HA Gene Of Virus Research Articles

Genomic epidemiology of highly pathogenic avian influenza A (H5N1) virus in wild birds in South Korea during 2021-2022: Changes in viral epidemic patterns.

Clade 2.3.4.4b highly pathogenic avian influenza A (HPAI) viruses have been detected in wild birds worldwide, causing recurrent outbreaks since 2016. During the winter of 2021-2022, we detected one H5N8 and forty-three H5N1 clade 2.3.4.4b HPAI viruses from wild birds in South Korea. Phylogenetic analysis revealed that HA gene of H5N1 viruses was divided into two genetically distinct groups (N1.G1 and N1.G2). Bayesian phylodynamic analysis demonstrated that wild birds play a vital role in viral transmission and long-term maintenance. We identified five genotypes (N1.G1.1, N1.G2, N1.G2.1, N1.G2.2, and N1.G2.2.1) having distinct gene segment constellations most probably produced by reassortments with low-pathogenic avian influenza viruses. Our results suggest that clade 2.3.4.4b persists in wild birds for a long time, causing continuous outbreaks, compared with previous clades of H5 HPAI viruses. Our study emphasizes the need for enhancing control measures in response to the changing viral epidemiology.

An efficient double-fluorescence approach for generating fiber-2-edited recombinant serotype 4 fowl adenovirus expressing foreign gene.

Recently, the infection of serotype 4 fowl adenovirus (FAdV-4) in chicken flocks has become endemic in China, which greatly threatens the sustainable development of poultry industry. The development of recombinant FAdV-4 expressing foreign genes is an efficient strategy for controlling both FAdV-4 and other important poultry pathogens. Previous reverse genetic technique for generating the recombinant fowl adenovirus is generally inefficient. In this study, a recombinant FAdV-4 expressing enhanced green fluorescence protein (EGFP), FA4-EGFP, was used as a template virus and directly edited fiber-2 gene to develop an efficient double-fluorescence approach to generate recombinant FAdV-4 through CRISPR/Cas9 and Cre-Loxp system. Moreover, using this strategy, a recombinant virus FAdV4-HA(H9) stably expressing the HA gene of H9N2 influenza virus was generated. Chicken infection study revealed that the recombinant virus FAdV4-HA(H9) was attenuated, and could induce haemagglutination inhibition (HI) titer against H9N2 influenza virus at early time points and inhibit the viral replication in oropharynx. All these demonstrate that the novel strategy for constructing recombinant FAdV-4 expressing foreign genes developed here paves the way for rapidly developing attenuated FAdV-4-based recombinant vaccines for fighting the diseases caused by both FAdV-4 and other pathogens.

Baculovirus-derived influenza virus-like particle confers complete protection against lethal H7N9 avian influenza virus challenge in chickens and mice

Currently, highly pathogenic avian influenza (HPAI) H7N9 viruses still pose a potential pandemic threat. Influenza virus-like particle (VLP) is one of the most promising vaccine strategies to complement traditional egg-dependent vaccines. Here, we generated a H7N9 VLP vaccine candidate by baculovirus expression system and evaluated its efficacy in chickens and mice. The H7N9 VLP was produced through co-infection of Sf9 insect cells with three recombinant baculoviruses expressing individual HA, NA and M1 gene of the HPAI H7N9 virus A/chicken/Guangdong/GD15/2016. Intramuscular immunization of the H7N9 VLP elicited robust antibody immune responses and conferred complete clinical protection against lethal H7N9 virus challenge both in chickens and mice. Meanwhile, H7N9 VLP significantly restrained virus shedding and dramatically alleviated pulmonary lesions caused by H7N9 virus infection in birds and mice. Interestingly, chicken antibodies induced by the H7N9 VLP also had a good cross-reactivity with H7N9 field strains isolated in different years. In addition, vaccination with the H7N9 VLP elicited high T cell immunity in mouse lung, evidenced by significantly upregulated expression of IL-2, IL-4 and IFN-γ. Furthermore, the H7N9 VLP significantly decreased the expression of some key inflammatory cytokines, such as IL6, RANTES and TNF-α in mouse lung, which may partially account for its contribution to alleviate lung pathology. Therefore, our study describes the good efficacy of the HA + NA + M1-containing H7N9 VLP both in chicken and mice models, highlighting the potential of VLP-based vaccine as a critical alternative of traditional egg-based vaccine for control of H7N9 influenza virus in both humans and poultry.

Assessment of exposure to influenza A viruses in pigs between weaning and market age

Influenza A viruses (IAVs) are common causes of respiratory infection in pigs. The objective of this study was to characterize the circulation of IAVs between weaning and market age on the basis of development of antibody response and molecular epidemiology of detected viruses. Two batches of weaned pigs were followed in the nursery and finisher barns with a sample of 81 and 75 pigs. Nasal swabs and blood samples were collected from individual pigs for virological and serological analyses. A H3N2 subtype virus, of cluster IV, was detected in Study 1, with a maximum of 97.9% identity to HA gene of viruses previously isolated in Ontario. In Study 2, a H1N1 subtype virus, of 2009 H1N1 pandemic lineage, was detected, with a maximum of 97.8% identity to HA gene of viruses previously isolated in Ontario. On the basis of HA gene, it was observed that pigs were being detected with the same virus over time. The existence of antibody titers for IAV other than the isolated one confirmed that more than one subtype can circulate in the same population. In Study 1, pigs with higher numbers of IAV detection had lower serological titers for the same virus that was confirmed to circulate in the nursery (P < 0.01). Thorough knowledge of all endemic viral strains is fundamental for development of infection and disease control, particularly in complex production systems. This may include consideration of sampling and testing strategies which could detect circulation of all IAV variants, even if they have low prevalence.

Genetic Characteristics of Avian Influenza Virus Isolated from Wild Birds in South Korea, 2019–2020

Wild aquatic birds, a natural reservoir of avian influenza viruses (AIVs), transmit AIVs to poultry farms, causing huge economic losses. Therefore, the prevalence and genetic characteristics of AIVs isolated from wild birds in South Korea from October 2019 to March 2020 were investigated and analyzed. Fresh avian fecal samples (3256) were collected by active monitoring of 11 wild bird habitats. Twenty-eight AIVs were isolated. Seven HA and eight NA subtypes were identified. All AIV hosts were Anseriformes species. The HA cleavage site of 20 representative AIVs was encoded by non-multi-basic amino acid sequences. Phylogenetic analysis of the eight segment genes of the AIVs showed that most genes clustered within the Eurasian lineage. However, the HA gene of H10 viruses and NS gene of four viruses clustered within the American lineage, indicating intercontinental reassortment of AIVs. Representative viruses likely to infect mammals were selected and evaluated for pathogenicity in mice. JB21-58 (H5N3), JB42-93 (H9N2), and JB32-81 (H11N2) were isolated from the lungs, but JB31-69 (H11N9) was not isolated from the lungs until the end of the experiment at 14 dpi. None of infected mice showed clinical sign and histopathological change in the lung. In addition, viral antigens were not detected in lungs of all mice at 14 dpi. These data suggest that LPAIVs derived from wild birds are unlikely to be transmitted to mammals. However, because LPAIVs can reportedly infect mammals, including humans, continuous surveillance and monitoring of AIVs are necessary, despite their low pathogenicity.

THE SPANISH INFLUENZA VIRUS: TREATS TO THE PORTRAIT AFTER 100 YEARS

The purpose of the study was to compare molecular characteristics of genes and proteins of pandemic influenza strains and find features of the 1918 Spanish influenza virus. Computer analysis has shown that the genes of the Spanish influenza virus in contrast to other pandemic strains contain optimal quantity of long complementary sequences that allow to obtain a supramolecular assembly of 8 virus RNA in according to a model ensuring selective packing of one copy of each virus RNA by the only possible scheme and high transmission to induce infection by single virions. Other pandemic strains contain redundant or insufficient quantity of complementary sequences that allow an assembly of its genome by means of some models including a stochastic one and occurrence of virions with incomplete genome, that is influenza virusts can exist primarily as a swarm of complementation-dependent semi-infectious virions. Analysis of an HA gene of the Spanish influenza virus found out exclusion from its translation code four triplets (CGG, CGA, CGC и CGU) coding arginine. This exclusion is observed in all the HlNl strains isolated during 100 years. Coding arginine in an HA gene of HlNl strains is provided by only triplets AGG and AGA. A NP gene of the Spanish influenza virus in contrast to other pandemics strains is avian-like and its NP protein is characterized by elevated quantity of arginine and decreased quantity of lysine that is considered as viral adaptation to avian body temperature. Prevalence of arginine provides more high positive charge for the Spanish influenza NP protein and its more powerful interaction with RNA and consequently more high thermal stability of the its RNP in comparison with the RNP of other pandemic strains. Potential consequence of existence of the avian-like NP in the Spanish influenza virus could be its high pathogenicity as infection fever creates optimal temperature for virus replication. These new data obtained by computer analysis of genomes in the Spanish influenza virus and other pandemic strains (altogether information about its proteins) can potentially be used to track pre-pandemic strains among circulating influenza A viruses and detect the formation of a possible trajectory of pandemic alert.

Vertical Transmission of H9N2 Avian Influenza Virus in Goose.

During a study on high mortality cases of goose embryo in Shandong Province, China (2014–2015), we isolated an H9N2 avian influenza virus (AIV) strain (A/goose/Shandong/DP01/2014, DP01), which was supposedly the causative agent for goose embryo death. Sequence analysis revealed that DP01 shared 99.9% homology in the HA gene with a classic immune suppression strain SD06. To study the potential vertical transmission ability of the DP01 strain in breeder goose, a total of 105 Taizhou breeder geese, which were 360 days old, were equally divided into five groups (A, B, C, D, and E) for experimental infection. H9N2 AIV (DP01) was used for inoculating through intravenous (group A), intranasal instillation (group B), and throat inoculation (group C) routes, respectively. The geese in group D were inoculated with phosphate buffer solution (PBS) and those in group E were the non-treated group. At 24 h post inoculation, H9N2 viral RNA could be detected at vitelline membrane, embryos, and allantoic fluid of goose embryos from H9N2 inoculated groups. Furthermore, the HA gene of H9N2 virus from vitelline membrane, embryo, allantoic fluid, and gosling shared almost 100% homology with an H9N2 virus isolated from the ovary of breeder goose, which laid these eggs, indicating that H9N2 AIV can be vertically transmitted in goose. The present research study provides evidence that vertical transmission of H9N2 AIV from breeding goose to goslings is possible.

Molecular and antigenic traits on hemagglutinin gene of avian influenza H9N2 viruses: Evidence of a new escape mutant in Egypt adapted in quails

The LPAI viruses of H9N2 subtype became widely distributed in Middle Eastern countries, causing great economic losses in poultry industry especially when complicated with other pathogens. The H9N2 viruses in Egypt have a wide spread nature since its first occurrence in 2011. In this study, we collected cloacal and tracheal samples from 19 flocks for detection and propagation of H9N2 virus using real-time RT-PCR and egg inoculation. We studied the molecular evolution of the Hemagglutinin gene of H9N2 viruses by full HA gene sequencing, then the antigenic characterization was implemented using the cross HI assay and analyzed using 3D Bioinformatics cartography software. The phylogenetic analysis of the HA gene of Egyptian H9N2 viruses clearly points out the presence of only one group (Egy/G1) of originally introduced viruses in 2011 related to the G1 lineage within group B, with the presence of multiple minor clusters includes viruses from 2011 to 2015. However, a new variant (Egy/G1var) cluster was detected in quails since 2012. Genetically, Egy/G1var viruses characterized by presence of 20 amino acid substitutions within and adjacent to the antigenic sites in comparison to other Egyptian viruses. In addition, two glycosylation sites at amino acid residues 127 and 189 were determined in close to the receptor binding and antigenic sites. The antigenic analysis based on 3D antigenic mapping showed that the Egy/G1var cluster was clearly distinct from the original Egy/G1 viruses. In conclusion, Egy/G1var is shown to be a new escape mutant variant cluster with an adaptive evolution in quails.

Identification of cis-acting packaging signals in the coding regions of the influenza B virus HA gene segment.

For influenza A and B viruses to be infectious, they require eight viral RNA (vRNA) genome segments to be packaged into virions. For efficient packaging, influenza A viruses utilize cis-acting vRNA sequences, containing both non-coding and protein coding regions of each segment. Whether influenza B viruses have similar packaging signals is unknown. Here we show that coding regions at the 3' and 5' ends of the influenza B virus vRNA segment 4 are required for genome packaging, with the first 30 nt at each end essential for this process. Synonymous mutation of these regions led to virus attenuation, an increase in defective particle production and a reduction in packaging of multiple vRNAs. Overall, our data suggest that the influenza B virus vRNA gene segments likely interact with each other during the packaging process, which is driven by cis-acting packaging signals that extend into protein coding regions of the vRNA.

Induction of Selected Deletion in HA Gene of Egyptian HPAI-H5N1 Viruses Using Site Directed Mutagenesis

Induction of Selected Deletion in HA Gene of Egyptian HPAI-H5N1 Viruses Using Site Directed Mutagenesis

Surveillance and molecular characterization of human influenza B viruses during 2006–2010 revealed co-circulation of Yamagata-like and Victoria-like strains in eastern India

Acute respiratory illness (ARI) is one of the major health problems in tropical countries of Asia, like India where approximately 0.5million children in the age group of <5years die annually. Previously we have reported the genetic characterization of influenza A (Inf-A) strains circulating in Kolkata, eastern India. This study was initiated to characterize the genetic diversity of the circulating influenza B (Inf-B) viruses. Of 3035 nasal/throat swabs, 494 (16.3%) samples were identified as influenza A/B positive by real time RT-PCR, of which 244 samples were confirmed having Inf-B infection. Comparison of nucleotide (nt) and amino acid (aa) sequences of HA and NA gene of Inf-B viruses revealed co-circulation of B/Yamagata and B/Victoria lineages. Of the 32 randomly selected Inf-B strains from Kolkata, seventeen strains possessed reassorted NA gene. There was a single Histidine to Asparagine substitution in the 131st position which is a part of 120 loop on HA1 region along with a deletion at position 178 in the Kolkata strains belonging to the Yamagata lineage. Amino acid substitution was observed at position 198 on NA gene in the strains B/Kol/542/2006, B/Kol/1373/2008, B/Kol/1880/2008, B/Kol/2044/2008 and in all the representative strains isolated during 2009 with respect to the circulating vaccine strains. This substitution is responsible for reduced sensitivity of neuraminidase inhibitors. The results highlight the importance of monitoring Inf-B viruses for development of antiviral resistance among circulating strains.

Influenza H1N1 surveillance and analysis of genetic characteristics of predominant strains in Quanzhou during 2009

Objective To investigate the influenza H1N1 virus surveillance of 2009 in Quanzhou,and analyze the HA and NA gene of influenza H1N1 virus, explore its genetic variation and molecular characteristics. Methods During the influenza H1N1 virus surveillance in Quanzhou,specimens of throat swabs from the patients with influenza were collected, and detected by real-time RT-PCR. Viruses were isolated with MDCK cells and identified with serological test. Two influenza virus isolates were extracted, and their HA and NA genes were amplified by RT-PCR. The purified PCR products were sequenced. The data obtained were analyzed with the software DNAMAN. Results Of 1020, influenza H1N1 virus RNA was detected in 200 specimens, seasonal influenza virus RNA was detected in 70 specimens. A total of 29 influenza A H1N1 virus strains were isolated. The nucleotide homology in the HA gene was highly homologous with that of pandemic influenza virus in North America. The amino acids sequences deduced from the nucleotide sequences in HA region of the isolated strain had 22 variations compared with A/Brisbane/59/2007 vaccine strain recommend by WHO,the characteristics of α2,6 sialic acid receptor binding remained. The analysis of amino acids sequences of NA indicated that this virus possessed Oseltamivir sensitivity. Conclusion The causative influenza H1N1 strains in Quanzhou is highly homologous with that of pandemic influenza in North America, and it is antigenically and genetically different from the vaccine strain. Key words: H1N1 influenza virus; Real-time RT-PCR; Sequence analysis

Protective Efficacy of H7 Subtype Avian Influenza DNA Vaccine

Highly pathogenic avian influenza viruses (HPAIV) have historically caused disastrous damage to the poultry industry, and recently they have shown their zoonotic potential by causing human infections and deaths. Control and prevention of HPAIV are therefore important issues for both veterinary and human public health. In this study, we constructed a plasmid, pCAGGoptiH7, encoding a codon-optimized HA gene of the H7N1 avian influenza virus A/FPV/Rostock/34 (RK/34). To evaluate the vaccine efficacy of pCAGGoptiH7, groups of specific-pathogen-free (SPF) chickens were intramuscularly inoculated with one or two doses of 100 microg, 50 microg, or 10 microg of the plasmid in 3-wk intervals. Four weeks after the single vaccination or 2 wk after the second dose, all chickens were challenged with 100CLD50 (chicken lethal dose) of highly pathogenic RK/34. After the single dose vaccination, only 90% of chickens were protected in all of the pCAGGoptiH7-immunized groups, although all of the chickens immunized generated detectable HI antibodies. After the second dose of vaccination, HI antibodies increased sharply, and chickens in the 100-microg and 50-microg pCAGGoptiH7-immunized groups were completely protected from virus challenge (no disease signs, no virus shedding, and no deaths). Low titers of virus shedding were detected in two out of ten chickens inoculated with two doses of 10-microg pCAGGoptiH7, although no disease or death was observed. These results provide a strong argument for the continued evaluation of this vaccine in field trials.

A real-time RT-PCR for detection of clade 1 and 2 H5N1 Influenza A virus using Locked Nucleic Acid (LNA) TaqMan probes

BackgroundThe emergence and co-circulation of two different clades (clade 1 and 2) of H5N1 influenza viruses in Vietnam necessitates the availability of a diagnostic assay that can detect both variants.ResultsWe developed a single real-time RT-PCR assay for detection of both clades of H5N1 viruses, directly from clinical specimens, using locked nucleic acid TaqMan probes. Primers and probe used in this assay were designed based on a highly conserved region in the HA gene of H5N1 viruses. The analytical sensitivity of the assay was < 0.5 PFU and 10 - 100 ssDNA plasmid copies. A total of 106 clinical samples (58 from patients infected with clade 1, 2.1 or 2.3 H5N1 viruses and 48 from uninfected or seasonal influenza A virus-infected individuals) were tested by the assay. The assay showed 97% concordance with initial diagnostics for H5 influenza virus infection with a specificity of 100%.ConclusionsThis assay is a useful tool for diagnosis of H5N1 virus infections in regions where different genetic clades are co-circulating.

MVA-Based H5N1 Vaccine Affords Cross-Clade Protection in Mice against Influenza A/H5N1 Viruses at Low Doses and after Single Immunization

Human infections with highly pathogenic avian influenza viruses of the H5N1 subtype, frequently reported since 2003, result in high morbidity and mortality. It is feared that these viruses become pandemic, therefore the development of safe and effective vaccines is desirable. MVA-based H5N1 vaccines already proved to be effective when two immunizations with high doses were used. Dose-sparing strategies would increase the number of people that can be vaccinated when the amount of vaccine preparations that can be produced is limited. Furthermore, protective immunity is induced ideally after a single immunization. Therefore the minimal requirements for induction of protective immunity with a MVA-based H5N1 vaccine were assessed in mice. To this end, mice were vaccinated once or twice with descending doses of a recombinant MVA expressing the HA gene of influenza virus A/Vietnam/1194/04. The protective efficacy was determined after challenge infection with the homologous clade 1 virus and a heterologous virus derived from clade 2.1, A/Indonesia/5/05 by assessing weight loss, virus replication and histopathological changes. It was concluded that MVA-based vaccines allowed significant dose-sparing and afford cross-clade protection, also after a single immunization, which are favorable properties for an H5N1 vaccine candidate.

Partial direct contact transmission in ferrets of a mallard H7N3 influenza virus with typical avian-like receptor specificity

BackgroundAvian influenza viruses of the H7 subtype have caused multiple outbreaks in domestic poultry and represent a significant threat to public health due to their propensity to occasionally transmit directly from birds to humans. In order to better understand the cross species transmission potential of H7 viruses in nature, we performed biological and molecular characterizations of an H7N3 virus isolated from mallards in Canada in 2001.ResultsSequence analysis that the HA gene of the mallard H7N3 virus shares 97% identity with the highly pathogenic avian influenza (HPAI) H7N3 virus isolated from a human case in British Columbia, Canada in 2004. The mallard H7N3 virus was able to replicate in quail and chickens, and transmitted efficiently in quail but not in chickens. Interestingly, although this virus showed preferential binding to analogs of avian-like receptors with sialic acid (SA) linked to galactose in an α2–3 linkage (SAα2–3Gal), it replicated to high titers in cultures of primary human airway epithelial (HAE) cells, comparable to an avian H9N2 influenza virus with human-like α2–6 linkage receptors (SAα2–6Gal). In addition, the virus replicated in mice and ferrets without prior adaptation and was able to transmit partially among ferrets.ConclusionOur findings highlight the importance and need for systematic in vitro and in vivo analysis of avian influenza viruses isolated from the natural reservoir in order to define their zoonotic potential.

Study on evolutionary origin of influenza A virus (H1N1) based on HA gene

To determine the evolutionary rate and divergence time of influenza A virus HA gene isolated recently worldwide pandemic and explore the origin and its transmission. A total of 344 H1 sequences available in the GenBank (including 248 isolated from human, 84 from swine, 11 from avian, and 1 from ferret) and 7 isolated in Shanghai were collected. The nucleotide substitution rate and time to most recent common ancestor (tMRCA) was calculated using molecular clock theory and Bayesian Skyline Plot (BSP) based on Markov chain Monte Carlo. Then genetic phylogeny was constructed referring to posterior distribution. It was found that H1 sequences in the US from human, swine and avian were clustered significantly with swine H1 ones from Asia phylogenetically (Cluster US). The second cluster (Cluster Eurasian Human) nearly consisted of human H1 sequences isolated in other regions. The third cluster (Cluster Eurasian Animal) consisted of swine and avian H1 sequences from China and Italy respectively. As for all the H1 sequences, the evolutionary rate was of 2.57 x 10(-3) substitutions/site per year averagely (95% Highest Posterior Density: 1.96 x 10(-3) - 3.03 x 10(-3)/site per year). The estimated dates for tMRCA of human H1 in Europe and swine H1 in the mainland of China were the earliest, with the corresponding rates of 6.46 x 10(-3)/site per year and 0.97 x 10(-3)/site per year respectively. The tMRCAs of human and swine H1 sequences from the US were similar, with the rates of 5.86 x 10(-3)/site per year and 5.02 x 10(-3)/site per year. The present flu outbreak was possibly induced by long-term circulation of influenza A virus (H1N1) in human population and swine herds in America. There was no evidence proving that influenza virus in China involved in the present outbreak.

An analysis on genetic characterization of HA1 gene of influenza virus subtype H3N2 circulated from 2001 to 2006 in Liaoning local area

To understand the HA1 genetic variation characterization of influenza virus subtype H3N2 circulated from 2001 to 2006 in Liaoning local area. Viral RNA was extracted and transcribed into cDNA by reverse transcriptase and amplified by PCR. The product of PCR was purified by QIAgen purification kits,and sequenced by ABI 3100avant. The sequence data were analyzed phylogenetically by Sequence software with epidemic records. Finally, the phylogenetic trees were drawn according to deduced amino acid sequences of influenza virus H3N2 from 2000 to 2006 in the NCBI database. The seven HA1domain sequences of H3N2 influenza viruses circulated from 2001 to 2006 in Liaoning local area had been analyzed. Compared with WHO 2004-2006 H3N2 vaccine A/California/7/2004, 12 bases had changed, 4 positions had amino acid substitution in 62 * > E, 182 T > 1,224 S > A,225 C > Y. 224 and 225 are RBS (Receptor binding site). The homology is lower than 98%. Phylogenetic tree showed Liaoning H3N2 2006 strains and Zhejiang 2005 strains were similar to WHO Northern hemisphere winter 2006-2007 Vaccine A/Wisconsin/67/2005 (H3N2)-like virus and grouped together to form an independent cluster even though several bases were still different. The HA1 domain of HA gene of influenza viruses (H3N2) isolated from 2001-2006 in Liaoning local area showed base mutation, amino acid sequence difference compared to A/California/7/2004 (2005-2006 vaccine), suggesting it might be the main cause leading to the spread of influenza. The sequence analysis showed Liaoning 2006 H3N2 strains were similar to those from Southern area which suggested that further surveullance should be conducted to monitor the virus mutation in circulation.

Antigenic and genetic study of hemagglutinin gene of influenza virus (H3N2) circulated in China in 2004

To study the antigenic and genetic characteristics of influenza (H3N2) virus circulated in China in 2004. Single-way and cross-way hemagglutination inhibition (HI) tests were firstly used to determine the reactivity with the reference serum of virus isolates. Based on the serological results, virus isolates were selected according to the different time and location in China in 2004. The HA1 domain of HA gene of those virus isolates were then sequenced in order to analyze the gene characterization. Single-way HI test results showed that 52.3% of isolates showed 4 folds or more HI titer difference compared to A/Fujian/411/2002 (H3N2) itself (international reference strain in 2004). Cross-way HI test results showed that the antigenic ratio was 4. The nucleic acid and amino acid sequence data of HA1 domain showed that the mutated virus appeared in early February of 2004, and became the dominant circulating strain gradually. There were four important mutant positions, they were 159 Y>F, 189 S>N, 145 K>N, 226 V>I, respectively. The results also indicated that the mutated viruses originated from southern China, then transmitted to northern China, according to the analysis of time and location distribution. The HA1 domain of HA gene of influenza virus (H3N2) isolated from 2004 in China showed mutation and antigenic drift, and the mutated viruses were becoming the dominant circulating strain in China, and showed amino acid sequence difference compared to A/Fujian/411/2002 (H3N2) A/Wellington/1/2004 (H3N2), the vaccine components pronounced by WHO for 2004-2005 northern hemisphere and 2005 southern hemisphere respectively, which suggested that further surveillance should be conducted to monitor the virus mutation in circulation.

Influenza B viruses with site-specific mutations introduced into the HA gene

We have succeeded in engineering changes into the genome of influenza B virus. First, model RNAs containing the chloramphenicol acetyltransferase gene flanked by the noncoding sequences of the HA or NS genes of influenza B virus were transfected into cells which were previously infected with an influenza B helper virus. Like those of the influenza A viruses, the termini of influenza B virus genes contain cis-acting signals which are sufficient to direct replication, expression, and packaging of the RNA. Next, a full-length copy of the HA gene from influenza B/Maryland/59 virus was cloned. Following transfection of this RNA, we rescued transfectant influenza B viruses which contain a point mutation introduced into the original cDNA. A series of mutants which bear deletions or changes in the 5' noncoding region of the influenza B/Maryland/59 virus HA gene were constructed. We were able to rescue viruses which contained deletions of 10 or 33 nucleotides at the 5' noncoding region of the HA gene. The viability of these viruses implies that this region of the genome is flexible in sequence and length.