R229I substitution from oseltamivir induction in HA1 region significantly increased the fitness of a H7N9 virus bearing NA 292K.

The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was high. These drug-resistant strains showed good replication capacity without serious loss of fitness. In the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. Pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased (1) replication ability in MDCK(P < 0.05) and A549(P < 0.05), (2) neuraminidase enzyme activity, (3) binding ability to both α2,3 and α2,6 receptor, (4) pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), (5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (>4-fold difference of HI titre). It indicated that through the fine-tuning of HA-NA balance, R229I increased the fitness and changed the antigenicity of H7N9 virus bearing NA 292K. Public health attention to this mechanism needs to be drawn.

Metagenomic Analysis of Environmental Samples from Wildlife Rescue Station at Poyang Lake, China.

Objective: To improve the understanding of the virome and bacterial microbiome in the wildlife rescue station of Poyang Lake, China. Methods: Ten smear samples were collected in March 2019. Metagenomic sequencing was performed to delineate bacterial and viral diversity. Taxonomic analysis was performed using the Kraken2 and Bracken methods. A maximum-likelihood tree was constructed based on the RNA-dependent RNA polymerase (RdRp) region of picornavirus. Results: We identified 363 bacterial and 6 viral families. A significant difference in microbial and viral abundance was found between samples S01-S09 and S10. In S01-S09, members of Flavobacteriia and Gammaproteobacteria were the most prevalent, while in S10, the most prevalent bacteria class was Actinomycetia. Among S01-S09, members of Myoviridae and Herelleviridae were the most prevalent, while the dominant virus family of S10 was Picornaviridae. The full genome of the pigeon mesivirus-like virus (NC-BM-233) was recovered from S10 and contained an open reading frame of 8,124 nt. It showed the best hit to the pigeon mesivirus 2 polyprotein, with 84.10% amino acid identity. Phylogenetic analysis showed that RdRp clustered into Megrivirus B. Conclusion: This study provides an initial assessment of the bacteria and viruses in the cage-smeared samples, broadens our knowledge of viral and bacterial diversity, and is a way to discover potential pathogens in wild birds.

Epidemiological and virological surveillance of influenza viruses in China during 2020-2021.

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, seasonal influenza activity declined globally and remained below previous seasonal levels, but intensified in China since 2021. Preventive measures to COVID-19 accompanied by different epidemic characteristics of influenza in different regions of the world. To better respond to influenza outbreaks under the COVID-19 pandemic, we analyzed the epidemiology, antigenic and genetic characteristics, and antiviral susceptibility of influenza viruses in the mainland of China during 2020-2021. METHODS: Respiratory specimens from influenza like illness cases were collected by sentinel hospitals and sent to network laboratories in Chinese National Influenza Surveillance Network. Antigenic mutation analysis of influenza virus isolates was performed by hemagglutination inhibition assay. Next-generation sequencing was used for genetic analyses. We also conducted molecular characterization and phylogenetic analysis of circulating influenza viruses. Viruses were tested for resistance to antiviral medications using phenotypic and/or sequence-based methods. RESULTS: In the mainland of China, influenza activity recovered in 2021 compared with that in 2020 and intensified during the traditional influenza winter season, but it did not exceed the peak in previous years. Almost all viruses isolated during the study period were of the B/Victoria lineage and were characterized by genetic diversity, with the subgroup 1A.3a.2 viruses currently predominated. 37.8% viruses tested were antigenically similar to reference viruses representing the components of the vaccine for the 2020-2021 and 2021-2022 Northern Hemisphere influenza seasons. In addition, China has a unique subgroup of 1A.3a.1 viruses. All viruses tested were sensitive to neuraminidase inhibitors and endonuclease inhibitors, except two B/Victoria lineage viruses identified to have reduced sensitivity to neuraminidase inhibitors. CONCLUSIONS: Influenza activity increased in the mainland of China in 2021, and caused flu season in the winter of 2021-2022. Although the diversity of influenza (sub)type decreases, B/Victoria lineage viruses show increased genetic and antigenic diversity. The world needs to be fully prepared for the co-epidemic of influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus globally.

Distribution of avian influenza viruses according to environmental surveillance during 2014-2018, China.

BACKGROUND: Recurrent infections of animal hosts with avian influenza viruses (AIVs) have posted a persistent threat. It is very important to understand the avian influenza virus distribution and characteristics in environment associated with poultry and wild bird. The aim of this study was to analyze the geographic and seasonal distributions of AIVs in the 31 provinces, municipalities and autonomous region (PMA) of China, compare the AIVs prevalence in different collecting sites and sampling types, analyze the diversity of AIVs subtypes in environment. METHODS: A total of 742 005 environmental samples were collected from environmental samples related to poultry and wild birds in different locations in the mainland of China during 2014-2018. Viral RNA was extracted from the environmental samples. Real-time RT-PCR assays for influenza A, H5, H7 and H9 subtypes were performed on all the samples to identify subtypes of influenza virus. The nucleic acid of influenza A-positive samples were inoculated into embryonated chicken eggs for virus isolation. Whole-genome sequencing was then performed on Illumina platform. SPSS software was used to paired t test for the statistical analysis. ArcGIS was used for drawing map. Graphpad Prism was used to make graph. RESULTS: The nucleic acid positivity rate of influenza A, H5, H7 and H9 subtypes displayed the different characteristics of geographic distribution. The nucleic acid positivity rates of influenza A were particularly high (25.96%-45.51%) in eleven provinces covered the Central, Eastern, Southern, Southwest and Northwest of China. The nucleic acid positivity rates of H5 were significantly high (11.42%-13.79%) in two provinces and one municipality covered the Southwest and Central of China. The nucleic acid positivity rates of H7 were up to 4% in five provinces covered the Eastern and Central of China. The nucleic acid positivity rates of H9 were higher (13.07%-2.07%) in eleven PMA covered the Southern, Eastern, Central, Southwest and Northwest of China. The nucleic acid positivity rate of influenza A, H5, H7 and H9 showed the same seasonality. The highest nucleic acid positivity rates of influenza A, H5, H7, H9 subtypes were detected in December and January and lowest from May to September. Significant higher nucleic acid positivity rate of influenza A, H5, H7 and H9 were detected in samples collected from live poultry markets (LPM) (30.42%, 5.59%, 4.26%, 17.78%) and poultry slaughterhouses (22.96%, 4.2%, 2.08%, 12.63%). Environmental samples that were collected from sewage and chopping boards had significantly higher nucleic acid positivity rates for influenza A (36.58% and 33.1%), H5 (10.22% and 7.29%), H7(4.24% and 5.69%)and H9(21.62% and 18.75%). Multiple subtypes of AIVs including nine hemagglutinin (HA) and seven neuraminidase (NA) subtypes were isolated form the environmental samples. The H5, H7, and H9 subtypes accounted for the majority of AIVs in environment. CONCLUSIONS: In this study, we found the avian influenza viruses characteristics of geographic distribution, seasonality, location, samples types, proved that multiple subtypes of AIVs continuously coexisted in the environment associated with poultry and wild bird, highlighted the need for environmental surveillance in China.

Development and Assessment of Two Highly Pathogenic Avian Influenza (HPAI) H5N6 Candidate Vaccine Viruses for Pandemic Preparedness.

OBJECTIVE: In China, 24 cases of human infection with highly pathogenic avian influenza (HPAI) H5N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5N6 candidate vaccine viruses (CVVs). METHODS: In accordance with the World Health Organization (WHO) recommendations, we constructed two reassortant viruses using reverse genetics (RG) technology to match the two different epidemic H5N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo. RESULTS: The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs. CONCLUSION: We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics, which may aid in our preparedness for a potential H5N6 pandemic.

Profile and generation of reduced neuraminidase inhibitor susceptibility in highly pathogenic avian influenza H7N9 virus from human cases in Mainland of China, 2016-2019.

Human infections with highly pathogenic avian influenza (HPAI) H7N9 virus were detected in late 2016. We examined the drug resistance profile of 30 HPAI H7N9 isolates from Mainland of China (2016-2019). Altogether, 23% (7/30) carried neuraminidase inhibitors (NAIs) - resistance mutations, and 13% (4/30) displayed reduced susceptibility to NAIs in neuraminidase (NA) inhibition test. An HPAI H7N9 reassortment virus we prepared was passaged with NAIs for 10 passages. Passage with zanamivir induced an E119G substitution in NA, whereas passage with oseltamivir induced R292K and E119V substitutions that simulated that seen in oseltamivir -treated HPAI H7N9 cases, indicating that the high frequency of resistant strains in the HPAI H7N9 isolates is related to NAIs use. In presence of NAIs, R238I, A146E, G151E and G234T substitutions were found in HA1 region of HA. No amino acid mutations were found in the internal genes of the recombinant virus.

Effectiveness of favipiravir (T-705) against wild-type and oseltamivir-resistant influenza B virus in mice.

The emergence of resistant mutants to the wildly used neuraminidase inhibitors (NAIs) makes the development of novel drugs necessary. Favipiravir (T-705) is one of the RNA-dependent RNA polymerase (RdRp) inhibitors developed in recent years. To examine the efficacy of T-705 against influenza B virus infections in vivo, C57BL/6 mice infected with wild-type or oseltamivir-resistant influenza B/Memphis/20/96 viruses were treated with T-705. Starting 2 h post inoculation (hpi), T-705 was orally administered to mice BID at dosages of 50, 150, or 300 mg/kg/day for 5 days. Oseltamivir was used as control. Here, we showed that T-705 protected mice from lethal infection in a dose-dependent manner. T-705 administration also significantly reduced viral loads and suppressed pulmonary pathology. In addition, phenotypic assays demonstrated that no T-705-resistant viruses emerged after T-705 treatment. In conclusion, T-705 can be effective to protect mice from lethal infection with both wild-type and oseltamivir-resistant influenza B viruses.

Response of Local Nitric Oxide Release to Manual Acupuncture and Electrical Heat in Humans: Effects of Reinforcement Methods.

This study was to examine the influences of manual acupuncture (MA) and electrical heat corresponding to reinforcing methods on nitric oxide (NO) release over the skin regions in humans. A device with collecting solution was taped to the skin surface along pericardium (PC) or lung (LU) meridian. Acupuncture needles were gently inserted into PC 4 with reinforcing stimulation (low force/rate) for 20 minutes in the MA group. LU11 on the finger was heated (43-44°C) by electrical heat for 20 minutes. Biocapture was consecutively conducted for two 20-minute intervals during and after each treatment. Total nitrite and nitrate (NO x-) in the collecting samples were quantified using chemiluminescence in blinded fashion. Baseline NO x- levels are higher and tended to be higher over PC and LU acupoints during the 1st biocapture. NO x- levels over PC regions were consistently increased by MA during both intervals. NO x- concentrations over LU acupoints were increased and tended to be increased by electrical heat in the 1st and 2nd biocapture. The results suggest that reinforcing MA and electrical heat induce NO released from the local skin regions with higher levels at acupoints, which improve local circulation and contribute to the beneficial effects of the therapies.

Impaired expression of neuronal nitric oxide synthase in the gracile nucleus is involved in neuropathic changes in Zucker Diabetic Fatty rats with and without 2,5-hexanedione intoxication.

These studies examined the influence of 2,5-hexanedione (2,5-HD) intoxication on expression of neuronal nitric oxide synthase (nNOS) in the brainstem nuclei in Zucker Diabetic Fatty (ZDF) vs. lean control (LC) rats. Functional neuropathic changes were also investigated following axonal damage and impaired axonal transport induced by the treatment. Animals were intoxicated by i.p. injection of 2,5-HD plus unilateral administration of 2,5-HD over the sciatic nerve. The mechanical thresholds and withdrawal latencies to heat and cold stimuli on the foot were measured at baseline and after intoxication. The medulla sections were examined by nNOS immunohistochemistry and NADPH-diaphorase histochemistry at the end of the treatments. The mechanical thresholds and withdrawal latencies were significantly decreased while nNOS immunostained neurons and NADPH-diaphorase positive cells were selectively reduced in the gracile nucleus at baseline in ZDF vs. LC rats. NADPH-diaphorase reactivity and nNOS positive neurons were increased in the ipsilateral gracile nucleus in LC rats following 2,5-HD intoxication, but its up-regulation was attenuated in ZDF rats. These results suggest that diabetic and chemical intoxication-induced nNOS expression is selectively reduced in the gracile nucleus in ZDF rats. Impaired axonal damage-induced nNOS expression in the gracile nucleus is involved in neuropathic pathophysiology in type II diabetic rats.

Influence of age, gender, and race on nitric oxide release over acupuncture points-meridians.

This study examined the influence of age, gender and race on nitric oxide (NO) release over acupuncture points, meridian without acupoint, and non-meridian regions of the Pericardium (PC) and Bladder (BL) meridian as well as aging on LU meridian in 61 healthy subjects. Biocapture tubes were attached to the skin surface, and total nitrite and nitrate was biocaptured and quantified using chemiluminescence. In elder ages compared to adults, NO levels over the ventral forearm were significantly decreased over LU on radial regions but not altered over PC on medial regions. Conversely, NO content was elevated over BL regions only in overweight/obesity of elder ages. NO levels over PC regions were marginally elevated in overweight/obese males compared to females but did not alter between races. These results suggest a selective reduction of NO release over LU meridian with aging, which is consistent with a progressive decline in lung function and increase in chronic respiratory disease in elder ages. Increased NO levels along the BL meridian in older obese subjects may reflect a modified NO level along somatic-bladder pathway for counteracting bladder dysfunctions with aging. Both of them support somatic-organ connections in the meridian system associated with potential pathophysiological changes with aging.

Transcutaneous Electrical Stimulation Increased Nitric Oxide-Cyclic GMP Release Biocaptured Over Skin Surface of Pericardium Meridian and Acupuncture Points in Humans.

OBJECTIVES: The purpose of this study was to consecutively capture and quantify nitric oxide (NO) and cGMP, the second messenger of NO, over the skin surface of acupuncture points (acupoints), meridian line without acupoint, and non-meridian control regions of the Pericardium meridian (PC) in humans, and investigate their response to transcutaneous electrical nerve stimulation (TENS) . DESIGN, SETTING, AND MAIN OUTCOME MEASURES: Adhesive biocapture tubes were attached to the skin surface along PC regions and injected with 2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl solution, an NO-scavenging compound, contacting the skin surface for 20 minutes each during 4 consecutive biocapture intervals. TENS (1.0 mA, 6 Hz, 1.0 msec duration) was applied over acupoints PC 8 and PC 3 during the 2nd biocapture for 20 min. Total nitrite and nitrate (NO(x)-), the stable metabolic products of NO, and cGMP in biocaptured samples were quantified using chemiluminescence and ELISA. RESULTS: NO(x)- levels in the 1st biocapture over PC regions are almost two fold higher compared to subsequent biocaptures and are higher over PC acupoints versus non-meridian control region. Following TENS, NO(x)- concentrations over PC regions were significantly increased, and cGMP is predominantly released from the skin surface of PC acupoints. CONCLUSIONS: TENS induces elevations of NO-cGMP concentrations over local skin region with a high level at acupoints. The enhanced signal molecules improve local circulation, which contributes to beneficial effects of the therapy.

Comparing the immunogenicity and safety of 3 Japanese encephalitis vaccines in Asia-Pacific area: A systematic review and meta-analysis.

Japanese encephalitis virus (JEV), a leading cause of Japanese encephalitis (JE) in children and adults, is a major public health problem in Asian countries. This study reports a meta-analysis of the immunogenicity and safety of vaccines used to protect infants or children from JE. Three types of JE vaccine were examined, namely, Japanese encephalitis live-attenuated vaccine (JEV-L), Japanese encephalitis inactivated vaccine (Vero cell) (JEV-I(Vero)), and Japanese encephalitis inactivated vaccine (primary hamster kidney cell) (JEV-I(PHK)). These vaccines are used to induce fundamental immunity against JE; however, few studies have compared their immunogenicity and safety in infants and young children less than 2 years of age. Data were obtained by searching 5 databases: Web of Science, PubMed, China National Knowledge Infrastructure, the China Wanfang database, and the Cochrane database. Fifteen articles were identified and scored using the Jadad score for inclusion in the meta-analysis. Random effect models were used to calculate the pooled seroconversion rate and adverse reaction rate when tests for heterogeneity were significant. The results showed that the pooled seroconversion rate for JEV-I(PHK) (62.23%) was lower than that for JEV-I(Vero) (86.49%) and JEV-L (83.52%), and that the pooled adverse reaction rate for JEV-L (18.09%) was higher than that for JEV-I(PHK) (10.08%) and JEV-I(Vero) (12.49%). The pooled relative risk was then calculated to compare the seroconversion and adverse reaction rates. The results showed that JEV-I(Vero) and JEV-L were more suitable than JEV-I(PHK) for inducing fundamental immunity to JE in infants and children less than 2 years of age.

[Selection pressure analysis of H3N2 influenza virus from China between 1992 and 2012].

OBJECTIVE: In order to investigate the relationship between selection pressure and the prevalence of antigenic clusters, we sequenced and analyzed the H3N2 influenza virus from China between 1992 and 2012. METHODS: The H3N2 influenza virus (n = 1206) in China from 1992 to 2012 was analyzed, include global selection pressure and sites positive selection pressure analysis. RESULTS: Considering all the H3N2 influenza viruses during these 21 years, a total of four amino acid sites subject to positive selection. The global selection pressure varies with the variation of different antigenic clusters and three years with peak bottom selection pressure were identified. CONCLUSION: The global selection pressure rise from the peak bottom, a new antigenic clusters will appear andprevalent in the population, indicating the best time to replace the vaccine strain.

[Development of a real-time reverse transcriptase PCR assay for detection of E119V amino acid change in neuraminidase of influenza A (H3N2) using the TaqMan-MGB probe].

OBJECTIVE: To develop a rapid duplex Real-time reverse transcription PCR (rRT-PCR) method to detect E119V mutation on neuraminidase (NA) of influenza A(H3N2) subtype with drug resistance to oseltamivir. METHODS: Twenty-six NA genes of influenza A(H3N2) virus between 2000 and 2012 in GenBank database were selected as the target genes, and specific TaqMan-MGB probe was designed to target the E119V amino acid change in neuraminidase protein. rRT-PCR was then performed and evaluated for the sensitivity, specificity and reproducibility using virus with E119V mutation and clinical samples. RESULTS: This study described the validation of a highly sensitive and specific duplex rRT-PCR for detection of substitutions leading to the E119V amino acid change in NA protein of influenza A(H3N2). Fluorescence signals could be detected even when diluted a A (H3N2) virus (HA = 8) into 10(-5) and linear correlation between the logarithm of the viral titer with the Ct values was observed. In addition, the assay was highly specific in that there was no cross-react with other respiratory viruses, nor did two TaqMan-MGB probes. E119V substitution in quasispecies with both sensitive and resistant viruses could be detected as well. The limit of detection was 5% for quasispecies with high concentrations and 50% for quasispecies with low concentrations. The average coefficient of variation (CV) for within-run assays was 2.32% and 0.57% for H3N2-119E and H3N2-119V primer/probe sets separately, 1.77% and 0.97% for average CV of between-run assays, which exhibited good repeatability. Sequence analysis of twenty NA genes verified glutamic acid (E) at amino acid site 119, which was in consistent with the results from our rRT-PCR method. CONCLUSION: The assay developed in this study is highly sensitive and specific, and easy to operate; thereby it could be used for identification of A(H3N2) virus with E119V amino acid change in NA protein.

[Virological characterization of influenza A(H3N2) virus in Mainland China during 2011-2012].

To study the prevalence and variation of influenza A(H3N2) viruses, the antigenic and genetic characteristics of influenza A(H3N2) viruses circulating in Mainland China during April 2011 to March 2012 were analyzed. The results showed that influenza A(H3N2) viruses increased gradually since 2012 and became the dominant strain since March. The viruses were antigenically closely related to the vaccine strain A/PER/16/09 (87.2%) and the representative virus A/FJ/196/09 (76.0%) in Mainland China. The genetic characteristics analysis results showed that recently isolated viruses belonged to the Vic/208 clade, and most of the low reaction strains also fell into the same clade. Crystal structure analysis of HA protein found that, compared with the vaccine strain A/PER/16/09, the recently isolated viruses had amino acid substitutions in the antigenic site A, B and C areas, in addition to gaining potential glycosylation sites at the amino acid position of 45 of HA and 367 of NA. Although the majority of circulating influenza A (H3N2) viruses in 2011-2012 season in Mainland China were antigeniclly matched by current influenza vaccine strain and the selected representative viruses, low reaction strains have increased since 2012, therefore it is necessary to strengthen the surveillance on the variation of influenza virus and to provide solid information for the vaccine strain selection.

[Emerged Pdm09 influenza virus increased purifying selection of seasonal H1N1 influenza virus].

Pdm09 virus outbreak occurred in Mainland China in May 2009, a few months later, the prevalence of seasonal H1N1(sH1N1) influenza virus that already circulated in human for tens of years began to decline and disappeared afterwards. To identify the reason for the rapid decline of sH1N1 in mainland China, we sequenced the HA1 of sH1N1 during 2006-2011, and then analyzed the selective pressure in different phases. Our results showed before Pdm09 outbreak, the omega value was 0. 36 while after Pdm09 outbreak the omega value was 0. 28 and significant difference (t test, P<0. 05) was identified. We concluded that sH1N1 obtained stronger purifying selection after Pdm09 outbreak in China. This might one of the major reasons causing the disappearance of sH1N1 in human.

[Virological characterization of influenza B virus in mainland China during 2011-2012].

In order to understand the prevalence and variation of influenza B viruses, the antigenic and genetic characteristics of influenza B viruses circulating in Mainland China during April, 2011 to March, 2012 were analyzed. The results showed the B Victoria lineage viruses were much more prevalent than B Yamagata lineage during this period, phylogenetic analysis showed vast majority of Victoria lineage viruses belong to genetic group 1, intra-clade reassortant between HA1 and NA gene was identified in a minor proportion of the viruses. 72.8% of the B/Victoria-lineage viruses were antigenically closely related to the vaccine strain B/Brisbane/60/2008. B Yamagata component was not included in the trivalent influenza vaccine in China during the study period, however vast majority of B Yamagata lineage viruses were antigenically and genetically closely related to the representative virus B/Hubei-Wujiagang/158/2009(97.8%) and B/Sichuan-Anyue/139/2011(85.2%) in China, reassortant between HA1 and NA was not identified in B Yamagata lineage viruses. Overall, the predominant circulating influenza B viruses in 2011-2012 season in China were matched by current influenza vaccine and the selected representative viruses were proved to represent the antigenic and genetic characteristics of the circulating viruses.

[Analysis of genetic features of influenza B virus in Hunan province from 2007 to 2010].

OBJECTIVE: To investigate the gene variations of influenza B virus isolated in Hunan province from 2007 to 2010. METHODS: A total of 42 strains of influenza B virus,which were isolated in the Influenza Surveillance Network Laboratories in Hunan province between year 2007 and 2010, were selected for the study. The hemagglutinin 1 (HA1) and neuraminidase (NA) genes of the selected strains were amplified by RT-PCR, and the sequence of the purified product were detected and homologically compared with the sequence of influenza vaccine strains isolated from Northern Hemisphere by WHO during the same period. In addition, the phylogenetic trees were constructed to characterize the molecular features. RESULTS: In the Victoria branch of the HA1 gene phylogenetic tree, the strains isolated from year 2007 to 2009 were included in the V1 sub-branch, as well as the vaccine strain Malaysia/2506/2004; the strains isolated in year 2010 were involved in the V2 sub-branch, similar to the vaccine strains Brisbane/60/2008. In the Yamagata branch,the strains isolated in year 2007 were in the Y1 sub-branch,different from the strains isolated between year 2008 and 2010, which were in the Y2 sub-branch, instead. All virus in NA gene phylogenetic tree were included in the Yamagata branch, indicated their Yamagata origin. The genetic sequence analysis of the 7 strains isolated in year 2010 revealed that the viruses were classified as genotype 2 and genotype 15. The results of homological comparison between HA1 molecule and the influenza vaccine strains recommended by WHO were as below: Victoria lineage, 98.6% - 99.1% in 2007, 98.6% - 99.1% in 2008, 98.1% - 99.1% in 2009, and 97.6% - 99.1% in 2010; and Yamagata lineage, 97.9% - 98.5% in 2007, 97.9% - 98.5% in 2009 and 97.9% - 98.2% in 2010. The major mutations of the strains isolated in year 2007 were found in sites R48K, K88R, P108A, D197N and S230G. While the major mutations of the strains isolated between year 2009 and 2010 were sited in K88R, S150I, N166Y, D197N and S230G. CONCLUSION: The prevalent influenza B virus isolated in Hunan province from 2007 to 2010 has mutated and evolved continuously.

[Establishment of a mouse-lethal model for pandemic H1N1 influenza virus].

To establish the mouse-lethal model for pandemic H1N1 influenza virus, provide an animal model for studying the pathogenicity and host adaptation of 2009 pandemic H1N1 influenza virus, and find out the key amino acid mutations which may affect viral virulence and replication. A pandemic H1N1 influenza virus strain, A/Sichuan/SWL1/2009 (H1N1, SC/1) was passaged in mouse lung by 15 cycles with intranasal infection. The passaged viruses were all propagated in MDCK cells and sequenced. Based on the sequencing results, four mice in each group were inoculated with 6 selected viruses and their weight and survival rate were monitored during the following 14 days after infection. Additionally, SC/1-MA P14 and P15 viruses were sequenced after purification by Plague Assay. Viral virulence was increased after serial passages and the mortality of 100% was detected after 7 passages. Several amino acid residue mutations of passaged viruses which may contribute to the enhanced virulence were observed. The increased virulence of passaged viruses and mammalian host adaptation maybe associated with amino acid mutations in viral functional proteins. Finally, we established a mouse-lethal model.