Molecular characterization of low pathogenic avian influenza viruses, isolated from food products imported into Singapore

We have completed the genetic characterization of all eight gene segments for four low pathogenic avian influenza (LPAI) viruses. The objective of this study was to detect the presence of novel signatures that may serve as early warning indicators of the conversion of LPAI viruses to high pathogenic avian influenza (HPAI) viruses. This study included three H5N2 and one H5N3 viruses that were isolated from live poultry imported into Singapore as part of the national avian influenza virus (AIV) surveillance program. Based on the molecular criterion of the World Organisation for Animal Health (OIE), sequence analysis with the translated amino acid (aa) sequence of the hemagglutinin (HA) gene revealed the absence of multibasic aa at the HA cleavage site, identifying all four virus isolates as LPAI. Detailed phylogenetic tree analyses using the HA and neuraminidase (NA) genes clustered these isolates in the Eurasian H5 lineage, but away from the HPAI H5 subtypes. This analysis further revealed that the internal genes clustered to different avian and swine subtypes, suggesting that the four isolates may possibly share their ancestry with these different influenza subtypes. Our results suggest that the four LPAI isolates in this study contained mainly avian signatures, and the phylogenetic tree for the internal genes further suggests the potential for reassortment with other different circulating avian subtypes. This is the first comprehensive report on the genetic characterization of LPAI H5N2/3 viruses isolated in South-East Asia.     

Surveillance for highly pathogenic avian influenza in wild birds in the USA

As part of the USA's National Strategy for Pandemic Influenza, an Interagency Strategic Plan for the Early Detection of Highly Pathogenic H5N1 Avian Influenza in Wild Migratory Birds was developed and implemented. From 1 April 2006 through 31 March 2009, 261 946 samples from wild birds and 101 457 wild bird fecal samples were collected in the USA; no highly pathogenic avian influenza was detected. The United States Department of Agriculture, and state and tribal cooperators accounted for 213 115 (81%) of the wild bird samples collected; 31, 27, 21 and 21% of the samples were collected from the Atlantic, Pacific, Central and Mississippi flyways, respectively. More than 250 species of wild birds in all 50 states were sampled. The majority of wild birds (86%) were dabbling ducks, geese, swans and shorebirds. The apparent prevalence of low pathogenic avian influenza viruses during biological years 2007 and 2008 was 9.7 and 11.0%, respectively. The apparent prevalence of H5 and H7 subtypes across all species sampled were 0.5 and 0.06%, respectively. The pooled fecal samples (n= 101 539) positive for low pathogenic avian influenza were 4.0, 6.7 and 4.7% for biological years 2006, 2007 and 2008, respectively. The highly pathogenic early detection system for wild birds developed and implemented in the USA represents the largest coordinated wildlife disease surveillance system ever conducted. This effort provided evidence that wild birds in the USA were free of highly pathogenic avian influenza virus (given the expected minimum prevalence of 0.001%) at the 99.9% confidence level during the surveillance period.     

Novel reassortant H5N6 highly pathogenic influenza A viruses in Vietnamese quail outbreaks

Avian influenza A H5N6 virus is a highly contagious infectious agent that affects domestic poultry and humans in South Asian countries. Vietnam may be an evolutionary hotspot for influenza viruses and therefore could serve as a source of pandemic strains. In 2015, two novel reassortant H5N6 influenza viruses designated as A/quail/Vietnam/CVVI01/2015 and A/quail/Vietnam/CVVI03/2015 were isolated from dead quails during avian influenza outbreaks in central Vietnam, and the whole genome sequences were analyzed. The genetic analysis indicated that hemagglutinin, neuraminidase, and polymerase basic protein 2 genes of the two H5N6 viruses are most closely related to an H5N2 virus (A/chicken/Zhejiang/727079/2014) and H10N6 virus (A/chicken/Jiangxi/12782/2014) from China and an H6N6 virus (A/duck/Yamagata/061004/2014) from Japan. The HA gene of the isolates belongs to clade 2.3.4.4, which caused human fatalities in China during 2014–2016. The five other internal genes showed high identity to an H5N2 virus (A/chicken/Heilongjiang/S7/2014) from China. A whole-genome phylogenetic analysis revealed that these two outbreak strains are novel H6N6-like PB2 gene reassortants that are most closely related to influenza virus strain A/environment/Guangdong/ZS558/2015, which was detected in a live poultry market in China. This report describes the first detection of novel H5N6 reassortants in poultry during an outbreak as well as genetic characterization of these strains to better understand the antigenic evolution of influenza viruses.     

Pathogenicity and transmission of H5N1 avian influenza viruses in different birds

In this study, we selected three H5N1 highly pathogenic avian influenza viruses (HPAIVs), A/Goose/Guangdong/1/1996 (clades 0), A/Duck/Guangdong/E35/2012 (clade 2.3.2.1) and A/Chicken/Henan/B30/2012 (clade 7.2) isolated from different birds in China, to investigate the pathogenicity and transmission of the viruses in terrestrial birds and waterfowl. To observe the replication and shedding of the H5N1 HPAIVs in birds, the chickens were inoculated intranasally with 10⁶ EID₅₀ of GSGD/1/96, 10³ EID₅₀ of DkE35 and CkB30, and the ducks and geese were inoculated intranasally with 10⁶ EID₅₀ of each virus. Meanwhile, the naive contact groups were set up to detect the transmission of the viruses in tested birds. Our results showed that DkE35 was highly pathogenic to chickens and geese, but not fatal to ducks. It could be detected from all the tested organs, oropharyngeal and cloacal swabs, and could transmit to the naive contact birds. GSGD/1/96 could infect chickens, ducks and geese, but only caused death in chickens. It could transmit to the chickens and ducks, but was not transmittable to geese. CkB30 was highly pathogenic to chickens, low pathogenic to ducks and not pathogenic to geese. It could be transmitted to the naive contact chickens, but not to the ducks or geese. Our findings suggested that H5N1 HPAIVs from different birds show different host ranges and tissue tropisms. Therefore, we should enhance serological and virological surveillance of H5N1 HPAIVs, and pay more attention to the pathogenic and antigenic evolution of these viruses.     

近年来中国H9亚型禽流感分离株谱系分析

从GenBank中下载所有来自中国（含港、澳、台）的H9亚型禽流感病毒血凝素基因885条核苷酸序列（长度≥900bp）,用MEGA5.0软件进行谱系分析。结果表明我国近年来H9亚型禽流感病毒以第h9.4.2.5分支为主（代表株为A/chicken/Guangxi/55/2005）,而不是WHO新近报告所列出的4株病毒（A/Quail/HongKong/G1/97、A/chicken/HongKong/G9/97、A/duck/HongKong/Y280/97、A/HongKong/33982/2009）所代表的分支。此分析结果对于研制针对这一病毒感染的疫苗有重要指导意义。     

A型禽流感病毒核蛋白研究进展

A型禽流感是禽类的重要疾病之一。近年来,随着分子生物学的研究深入,对A型禽流感病毒核蛋白的研究也取得了显著的进展,本文概要综述了A型禽流感病毒核蛋白在诊断A型禽流感方面的研究进展。     

野生鸟类禽流感病毒感染情况的调查

为了解野生鸟类禽流感病毒(AIV)的携带感染情况,2006年～2010年,本研究在湖南省主要候鸟迁徙地收集115只野鸟组织或拭子样品、75份野鸟的新鲜粪便样品和72份血清样品。组织或拭子样品采用RT-PCR方法检测和鸡胚接种病毒分离鉴定,血清样品分别进行H5(含Re-5和Re-4)、H6、H7、H9、H10和H11抗体检测。结果表明,从斑鸠和绿头鸭组织中分别分离到H5N1亚型和H3N2亚型AIV;72份血清中有17份抗体为阳性,其中H5(Re-5)亚型5份、H5(Re-4)亚型1份、H6亚型1份、H7亚型2份和H9亚型8份,阳性率分别为6.94%、1.39%、1.39%、2.78%和11.11%。H10和H11亚型未检测到抗体阳性。     

一株野鸭源H6N1亚型禽流感病毒HA基因的克隆及分子进化分析

根据禽流感病毒的HA基因,设计特异性的引物,而后从尿囊液提取病毒的RNA,应用RT-PCR技术扩增该流感病毒的HA全基因序列,将该基因克隆到pMD-18T载体,并进行鉴定和测序,应用生物学分析软件进行同源性比对和绘制HA基因的进化树,并推导其氨基酸序列。结果表明：HA基因全长1701 bp,共编码567个氨基酸,与Genbank中选择的27株禽流感病毒基因的HA基因序列相比较,核苷酸序列和氨基酸序列同源性均在92%以上。通过对HA基因的同源性比对,可以明确毒株间的进化关系,从而为禽流感的遗传进化研究提供参考。     

2009—2010年华东地区家禽低致病性禽流感病毒的流行病学调查与分析

为了解华东地区家禽中低致病性禽流感病毒（low pathogenic avian influenza viruses,LPAIVs）的分布规律,从2009年10月到2010年9月在华东地区某活禽市场采集鸡、鸭、鹅等家禽的泄殖腔拭子共1 650份,经鸡胚接种和HA、HI试验鉴定,结果从58份样品中分离到了LPAIVs,总分离率为3.51%。所分离到的6种HA亚型及各HA亚型分离率从高到底依次为：H6、H3、H1、H4、H9、H11。从这些样品中鉴定出7种NA亚型,包括N1、N2、N3、N4、N5、N6、N8,二者之间有11种组合。家鸭样品中LPAIVs的分离率为7.28%,显著高于鸡源样品的分离率1.00%和鹅源样品的分离率1.02%。LPAIVs的季节性分布较为明显,3～6月份和10～12月份的分离率较高,而冬季最冷的1月份和夏季最热的7月份则没有分离到。2种或2种以上不同HA亚型混合感染的样品有6份,全部为水禽源样品,占总阳性样品数的10.34%。这些数据表明活禽市场可以作为AIV的一个重要储存库,而家养水禽可作为AIV的一个重要储存宿主,应该继续加强对活禽市场,尤其是家养水禽中AIV的监测。     

Australian surveillance for avian influenza viruses in wild birds between July 2005 and June 2007

Objective   To identify and gain an understanding of the influenza viruses circulating in wild birds in Australia.
Design   A total of 16,303 swabs and 3782 blood samples were collected and analysed for avian influenza (AI) viruses from 16,420 wild birds in Australia between July 2005 and June 2007. Anseriformes and Charadriiformes were primarily targeted.
Procedures   Cloacal, oropharyngeal and faecal (environmental) swabs were tested using polymerase chain reaction (PCR) for the AI type A matrix gene. Positive samples underwent virus culture and subtyping. Serum samples were analysed using a blocking enzyme-linked immunosorbent assay for influenza A virus nucleoprotein.
Results   No highly pathogenic AI viruses were identified. However, 164 PCR tests were positive for the AI type A matrix gene, 46 of which were identified to subtype. A total of five viruses were isolated, three of which had a corresponding positive PCR and subtype identification (H3N8, H4N6, H7N6). Low pathogenic AI H5 and/or H7 was present in wild birds in New South Wales, Tasmania, Victoria and Western Australia. Antibodies to influenza A were also detected in 15.0% of the birds sampled.
Conclusions   Although low pathogenic AI virus subtypes are currently circulating in Australia, their prevalence is low (1.0% positive PCR). Surveillance activities for AI in wild birds should be continued to provide further epidemiological information about circulating viruses and to identify any changes in subtype prevalence.     

Genetic characterization of H9N2 avian influenza virus previously unrecognized in Korea

In this study, we describe the isolation and characterization of previously unreported Y280-lineage H9N2 viruses from two live bird markets in Korea in June 2020. Genetic analysis revealed that they were distinct from previous H9N2 viruses circulating in Korea and had highest homology to A/chicken/Shandong/1844/2019(H9N2) viruses. Their genetic constellation showed they belonged to genotype S, which is the predominant genotype in China since 2010, where genotype S viruses have infected humans and acted as internal gene donors to H5 and H7 zoonotic influenza viruses. Active surveillance and control measures need to be enhanced to protect the poultry industry and public health.     

Evolution of highly pathogenic H7N3 avian influenza viruses in Mexico

Highly pathogenic H7N3 influenza A viruses have persisted in poultry in Mexico since 2012, diversifying into multiple lineages that have spread to three Mexican states, as of 2016. The H7N3 viruses segregate into three distinct clades that are geographically structured. All 2016 viruses are resistant to adamantane antiviral drugs and have an extended 24‐nucleotide insertion at the HA cleavage site that was acquired from host 28S ribosomal RNA.     

1株野鸭源H6N2亚型AIV对SPF鸭的致病性

本研究选取2周龄SPF鸭（绍兴麻鸭）自天然孔感染1株野鸭源H6N2亚型LPAIV,评价其对幼龄鸭的致病性。结果显示,试验感染鸭临床症状较轻微,病毒在鸭消化道复制能力较呼吸道内更强,且具有水平传播的能力。仅能在盲肠扁桃体和法氏囊2个组织器官中检测到病毒,但可对法氏囊等多个组织器官造成不同程度的损伤。此外感染鸭可产生HI抗体,并在第21天达到高峰。结果表明,该株H6N2亚型LPAIV对幼龄鸭的致病力较低,在AIV病毒传播中幼龄鸭起到了一定作用,为研究H6N2亚型LPAIV的致病机制及AIV发病机理提供了理论数据。     

Recent developments in avian influenza research: epidemiology and immunoprophylaxis

Influenza A viruses have been isolated from humans, from several other mammalian species and a wide variety of avian species, among which, wild aquatic birds represent the natural hosts of influenza viruses. The majority of the possible combinations of the 15 haemagglutinin (HA) and nine neuraminidase (NA) subtypes recognized have been identified in isolates from domestic and wild birds. Infection of birds can cause a wide range of clinical signs, which may vary according to the host, the virus strain, the host's immune status, the presence of any secondary exacerbating microorganisms and environmental factors. Most infections are inapparent, especially in waterfowl and other wild birds. In contrast, infections caused by viruses of H5 and H7 subtypes can be responsible for devastating epidemics in poultry. Despite the warnings to the poultry industry about these viruses, in 1997 an avian H5N1 influenza virus was directly transmitted from birds to humans in Hong Kong and resulted in 18 confirmed infections, thus strengthening the pandemic threat posed by avian influenza (AI). Indeed, reassortant viruses, harbouring a combination of avian and human viral genomes, have been responsible for major pandemics of human influenza. These considerations warrant the need to continue and broaden efforts in the surveillance of AI. Control programmes have varied from no intervention, as in the case of the occurrence of low pathogenic (LP) AI (LPAI) viruses, to extreme, expensive total quarantine-slaughter programmes carried out to eradicate highly pathogenic (HP) AI (HPAI) viruses. The adoption of a vaccination policy, targeted either to control or to prevent infection in poultry, is generally banned or discouraged. Nevertheless, the need to boost eradication efforts in order to limit further spread of infection and avoid heavy economic losses, and advances in modern vaccine technologies, have prompted a re-evaluation of the potential use of vaccination in poultry as an additional tool in comprehensive disease control strategies. This review presents a synthesis of the most recent research on AI that has contributed to a better understanding of the ecology of the virus and to the development of safe and efficacious vaccines for poultry.     

Risk profile of low pathogenicity avian influenza virus infections in farms in southern Vietnam

The impact of low pathogenicity avian influenza (LPAI) has been confirmed mainly in farms. Unlike apparent losses caused by the high pathogenicity avian influenza (HPAI), the LPAI impact has been hardly evaluated due to underestimating its spread and damage. In 2019, a questionnaire study was conducted in southern Vietnam to identify the specific risk factors of LPAI virus (LPAIV) circulation and to find associations between husbandry activities and LPAI prevalence. A multilevel regression analysis indicated that keeping Muscovy ducks during farming contributed to LPAIV positivity [Odds ratio=208.2 (95% confidence interval: 13.4–1.1 × 10⁴)]. In cluster analysis, farmers willing to report avian influenza (AI) events and who agreed with the local AI control policy had a slightly lower risk for LPAIV infection although there was no significance in the correlation between farmer characteristics and LPAI occurrence. These findings indicated that keeping Muscovy ducks without appropriate countermeasures might increase the risk of LPAIV infection. Furthermore, specific control measures at the local level are effective for LPAIV circulation, and the improvement of knowledge about biosecurity and attitude contributes to reducing LPAI damage.