| 1. Background and ObjectiveInfluenza is Orthomyxoviridae which is a single-stranded, negative-strand RNA, spherical or filamentous, diameter80-120nm. It is made of three layers, the inner layer is the nucleocapsid that contains nucleoprotein (NP) and RNA, the second layer is virus envelope by a film layer of lipids and proteins (M), protein M antigen is stability, but also has a specific type. The outer glycoprotein is composed of two hemagglutinin (HA) and neuraminidase (NA). Depending on the HA and NA antigens can be divided into many subtypes, H have17subtypes (H1～H17), N have9subtypes (N1～N9). H1N1, H2N2, H3N2, and the H1N1which is prevalent in2009can infect human, the natural host of the other subtypes are birds and animals. H5, H7and H9subtypes are the most harmful to birds. They are all highly pathogenic infection in humans, when mutated and communicated with people, that can lead to prevalent in humans. Antigenic variation of influenza viruses is mainly refer to changing the structure of the HA and NA antigens, when occurs small variation in subtypes, that is antigenic drift, when occurs a major antigenic variation, that is antigenic shift. When HA and NA have a large variation, that will lead to the emergence of new subtypes and cause a worldwide pandemic.In the twentieth century, there are three major epidemic, one is the Spanish flu in1918, caused20-40million people deaths by the H1N1.It is the biggest disaster in the human infectious diseases in history. Second, the Asian flu in1957, which is caused by the H2N2, is maded of human influenza and avian influenza gene recombination. Third, the Hong Kong flu in1968, which is caused by the H3N2, also is maded of human influenza and avian influenza gene recombination. From the global and domestic epidemiological analysis can be seen in recent years, H3N2is the advantage of strains in many countries. The H3N2and B Victoria also are the advantage of strains in our country. Seven of ten small epidemic are caused by H3N2in China between1968-1992, and have taken place a wider range of H3N2subtype of influenza pandemic in our country in1998and2002. From November to December1998in Beijing, the overall incidence is up to10%, the total incidence is up to100million. There has not large influenza epidemic occurred in Guangdong Province in2005, but local outbreak still relatively active, and mainly strain is A (H3N2) in primary and secondary schools. In influenza monitoring in our country, H3N2caused a small outbreak in the southern province in2012. China is large population base, and is a high incidence of influenza, and several new variants were first in southern of China. It is the multiple centers of influenza incidence. The flu pathogen monitoring can be found H3N2is the predominant strains in Guangdong Province in recent years.Influenza has been prevalent in the population what the major factor is the major antigenic variation continue to occur, and thus escape the host immune response, continue to trigger a new pandemic. The main antigens are HA and NA proteins, and also is a hot research field. Through the analysis of HA and NA sequences and amino acid sequences, we can understand the evolution of influenza in Shenzhen, and play an important role in the prevention of influenza.2. Methods2.1People infected H3N2and detected serum antibody in Shenzhen during2007-2011(1) Influenza surveillance NetworkShenzhen Influenza Surveillance Network consists of:the Center for Disease Control and Prevention of Shenzhen and eight districts CDC, is mainly responsible for collecting ELI and outpatient, emergency department cases, such as data collection, separation and identification of influenza virus and outbreaks deal work. There are the First People’s Hospital of Shenzhen, maternal and children health hospital of Shenzhen and eight District People’s Hospitals, while the eight districts chooseed a community health service center. Then collect the influenza-like cases and the treatment of cases. Choose a college, secondary and elementary schools as monitoring point and two homes for the elderly as the elderly people monitoring point, to collect influenza-like illness and treatment of cases. Monitoring units send the monitoring data of last week to the Center for Disease Control and Prevention of Shenzhen every monday through Network or Fax.ILI collection; Nurse of monitoring hospital acquisition nasal swab specimens of influenza-like illness patient that ill within three days and had not taken antiviral, while collecting basic information and clinical data. The samples should be refrigerated (4-8℃) and transported to the laboratory for testing and separating virus within24hours.Sampling object is the people who have not respiratory symptoms within a week. Using cluster random sampling method,as follow:the streets of eight districts were numbered, the application randomly giving each street a random number; and predetermined random number selected the largest, if two street are the same maximum random number, then given their random number drawn again. Every district CDC collected serum of five age groups0～5～15～25～60～in the street which selected during influenza epidemic before and after (3,9month), each age group should collect at least30parts every distrct, each street sampling points should be at least3.And to detect serum antibodies by hemagglutination inhibition assay (HAI). The result is analyzed with SPSS13.0.2.2Analysis on mutation of HA and NA genes of H3N2subtype in Shenzhen during2007-2011(1) Primer design and synthesisThe HA and NA fragment of H3N2subtype were downloaded10-20from the Genbank and use DNAstar to alignment, used the Primer Primer5.0to design HA and NA primer by TAKARA to synthetize.(2) Amplification of HA and NA geneExtracted viral RNA by the High Pure Viral RNA Kit of the Roche of the United States, and amplified the HA, NA by the One Step RT-PCR DRR024A kit, used1%agarose gel to identify the PCR products for placing under the gel imaging system analyzer to observe specific bands that send the PCR product to sequencing forTakara,(3) Sequence analysisWith BioEdit to alignment nucleic acid sequences. Use the HA and NA of A/HongKong/1/1968as a reference sequence, to analysed nucleotide evolution and constructed evolutionary trees by Mega5.0. Use the HA and NA of A/Brisbane/10/2007as a reference amino acid sequence to analysis the variance.2.3The immunization of mice(1) From6-8week old female Balb/c mice immunized with the first inactivated H3N2antigen with an equal volume of Freund’s incomplete adjuvant mixing, emulsion to drop not after subcutaneous injection, the injection volume was200μl/mice. After1week, the inactivated H3N2antigen with an equal volume of Freund’s incomplete adjuvant mixing, emulsifying to drop not after subcutaneous injection, the injection volume was200μl/mice. After1week, removal of the eyeball blood, stand for1hour,3000rpm centrifuged for20minutes, the supernatant carefully in EP tube and stored at-20℃.(2) Cross-determination of hemagglutination inhibition and antigen ratio calculation, Antigen ratio (R) is calculated as follows: r1=A antigen in B serum hemagglutination inhibition titers/A antigen in A serum hemagglutination inhibition titers r2=B antigen in A serum hemagglutination inhibition titers of B antigen in B serum hemagglutination inhibition titers. R (antigen ratio)=(r1*r2)1/23. Results 3.1H3N2-positive rate and antibody of anti-HA antigen rate during2007-2011The percentage of the ILI in Shenzhen from2007to2011were respectively6.66%(151,890/2,278,884),5.91%(159,592/2,696,653),7.06%(221,552/3,137,273)5.43%(160,774/2,959,464),4.69%(147,298/3,138,229). In ILI patients, the positive rate of Nucleic acid of H3N2subtype were6.9%(174/2505),1.5%(50/3179),9.3%(479/5144),1.0%(42/4031),0.8%(42/4257), respectively from2007to2011. Antibody of anti-HA antigen in2007compare with2008%2009、2010, respectively, there was no significant difference. Antibody of anti-HA antigen in2011compare with2008%2009%2010, respectively, there have significant.And comparing between2007and2011,2008and2009, two years there was no significant difference.3.2Analyzing HA and NA sequences(1) Analyzed HA sequenceThe HA of H3N2was evoluted in constant and gradual evolution in chronological order, but there appeared in the evolutionary tree of alternating transition during2007and2008. The homologies that the HA of A/Shenzhen/330/2011is minimum (85.8%) with A/HongKong/1/1968, and the homology is largest (90.6%) with A/Wuhang/359/1995. Compared with HA of vaccine strain (A/Brisbane/10/2007), the genetic homologies of shenzhen strains in2007reached to98.5%-99.6%, and of shenzhen strains in2008reached to98.6%-99.2%,and of shenzhen strains in2009reached to98.6%-98.7%, and of shenzhen strains in2010reached to97.4%-98.2%,and of shenzhen strains in2011reached to97.9%-98.2%.(2) Analyzed NA sequencesThe NA of H3N2was similar evolution with HA. The homologies that the NA of A/Shenzhen/330/2011is minimum (86.9%) with A/HongKong/1/1968, and the homology is largest (91%) with A/Wuhang/359/1995. Compared with NA of vaccine strain (A/Brisbane/10/2007), the genetic homologies of shenzhen strains in2007reached to98.7%-99.6%,and of shenzhen strains in2008reached to 98.5%～99.5%,and of shenzhen strains in2009reached to98.5%～99%, and of shenzhen strains in2010reached to97.4%-98.2%,and of shenzhen strains in2011reached to98.2%～98.6%.3. Antigen ratio between in the stains of2007、2009and2011Selected one of the strains of2007,2009and2011to do hemagglutination inhibition test, according to the result to calculate antigen ratio are2.8between A/shenzhen/84/2007and A/shenzhen/360/2009, A/shenzhen/218/2011. That explain their antigenicity have some differences. Antigen ratio is1.4between A/shenzhen/360/2009and A/shenzhen/218/2011, indicating no significant difference between the two strains of antigens.4. ConclusionThis study was designed primers to amplify HA and NA gene of H3N2. Analyzed the sequences by BioEdit and Mega5.0. Then there is some variation in HA and NA gene. Compared with HA, NA of vaccine strain (A/Brisbane/10/2007), the genetic homologies of shenzhen strains in2007is the highest, and reduced year by year. There were some amino acid substitutions in five epitope regions of HA during2007-2011, especially in A region (S140N/R), B region (K189E/N/Q) and D region (E174K) in2008; B region (N160K、K189Q) and E region (E78K) in2009-2011. Antibody of anti-HA antigen in in people in2007and2011was a higher level, antibody positive rate were69.4%and69.4%two years there was no significant difference. And low antibody levels in2008and2009,40.4%and38.4%, respectively, comparing between two years there was no significant difference, but with2007or2011have significant difference. H3N2virus is evolving in the continuous, the five epitope regions of HA in2009was easy change. The antigenicity was a smaller difference between A/shenzhen/84/2007and A/shenzhen/218/2009, A/shenzhen/360/2011. Lower antibody was related to the prevalence of the H3N2virus subtype in shenzhen. |
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