Measurement of neutralizing antibody responses against H5N1 clades in immunized mice and ferrets using pseudotypes expressing influenza hemagglutinin and neuraminidase

Neutralizing antibody is associated with the prevention and clearance of influenza virus infection. Microneutralization (MN) and hemagglutination inhibition (HI) assays are currently used to evaluate neutralizing antibody responses against human and avian influenza viruses, including H5N1. The MN assay is somewhat labor intensive, while HI is a surrogate for neutralization. Moreover, use of replication competent viruses in these assays requires biosafety level 3 (BSL-3) containment. Therefore, a neutralization assay that does not require BSL-3 facilities would be advantageous. Toward this goal, we generated a panel of pseudotypes expressing influenza hemagglutinin (HA) and neuraminidase (NA) and developed a pseudotype-based neutralization (PN) assay. Here we demonstrate that HA/NA pseudotypes mimic release and entry of influenza virus and that the PN assay exhibits good specificity and reveals quantitative difference in neutralizing antibody titers against different H5N1 clades and subclades. Using immune ferret sera, we demonstrated excellent correlation between the PN, MN, and HI assays. Thus, we conclude that the PN assay is a sensitive and quantifiable method to measure neutralizing antibodies against diverse clades and subclades of H5N1 influenza virus.     

浙江省与日本A3型流行性感冒病毒株的血凝特性及HA1序列比较

目的：比较近年来在浙江省与日本流行的A3（H3N2）型流流行性感冒（流感）毒株的血凝特征与血凝素重链（HA1）区域的序列差异,方法：对浙江省不同性状的流感代表进行了抗原性分析,并与日本同期毒株作了血凝特性及HA1区域的氨基酸序列比较分析,结果：近年在浙江流行的A3型流行株,其抗原性已发生较大变异,其中绝大部分是对鸡红细胞缺乏凝集特性的O相毒株,它与A／武汉／359／95株二者在HA1区域的氨基酸同源性仅为94．82％,存在较大差异,这些O相毒株可以通过在MDCK细胞上的传代而转变为D相毒株,日本的同期毒株,其情况也相似,结论：A3型流感病毒在HA1区域发生的变异,是近年来引起浙江省流感流行的主要因素。     

Pseudoparticle neutralization is a reliable assay to measure immunity and cross-reactivity to H5N1 influenza viruses

The standard serological methods present limitations for the measurement of immunity against H5N1 influenza strains. The hemagglutination inhibition (HI) assay lacks sensitivity and requires standardization, while the viral micro-neutralization (MN) assay needs handling of live virus. We produced pseudoparticles expressing hemagglutinin from clades 1 or 2 H5N1 in order to measure neutralizing antibodies in human sera after prime-boost vaccination with plain or MF59-adjuvanted H5N1 clade 1 subunit vaccines. Titers measured by pseudoparticle neutralization (PPN) assay significantly correlated with those measured by HI, single radial haemolysis or MN, with a PPN titer of 1:357 corresponding to an MN titer of 1:80. Notably, results from the PPN assay, confirm that MF59-H5N1 vaccine induces potent and long-lasting neutralizing antibody responses not only against the vaccine strain, but also against several heterologous clade 2 strains. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of H5N1 vaccine immunogenicity.     

Systematic evaluation of suspension MDCK cells,adherent MDCK cells,and LLC-MK2 cells for preparing influenza vaccine seed virus

Suspension Madin–Darby canine kidney (MDCK) cells (MDCK-N), adherent MDCK cells (MDCK-C), and adherent rhesus monkey kidney LLC-MK2 cells (LLC-MK2D) were systematically evaluated for the preparation of influenza vaccine seed viruses for humans on the basis of primary virus isolation efficiency, growth ability, genetic stability of the hemagglutinin (HA) and neuraminidase (NA) genes, and antigenic properties in hemagglutination inhibition (HI) test of each virus isolate upon further passages. All the subtypes/lineages of influenza viruses (A(H1N1), A(H1N1)pdm09, A(H3N2), B-Victoria, and B-Yamagata) were successfully isolated from clinical specimens by using MDCK-N and MDCK-C, whereas LLC-MK2D did not support virus replication well. Serial passages of A(H1N1) viruses in MDCK-N and MDCK-C induced genetic mutations of HA that resulted in moderate antigenic changes in the HI test. All A(H1N1)pdm09 isolates from MDCK-C acquired amino acid substitutions at the site from K153 to N156 of the HA protein, which resulted in striking antigenic alteration. In contrast, only 30% of MDCK-N isolates showed amino acid changes at this site. The frequency of MDCK-N isolates with less than two-fold reduction in the HI titer was as high as 70%. A(H3N2) and B-Yamagata isolates showed high antigenic stability and no specific amino acid substitution during passages in MDCK-N and MDCK-C. B-Victoria isolates from MDCK-N and MDCK-C acquired genetic changes at HA glycosylation sites that greatly affected their antigenicity. When these cell isolates were applied to passages in hen eggs, A(H1N1), B-Victoria, and B-Yamagata viruses grew well in eggs, while none of the cell isolates of A(H3N2) viruses did. Thus, we demonstrate that MDCK-N might be useful for the preparation of influenza vaccine seed viruses.     

2000—2007年佛山市甲1亚型（H1N1）流感病毒监测情况及其HA1基因的变异

目的分析2000—2007年佛山市甲1亚型（H1N1）毒株流行情况及其HA1基因变异情况。方法用MDCK细胞分离流感病毒，采用血球凝集抑制试验进行型别鉴定，选取每年2～4株甲1亚型毒株的细胞培养物提取病毒核酸，进行HA1基因的逆转录，对其核苷酸和氨基酸序列及亲缘关系进行分析。结果病毒分离结果显示2000—2007年间甲1亚型流感病毒在佛山市人群中的流行是间断性的，只在2000、2001、2005、2006年4个年份分离到甲1亚型流感毒株。毒株的HAl区氨基酸序列与流感疫苗推荐株A／NewCMedonia／20／99和A／Solomon Islands／3／2006相比，同源性分别为97．2％～99．7％和97．2％-98．5％。氨基酸残基平均替换数随年份的推移而增加，只有2006年的毒株在抗原决定簇发生了替换。结论与疫苗株比较，佛山市的甲1亚型流感毒株抗原性已逐渐发生了改变，应密切注意疫苗对毒株的免疫效果。     

2000-2007年佛山市甲1亚型(H1N1)流感病毒监测情况及其HA1基因的变异

目的分析2000-2007年佛山市甲1亚型(H1N1)毒株流行情况及其HA1基因变异情况。方法用MDCK细胞分离流感病毒,采用血球凝集抑制试验进行型别鉴定,选取每年2~4株甲1亚型毒株的细胞培养物提取病毒核酸,进行HA1基因的逆转录,对其核苷酸和氨基酸序列及亲缘关系进行分析。结果病毒分离结果显示2000-2007年间甲1亚型流感病毒在佛山市人群中的流行是间断性的,只在2000、2001、2005、2006年4个年份分离到甲1亚型流感毒株。毒株的HA1区氨基酸序列与流感疫苗推荐株A/New Caledonia/20/99和A/Solomon Islands/3/2006相比,同源性分别为97.2%~99.7%和97.2%~98.5%。氨基酸残基平均替换数随年份的推移而增加,只有2006年的毒株在抗原决定簇发生了替换。结论与疫苗株比较,佛山市的甲1亚型流感毒株抗原性已逐渐发生了改变,应密切注意疫苗对毒株的免疫效果。     

Development of broadly reactive H5N1 vaccine against different Egyptian H5N1 viruses

The H5N1 highly pathogenic avian influenza (HPAI) virus was isolated for the first time in Egypt in 2006, since then, the virus has become endemic causing a significant threat to the poultry industry and humans. H5N1 HPAI outbreaks continue to occur despite extensive vaccination programs that have been implemented nationwide in different poultry species. Several studies showed that the co-circulating H5N1 viruses in Egypt are genetically and antigenically distant raising a question on the cross protective efficacy of commercial vaccines. In this study, we introduced mutations at the antigenic sites of the hemagglutinin (HA) to broaden reactivity of the Egyptian H5N1 virus. A reverse genetically created variant H5N1 virus (A/chicken/Egypt/1063/2010) with five amino acid mutations (G140R, Y144F, I190L, K192Q, D43N) in the HA gene showed enhanced cross reactivity. This virus showed up to 16 fold increase in reactivity to the classic-lineageH5N1viruses measured by hemagglutination inhibition (HI) assay while maintaining similar level of reactivity with the variant-lineage viruses compared to wild-type virus. In addition, a single amino acid substitution (N165H), which removes potential glycosylation site at the HA globular head of two classic strains (A/chicken/Egypt/527/2012 and A/chicken/Egypt/102d/2010) broadened the reactivity to antisera generated against H5N1 viruses from different clusters. The broadened reactivity of the mutant viruses were also confirmed by testing reactivity of antisera prepared from the mutant viruses against reference viruses from both classic and variant clades. The virus neutralization test using selected antisera and viruses further confirmed the cross HI results. This study highlights that targeted mutation in the HA may be effectively used as a tool to develop broadly reactive influenza vaccines to cope with the continuous antigenic evolution of viruses.     

Novel hemagglutinin nanoparticle influenza vaccine with Matrix-M™ adjuvant induces hemagglutination inhibition,neutralizing, and protective responses in ferrets against homologous and drifted A(H3N2) subtypes

Influenza viruses frequently acquire mutations undergoing antigenic drift necessitating annual evaluation of vaccine strains. Highly conserved epitopes have been identified in the hemagglutinin (HA) head and stem regions, however, current influenza vaccines induce only limited responses to these conserved sites. Here, we describe a novel seasonal recombinant HA nanoparticle influenza vaccine (NIV) formulated with a saponin-based adjuvant, Matrix-M™. NIV induced hemagglutination inhibition (HAI) and microneutralizing (MN) antibodies against a broad range of influenza A(H3N2) subtypes. In a comparison of NIV against standard-dose and high-dose inactivated influenza vaccines (IIV and IIV-HD, respectively) in ferrets NIV elicited HAI and MN responses exceeding those induced by IIV-HD against homologous A(H3N2) by 7 fold, A(H1N1) by 26 fold, and B strain viruses by 2 fold. NIV also induced MN responses against all historic A/H3N2 strains tested, spanning more than a decade of viral evolution from the 2000–2017 influenza seasons whereas IIV and IIV-HD induced HAI and MN responses were largely directed against the homologous A(H3N2), A(H1N1), and B virus strains. NIV induced superior protection compared to IIV and IIV-HD in ferrets challenged with a homologous or 10-year drifted influenza A(H3N2) strain. HAI positive and HAI negative neutralizing monoclonal antibodies derived from mice immunized with NIV were active against homologous and drifted influenza A(H3N2) strains. Taken together these observations suggest that NIV can induce responses to one or more highly conserved HA head and stem epitopes and result in highly neutralizing antibodies against both homologous and drift strains.     

Antigenic sites of H1N1 influenza virus hemagglutinin revealed by natural isolates and inhibition assays

The antigenic sites of hemagglutinin (HA) are crucial for understanding antigenic drift and vaccine strain selection for influenza viruses. In 1982, 32 epitope residues (called laboratory epitope residues) were proposed for antigenic sites of H1N1 HA based on the monoclonal antibody-selected variants. Interestingly, these laboratory epitope residues only cover 28% (23/83) mutation positions for 9 H1N1 vaccine strain comparisons (from 1977 to 2009). Here, we propose the entropy and likelihood ratio to model amino acid diversity and antigenic variant score for inferring 41 H1N1 HA epitope residues (called natural epitope residues) with statistically significant scores according to 1572 HA sequences and 197 pairs of HA sequences with hemagglutination inhibition (HI) assays of natural isolates. By combining both natural and laboratory epitope residues, we identified 62 (11 overlapped) residues clustered into five antigenic sites (i.e., A-E) which are highly correlated to the antigenic sites of H3N2 HA. Our method recognizes sites A, B and C as critical sites for escaping from neutralizing antibodies in H1N1 virus. Experimental results show that the accuracies of our models are 81.2% and 82.2% using 41 and 62 epitope residues, respectively, for predicting antigenic variants on 197 paring HA sequences. In addition, our model can detect the emergence of epidemic strains and reflect the genetic diversity and antigenic variant between the vaccine and circulating strains. Finally, our model is theoretically consistent with the evolution rates of H3N2 and H1N1 viruses and is often consistent to WHO vaccine strain selections. We believe that our models and the inferred antigenic sites of HA are useful for understanding the antigenic drift and evolution of influenza A H1N1 virus.     

Generation and evaluation of the trivalent inactivated reassortant vaccine using human, avian, and swine influenza A viruses

Reassortant technology was used to obtain three interspecific reassortant influenza viruses using three influenza viruses of A/Puerto Rico/8/34(H1N1), A/swine/Hebei/1/2005(H3N2) and A/chicken/Guangdong/126/2002(H9N2). The high-growth reassortant strains were H9/PR8, H3/H9N2 and H1/H9N2 that contained hemagglutinin (HA) and neuraminidase (NA) genes from the inactivated parental viruses and the other 6 internal genes from the live parental viruses. The trivalent formalin-inactivated vaccine, containing H1, H3 and H9 subtype antigens from human, swine and avian influenza viruses respectively, was prepared using these reassortant viruses. Animal studies showed that the vaccine was safe and immunogenic. Two-dosing regimen of the influenza vaccine induced high titers of hemagglutination inhibiting (HI) antibodies and influenza-specific IgG antibodies without antigenic cross-interference. It protected 100% chickens from challenge of A/chicken/Guangdong/126/2002 virus and protected 100% mice against challenges with different combinations of the three infective parental viruses. These results indicated that the trivalent vaccine could offer multi-protection against multi-influenza viruses synchronously. This kind of multivalent inactivated reassortant influenza vaccine maybe enlightens the pandemic influenza preparedness as the emergency measure.     

The impact of antigenic drift of influenza A virus on human herd immunity: Sero-epidemiological study of H1N1 in healthy Thai population in 2009

To examine the effect of the antigenic drift of H1N1 influenza viruses on herd immunity, neutralization antibodies from 744 sera from Thai healthy volunteers in 2008–2009, who had not been vaccinated for at least the last 5 years, were investigated by microneutralization (MN) and hemagglutination inhibition (HI) assays. Significantly higher MN titers were observed for the H1N1 Thai isolate in 2006 than in 2008. The results indicate that the antigenically drifted virus effectively escaped herd immunity. Since the low neutralization activity of herd immunity against drifted viruses is an important factor for viruses to spread efficiently, continuous sero-epidemiological study is required for public health.     

Use of GFP-expressing influenza viruses for the detection of influenza virus A/H5N1 neutralizing antibodies

The hemagglutination inhibition (HI) assay is used most commonly for the detection of antibodies to influenza viruses. However, for the detection of antibodies to avian influenza viruses of the H5N1 subtype either induced by infection or by vaccination, the HI assay is insensitive. Therefore, the virus neutralization (VN) assay has become the method of choice to detect human serum antibodies directed to these viruses. However, this assay requires a second assay for the detection of residual virus replication, which makes it laborious to perform and less suitable for high throughput testing of large numbers of samples. Here we describe an alternative method for the detection of these antibodies, which is based on the use of reporter viruses that express the green fluorescent protein (GFP) upon infection of target cells. GFP-expressing viruses were generated carrying the HA of a variety of antigenically distinct H5N1 influenza viruses. The method proved easy to perform and could be carried out rapidly. Using a panel of antisera raised against H5N1 influenza viruses, the assay based on GFP expressing viruses was compared with the classical virus neutralization assay and the hemagglutination inhibition assay. In general, the results obtained in these assays correlated well. It was concluded that the assay based on the reporter viruses is an attractive alternative for the classical virus neutralization assay and suitable for large sero-epidemiological studies or for the assessment of vaccine efficacy in clinical trials.     

Biochemical and antigenic analysis using monoclonal antibodies of a series of of influenza A (H3N2) and (H1N1) virus reassortants

Reassortant influenza A viruses with high growth capacity in eggs and suitable as candidate vaccine strains or as standard reagents for influenza HA quantification were prepared using the high yielding A/PR/8/34 (H1N1) as one parent and a number of 'wild' strains of influenza A (H1N1) or (H3N2) viruses as the other parent. The genetic and antigenic composition of the reassortants was determined. The parental derivation of genes in the reassortants was established by electrophoretic analysis of virus RNA and virus induced polypeptides. The haemagglutinin (HA) antigens of the three H1N1 viruses (NIB-6, NIB-7 NIB-12) were found to resemble those of the parental viruses when tested against a panel of monoclonal antibodies and using the HI test. A similar correspondence between the antigenic characteristics of the HA of the influenza A (H3N2) reassortants (NIB-1, NIB-4, NIB-5, NIB-8 and NIB-11) and parental viruses was noted. Therefore laboratory manipulations to produce the reassortants did not result in the selection of significant antigenic variants.     

2010-2012年长沙市流感流行情况及B型流感病毒基因特性分析

目的分析2010-2012年长沙市流感流行情况,并探讨B型流感病毒分离株血凝素(HA)和神经氨酸酶(NA)基因的分子流行病学特征。方法采集长沙市流感网络监测哨点医院及暴发点流感样病例(ILI)咽拭子样本,狗肾传代细胞(MDCK)进行病毒分离,血凝和血凝抑制实验进行型别和亚型鉴定;提取B型流感病毒核酸,一步法RT-PCR扩增HA和NA基因片段,双向测定扩增产物核苷酸序列,对基因序列和氨基酸序列进行分析。结果 2010-2012年间,长沙市甲型H1N1、H3N2、B型流感交替流行,流行高峰为冬春季。3年间共分离到B型流感病毒78株,其中79.5%为Victoria系,20.5%为Yamagata系,以Victoria系为主。与WHO推荐疫苗株B/Brisbane/60/2008相比,HA基因的同源性在87.2%~98.4%之间,NA基因的同源性在94.5%~97.5%之间。氨基酸序列分析发现,与疫苗株相比,HA蛋白主要存在单个氨基酸的突变;种系进化分析发现,所有毒株HA和NA蛋白位于相应的谱系内,无抗原重排发生。结论 2010-2012年间长沙市甲型H1N1、H3N2、B型流感交替流行,B型流感病毒HA基因出现抗原漂移,但无重组毒株的出现。     

Eight-plasmid system for rapid generation of influenza virus vaccines

The antigenic variation of influenza A virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins requires frequent changes in vaccine formulation. The classical method of creating influenza virus seed strains for vaccine production is to generate 6 + 2 reassortants that contain six genes from a high-yield virus, such as A/PR/8/34 (H1N1) and the HA and NA genes of the circulating strains. The techniques currently used are time-consuming because of the selection process required to isolate the reassortant virus. We generated the high-yield virus A/PR/8/34 (H1N1) entirely from eight plasmids. Its growth phenotype in embryonated chicken eggs was equivalent to that of the wild-type virus. By using this DNA-based cotransfection technique, we generated 6 + 2 reassortants that had the antigenic determinants of the influenza virus strains A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), A/teal/HK/W312 (H6N1), and A/quail/HK/G1/97 (H9N2). Our findings demonstrate that the eight-plasmid system allows the rapid and reproducible generation of reassortant influenza A viruses for use in the manufacture of vaccines.     

Neutralizing antibody but not hemagglutination antibody provides accurate evaluation for protective immune response to H5N1 avian influenza virus in vaccinated rabbits

In order to develop an animal model and an assay method to evaluate protective immune response to H5N1 avian influenza vaccination, H5N1 avian influenza vaccine was prepared. New Zealand rabbits were assigned to receive two doses of vaccine with different hemagglutinin (HA) dosage. The sera from vaccinated rabbits was evaluated to determine antibody titer and specificity using different tested methods including hemagglutination inhibition assay (HI), neutralizing assay (NT), cross-HI assay, cross-single immunodiffusion assay and cross-neutralization assay. The titer of HI antibody from rabbits immunized with different doses of HA were no less than 1:40 among groups 14 days after the first immunization. Whereas the NT antibody titer was less than 1:10 among groups 14 days after the first immunization. NT antibodies can be detected 14 days after the second immunization in rabbits immunized at HA doses higher than 6 μg, and the NT antibody titers were equal to or higher than 1:40. A good concentration-dependent NT antibody response can be detected in the vaccinated rabbits 14 days after the second immunization, and in contrast, no concentration-dependent relationship can be seen for HA antibody. The cross-HI test showed sera from vaccinated rabbits could cross react with influenza A H5N1 virus with the titers higher than 1:40. No cross reaction among different types (influenza A/H1N1 virus, influenza A/H3N2 virus, influenza B virus and influenza A/H5N1 virus) can be detected in the sera using the single immunodiffusion assay and using NT antibody test. This showed NT antibody test was demonstrated as a more accurate assay method for evaluating vaccination and quality of the vaccine than HI antibody test.     

郴州市2007年流感病原学监测及A/H3N2亚型血凝素基因特性研究

目的对郴州市2007年流行性感冒的病原学监测结果进行分析,了解流感病毒优势流行毒株A(H3N2)亚型的基因变异特征。方法采用流感样病例(ILI)的咽拭子标本用狗肾细胞(MDCK)进行病毒分离培养,采用血凝(HA)及血凝抑制(HI)方法进行流感病毒初筛及分型鉴定;取病原学监测中分离到的4株A(H3N2)亚型毒株,对其血凝素基因进行逆转录-聚合酶链反应,扩增产物纯化后测序,测序结果与WHO全球流感疫苗序列进行同源比对。结果共检测4所哨点医院ILI咽拭子标本1435份,分离到流感病毒277株;阳性率19.30%,其中A(H3N2)亚型毒株156株。2007年分离株与北半球疫苗株A/Wisconsin/67/05比较,发生了变异。结论 A(H3N2)亚型流感病毒为郴州市2007年优势毒株,A(H3N2)亚型流感流行株的HA1基因发生了变异,可能是导致其流行的重要原因。     

郴州市2009年流感病原学监测及A(H1N1)NA基因特征研究

[目的]了解2009年郴州市流感病毒流行株的病原学特征,为流感防控提供科学依据。[方法]采集哨点医院ILI咽拭子标本,用MDCK细胞进行病毒分离,采用HA和HI试验对流感病毒进行分型鉴定;采集暴发疫情ILI咽拭子用PCR法检测流感病毒核酸;随机抽取4、10月份的季节性A(H1N1)流感毒株进行NA基因测序,测序结果与国内各省市流行株及WHO推荐的北半球疫苗株作同源性比对,绘制种系进化树。[结果]分离哨点医院监测标本2284份,阳性254份:其中季节性A(H1N1)109株、A(H3N2)97株、甲型H1N130株、B型18株;PCR检测3024份标本,甲型H1N1核酸阳性1002份,阳性率33.13%;BLAST比对结果显示,4、10月份的季节性A(H1N1)毒株与2009年WHO推荐的北半球疫苗毒株A/Brisbane/59/2007的核酸同源性分别为99%和98%;种系进化分析结果表明4月份分离株与疫苗株亲缘关系最近,而10月份分离株较4月份分离株已明显发生变异。[结论]2009年郴州市甲型流感病毒异常活跃,其中1～6月季节性A(H1N1)为优势毒株,7～9月A(H3N2)为优势毒株,10～12月甲型H1N1为优势毒株,且出现甲型H1N1流感大流行。季节性A(H1N1)病毒在流行过程中NA基因已经发生了一定的变异,有必要持续跟踪和监测其变异情况。     

Dynamics of antigenic and genetic changes in the hemagglutinins of influenza A/H3N2 viruses of three consecutive seasons (2002/2003 to 2004/2005) in Austria

Human influenza viruses are subject to continuous antigenic drift and this phenomenon poses great problems for the annual production of vaccines which should ideally be manufactured from strains closely matching the predominant strains of the coming influenza season. We have investigated the dynamics of antigenic and genetic changes in the hemagglutinins of circulating influenza A/H3N2 strains in three consecutive seasons (2002/2003 to 2004/2005) in Austria by sequence analysis of the HA1 domain and by antigenic characterization using a hemagglutination inhibition assay. Each of the three seasons was dominated by a single and different H3N2 variant, but in all cases sequencing revealed the co-circulation of a drift variant which would have been missed by conventional antigenic analysis. These emerging strains always showed already a close genetic relationship to the dominating strain of the following season. Our results underscore the value of monitoring seasonal influenza strain dynamics by sequence analysis as an instrument that can provide important and timely information on the appearance of strains with epidemiologic significance.     

青海省流感病毒病原学监测及H3亚型血凝素基因变异研究

目的 监测青海省2007～2008年度季节性流感病毒型与亚型分布,了解2007～2008年度部分A/H3N2分离株血凝素(HA)基因变异情况.方法 采集监测哨点医院流感样病例的鼻咽双拭子标本,接种MDCK细胞,采用标准抗血清鉴定阳性分离株型与亚型,并对2007～2008年度部分H3亚型流感病毒进行HA基因测序,分析HA基因变异情况.结果 2007～2008年度在哨点医院送检标本中共分离到144株流感病毒,其中124株为A/H3N2,B型20株,型与亚型有明显分布强弱特征.H3亚型HA序列分析,青海省6株分离株与2008、2009年部分省份分离株及江西同期分离株接近,与WHO 2008～2009年度疫苗推荐株相近.结论 WHO推荐的2006～2007年度疫苗株A/Hiroshima/52/2005滞后于在我省流行的流感病毒毒株.