Genetic Conservation of Hemagglutinin Gene of H9 Influenza Virus in Chicken Population in Mainland China

The hemagglutinin (HA) genes of 12 H9N2 influenza virus strains isolated from chickens in Mainland China during the period 1995–2002 were genetically analyzed. All the isolates possessed the same amino acid motif -R-S-S-R/G-L- at the cleavage site of HA. Except for the conserved amino acids, as is the case in the other avian influenza viruses, located in the receptor binding site, all of the 12 isolates possessed N at amino acid position 183; A, T, or V at position 190; K at position 137, whereas the representative strains of the other lineage (except Dk/HK/Y280/97-like lineage) virus of H9N2 viruses had H, E, and R at these positions respectively. These could be considered as the partial molecular markers of the H9 viruses isolated from chickens in Mainland China. Phylogenetic analyses showed HA genes of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. No A/quail/Hong Kong/Gl/97-like virus was found in chicken, population since the outbreak of H9N2 influenza in Mainland China in 1992. The available evidence indicates that HA genes of H9 influenza virus circulating in Mainland China during the past years were well conserved.     

Amplification of four genes of influenza A viruses using a degenerate primer set in a one step RT-PCR method

We designed a degenerate primer set that yielded full-length amplification of hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural protein (NSP) genes of influenza A viruses in a single reaction mixture. These four genes were amplified from 15 HA (1–15) and 9 NA (1–9) subtypes of influenza A viruses of avian (n = 16) origin. In addition, 272 field isolates of avian origin were tested by this method. Full-length amplification of HA, NA, M, and NSP genes was obtained in 242 (88.9%), 254 (93.4%), 268 (98.5%), and 268 (98.5%) isolates, respectively. No gene was amplified in four isolates. Of these four isolates, two were subtyped as H4N6, one as H7N7, and one as H10N7. Amplification was successful for all 4 genes of H1N1, H2N3, and H3N2 isolates of swine influenza. Also, all four genes were amplified in one equine influenza (H3N8) isolate and seven isolates of human origin (H1N1 and H3N2). This appears to be the first study using degenerate primer set for full-length amplification of four genes of influenza A viruses in a single reaction. Further studies are needed to determine if this primer set can be used for subtyping of influenza virus isolates.     

Genetic and antigenic variation in the haemagglutinin of recently circulating human influenza A (H3N2) viruses in the United Kingdom

Summary Variation in the haemagglutinin (HA) gene of influenza A (H₃N₂) viruses isolated in the U. K. and abroad from 1992–1994, was determined by nucleotide sequencing of the HA1 domain of the HA gene. Viruses isolated in the U.K. early in the 1992–93 season were from the A/Beijing/353/89 lineage and were replaced later that season by viruses from the A/Beijing/32/92 lineage. Viruses from the new lineage continued to be isolated during the 1993–94 season, but were heterogeneous. Most of these isolates were more closely related to an A/Beijing/32/92 variant, A Hong Kong/23/92, but could be distinguished into three groups by serology (of which one group was circulating during the previous season) and four groups based on sequence variation in the HA gene. However, phylogenetic analysis of antigenically-distinct isolates showed that the HA gene is evolving along one lineage. Sequence analysis identified mainstream, subgroup and strain specific amino acid substitutions. There was a broad correlation between the observed amino acid changes and the antigenic sites of the HA. The results of this study highlight the value of regular molecular analysis of circulating viruses.     

甲1(H1N1)亚型流感病毒相变异分子生物学基础的研究

目的 阐明甲１（Ｈ１Ｎ１）亚型株相变异的分子生物学基础。方法 病毒ＲＮＡ经逆转录合成ｃＤＮＡ,利用聚合酶链反应（ＰＣＲ）进行扩增,产物纯化,采用双脱链末端终止法进行核苷酸序列测定,最后用ＤＮＡ ＳＴＡＲ公司出口的分析软件ＭｅｇＡｌｉｎｇ（１．０３版）和Ｅｄｉｔｓｅｑ（３．６９版０对核苷酸序列进行分析。结果 见不到“Ｏ”、“Ｄ”相毒株ＨＡ１蛋白分子间有特殊氨基酸的差异。但１９９５年前后毒株在－２,－     

Molecular and phylogenetic analysis and vaccine strain match of human influenza A(H3N2) viruses isolated in Northern Greece between 2004 and 2008

Influenza A viruses are characterized by a unique genome structure, causing genetic instability, especially to the genes of haemagglutinin and neuraminidase. The objectives of this research was molecular and phylogenetic analysis of influenza A(H3N2) strains that circulated in Northern Greece since 2004, particularly the identification of sequence variations and the comparison of circulating viruses with vaccine strains. Since 2004 in Northern Greece, a total of 216 clinical samples were positive for influenza virus infections, of which 83 (38.4%) were attributed to influenza A(H3N2). Molecular analysis of the HA genes of 23 isolates showed that all circulating strains had variations at antigenic sites. Receptor binding sites were conserved in 2004–2005 and 2005–2006 strains whereas a variation was observed in all 2006–2007 strains (H195Y). Furthermore, alternative amino acids for sialic acid receptor binding sites were observed in most of the 2004–2006 isolates. Some amino acid substitutions were also observed at the neuraminidase sequences, which however had no effect on the antigenicity of the viruses. Phylogenetic analysis of each year's circulating strains revealed a relatively low match with the vaccine strains A/Fujian/411/02 and A/California/7/04 for 2004–2005 and 2005–2006, respectively, whereas most 2006–2007 isolates match with the vaccine strain, A/Wisconsin/67/05. This year, unique variations were observed at antigenic and glycosylation sites of A/Serres/77/07-like stains. Constant surveillance of yearly variations is of great importance, so that vaccine strains can be evaluated.     

HA1 domain of influenza A (H3N2) viruses in Finland in 1989–1995: evolution,egg-adaptation and relationship to vaccine strains

Summary The HA1 gene sequences of 22 MDCK cell-derived influenza A (H3N2) strains, ten of their egg-derived counterparts and three vaccine strains were determined. Antigenic and sequence differences between the epidemic and vaccine strains were recorded, most striking in 1992/93; a minority of the amino acid differences in 1989–95 was involved in egg-adaptation. Changes in the assortment of amino acid substitutions produced during egg-adaptation of field strains may account for the difficulty encountered in isolating these viruses in embryonated eggs. Six revertant amino acids, characteristic of field strains prevalent in 1969–71 were recorded in 1994/95. Their genome sequence was interpreted to have been maintained over the interval years among low abundant sequences of the viral quasispecies. Potential changes of carbohydrate moieties were recorded in two glycosylation sites, suggesting that oligosaccharides at these sites are not necessarily advantageous for the H3N2 subtype virus currently.     

Emergence of amantadine-resistant avian influenza H5N1 virus in India

This study reports the genetic characterization of highly pathogenic avian influenza (HPAI) virus (subtype H5N1) isolated from poultry in West Bengal, India. We analyzed all the eight genome segments of two viruses isolated from chickens in January 2010 to understand their genetic relationship with other Indian H5N1 isolates and possible connection between different outbreaks. The hemagglutinin (HA) gene of the viruses showed multiple basic amino acids at the cleavage site, a marker for high virulence in chickens. Of greatest concern was that the viruses displayed amino acid substitution from serine-to-asparagine at position 31 of M2 ion channel protein suggesting emergence of amantadine-resistant mutants not previously reported in HPAI H5N1 outbreaks in India. Amino acid lysine at position 627 of the PB2 protein highlights the risk the viruses possess to mammals. In the phylogenetic trees, the viruses clustered within the lineage of avian isolates from India (2008-2009) and avian and human isolates from Bangladesh (2007-2009) in all the genes. Both these viruses were most closely related to the viruses from 2008 in West Bengal within the subclade 2.2.3 of H5N1 viruses.     

Phylogenetic Analysis of Neuraminidase Gene of H9N2 Influenza Viruses Prevalent in Chickens in China during 1995–2002

The neuraminidase (NA) genes of 12 H9N2 influenza virus strains isolated from diseased chickens in different farms in mainland China during 1995–2002 were amplified and sequenced. Amino acids at hemadsorbing (HB) site of these isolates are different from those of A/quail/Hong Kong/G1/97-like viruses and A/chicken/Korea/96-like viruses. Neuraminidases of the 12 strains had a deletion of 3 amino acid residues at positions 63–65 as compared to that of A/turkey/Wisconsin/189/66, while those of Korea and Pakistan H9N2 isolates had no deletion. Phylogenetic analyses showed NA gene of these isolates belonged to that of A/duck/Hong Kong/Y280/97-like virus lineage. NA gene of the H9N2 viruses isolated in Korea and Pakistan belonged to lineage different from those of the 12 isolates. The present results indicate that the NA of H9N2 strains isolated in mainland China during the past 8 years were well preserved and the geographical distribution play a significant role in the evolution of the H9N2 influenza viruses.     

Genetic variation of the hemagglutinin of avian influenza virus H9N2

Avian influenza virus H9N2 has become the dominant subtype of influenza which is endemic in poultry. The hemagglutinin, one of eight protein-coding genes, plays an important role during the early stage of infection. The adaptive evolution and the positively selected sites of the HA (the glycoprotein molecule) of H9N2 subtype viruses were investigated. Investigating 68 hemagglutinin H9N2 avian influenza virus isolates in China and phylogenetic analysis, it was necessary that these isolates were distributed geographically from 1994, and were all derived from the Eurasian lineage. H9N2 avian influenza virus isolates from domestic poultry in China were distinct phylogenetically from those isolated in Hong Kong, including viruses which had infected humans. Seven amino acid substitutions (2T, 3T, 14T, 165D, 197A, 233Q, 380R) were identified in the HA possibly due to positive selection pressure. Apart from the 380R site, the other positively selected sites detected were all located near the receptor-binding site of the HA1 strain. Based on epidemiological and phylogenetics analysis, the H9N2 epidemic in China was divided into three groups: the 1994-1997 group, the 1998-1999 group, and the 2000-2007 group. By investigating these three groups using the maximum likelihood estimation method, there were more positive selective sites in the 1994-1997 and 1998-1999 epidemic group than the 2000-2007 groups. This indicates that those detected selected sites are changed during different epidemic periods and the evolution of H9N2 is currently slow. The antigenic determinant or other key functional amino acid sites should be of concern because their adjacent sites have been under positive selection pressure. The results provide further evidence that the pathogenic changes in the H9N2 subtype are due mainly to re-assortment with other highly pathogenic avian influenza viruses.     

我国猪群中H9N2亚型毒株HA和NA基因特性的研究

目的 了解我国内地从猪中分离到H9N2亚型毒株HA和NA基因来源及它们使猪致病的原因。方法 用PCR扩增目的基因，与P^GEM-T Easy Vector4℃过夜连接，重组质粒转化DH-10β细菌，筛选阳性菌落，酶切鉴定，测序。然后，进行进化树分析。结果 两株猪H9N2毒株HA蛋白分子上第226位上氨基酸为L，这与从人和猪所分离出的H9N2毒株相同，其连接肽属对禽致病的毒株，但它们的序列为R-L-S-R，而不是R-S-S-R；其NA蛋白茎区第62～64位存在掉失，这与A／Shaoguarn/408／98，A／Swine／Hong Kong／9／98及A／Duck／Hong Kong／y280／97(H9N2)毒株相同；HA与NA基因进化树分析表明，两株猪H9N2毒株的HA基因接近于A／Chicken／Hong Kong／G23／97和A／Chicken／Hong Kong／G9／97．而NA基因接近于A／Shaoguan／408／98毒株。结论 两株猪H9N2亚型毒株的HA和NA基因可能性最大来自禽H9N2毒株。由于其HA蛋白分子上连接肽氨基酸序列发生替换，可能造成了它们对猪具有致病性。禽H9N2毒株NA蛋白茎区氨基酸掉失，造成了它们能直接感染猪。     

Phylogenetic analysis of hemagglutinin genes of 40 H9N2 subtype avian influenza viruses isolated from poultry in China from 2010 to 2011

Avian influenza virus (H9N2) infection is a major problem of product performance in poultry worldwide. Vaccination is used to limit spread, but more knowledge is needed on the epidemiology of virus subtypes to improve vaccine design. In this study, 40 H9N2 subtype avian influenza viruses (AIVs) were isolated from vaccinated poultry flocks in China from 2010 to 2011. Hemagglutinin (HA) from different virus strains was sequenced and analyzed. We found that the HA genes of these strains shared nucleotide and deduced amino acid homologies that ranged from 90.1 to 92.9 and 91.4 to 95.0 %, respectively, when compared with vaccine strains. Phylogenetic analysis showed that the strains tested could be divided into two major groups. Group I consisted of 24 strains isolated mainly from Eastern and Central China. Group II consisted of 20 strains isolated from Southern China. The cleavage site within the HA protein contained two basic motifs, PSRSSR↓GLF for group I, and PARSSR↓GLF for group II. Additional potential glycosylation sites were found at amino acid position 295 in the HA1 of the isolates in group I, compared with isolates in group II and the vaccine strains. Furthermore, 38 out of the 40 isolates had a leucine residue at position 216 (aa 226 in H3), which was characteristic of human influenza virus-like receptor specificity. In the present study we found that geographical factors play a significant role in virus evolution, and emphasize the importance of continuing surveillance of H9N2 AIVs in chickens in China.     

深圳地区甲1(H1N1)亚型流感病毒基因特性的研究

目的　了解近几年H1N1亚型毒株在深圳地区人群中活动加强及“O”相特性出现的分子生物学基础及其基因演变的特性。方法　病毒粒RNA经逆转录合成cDNA,用PCR扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定并推导出其所编码的氨基酸序列。进化树分析用DNASTAR公司出品的序列分析软件,MegAlign(103版)的Editseq(369版)。结果　根据病毒粒HA1基因特性,至少1995年以来深圳地区人群中同时流行着基因特性不同的三系毒株;1995～1997年H1N1毒株与A新加坡686(H1N1)病毒相比较,其HA1蛋白分子上第54和155位上分别插入和缺失一个糖基化点,同时氨基酸序列发生了替换。结论　近年来深圳地区人群中同时流行着HA1基因不同的三系H1N1亚型毒株。由于HA1蛋白分子上氨基酸序列发生了替换,尤其糖基化位点插入和缺失,造成1995年以来H1N1毒株活动加强,这些可能与毒株“O”相特性再现密切相关。     

Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand

The H5N1 avian influenza virus outbreak among zoo tigers in mid-October 2004, with 45 animals dead, indicated that the avian influenza virus could cause lethal infection in a large mammalian species apart from humans. In this outbreak investigation, six H5N1 isolates were identified and two isolates (A/Tiger/Thailand/CU-T3/04 and A/Tiger/Thailand/CU-T7/04) were selected for whole genome analysis. Phylogenetic analysis of the 8 gene segments showed that the viruses clustered within the lineage of H5N1 avian isolates from Thailand and Vietnam. The hemagglutinin (HA) gene of the viruses displayed polybasic amino acids at the cleavage site, identical to those of the 2004 H5N1 isolates, which by definition are highly pathogenic avian influenza (HPAI). In addition, sequence analyses revealed that the viruses isolated from tigers harbored few genetic changes compared with the viruses having infected chicken, humans, tigers and a leopard isolated from the early 2004 H5N1 outbreaks. Sequence analyses also showed that the tiger H5N1 isolated in October 2004 was more closely related to the chicken H5N1 isolated in July than that from January. Interestingly, all the 6 tiger H5N1 isolates contained a lysine substitution at position 627 of the PB2 protein similar to the human, but distinct from the original avian isolates.     

甲1型流感病毒新分离株HA基因的序列分析

目的 研究新分离的H1N1亚型流感病毒株的HA1基因序列。方法 甲型流感病毒通过鸡胚增殖后提取RNA、逆转录合成cDNA,经PCR扩增和产物纯化构建重组质粒,用双脱氧链终止法进行核苷酸序列测定;并进行基因特性分析。结果 新分离到的3株流感病毒株（H1N1）HA1区基因长度为981bp,编码327个氨基酸;与A/桂防/10/94和A/Bayern/07/95（H1N1）标准株比较其同源性分别为92.8%和91.3%,丢失了第130位氨基酸和304位糖基化位点;新分离的3株甲型流感病毒（H1地）标准株比较其同源性分别为92.8%和91.3%,丢失了第130位氨基酸和304位糖基化位点,新分离的3株甲型流感病毒株（H1N1）HA1区氨基酸同源性高达98%;A/桂防/10/94和A/Bayern/07/95(H1N1)毒株HN1氨基酸的同源性高达96%。结论 新分离到的3株H1N1毒株HA编码氨基酸不同于A/Baydrn/07/95(H1N1)和A/桂防/10/94（H1N1）标准株,它们可能为新的甲型流感病毒变异性。     

Molecular characterization of the influenza A(H1N1)pdm09 isolates collected in the 2015-2016 season and comparison of HA mutations detected in Turkey since 2009

Influenza A(H1N1)pdm09 pandemic virus causing the 2009 global outbreak moved into the post-pandemic period, but its variants continued to be the prevailing subtype in the 2015-2016 influenza season in Europe and Asia. To determine the molecular characteristics of influenza A(H1N1)pdm09 isolates circulating during the 2015-2016 season in Turkey, we identified mutations in the hemagglutinin (HA) genes and investigated the presence of H275Y alteration in the neuraminidase genes in the randomly selected isolates. The comparison of the HA nucleotide sequences revealed a very high homology (>99.5%) among the studied influenza A(H1N1)pdm09 isolates, while a relatively low homology (96.6%-97.2%), was observed between Turkish isolates and the A/California/07/2009 vaccine virus. Overall 14 common mutations were detected in HA sequences of all 2015-2016 influenza A(H1N1)pdm09 isolates with respect to the A/California/07/2009 virus, four of which located in three different antigenic sites. Eleven rare mutations in 12 HA sequences were also detected. Phylogenetic analysis revealed that all characterized influenza A(H1N1)pdm09 isolates formed a single genetic cluster, belonging to the genetic subclade 6B.1, defined by HA amino acid substitutions S84N, S162N, and I216T. Furthermore, all isolates showed an oseltamivir-sensitive genotype, suggesting that Tamiflu (Oseltamivir) could still be the drug of choice in Turkey.     

Genetic analysis of avian influenza A viruses isolated from domestic waterfowl in live-bird markets of Hanoi, Vietnam, preceding fatal H5N1 human infections in 2004

The first known cases of human infection with highly pathogenic avian influenza (HPAI) H5N1 viruses in Vietnam occurred in late 2003. However, HPAI H5N1 and low-pathogenic avian influenza (LPAI) H5N2 and H9N3 viruses were isolated from domestic waterfowl during live-bird market (LBM) surveillance in Vietnam in 2001 and 2003. To understand the possible role of these early viruses in the genesis of H5N1 strains infecting people, we performed sequencing and molecular characterization. Phylogenetic analysis revealed that the hemagglutinin (HA) genes of two geese HPAI H5N1 strains belonged to clade 3, and their surface glycoprotein and replication complex genes were most closely related (98.5–99.7% homologous) to A/duck/Guangxi/22/01 (H5N1) virus, detected contemporarily in southern China, whilst the M and NS genes were derived from an A/duck/Hong Kong/2986.1/00 (H5N1)-like virus. The H5 HA gene of the duck HPAI H5N1 strain belonged to clade 5 and acquired a gene constellation from A/quail/Shantou/3846/02 (H5N1), A/teal/China/2978.1/02 (H5N1) and A/partridge/Shantou/2286/03 (H5N1)-like viruses. The phylogenetic analysis further indicated that all eight gene segments of goose and duck HPAI H5N1 and LPAI H5N2 viruses were distinct from those of H5N1 clade-1 viruses known to have caused fatal human infections in Vietnam since late 2003. The duck H9N3 isolates derived genes from aquatic-bird influenza viruses, and their H9 HA belonged to the Korean lineage. The PB2 gene of A/duck/Vietnam/340/01 (H9N3) virus had lysine at position 627. Based on the molecular characterization of specific amino acid residues in the surface and relevant internal protein-coding genes, the Vietnamese H5N1 and H9N3 virus isolates indicated specificity to avian cell surface receptor and susceptibility for currently licensed anti-influenza A virus chemotherapeutics. Our findings suggest that the H5N1 and H5N2 viruses that circulated among geese and ducks in LBMs in Hanoi, Vietnam, during 2001 and 2003 were not the immediate ancestors of the clade-1 viruses associated with fatal human infections in Vietnam. The clade-1 HPAI H5N1 viruses were independently introduced into Vietnam.     

一株鹅H5N1亚型流感病毒基因特性的分析

目的 弄清了Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）毒株对鹅致病的分子生物学基础 ，研究香港区人群中发生的禽（Ｈ５Ｎ１）流感的病因，方法 病毒ＲＮＡ经逆转录合成ｃＤＮＡ经聚合酶链反应（ＰＣＲ）扩增，产物纯化，采用双脱链末端终止法测定核苷酸序列，结果 Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）与Ａ／ＨＫ／１５６／９７（Ｈ５Ｎ１）毒株ＲＮＡ４核苷酸序列有２２个位点不同（同源性为９８．８％）无任何掉失或插入。它与人和     

Identification of amino acids in highly pathogenic avian influenza H5N1 virus hemagglutinin that determine avian influenza species specificity

To test the role of neutralizing antibodies (nAbs) and receptor adaptation in interspecies transmission of influenza virus, two H5N1 strains, isolated from human and avian hosts, with four amino acid differences in hemagglutinin (HA) and seven HA mutations were studied. We found that a mutation at amino acid position 90 in the H5N1 HA, outside the receptor-binding domain (RBD), could simultaneously induce changes in the RBD conformation to escape from nAb binding and alter the receptor preference through long-range regulation. This mutation was deemed a “key event” for interspecies transmission. It is likely a result of positive selection caused by antibodies, allowing the original invasion by new species-specific variants. A mutation at amino acid position 160 in the RBD only induced a change in receptor preference. This mutation was deemed a “maintaining adaptation”, which ensured that influenza virus variants would be able to infect new organisms of a different species successfully. The mutation is the result of adaptation caused by the receptor. Our results suggest that continuing occurrence of these two types of mutations made the variants persist in the new host species.     

Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs

To study the receptor specificity of modern human influenza H1N1 and H3N2 viruses, the analogs of natural receptors, namely sialyloligosaccharides conjugated with high molecular weight (about 1500 kDa) polyacrylamide as biotinylated and label-free probes, have been used. Viruses isolated from clinical specimens were grown in African green monkey kidney (Vero) or Madin-Darby canine kidney (MDCK) cells and chicken embryonated eggs. All Vero-derived viruses had hemagglutinin (HA) sequences indistinguishable from original viruses present in clinical samples, but HAs of three of seven tested MDCK-derived isolates had one or two amino acid substitutions. Despite these host-dependent mutations and differences in the structure of HA molecules of individual strains, all studied Vero- and MDCK-isolated viruses bound to Neu5Ac alpha2-6Galbeta1-4GlcNAc (6'SLN) essentially stronger than to Neu5Acalpha2-6Galbeta1-4Glc (6'SL). Such receptor-binding specificity has been typical for earlier isolated H1N1 human influenza viruses, but there is a new property of H3N2 viruses that has been circulating in the human population during recent years. Propagation of human viruses in chicken embryonated eggs resulted in a selection of variants with amino acid substitutions near the HA receptor-binding site, namely Gln226Arg or Asp225Gly for H1N1 viruses and Leu194Ile and Arg220Ser for H3N2 viruses. These HA mutations disturb the observed strict 6'SLN specificity of recent human influenza viruses.