocks, an effective vaccine against HPAI (highly pathogenic avian influenza) is highly desirable. Using reverse genetics, a set of experimental live attenuated vaccine strains based on recombinant H5N1 influenza virus A/Viet Nam/1203/04 was generated. Each virus was attenuated through expression of a hemagglutinin protein in which the polybasic cleavage site had been removed. Viruses were generated which possessed a full-length NS1 or a C-terminally truncated NS1 protein of 73, 99, or 126 amino acids. Viruses with each NS genotype were combined with a PB2 polymerase gene which carried either a lysine or a glutamic acid at position 627. We predicted that glutamic acid at position 627 of PB2 would attenuate the virus in mammalian hosts, thus increasing the safety of the vaccine. All recombinant viruses grew to high titers in 10-day-old embryonated chicken eggs but were attenuated in mammalian cell culture. Induction of high levels of beta interferon by all viruses possessing truncations in the NS1 protein was demonstrated by interferon bioassay. The viruses were each found to be highly attenuated in a mouse model. Vaccination with a single dose of any virus conferred complete protection from death upon challenge with a mouse lethal virus expressing H5N1 hemagglutinin and neuraminidase proteins. In a chicken model, vaccination with a single dose of a selected virus encoding the NS1 1-99 protein completely protected chickens from lethal challenge with homologous HPAI virus A/Viet Nam/1203/04 (H5N1) and provided a high level of protection from a heterologous virus, A/egret/Egypt/01/06 (H5N1). Thus, recombinant influenza A/Viet Nam/1203/04 viruses attenuated through the introduction of mutations in the hemagglutinin, NS1, and PB2 coding regions display characteristics desirable for live attenuated vaccines and hold potential as vaccine candidates in poultry as well as in mammalian hosts.     

Growth and Pathogenic Potential of Naturally Selected Reassortants after Coinfection with Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Viruses

Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants.     

一株鹅源H5N2亚型高致病性禽流感病毒的分离鉴定及生物学特性分析

分离到一株鹅源 H5N2亚型高致病性禽流感病毒,SPF鸡静脉接种致病指数为2.99,但鸭子对该病毒不敏感．病毒感染小鼠后不致病,但能够在肺内有效复制,表明其具有感染哺乳动物的潜在风险．血凝素(hemagglutinin, HA)蛋白裂解位点上插入有多个连续的碱性氨基酸(-RRRKKR-),从分子上证实这是一株高致病性禽流感病毒．核酸序列比较分析表明,分离的流感病毒HA基因与A/chicken/Hubei/489/2004 (H5N1)同源率达到99.4%,神经氨酸酶(neuraminidase, NA)基因与A/chicken/Jilin/53/01(H9N2)同源率达到99.8%;氨基酸水平上,HA与2004年分离到的A/chicken/Hubei/489/2004(H5N1)、A/swan/Guangxi/307/2004(H5N1)、A/wildduck/Guangdong/314/　2004(H5N1)和A/chicken/Henan/210/2004(H5N1)同源率均为99.3%,NA 与A/chicken/Jilin/53/01(H9N2)同源率为99.6%．进化树分析结果表明,该流感病毒分离株可能是由H5N1和H9N2两个亚型病毒重排而来．     

Immunization of Chickens with Newcastle Disease Virus Expressing H5 Hemagglutinin Protects against Highly Pathogenic H5N1 Avian Influenza Viruses

Background

Highly-pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are the two most important poultry viruses in the world. Natural low-virulence NDV strains have been used as vaccines over the past 70 years with proven track records. We have previously developed a reverse genetics system to produce low-virulent NDV vaccine strain LaSota from cloned cDNA. This system allows us to use NDV as a vaccine vector for other avian pathogens.

Methodology/Principal Finding

Here, we constructed two recombinant NDVs (rNDVs) each of which expresses the hemagglutinin (HA) gene of HPAIV H5N1strain A/Vietnam/1203/2004 from an added gene. In one, rNDV (rNDV-HA), the open reading frame (ORF) of HA gene was expressed without modification. In the second, rNDV (rNDV-HAF), the ORF was modified so that the transmembrane and cytoplasmic domains of the encoded HA gene were replaced with those of the NDV F protein. The insertion of either version of the HA ORF did not increase the virulence of the rNDV vector. The HA protein was found to be incorporated into the envelopes of both rNDV-HA and rNDV-HAF. However, there was an enhanced incorporation of the HA protein in rNDV-HAF. Chickens immunized with a single dose of either rNDV-HA or rNDV-HAF induced a high titer of HPAIV H5-specific antibodies and were completely protected against challenge with NDV as well as lethal challenges of both homologous and heterologous HPAIV H5N1.

Conclusion and Significance

Our results suggest that these chimeric viruses have potential as safe and effective bivalent vaccines against NDV and. HPAIV. These vaccines will be convenient and affordable, which will be highly beneficial to the poultry industry. Furthermore, immunization with these vaccines will permit serological differentiation of vaccinated and avian influenza field virus infected animals.     

I222 Neuraminidase Mutations Further Reduce Oseltamivir Susceptibility of Indonesian Clade 2.1 Highly Pathogenic Avian Influenza A(H5N1) Viruses

We have tested the susceptibility to neuraminidase inhibitors of 155 clade 2.1 H5N1 viruses from Indonesia, isolated between 2006–2008 as well as 12 clade 1 isolates from Thailand and Cambodia from 2004–2007 using a fluorometric MUNANA-based enzyme inhibition assay. The Thailand and Cambodian clade 1 isolates tested here were all susceptible to oseltamivir and zanamivir, and sequence comparison indicated that reduced oseltamivir susceptibility we observed previously with clade 1 Cambodian isolates correlated with an S246G neuraminidase mutation. Eight Indonesian viruses (5%), all bearing I222 neuraminidase mutations, were identified as mild to extreme outliers for oseltamivir based on statistical analysis by box plots. IC₅₀s were from 50 to 500-fold higher than the reference clade 1 virus from Viet Nam, ranging from 43–75 nM for I222T/V mutants and from 268–349 nM for I222M mutants. All eight viruses were from different geographic locales; all I222M variants were from central Sumatra. None of the H5N1 isolates tested demonstrated reduced susceptibility to zanamivir (IC₅₀s all <5 nM). All I222 mutants showed loss of slow binding specifically for oseltamivir in an IC₅₀ kinetics assay. We identified four other Indonesian isolates with higher IC₅₀s which also demonstrated loss of slow binding, including one virus with an I117V mutation. There was a minimal effect on the binding of zanamivir and peramivir for all isolates tested. As H5N1 remains a potential pandemic threat, the incidence of mutations conferring reduced oseltamivir susceptibility is concerning and emphasizes the need for greater surveillance of drug susceptibility.     

Persistence of Highly Pathogenic Avian Influenza H5N1 Virus Defined by Agro-Ecological Niche

The highly pathogenic avian influenza (HPAI) H5N1 virus has spread across Eurasia and into Africa. Its persistence in a number of countries continues to disrupt poultry production, impairs smallholder livelihoods, and raises the risk a genotype adapted to human-to-human transmission may emerge. While previous studies identified domestic duck reservoirs as a primary risk factor associated with HPAI H5N1 persistence in poultry in Southeast Asia, little is known of such factors in countries with different agro-ecological conditions, and no study has investigated the impact of such conditions on HPAI H5N1 epidemiology at the global scale. This study explores the patterns of HPAI H5N1 persistence worldwide, and for China, Indonesia, and India includes individual provinces that have reported HPAI H5N1 presence during the 2004–2008 period. Multivariate analysis of a set of 14 agricultural, environmental, climatic, and socio-economic factors demonstrates in quantitative terms that a combination of six variables discriminates the areas with human cases and persistence: agricultural population density, duck density, duck by chicken density, chicken density, the product of agricultural population density and chicken output/input ratio, and purchasing power per capita. The analysis identifies five agro-ecological clusters, or niches, representing varying degrees of disease persistence. The agro-ecological distances of all study areas to the medoid of the niche with the greatest number of human cases are used to map HPAI H5N1 risk globally. The results indicate that few countries remain where HPAI H5N1 would likely persist should it be introduced.     

H5N1高致病性禽流感病毒的危害及生态问题

高致病性禽流感(HPAI)H5N1病毒亚型已对人类健康、养殖业发展、野生鸟类及生态环境带来极大危害,引起国内外广泛关注。研究发现,禽流感病毒通过发生重组或者突变,可产生感染人类或其他生物的新病毒亚型,或产生更高的致病性,而人类亦具有丰富的与H5N1结合的受体。对候鸟迁徙停歇地禽流感调查表明,湿地、湖泊可能是HPAI病毒存活、散播的疫源地,病毒可随着鸟类的迁徙到处传播。因此,野生鸟类及其赖以生存的主要湿地环境处于感染HPAI的风险之中。     

人源中和性抗高致病性禽流感病毒H5N1基因工程全抗体的研制

运用噬菌体表面呈现技术,从禽流感病人恢复期血中获得淋巴细胞,通过基因工程手段,构建了人源抗H5NI禽流感病毒基因工程抗体文库.用纯化的人源H5N1禽流感病毒颗粒(A/Anhui/1/2005)及重组血凝素蛋白HA(A/Viet Nam/1203/2004)对Fab噬菌体抗体库进行富集筛选,成功地获得了抗禽流感病毒H5N1血凝素蛋白HA的人源单抗Fab段基因,并在大肠杆菌中获得有效表达.通过序列测定确定抗体轻重链型别,然后将阳性克隆的轻链和重链Fd段基因分别克隆入全抗体表达载体pAC-L-Fc后转染昆虫Sf9细胞,利用杆状病毒/昆虫细胞系统实现全抗体的分泌型表达.用ELISA、IFA和流式细胞术对所获人源单抗的功能特性进行鉴定.结果表明,我们获得了2株特异性针对H5N1禽流感病毒血凝素蛋白HA而与甲1型和甲3型人流感病毒无交叉反应的人源单抗(AVFlulgG01、AVFlulgG03).微量中和试验结果表明,除A/Guangdong/1/2006外,AVFlu-IgG01能够广泛地中和HA基因进化上属于Clade 2的中国南方、北方及中部地区的H5N1禽流感病毒分离株,同时还对属于Clade Ⅰ的越南H5N1分离株A/Viet Nam/1203/2004具有中和活性;AVFluIgG03虽然不能中和A/Viet Nam/1203/2004,但是对属于Clade 2的所有中国H5N1分离株均具有中和作用.人源中和性抗禽流感病毒H5N1基因工程全抗体的获得不仅为高致病性禽流感病毒H5N1的预防和治疗带来了希望,同时也为其疫苗研制提供了新的思路.     

Experimental Infection of Macaques with a Wild Water Bird-Derived Highly Pathogenic Avian Influenza Virus (H5N1)

Highly pathogenic avian influenza virus (HPAIV) continues to threaten human health. Non-human primate infection models of human influenza are desired. To establish an animal infection model with more natural transmission and to determine the pathogenicity of HPAIV isolated from a wild water bird in primates, we administered a Japanese isolate of HPAIV (A/whooper swan/Hokkaido/1/2008, H5N1 clade 2.3.2.1) to rhesus and cynomolgus monkeys, in droplet form, via the intratracheal route. Infection of the lower and upper respiratory tracts and viral shedding were observed in both macaques. Inoculation of rhesus monkeys with higher doses of the isolate resulted in stronger clinical symptoms of influenza. Our results demonstrate that HPAIV isolated from a water bird in Japan is pathogenic in monkeys by experimental inoculation, and provide a new method for HPAIV infection of non-human primate hosts, a good animal model for investigation of HPAIV pathogenicity.