Recent changes among human influenza viruses

Recurrent epidemics of influenza are due to the frequent emergence of antigenic variants. With co-circulation of two influenza A subtypes and two antigenically distinct lineages of B viruses, genetic reassortment also has an important role in antigenic drift, as illustrated by recent changes in both A and B viruses. The H1N2 subtype viruses, which emerged during 2001, possessed a H1 HA similar to those of contemporary A/New Caledonia/20/99 (H1N1)-like viruses and seven genes closely related to those of recent H3N2 viruses, and did not represent a significant increase in the antigenic diversity of circulating viruses. The re-emergence of B/Victoria/2/87-lineage viruses, previously prevalent during the 1980s, in 2000 has been followed by the predominant circulation of reassortant B viruses possessing a B/Victoria-lineage HA and a B/Yamagata-lineage NA similar in sequence to those of recent B/Sichuan/379/99-like viruses. These events emphasize not only the lack of divergence in the complementary functional characteristics of the HA and NA of divergent influenza B lineages, but also the apparent convergence in compatibility between the H1 and N2 components of the two influenza A subtypes.     

Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983

During 1988-1989 two highly distinct antigenic variants of influenza type B were recognized in hemagglutination-inhibition tests with postinfection ferret serum. These viruses were antigenically related to either B/Victoria/2/87, the most recent reference strain, or B/Yamagata/16/88, a variant that was isolated in Japan in May 1988. All influenza B viruses isolated in the United States during an epidemic in the winter of 1988-1989 were antigenically related to B/Victoria/2/87. However, in several countries in Asia, both B/Victoria/2/87-like viruses and B/Yamagata/16/88-like viruses were isolated. Sequence analysis of the hemagglutinin (HA) genes of several influenza B isolates from 1987 to 1988 indicated that the HA1 domains of the B/Yamagata/16/88-like viruses and B/VI/87-like viruses isolated in 1988 differed by 27 amino acids. Evolutionary relationships based on this sequence data indicated that the B/Yamagata/16/88-like viruses were more closely related to epidemic viruses from 1983 (B/USSR/100/83-like viruses) than to more recent reference strains such as B/Victoria/2/87. All other Asian strains, as well as selected isolates from the United States in 1988, were confirmed by sequence analysis as being genetically related to B/Victoria/2/87. These data provide clear evidence that two parallel evolutionary pathways of influenza type B have existed since at least 1983 and that viruses from each of the separate lineages were isolated from cases of influenza B in 1988. This finding is similar to earlier observations for type A H1N1 and H3N2 influenza viruses.     

Phylogenetic analysis of H1N2 isolates of influenza A virus from pigs in the United States

Twenty-four H1N2 influenza A viruses were newly isolated from pigs in the United States. These isolates originated from 19 farms in 9 different swine producing states between 1999 and 2001. All farms had clinical histories of respiratory problem and/or abortion. The viral isolates were characterized genetically to determine the origin of all eight gene segments. The results showed that all H1N2 isolates were reassortants of classical swine H1N1 and triple reassortant H3N2 viruses. The neuraminidase (NA) and PB1 genes of the H1N2 isolates were of human origin, while the hemagglutinin (HA), nucleoprotein (NP), matrix (M), non-structural (NS), PA and PB2 polymerase genes were of avian or swine origin. Fifteen of the 24 H1N2 isolates were shown to have a close phylogenic relationship and high amino acid homology with the first US isolate of H1N2 (A/SW/IN/9K035/99). The remaining nine isolates had a close phylogenic relationship with classical swine influenza H1N1 in the HA gene. All other genes including NA, M, NP, NS, PA, PB1 and PB2 showed a close phylogenic relationship with the H1N2 (A/SW/IN/9K035/99) strain and triple reassortant H3N2 viruses. However, PB1 genes of two isolates (A/SW/KS/13481-S/00, A/SW/KS/13481-T/00) were originated from avian influenza A virus lineage. These results suggest that although there are some variations in the HA genes, the H1N2 viruses prevalent in the US swine population are of a similar genetic lineage.     

Genetic analysis and evaluation of the reassortment of influenza B viruses isolated in Taiwan during the 2004-2005 and 2006-2007 epidemics

Influenza B viruses were predominant in Taiwan during the 2004-2005 epidemic and both Victoria and Yamagata lineage viruses co-circulated. A reassortant influenza B virus that contained a Victoria lineage hemagglutinin (HA) gene and Yamagata lineage neuraminidase (NA) gene appeared first in 2002 and became predominant during the 2004-2005 epidemic. During the 2006-2007 epidemic, an influenza B outbreak occurred in Taiwan and only Victoria lineage viruses circulated. We characterized the viruses isolated in the 2006-2007 epidemic and found that the HA genes of influenza B viruses from that epidemic were highly similar to those from the 2004-2005 epidemic. We also analyzed the NA genes of isolates from the 2006-2007 epidemic and found that they all belonged to the Yamagata lineage and formed a new genetic subclade. Comparison of isolates from the 2004-2005 and 2006-2007 epidemics revealed four substitutions, N220K, E320D, K343R and E404K in NA genes. Although the HA sequences from the 2006-2007 epidemic were similar to those from the 2004-2005 epidemic, the NA sequences differed, suggesting distinct patterns of evolution of the HA and NA genes from 2004-2007 in Taiwan. This study emphasizes that the evolution of the NA genes may contribute to reemergence of influenza B viruses.     

Molecular analysis of isolates from influenza B outbreaks in the U.S. and Nepal, 2005

Summary. Currently circulating influenza B viruses can be divided into two antigenically and genetically distinct lineages referred to by their respective prototype strains, B/Yamagata/16/88 and B/Victoria/2/87, based on amino acid differences in the hemagglutinin surface glycoprotein. During May and July 2005, clinical specimens from two early season influenza B outbreaks in Arizona and southeastern Nepal were subjected to antigenic (hemagglutinin inhibition) and nucleotide sequence analysis of hemagglutinin (HA1), neuraminidase (NA), and NB genes. All isolates exhibited little reactivity with the B/Shanghai/361/2002 (B/Yamagata-like) vaccine strain and significantly reduced reactivity with the previous 2003/04 B/Hong Kong/330/2001 (B/Victoria-like) vaccine strain. The majority of isolates were antigenically similar to B/Hawaii/33/2004, a B/Victoria-like reference strain. Sequence analysis indicated that 33 of 34 isolates contained B/Victoria-like HA and B/Yamagata-like NA and NB proteins. Thus, these outbreak isolates are both antigenically and genetically distinct from the current Northern Hemisphere vaccine virus strain as well as the previous 2003–04 B/Hong Kong/330/2001 (B/Victoria lineage) vaccine virus strain but are genetically similar to B/Malaysia/2506/2004, the vaccine strain proposed for the coming seasons in the Northern and Southern Hemispheres. Since these influenza B outbreaks occurred in two very distant geographical locations, these viruses may continue to circulate during the 2006 season, underscoring the importance of rapid molecular monitoring of HA, NA and NB for drift and reassortment.     

Changes in the hemagglutinins and neuraminidases of human influenza B viruses isolated in Italy during the 2001-02, 2002-03, and 2003-04 seasons

Throughout most of the last decade, B/Yamagata/16/88-lineage influenza viruses were predominant among the B isolates circulating worldwide, whereas B/Victoria/2/87-lineage viruses were isolated infrequently and restricted geographically to eastern Asia. During the 2001-02 influenza season, B/Victoria/2/87-lineage viruses re-emerged in North America and Europe and spread worldwide. Virological surveillance in Italy during that season showed wide circulation of influenza B viruses, of which most were antigenically related to the B/Sichuan/379/99 (Yamagata-lineage) vaccine strain, together with a smaller number of B viruses antigenically similar to B/HongKong/330/01, a recent B/Victoria/2/87-lineage antigenic variant. In the subsequent 2002-03 epidemic season, B viruses with a Victoria-lineage hemagglutinin (HA), more closely related to that of B/Shandong/7/97, were isolated exclusively. Similar strains have continued to predominate among the few B viruses isolated in Italy during last season (2003-04), although most influenza B viruses, isolated sporadically elsewhere in Europe, again belong to the Yamagata-lineage. In the present study, phylogenetic analyses of the HA and neuraminidase (NA) genes of representative B strains, isolated throughout Italy during 2001-04, showed that during the first influenza season the NA genes, as well as the HA genes, separated into the two distinct clades, the Yamagata- and Victoria-lineages, and showed no evidence of genetic reassortment. On the contrary, all the B viruses isolated in the 2002-03 and most of those isolated in the 2003-04 epidemic season were "Victoria HA-Yamagata NA" reassortants similar to those isolated in other parts of the world, showing that these reassortants became established in the human population. The frequency of reassortment between HA and NA of distinct lineages and sublineages highlights again the importance of detailed molecular analyses of both surface glycoproteins in understanding the evolution of influenza B viruses.     

Reassortants in recent human influenza A and B isolates from South East Asia and Oceania

From 2000 to 2002, human influenza A and B viruses that were genetic reassortants of contemporary circulating human strains, were isolated in South East Asia and Oceania. Similar to reports from other regions, A(H1N2) isolates were found to be reassortants of circulating A(H3N2) viruses that had acquired only the haemagglutinin gene of an A(H1N1) virus. Some of these reassortants from Thailand and Singapore predate those previously recorded during the winter of 2001-2002 in Europe and the Middle East and may be precursors of these viruses. The B reassortants had a haemagglutinin similar to an earlier B strain, B/Shangdong/7/97 (B/Victoria/2/87-lineage) and a neuraminidase similar to the recently circulating B/Sichuan/379/99 virus (B/Yamagata/16/88-lineage). Despite the early occurrences of A(H1N2) reassortants and the extensive circulation of A(H1) viruses in South East Asia and Oceania during 2000-2001, these reassortant influenza A viruses have to date not been prominent unlike Europe and the Middle East where they were common in the 2001-2002 winter. In contrast the reassortant B viruses, which first emerged in this region in early 2002, rapidly became the predominant strains isolated from patients with influenza B in South East Asia and Oceania.     

Evaluation of Influenza Virus A/H3N2 and B Vaccines on the Basis of Cross-Reactivity of Postvaccination Human Serum Antibodies against Influenza Viruses A/H3N2 and B Isolated in MDCK Cells and Embryonated Hen Eggs

The vaccine strains against influenza virus A/H3N2 for the 2010-2011 season and influenza virus B for the 2009-2010 and 2010-2011 seasons in Japan are a high-growth reassortant A/Victoria/210/2009 (X-187) strain and an egg-adapted B/Brisbane/60/2008 (Victoria lineage) strain, respectively. Hemagglutination inhibition (HI) tests with postinfection ferret antisera indicated that the antisera raised against the X-187 and egg-adapted B/Brisbane/60/2008 vaccine production strains poorly inhibited recent epidemic isolates of MDCK-grown A/H3N2 and B/Victoria lineage viruses, respectively. The low reactivity of the ferret antisera may be attributable to changes in the hemagglutinin (HA) protein of production strains during egg adaptation. To evaluate the efficacy of A/H3N2 and B vaccines, the cross-reactivities of postvaccination human serum antibodies against A/H3N2 and B/Victoria lineage epidemic isolates were assessed by a comparison of the geometric mean titers (GMTs) of HI and neutralization (NT) tests. Serum antibodies elicited by the X-187 vaccine had low cross-reactivity to both MDCK- and egg-grown A/H3N2 isolates by HI test and narrow cross-reactivity by NT test in all age groups. On the other hand, the GMTs to B viruses detected by HI test were below the marginal level, so the cross-reactivity was assessed by NT test. The serum neutralizing antibodies elicited by the B/Brisbane/60/2008 vaccine reacted well with egg-grown B viruses but exhibited remarkably low reactivity to MDCK-grown B viruses. The results of these human serological studies suggest that the influenza A/H3N2 vaccine for the 2010-2011 season and B vaccine for the 2009-2010 and 2010-2011 seasons may possess insufficient efficacy and low efficacy, respectively.     

Protective efficacy of intranasal cold-adapted influenza A/New Caledonia/20/99 (H1N1) vaccines comprised of egg- or cell culture-derived reassortants

Live, cold-adapted, temperature-sensitive (ca/ts) Russian influenza A vaccines are prepared in eggs by a 6:2 gene reassortment of the ca/ts donor strain A/Leningrad/134/17/57 (H2N2) (Len/17) with a current wild-type (wt) influenza A strain contributing hemagglutinin (HA) and neuraminidase (NA) genes. However, egg-derived reassortant vaccines are potentially more problematic to manufacture in large quantities than vaccines from cell-based procedures. To compare egg- and cell culture-derived reassortant vaccines, we prepared in Madin Darby canine kidney (MDCK) cells two cloned, ca/ts reassortants (25M/1, 39E/2) derived from Len/17 and a wt reference strain A/New Caledonia/20/99 (H1N1) (NC/wt). Both 25M/1 and 39E/2 reassortants preserved the ca/ts phenotype and mutations described for internal genes of the A/Len/17 parent. When compared to a commercial, egg-derived ca/ts Russian A/17/NC/99/145 (H1N1) New Caledonia vaccine (NC/145), the MDCK-derived reassortant 39E/2 vaccine conferred similar levels of protection in ferrets challenged i.n. with 7 x 10(10) pfu of NC/wt. In a dose-ranging study, the protective vaccine dose for 50% of ferrets (PD50) was less than 1.2 x 10(4) pfu for the 25M/1 vaccine derived by recombination and amplification in MDCK cells. Clonal isolates of ca/ts influenza A/New Caledonia/20/99 (H1N1) obtained by recombination and amplification entirely in MDCK cells can be highly protective i.n. vaccines.     

Antigenic and genetic analysis of H5 influenza viruses isolated from water birds for the purpose of vaccine use

In order to prepare H5N1 influenza virus vaccine, the hemagglutinins (HAs) of 14 H5 virus isolates from water birds in Asia were antigenically and genetically analyzed. Phylogenetic analysis of the H5 HA genes revealed that 13 isolates belong to Eurasian and the other one to North American lineages. Each of the deduced amino acid sequences of the HAs indicated a non-pathogenic profile. Antigenic analysis using a panel of monoclonal antibodies recognizing six different epitopes on the HA of A/duck/Pennsylvania/10218/1984 (H5N2) and chicken antiserum to an H5N1 reassortant strain generated between A/duck/Mongolia/54/2001 (H5N2) and A/duck/Mongolia/47/2001 (H7N1), [R(Dk/Mong-Dk/Mong) (H5N1)] showed that the HAs of highly pathogenic avian influenza (HPAI) viruses currently circulating in Asia were antigenically closely related to those of the present isolates from water birds. Mice subcutaneously injected with formalin-inactivated R(Dk/Mong-Dk/Mong) were protected from challenge with 100 mouse lethal dose of A/Viet Nam/1194/2004 (H5N1). The present results support the notion that the H5 isolates and the reassortant H5N1 strain should be useful for vaccine preparation.     

Increasing appearance of reassortant influenza B virus in Taiwan from 2002 to 2005

Genetic and antigenic analyses of influenza B virus field strains isolated in Taiwan from 1998 to 2005 were performed. To investigate the molecular evolution of influenza B viruses, sequence analysis of the hemagglutinin (HA1 subunit) and neuraminidase genes was performed. All influenza B viruses isolated between 1998 and 2000 belonged to the B/Yamagata/16/88 lineage. The B/Victoria/2/87 lineage, which was cocirculating with the Yamagata lineage, was identified in Taiwan in March 2001. Concurrently, there was an increasing prevalence of this lineage in many parts of the world, including North America and Europe, during the 2001-2002 season. Since 2002, genetic reassortants of influenza B virus with the Victoria lineage of hemagglutinin and the Yamagata lineage of neuraminidase have been found at a rate of 46%. Therefore, in 2002, at least three sublineages of influenza B virus strains, the B/Shanghai/361/2002-like strain (Yamagata lineage), the B/Hong Kong/330/01-like strain (Victoria lineage), and the B/Hong Kong/1351/02-like strain (B reassortant lineage), were identified in Taiwan. The results showed that genetically distinct lineages can cocirculate in the population and that the reassortment among these strains plays a role in generating the genetic diversity of influenza B viruses. Interestingly, from January to April 2005, B reassortant viruses became dominant (73%) in Taiwan, which indicated that a mismatch had occurred between the influenza B vaccine strain recommended for the 2004-2005 season in the Northern hemisphere by the World Health Organization and the epidemic strain.     

Genetic evolution of swine influenza A (H3N2) viruses in China from 1970 to 2006

Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts, or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of wholly human-like H3N2 viruses, double-reassortant H3N2 viruses, and triple-reassortant H3N2 viruses in pigs in China by analyzing the eight genes of swine influenza A (H3N2) viruses found in China from 1970 to 2006. In 1970, the first wholly human-like H3N2 (Hong Kong/68-like) viruses were isolated from pigs in Taiwan, and then in the next years Victoria/75-like, Sydney/97-like, New York/99-like, and Moscow/99-like swine H3N2 viruses were regularly isolated in China. In the 1980s, two triple-reassortant viruses were isolated from pigs. Recently, the double-reassortant viruses containing genes from the human (HA and NA) and avian (PB2, PB1, PA, NP, M, and NS) lineages and the triple-reassortant viruses containing genes from the human (HA and NA), classical swine (NP), and avian (PB2, PB1, PA, M, and NS) lineages emerged in pigs in China. The coexistence of wholly human-like and reassortant viruses provides further evidence that pigs serve as intermediate hosts, or mixing vessels, and emphasizes the importance of reinforcing swine influenza virus surveillance in China.     

Influenza viruses which preconditioned the epidemic rise in Russia in 2002-2003. A resumed circulation of influenza viruses similar to V/Victoria/2/87

According to research, the epidemic rise of influenza was preconditioned, during 2002-2003, in Russia by the circulation of influenza A(H1N1), A(H3N2) and B viruses. The Center of Influenza Ecology and Epidemiology undertook a study of 178 epidemic strains: 41 strains A(H1N1), 116 strains A(H3N2) and 21 strains of influenza B were among them. All strains were isolated in the MDCK cell culture. A simultaneous isolation in embryonated eggs as well as changing of the isolation system from MDCK to embryonated eggs were found to be effective only for influenza A(H1N1) viruses. According to the antigenic analysis, all A(H1N1) viruses were variants of the etalon A/New Caledonia/20/99. The A(H3N2) viral strains' population was heterogeneous by its antigenic properties: among its isolates, there were variants similar to the etalons of A/Moscow/10/99 and of A/Panama/200/99 as well as strains, which weakly reacted with sera of both above etalons; possibly the latter were close to the etalon of A/Fujian/411/02. All epidemic strains of influenza B virus belonged, according to the antigenic properties of hemagglutinin, to the virus group of B/Victoria/2/87-like and were antigenic variants of the etalon of B/Hong Kong/22/01. This confirmed that influenza B viruses with the antigenic hemagglutinin structure of the virus group of B/Victoria/2/87-like, which were not present in Russia for more than 10 years, re-entered the active circulation. An analysis of antigenic properties of neuraminidases (NA) of the mentioned epidemic strains showed their different degrees of relationship with the NA etalons of both evolutionary groups, i.e. B/Victoria/2/87 and B/Yamagata/16/88-like. A study of paired sera obtained from patients showed a growth of antibodies to the etalons of influenza A(H1N1), A(H3N2) and B viruses of the season in question, which confirmed the virology data.     

Detection and characterization of new influenza B virus variants in 2002

One-hundred five influenza B-positive specimens obtained from southeast Asia in 2002 were categorized on the basis of DNA sequencing of HA1 gene as well as real-time PCR analysis of the NA gene. Phylogenetic analysis of the HA1 gene sequences showed that the majority of the viruses (96.2%) belonged to the B/Victoria/2/87 lineage, while a smaller percentage of the viruses (3.8%) belonged to the B/Yamagata/16/88 lineage. The B/Yamagata/16/88 viruses displayed significant antigenic drift in the deduced amino acid sequences of the HA1 protein, and the B/Victoria/2/87-like viruses consisted of B/Hong Kong/1351/02-like (72.3%) and B/Hong Kong/330/01-like (27.7%) viruses. The B/Hong Kong/1351/02-like viruses were reassortants with the HA gene belonging to the B/Victoria/2/87 lineage and the NA gene belonging to the B/Yamagata/16/88 lineage, whereas both the HA and NA genes of B/Hong Kong/330/01 virus belonged to the B/Victoria/2/87 lineage. In this study, however, all the B/Hong Kong/330/01-like isolates exhibited the B/Yamagata/16/88-like NA gene, which likely resulted from reassortment of B/Hong Kong/330/01 and B/Hong Kong/1351/02 viruses during coinfection. Additional molecular characterization of the six internal genes showed that the M, NS, PA, and PB2 genes of the new variants were B/Hong Kong/1351/02 in origin, whereas the NP and PA genes retained the B/Hong Kong/330/01 origin. Interestingly, these new variants all appeared late in the year 2002. These results support the notion that influenza B viruses continued to evolve through antigenic drift and shift.     

The use of bromelain in obtaining the subviral particles of influenza A and B viruses

Subviral particles of modern strains of influenza A viruses, i.e. A/New Caledonia/20/99 (H1N1), A/Moscow/10/99 (H3N2), reassortant X-31 (subtype H3N2) and B/Sichuan/379/99, were obtained by using two preparations of bromeline ("Sigma Co., Catalogues' Nos. B2252 and B5144). A selective ability of bromeline B5144 was detected to the proteolytic splitting of hemagglutinin of influenza A and B viruses. An influence of enzyme B5144 produced on influenza B viruses brought about an appearance of subviral particles. As for influenza A(H1N1) virus, the above enzyme did not have any impact on it under the similar experimental conditions. An incomplete effect was noted for the influenza A(H3N2) virus with particles (with intact external coatings) being found in the reaction mixture. Enzyme B2252 was found to be effective in respect to all viruses selected for testing, however, the highest effect was noted for influenza A(H1N1) and B viruses.     

Clinical and genetic characterization of severe influenza B-associated diseases during an outbreak in Taiwan.

BACKGROUND: Mismatches between circulating and vaccine strains of influenza virus had been observed in Taiwan. A comprehensive clinical and genetic analysis of influenza B viruses-associated important diseases was lacking. OBJECTIVES: Clinical and phylogenetic analysis of influenza B viruses during an outbreak in Taiwan. STUDY DESIGNS: Clinical manifestations of hospitalized, culture-confirmed patients were analyzed from July 2004 to June 2005. Partial genome sequence analysis of hemagglutinin (HA), neuraminidase (NA), and nonstructural (NS) genes were performed in 54 influenza B isolates during the study period, and nine srandomly chosen isolates during 2000 and 2003. RESULTS: Three specific diseases were found in these patients, including 13 of encephalitis/encephalopathy, 28 of influenza-associated myositis (IAM), and one of acute respiratory distress syndrome (ARDS). Three phylogenetic groups were identified, including reassortant strains-group 1 (Victoria lineage of HA, Yamagata lineage of NA, clade A of NS), group 2 (Yamagata lineage of HA, Yamagata lineage of NA, clade A of NS), and group 3 (Yamagata lineage of HA, Yamagata lineage of NA, clade B of NS). CONCLUSIONS: Severe influenza B-associated disease in children was not rare and might be fatal. We offered the evidence of co-circulation of the two HA lineages in the same outbreak.     

Internal epidemic influenza virus proteins: isolation and investigation

The internal influenza virus proteins M1 and RNP free from surface protein impurities were isolated from subviral particles (virions free from HA and NA ectomenes). The spikeless particles had no propensity to aggregate in the solution at pH 5.0 as compared with native viruses. The subviral particles of B/Hong Kong/330/01 influenza virus, which belonged to B/Victoria/2/87-lineage, were obtained by proteolytic treatment with the enzyme bromelain under the same conditions as in cases of influenza B viruses of B/Jamagata/16/88 lineage. A chromatographic analysis of the tryptic hydrolyzates obtained for matrix (M1) proteins of A(H1N1) and A(H3N2) influenza viruses revealed differences that were greatest between the protein M1 molecules isolated from influenza viruses of different subtypes of hemagglutinine. These findings suggest there are variations in the structure of this conservative internal viral protein M1 during evolution.     

The properties of the epidemic influenza viruses A and B strains circulating in Russia in the 2004-2005 epidemic season

The epidemic upsurge of influenza morbidity in Russia in 2004-2005 was caused by the active circulation of influenza A(H3N2) and B viruses. A hundred and sixty-six epidemic strains were studied. All the strains were isolated in the MCK cell culture. Influenza A(H3N2) viruses (n=77) were antigenic variants of the reference A/Fujian/411/ 2002 and A/California/7/2004 strains. Three influenza A(H1N1) viral strains that were antigenic variants of A/New Caledonia/20/99 strains were isolated in sporadic cases. Influenza B virus strains (n=83) were antigenic variants of the reference B/Shanghai/361/02--lineage B/Yamagata/l6/88. In addition, 3 antigenic variants of B/Hong Kong/ 330/2002 (lineage B/Victoria/2/87) strains were isolated. Nine (20%) strains resistant to rimantadine at a concentration of 5 microg/ml were identified. Chromatographic analysis of B/Shanghai/361/02 and BIHong Kong/330/01 viral protein M1 trypsin hydrolysates revealed differences in the profiles of chromatograms of influenza A virus proteins M1. Examination of 121 pair sera from patients revealed an increase in antibodies to influenza A(H3N2) viruses in 10-21% of cases and to influenza B viruses in 20-36% of cases.     

流感病毒A/广州/333/99(H9N2)毒株基因组特性的研究

目的 了解一株再次从流感患儿中分离出禽H9N2流感毒株的基因组特性，并弄清它的来源。方法 病毒在鸡胚中传代，从收获的尿囊液中提取RNA，通过逆转录合成cDNA，cDNA用PCR扩增。PCR产物用纯化试剂盒纯化，接着做核苷酸序列测定，然后用Meg Align(Version 1.03)和Editseg(Version 3.69)软件进行基因进化树分析。结果 A／广州／333／99（H9N2）毒株的基因组属于禽流感病毒，但它明显不同于A／Duck/Hong Kong/Y439/97毒株。同时不含有任何人流感病毒基因节段，其基因组中有4个基因节段（分别编码HA、NA、NP和NS蛋白）来自G9毒株基因系，而其余4个基因节段（分别编码PB2、PB1、PA和M蛋白）来自G1毒株基因系。结论 A／广州／333／99（H9N2）病毒是G9和G1毒株通过基因重配而来的重配株，它最大可能性直接来自禽。进一步证实了禽H9N2毒株能感染人，同时首次证实了H9N2不同基因系毒株间，在自然界中也能发生基因重配。     

High-yield reassortant virus containing hemagglutinin and neuraminidase genes of pandemic influenza A/Moscowl/01/2009 (H1N1) virus

The crossing of influenza A/Moscow/01/2009 (H1N1) virus and reassortant strain X31 (H3N2) containing the genes of internal and non-structural proteins of A/Puerto Rico/8/34 (H1N1) strain gave rise to reassortant virus ReM8. The reassortant contained hemagglutinin (HA) and neuraminidase (NA) genes of pandemic 2009 influenza virus and 6 genes of high-yield A/Puerto Rico/8/34 (H1N1) strain. The reassortant ReM8 produced higher yields in the embryonated chicken eggs than the parent pandemic virus, as suggested by infectivity and HA activity titration as well as by ELISA and the measurement of HA protein content by scanning electrophoresis in polyacrylamide gel slabs. High immunogenicity of ReM8 reassortant was demonstrated by immune protection studies in mice. The reassortant virus ReM8 is suitable as a candidate strain for the production of inactivated and subunit influenza vaccines.