Isolation and pathotyping of H9N2 avian influenza viruses in Indian poultry

A total of 1246 faecal and tissue samples collected/received from 119 farms located in various states of India were processed for isolation of avian influenza viruses (AIV) during 2003-2004 as part of a program to monitor AIV infection in Indian poultry population. Avian influenza virus was isolated for the first time in India from poultry farms with history of drop in egg production, respiratory illness and increased mortality in Haryana state. A total of 29 H9N2 AIV isolates were obtained from the states of Punjab, Haryana, Uttar Pradesh, Gujarat, and Orissa and Union Territory Delhi. Subtyping was done by HI, RT-PCR and neuraminidase inhibition assay. Pathotyping of six representative isolates by intravenous pathogenicity index (0.0/3.0) in 6-8 weeks old chicken, trypsin dependency in cell culture and HA cleavage site analysis (335RSSR*GLF341) confirmed that these isolates are low pathogenic. Nucleotide sequence analysis of the HA gene showed that the Indian isolates are very closely related (95.0-99.6%) and shared a homology of 92-96% with H9N2 isolates from Germany and Asian regions other than that of mainland China. Deduced amino acid sequences showed the presence of L226 (234 in H9 numbering) which indicates a preference to binding of alpha (2-6) sialic acid receptors. Two of the six isolates had 7 glycosylation sites in the HA1 cleaved protein and the remaining four had 5 sites. Phylogenetic analysis showed that they share a common ancestor Qa/HK/G1/97 isolate which had contributed internal genes of H5N1 virus circulating in Vietnam. Further characterization of Indian H9N2 isolates is required to understand their nature and evolution.     

The pathogenicity of an avian influenza virus isolated in Victoria

An influenza virus (H7N7) isolated from an outbreak of disease in chickens in Victoria, was examined for its ability to cause disease in inoculated chickens, turkeys and ducks. The virus was highly pathogenic in chickens and turkeys but produced no clinical disease in ducks. Transmission of infection occurred from inoculated chickens to those in direct contact but other chickens separated by a distance of 3m directly downwind developed neither clinical disease nor antibody to the virus.     

Molecular characterization of low pathogenicity H7N3 avian influenza viruses isolated in Italy

The complete coding regions of the surface glycoproteins, nucleoprotein (NP), polymerase 2 (PB2), and matrix (M) of A/turkey/214845/02 and A/turkey/220158/99 (H7N3) low pathogenicity avian influenza (LPAI) viruses isolated in October 2002 in Italy were amplified and sequenced to determine the epidemiologic relationships with an A/turkey/Italy/4603/99 (H7N1/4603/99) LPAI virus isolated during the 1999-2001 epizootic in Italy. The hemagglutinin (HA) of H7N3 viruses showed 97.8% nucleotide similarity with A/turkey/Italy/4603/99 (H7N1), and NP, M, and PB2 gene similarities were 93.6%, 98.2%, and 96.2%, respectively. Phylogenetic analyses of HA, PB2, and M genes showed that H7N3 and H7N1 viruses were closely related. Sequence analysis revealed a 23 amino acid deletion in the stalk of the neuraminidase of H7N3 viruses and a unique deletion of amino acid glycine in position 17 in the NP gene of H7N1 virus.     

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.     

Evolution of avian influenza viruses

Although influenza viruses can infect a wide variety of birds and mammals, the natural host of the virus is wild waterfowl, shorebirds, and gulls. When other species of animals, including chickens, turkeys, swine, horses, and humans, are infected with influenza viruses, they are considered aberrant hosts. The distinction between the normal and aberrant host is important when describing virus evolution in the different host groups. The evolutionary rate of influenza virus in the natural host reservoirs is believed to be slow, while in mammals the rate is much higher. The higher rate of evolution in mammals is thought to be a result of selective pressure on the virus to adapt to an aberrant host species. Chickens and turkey influenza virus isolates have previously and incorrectly been lumped together with wild waterfowl, gull, and shorebird influenza viruses when determining rates of evolutionary change. To determine mutational and evolutionary rates of a virus in any host species, two primary assumptions must be met: first, all isolates included in the analysis must have descended from a single introduction of the virus, and second, the outbreak must continue long enough to determine a trend. For poultry, three recent outbreaks of avian influenza meet these criteria, and the sequences of the hemagglutinin and nonstructural genes were compared. Sequences from all three outbreaks were compared to an avian influenza virus consensus sequence, which at the amino acid level is highly conserved for all the internal viral proteins. The consensus sequence also provides a common point of origin to compare all influenza viruses. The evolutionary rates determined for all three outbreaks were similar to what is observed in mammals, providing strong evidence of adaptation of influenza to the new host species, chickens and turkeys.     

Molecular characterization of H5N2 avian influenza viruses isolated from South African ostriches in 2006

Highly pathogenic avian influenza (HPAI) H5N2 reemerged in ostriches in South Africa during 2006, and a low-pathogenic AI H5N2 virus was also isolated. Molecular and phylogenetic characterization was performed to determine whether the outbreak strains were genetically derived from the supposedly eradicated Eastern Cape ostrich outbreak HPAI H5N2 strain of 2004. It was demonstrated that although the 2004 and 2006 South African H5N2 strains shared a common ancestor, the two outbreaks were not related. Not only were extensive reassortments with wild bird viruses involved in the evolution of the 2006 strains, but the precursor HA molecule HA0 cleavage site sequence of the 2006 HPAI H5N2 virus also contained fewer basic amino-acid insertions. Multiple transmission events occurred from wild birds to ostriches in 2006, and it appears that a reservoir of H5N2 with pathogenic potential for poultry is established in the South African wild duck population.     

2013—2014年H7亚型禽流感病毒遗传进化分析

对2013—2014年15株不同来源的H7亚型禽流感病毒国内分离株进行了基因组测序与遗传进化分析。结果显示,15株H7亚型禽流感病毒具有遗传多样性,可分为2种亚型:13株为H7N9亚型,2株为H7N3亚型。HA蛋白裂解位点附近氨基酸分析显示所有H7亚型流感分离株均为低致病性毒株。基因组遗传进化分析显示,13株H7N9亚型禽流感病毒均与2013年人源分离株同源性较高,但具有2种遗传学特性,13株病毒的PB2、PA、HA、NP、NA、M和NS基因高度同源,而PB1基因来源于2个不同分支。2株H7N3亚型流感病毒与国内鸭源分离株同源性较高。15株H7亚型禽流感病毒PB2蛋白均未出现627K、701N等与哺乳动物适应性相关的氨基酸变异。13株H7N9亚型禽流感病毒HA裂解位点附近氨基酸(EIPKGR/GL)与人源H7N9病毒一致,且HA蛋白和M2蛋白分别具有与哺乳动物适应性和金刚烷胺耐药性相关的标志性变异,而2株H7N3亚型禽流感病毒未出现上述氨基酸变异。     

Pathogenicity of avian influenza viruses isolated from wild mallard ducks and domestic turkeys

Groups of turkeys were exposed to different isolates of avian influenza virus from wild mallard ducks and domestic turkeys by the intracerebral, intravenous, intratracheal, and intra-airsac routes, and pathogenicity indices were calculated. For the intracerebral pathogenicity study, body weight was also measured. For intravenous, intratracheal, and intra-airsac pathogenicity studies, necropsy lesions were scored and serological responses were recorded. Only the intracerebral pathogenicity index and body weight gain post intracerebral infection demonstrated any differences between isolates. The other procedures failed to demonstrate any pathogenicity whatsoever. There was a correlation (R = 0.73) between intracerebral pathogenicity index and reduced weight gain postinfection. These studies suggest that growth suppression may be an objective measure of pathogenic potential of influenza viruses found to be nonpathogenic by other methods.     

Persistence of avian influenza viruses in water

Persistence of five avian influenza viruses (AIVs) derived from four waterfowl species in Louisiana and representing five hemagglutinin and neuraminidase subtypes was determined in distilled water at 17 C and 28 C. Infectivity was determined over 60 days by microtiter endpoint titration. One AIV was tested over 91 days at 4 C. Linear regression models for these viruses predicted that an initial concentration of 1 x 10(6) TCID50/ml water could remain infective for up to 207 days at 17 C and up to 102 days at 28 C. Significant differences in slopes for AIV persistence models were detected between treatment temperatures and among viruses. Results suggest that these viruses are adapted to transmission on waterfowl wintering habitats. Results also suggest a potential risk associated with waterfowl and domestic poultry sharing a common water source.     

Intracerebral pathogenicity for chickens of avian influenza viruses isolated from free-living waterfowl in Japan

The pathogenicity for chickens of 91 strains of avian influenza A virus isolated from such free-living waterfowl as whistling swan, pintail, tufted duck, mallard and black-tailed gull in Japan was tested. The majority of the virus strains infected and were pathogenic for the chickens. The virulence of these viruses seemed not to be as high as that of fowl plague virus. There were no significant differences in the intracerebral index score among the viruses belonging to the same subtype, irrespective of year of isolation or host.     

Public health risk from avian influenza viruses

Since 1997, avian influenza (AI) virus infections in poultry have taken on new significance, with increasing numbers of cases involving bird-to-human transmission and the resulting production of clinically severe and fatal human infections. Such human infections have been sporadic and are caused by H7N7 and H5N1 high-pathogenicity (HP) and H9N2 low-pathogenicity (LP) AI viruses in Europe and Asia. These infections have raised the level of concern by human health agencies for the potential reassortment of influenza virus genes and generation of the next human pandemic influenza A virus. The presence of endemic infections by H5N1 HPAI viruses in poultry in several Asian countries indicates that these viruses will continue to contaminate the environment and be an exposure risk with human transmission and infection. Furthermore, the reports of mammalian infections with H5N1 AI viruses and, in particular, mammal-to-mammal transmission in humans and tigers are unprecedented. However, the subsequent risk for generating a pandemic human strain is unknown. More international funding from both human and animal health agencies for diagnosis or detection and control of AI in Asia is needed. Additional funding for research is needed to understand why and how these AI viruses infect humans and what pandemic risks they pose.     

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。     

Emergency response following suspicion of an avian influenza outbreak

The management of a suspected index case of highly pathogenic avian influenza (AI) is crucial to the subsequent actions aimed at limiting the spread of infection and ultimately in prompt eradication of the virus. In this study, the legislative basis, basic concepts and actions behind a successful disease management programme are reviewed. These include actions at local veterinary headquarters and at farm level which must be coordinated centrally to ensure the flow of information essential to decision making. The success of any emergency intervention strategy is dependent upon the level of preparedness, including action plans to source manpower, equipment and on the degree of communication between relevant parties. Availability of information on the successes and failures in field outbreak management, will inevitably result in improved AI control worldwide.     

The susceptibility of culture cells to avian influenza viruses

The susceptibilities of culture cells to twelve avian influenza virus strains were determined with ten established cell lines including MDCK and ESK cells and three primary culture cells. The established cell lines derived from embryonic swine kidney (ESK) and chicken kidney (CK) primary culture cells were more sensitive to the avian influenza viruses than the other eleven cells. The ESK cell had a particularly higher infective titer than the MDCK cell with and without trypsin supplement in culture medium, and dispersion of the infective titers was narrower than that of the MDCK cell. The ESK cell is a suitable candidate for routine work on avian influenza viruses in laboratories.     

Pathogenicity of two recombinant avian leukosis viruses

We have recently described the isolation and molecular characteristics of two recombinant avian leukosis subgroup J viruses (ALV J) with an avian leukosis virus subgroup A envelope (r5701A and r6803A). In the present study, we examined the role of the subgroup A envelope in the pathogenesis of these recombinant viruses. Chickens of line 151(5) x 7(1) were inoculated at 1 day of age with r5701A, r6803A, Rous-associated virus type 1 (RAV-1), or strain ADOL-Hcl of ALV-J. At 2, 4, 10, 18, and 32 wk postinoculation (PI), chickens were tested for avian leukosis virus (ALV)-induced viremia, shedding, and neutralizing antibodies. All except one chicken inoculated with the recombinant viruses (98%) developed neutralizing antibodies by 10 wk PI compared with only 16% and 46% of the ADOL-Hcl and RAV-1-inoculated birds, respectively. ALV-induced tumors and mortality in the two groups inoculated with recombinant viruses were different. The incidence of tumors in groups inoculated with r5701A or RAV-1 was 100% compared with only 9% in the groups inoculated with r6803A or ADOL-Hcl. The data suggest that differences in pathogenicity between the two recombinant viruses might be due to differences in the sequence of the 3' untranslated region (presence or absence of the E element), and, therefore, not only the envelope but also other elements of the viral genome play an important role in the pathogenesis of ALV.