Generation of a reassortant avian influenza virus H5N2 vaccine strain capable of protecting chickens against infection with Egyptian H5N1 and H9N2 viruses

BackgroundAvian influenza H5N1 viruses have been enzootic in Egyptian poultry since 2006. Avian influenza H9N2 viruses which have been circulating in Egyptian poultry since 2011 showed high replication rates in embryonated chicken eggs and mammalian cells.MethodsTo investigate which gene segment was responsible for increasing replication, we constructed reassortant influenza viruses using the low pathogenic H1N1 PR8 virus as backbone and included individual genes from A/chicken/Egypt/S4456B/2011(H9N2) virus. Then, we invested this finding to improve a PR8-derived H5N1 influenza vaccine strain by incorporation of the NA segment of H9N2 virus instead of the NA of H5N1. The growth properties of this virus and several other forms of reassortant H5 viruses were compared. Finally, we tested the efficacy of this reassortant vaccine strain in chickens.ResultsWe observed an increase in replication for a reassortant virus expressing the neuraminidase gene (N2) of H9N2 virus relative to that of either parental viruses or reassortant PR8 viruses expressing other genes. Then, we generated an H5N2 vaccine strain based on the H5 from an Egyptian H5N1 virus and the N2 from an Egyptian H9N2 virus on a PR8 backbone. This strain had better replication rates than an H5N2 reassortant strain on an H9N2 backbone and an H5N1 reassortant on a PR8 backbone. This virus was then used to develop a killed, oil-emulsion vaccine and tested for efficacy against H5N1 and H9N2 viruses in chickens. Results showed that this vaccine was immunogenic and reduced mortality and shedding.DiscussionOur findings suggest that an inactivated PR8-derived H5N2 influenza vaccine is efficacious in poultry against H5N1 and H9N2 viruses and the vaccine seed replicates at a high rate thus improving vaccine production.     

Generation and evaluation of a high-growth reassortant H9N2 influenza A virus as a pandemic vaccine candidate

H9N2 subtype avian influenza viruses (AIVs) are widely distributed in avian species and were isolated from humans in Hong Kong and Guangdong province, China in 1999 raising concern of their potential for pandemic spread. We generated a high-growth reassortant virus (G9/PR8) that contains the hemagglutinin (HA) and neuraminidase (NA) genes from the H9N2 avian influenza virus A/chicken/Hong Kong/G9/97 (G9) and six internal genes from A/Puerto Rico/8/34 (PR8) by genetic reassortment, for evaluation as a potential vaccine candidate in humans. Pathogenicity studies showed that the G9/PR8 reassortant was not highly pathogenic for mice or chickens. Two doses of a formalin-inactivated G9/PR8 virus vaccine induced hemagglutination inhibiting antibodies and conferred complete protection against challenge with G9 and the antigenically distinct H9N2 A/Hong Kong/1073/99 (G1-like) virus in a mouse model. These results indicate that the high growth G9/PR8 reassortant has properties that are desirable in a vaccine seed virus and is suitable for evaluation in humans for use in the event of an H9 pandemic.     

Generation and characterization of a cold-adapted influenza A H9N2 reassortant as a live pandemic influenza virus vaccine candidate

H9N2 subtype influenza A viruses have been identified in avian species worldwide and were isolated from humans in 1999, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. Reassortant H1N1 and H3N2 human influenza A viruses with the internal genes of the influenza A/Ann Arbor/6/60 (H2N2) (AA) cold-adapted (ca) virus have proven to be attenuated and safe as live virus vaccines in humans. Using classical genetic reassortment, we generated a reassortant virus (G9/AA ca) that contains the hemagglutinin and neuraminidase genes from influenza A/chicken/Hong Kong/G9/97 (H9N2) (G9) and six internal gene segments from the AA ca virus. When administered intranasally, the reassortant virus was immunogenic and protected mice from subsequent challenge with wild-type H9N2 viruses, although it was restricted in replication in the respiratory tract of mice. The G9/AA ca virus bears properties that are desirable in a vaccine for humans and is available for clinical evaluation and use, should the need arise.     

Improvement of H5N1 influenza vaccine viruses: influence of internal gene segments of avian and human origin on production and hemagglutinin content

The H5N1-clade 1 influenza vaccine strain NIBRG-14 produces exceptionally low amounts of antigen, a problem recently encountered also for initial pandemic H1N1-2009 vaccine seeds. Here, we report on a strategy that may contribute to overcome this obstacle. Influenza vaccine viruses usually consist of two segments coding for the antigenic HA and NA proteins of a wild-type strain and the six residual internal gene segments of the vaccine donor strain A/PR/8/34 (PR8). To enhance the antigen yield from H5N1 vaccine virus we generated by reverse genetics a set of PR8-based reassortant viruses expressing the HA and NA segments of the prototypic strain A/Vietnam/1203/2004 and additional replacements of the internal M or PB1 genes of PR8. The reassortants were compared to the parental PR8 and H5N1 viruses in terms of growth in embryonated chicken eggs and the amount of incorporated antigenic HA protein. Compared to NIBRG-14, three out of six viruses displayed an increased replication in embryonated chicken eggs and higher HA content that was also maintained after ether/detergent extraction of virions. Electron microscopic analysis showed that the reassortment hardly affected particle shape and size. Two selected H5N1 reassortant viruses were investigated concerning their pathogenicity in ferrets and found to behave as low pathogenic as the PR8 donor strain. In conclusion, this study shows that replication and antigen content of PR8-derived H5N1 influenza vaccine viruses can be improved by incorporation of heterologous internal gene segments without compromising their attenuated character.     

Intanza(®) 15 mcg intradermal influenza vaccine elicits cross-reactive antibody responses against heterologous A(H3N2) influenza viruses

The aim of the present study was to explore the ability of Intanza^® 15 μg, the intradermal (ID) trivalent inactivated split-virion influenza vaccine containing 15 μg hemagglutinin per strain, to enhance the antibody responses against heterologous circulating H3N2 strains in adults 60 years and older. During the 2006–2007 influenza season, subjects aged 60 years or older were randomly assigned to receive one dose of ID or an intramuscular (IM, Vaxigrip^®) influenza vaccine, which contained the reassortant A/Wisconsin/67/05(H3N2) strain as the H3N2 component. Antibody responses were assessed against the homologous vaccine strain, against the A/Brisbane/10/07(H3N2) reassortant strain and against four heterologous H3N2 field isolates (A/Genoa/62/05(H3N2), A/Genoa/3/07(H3N2), A/Genoa/2/07(H3N2), A/Genoa/3/06(H3N2)). The viruses tested belonged to three different clades that were closely related antigenically to A/California/7/04(H3N2), A/Nepal/921/06(H3N2) and A/Brisbane/10/07(H3N2). Antibody responses to these viruses were measured in 25 subjects per group using both haemagglutination inhibition (HI) and neutralization (NT) assays.     

Emergence of novel clade 2.3.4 influenza A (H5N1) virus subgroups in Yunnan Province,China

From December 2013 to March 2014, a major wave of highly pathogenic avian influenza outbreak occurred in poultry in Yunnan Province, China. We isolated and characterized eight highly pathogenic avian influenza A (H5N1) viruses from poultry. Full genome influenza sequences and analyses have been performed.Sequence analyses revealed that they belonged to clade 2.3.4 but did not fit within the three defined subclades. The isolated viruses were provisional subclade 2.3.4.4e. The provisional subclade 2.3.4.4e viruses with six internal genes from avian influenza A (H5N2) viruses in 2013 were the novel reassortant influenza A (H5N1) viruses which were associated with the outbreak of H5N1 occurred in egg chicken farms in Yunnan Province. The HA genes were similar to subtype H5 viruses isolated from January to March of 2014 in Asia including H5N6 and H5N8. The NA genes were most closely related to A/chicken/Vietnam/NCVD-KA423/2013 (H5N1) from the subclade 2.3.2. The HI assay demonstrated a lack of antigenic relatedness between clades 2.3.4.4e and 2.3.4.1 (RE-5 vaccine strain) or 2.3.2.2 (RE-6 vaccine strain).     

Generation of an attenuated H5N1 avian influenza virus vaccine with all eight genes from avian viruses

In the face of disease outbreaks in poultry and the potential pandemic threat to humans caused by the highly pathogenic avian influenza viruses (HPAIVs) of H5N1 subtype, improvement in biosecurity and the use of inactivated vaccines are two main options for the control of this disease. Vaccine candidates of influenza A viruses of H5N1 subtype have been generated in several laboratories by plasmid-based reverse genetics with hemagglutinin (HA) and neuraminidase (NA) genes from the epidemic strains of avian viruses in a background of internal genes from the vaccine donor strain of human strains, A/Puerto Rico/8/34 (PR8). These reassortant viruses containing genes from both avian and human viruses might impose biosafety concerns, also may be do if C4/F AIV would be a live attenuated vaccine or cold-adaptive strain vaccine. In order to generate better and safer vaccine candidate viruses, we genetically constructed attenuated reassortant H5N1 influenza A virus, designated as C4/F AIV, by plasmid-based reverse genetics with all eight genes from the avian strains. The C4/F AIV virus contained HA and NA genes from an epidemic strain A/Chicken/Huadong/04 (H5N1) (C4/H5N1) in a background of internal genes derived from a low pathogenic strain of A/Chicken/F/98(H9N2). The reassortant virus was attenuated by removal of the multibasic amino acid motif in the HA gene by mutation and deletion (from PQRERRRKKR (downward arrow) G to PQIETR (downward arrow) G). The intravenous pathogenicity index (IVPI) of C4/F AIV virus was 0, whereas that of the donor virus C4/H5N1 was 3.0. The virus HA titer of C4/H5N1 in the allantoic fluid from infected embryonated eggs was as high as 1:2048. The inactivated vaccine prepared from the reassortant virus C4/F AIV-induced high HI titer in vaccinated chickens and gave 100% protection when challenged with highly pathogenic avian influenza virus of H5N1 subtype.     

Genetic characterization and protective immunity of cold-adapted attenuated avian H9N2 influenza vaccine

H9N2 influenza viruses are endemic in many Asian countries including China and Korea, and cause a considerable economic loss to chicken industry by reduction in egg production and about 30% mortality. Here we developed live cold-adapted attenuated H9N2 influenza vaccine by adaptation of viruses in hen's eggs at 25 degrees C. Genetic analysis shows that the cold-adapted H9N2 (A/Chicken/Korea/S1/03) viruses contain a total of 44 amino acid substitutions, of which 7 amino acids are identical to the loci identified in the cold-adapted H2N2 (A/Ann Arbor/6/60) vaccine strain compared to genes in wild-type H9N2 (A/Chicken/Korea/S1/03) influenza viruses. When cold-adapted H9N2 (A/Chicken/Korea/S1/03) influenza viruses were inoculated in layers viruses were detectable in the tracheas, not in the lungs, no reduction of egg production and mortality was observed in contrast to the infection of wild-type H9N2 influenza viruses, and CD8+ T lymphocytes expressing IFN-gamma were induced. When layers vaccinated with cold-adapted attenuated H9N2 (A/Chicken/Korea/S1/03) influenza viruses were challenged with wild-type H9N2 (A/Chicken/Korea/521/04) influenza viruses, they were protected from the loss of egg production and mortality. Our results suggest that cold-adapted attenuated H9N2 vaccine can be used for controlling the infection of H9N2 influenza viruses in chickens.     

Poly I:C adjuvanted inactivated swine influenza vaccine induces heterologous protective immunity in pigs

Swine influenza is widely prevalent in swine herds in North America and Europe causing enormous economic losses and a public health threat. Pigs can be infected by both avian and mammalian influenza viruses and are sources of generation of reassortant influenza viruses capable of causing pandemics in humans. Current commercial vaccines provide satisfactory immunity against homologous viruses; however, protection against heterologous viruses is not adequate. In this study, we evaluated the protective efficacy of an intranasal Poly I:C adjuvanted UV inactivated bivalent swine influenza vaccine consisting of Swine/OH/24366/07 H1N1 and Swine/CO/99 H3N2, referred as PAV, in maternal antibody positive pigs against an antigenic variant and a heterologous swine influenza virus challenge. Groups of three-week-old commercial-grade pigs were immunized intranasally with PAV or a commercial vaccine (CV) twice at 2 weeks intervals. Three weeks after the second immunization, pigs were challenged with the antigenic variant Swine/MN/08 H1N1 (MN08) and the heterologous Swine/NC/10 H1N2 (NC10) influenza virus. Antibodies in serum and respiratory tract, lung lesions, virus shedding in nasal secretions and virus load in lungs were assessed. Intranasal administration of PAV induced challenge viruses specific-hemagglutination inhibition- and IgG antibodies in the serum and IgA and IgG antibodies in the respiratory tract. Importantly, intranasal administration of PAV provided protection against the antigenic variant MN08 and the heterologous NC10 swine influenza viruses as evidenced by significant reductions in lung virus load, gross lung lesions and significantly reduced shedding of challenge viruses in nasal secretions. These results indicate that Poly I:C or its homologues may be effective as vaccine adjuvants capable of generating cross-protective immunity against antigenic variants/heterologous swine influenza viruses in pigs.     

Protective efficacy of an inactivated Eurasian avian-like H1N1 swine influenza vaccine against homologous H1N1 and heterologous H1N1 and H1N2 viruses in mice

Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses are prevalent in pigs in Europe and Asia, but occasionally cause human infection, which raises concern about their pandemic potential. Here, we produced a whole-virus inactivated vaccine with an EA H1N1 strain (A/swine/Guangxi/18/2011, SW/GX/18/11) and evaluated its efficacy against homologous H1N1 and heterologous H1N1 and H1N2 influenza viruses in mice. A strong humoral immune response, which we measured by hemagglutination inhibition (HI) and virus neutralization (VN), was induced in the vaccine-inoculated mice upon challenge. The inactivated SW/GX/18/11 vaccine provided complete protection against challenge with homologous SW/GX/18/11 virus in mice and provided effective protection against challenge with heterologous H1N1 and H1N2 viruses with distinctive genomic combinations. Our findings suggest that this EA H1N1 vaccine can provide protection against both homologous H1N1 and heterologous H1N1 or H1N2 virus infection. As such, it is an excellent vaccine candidate to prevent H1N1 swine influenza.     

Increase in viral yield in eggs and MDCK cells of reassortant H5N1 vaccine candidate viruses caused by insertion of 38 amino acids into the NA stalk

Background

The H5N1 subtype of highly pathogenic avian influenza viruses has spread to over 63 countries in Asia, Europe, and Africa and has become endemic in poultry. Since 2004, vaccination against H5N1 influenza has become common in domestic poultry operations in China. Most influenza vaccines have been produced in embryonated chicken eggs. High yield is the essential feature of a good vaccine candidate virus.

Objective

Therefore, the large-scale manufacture of such a vaccine requires that the viral yield of H5N1 reassortant vaccine viruses in eggs and MDCK cells be increased.

Methods

We generated two sets of reassortant H5N1 viruses based on backbone viruses A/Chicken/F/98 (H9N2) and A/Puerto Rico/8/34 (H1N1) using reverse genetics. The HAs and NAs of the reassortants were derived from the three epidemic H5N1 strains found in China. We compared the replication properties of these recombinant H5N1 viruses in embryonated chicken eggs and MDCK cells after inserting either 20 or 38 amino acids into their NA stalks.

Results

In this study, we demonstrated that inserting 38 amino acids into the NA stalks can significantly increase the viral yield of H5N1 reassortant viruses in both embryonated chicken eggs and MDCK cells, while inserting only 20 amino acids into the same NA stalks does not. Hemagglutinin inhibition testing and protection assays indicated that recombinant H5N1 viruses with 38 aa inserted into their NA stalks had the same antigenicity as the viruses with wt-NA.

Conclusion

These results suggest that the generation of an H5N1 recombinant vaccine seed by the insertion of 38 aa into the NA stalk may be a suitable and more economical strategy for the increase in viral yield in both eggs and MDCK cells for the purposes of vaccine production.     

Characterization of an influenza A H5N2 reassortant as a candidate for live-attenuated and inactivated vaccines against highly pathogenic H5N1 viruses with pandemic potential

We generated a high-growth 7:1 reassortant (Len17/H5) that contained the hemagglutinin (HA) gene from non-pathogenic A/Duck/Potsdam/1402-6/86 (H5N2) virus and other genes from the cold-adapted (ca) attenuated A/Leningrad/134/17/57 (H2H2) strain. Len17/H5 demonstrated an attenuated phenotype in mice and did not infect chickens. Mice administered Len17/H5 either as a live-attenuated intranasal vaccine or as an inactivated intramuscular vaccine were substantially protected from lethal challenge with highly pathogenic A/Hong Kong/483/97 (H5N1) virus and were protected from pulmonary infection with antigenically distinct A/Hong Kong/213/2003 (H5N1) virus. The cross-protective effect correlated with the levels of virus-specific mucosal IgA and/or serum IgG antibodies. Our results suggest a new strategy of using classical genetic reassortment between a high-growth ca H2N2 strain and antigenically related non-pathogenic avian viruses to prepare live-attenuated and inactivated vaccines for influenza pandemic.     

Genetic and antigenic relatedness of H3 subtype influenza A viruses isolated from avian and mammalian species

In 2004, we isolated triple reassortant H3N2 influenza viruses from turkey breeder hens in Ohio and Illinois. The Illinois flock was vaccinated twice with an inactivated H3N2 vaccine containing a swine origin virus before the outbreak. Additionally, a commercial inactivated vaccine containing an H3N4 virus of duck origin is being used in some turkey breeders. This prompted us to initiate a comparative study on the antigenic and genetic relatedness of various H3 subtype influenza viruses isolated from turkeys, ducks, pigs and humans. The antigenic relatedness between the different viruses was evaluated with the Archetti and Horsfall formula, while nucleotide genetic similarities were calculated using pairwise alignments. Results obtained indicated a high degree of antigenic (>90%) and genetic (>99%) similarities among the turkey-origin H3N2 viruses. However, the turkey viruses were antigenically distantly related to the swine-origin vaccine virus (<30%), although they had approximately 95% genetic similarity in the HA1 gene. Additionally, major genetic and antigenic changes were observed between the turkey viruses and the H3N4 duck vaccine virus as well as the H3N2 human virus. Such genetic and antigenic differences between the turkey-origin viruses and other H3 subtype viruses including vaccine strains could be the reason for the failure in protection in the Illinois turkey breeders vaccinated with swine origin virus. This also emphasizes the importance of using viruses for vaccines that are antigenically similar to the field strains.     

Supplementation of conventional trivalent influenza vaccine with purified viral N1 and N2 neuraminidases induces a balanced immune response without antigenic competition

Influenza viruses neuraminidase (NA) were chromatographically extracted from influenza viruses A/Nanchang/933/95 H3(NC)N2(NC) [R] and A/Johannesburg/82/96 H1(JH)N1(JH) [R] and used to supplement conventional inactivated trivalent influenza vaccine. Immunization of mice with this preparation resulted in high titers of antibodies to both hemagglutinins (HA) and neuraminidases (NA); there were no significant differences in the anti-HA antibody titers between the conventional and the supplemented vaccine preparation. Likewise, there were no significant differences in anti-NA antibody titers between the supplemented vaccine and titers from mice immunized with a neuraminidase vaccine containing a mixture of N1-NA and N2-NA. There was no evidence of a diminution of the immune response to the HA components of the vaccine despite the presence of antigenically equivalent amounts of both N1-NA and N2-NAs. Homotypic and distantly related heterotypic infections for both H1, N1 and H3N2 subtypes were suppressed and greater reduction in pulmonary virus titers (PVT) were observed in the trivalent vaccine supplemented with purified neuraminidase from each subtype, N1 and N2. Effects on the influenza B viral components were not studied. Previous studies on supplementation of conventional influenza vaccine focused only on monovalent H3N2 vaccine preparations; this study demonstrates in a mouse model system that supplementation of trivalent influenza vaccine with both influenza A subtype neuraminidases, N1 and N2 is highly immunogenic for HA and NA of each subtype and efficacious in protecting against influenza from homotypic and heterotypic infectious challenges of either subtype.     

甲型H1N1流感病毒基因组序列分析及其特性研究

目的 分析甲型H1N1流感病毒的基因组序列特征,阐明该毒株的遗传变异及分子特性.方法 GenBank中获取流感病毒全序列,对各段基因与已知序列进行分析比较,绘制进化树,并分析和预测甲型毒株的致病性、药物敏感性和现有疫苗的预防保护作用.结果 甲型H1N1病毒的HA、PB2、PB1、PA、NP、NS基因与美国本土的猪流感病毒序列具有高度同源性,NA和M基因具有典型的欧亚株系猪流感病毒特征.该病毒具有人传人的分子基础,HA上HA1和HA2裂解位点序列为PSIQSR↓+GLFGAI,尚不具备高致病性流感病毒的特征.病毒对金刚烷胺类药物耐药,而对达菲和扎那米韦敏感.HA片段5个抗原决定区氨基酸序列与人用流感疫苗具有较大差异,推测现有疫苗对预防本次疫情基本无效.结论 甲型H1N1是一种北美和欧亚两种猪流感病毒的混合体,开发针对本病毒的流感疫苗有助于进一步控制疫情蔓延.     

Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses

Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin-Darby canine kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 2(9) HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems.     

Multiple reassortment between pandemic (H1N1) 2009 and endemic influenza viruses in pigs, United States

As a result of human-to-pig transmission, pandemic influenza A (H1N1) 2009 virus was detected in pigs soon after it emerged in humans. In the United States, this transmission was quickly followed by multiple reassortment between the pandemic virus and endemic swine viruses. Nine reassortant viruses representing 7 genotypes were detected in commercial pig farms in the United States. Field observations suggested that the newly described reassortant viruses did not differ substantially from pandemic (H1N1) 2009 or endemic strains in their ability to cause disease. Comparable growth properties of reassortant and endemic viruses in vitro supported these observations; similarly, a representative reassortant virus replicated in ferrets to the same extent as did pandemic (H1N1) 2009 and endemic swine virus. These novel reassortant viruses highlight the increasing complexity of influenza viruses within pig populations and the frequency at which viral diversification occurs in this ecologically important viral reservoir.     

Potential Threats to Human Health from Eurasian Avian-Like Swine Influenza A(H1N1) Virus and Its Reassortants

During 2018–2020, we isolated 32 Eurasian avian-like swine influenza A(H1N1) viruses and their reassortant viruses from pigs in China. Genomic testing identified a novel reassortant H3N1 virus, which emerged in late 2020. Derived from G4 Eurasian H1N1 and H3N2 swine influenza viruses. This virus poses a risk for zoonotic infection.     

Preliminary assessment of the effectiveness of the 2003-04 inactivated influenza vaccine--Colorado, December 2003

Influenza activity started earlier than usual in the United States this season, with widespread influenza activity reported in 10 states by November 22, 2003. The predominant influenza viruses (A/Fujian/411/2002 [H3N2]-like viruses) circulating this season differ antigenically from the 2003-04 influenza A (H3N2) vaccine strain. A retrospective cohort study was conducted among workers at a Colorado hospital to provide preliminary data on the effectiveness of trivalent inactivated influenza vaccine (TIV) against influenza-like illness (ILI). This report summarizes the results of that study, which indicated that TIV had no or low effectiveness against ILI. However, additional studies are needed to evaluate the effectiveness of the 2003-04 vaccine against laboratory-confirmed influenza and influenza-related complications, including hospitalization and death. Influenza vaccine continues to be recommended, particularly for persons at increased risk for influenza-related complications, their household contacts, and health-care personnel.     

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.