Avian influenza viruses (AIVs) H9N2 are in the course of reassorting into novel AIVs / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

In 2013, two episodes of influenza emerged in China and caused worldwide concern. A new H7N9 avian influenza virus (AIV) first appeared in China on February 19, 2013. By August 31, 2013, the virus had spread to ten provinces and two metropolitan cities. Of 134 patients with H7N9 influenza, 45 died. From then on, epidemics emerged sporadically in China and resulted in several victims. On November 30, 2013, a 73-year-old woman presented with an influenza-like illness. She developed multiple organ failure and died 9 d after the onset of disease. A novel reassortant AIV, H10N8, was isolated from a tracheal aspirate specimen that was obtained from the patient 7 d after onset. This case was the first human case of influenza A subtype H10N8. On 4 February, 2014, another death due to H10N8 avian influenza was reported in Jiangxi Province, China.

Evolution, global spread, and pathogenicity of highly pathogenic avian influenza H5Nx clade 2.3.4.4

Novel subtypes of Asian-origin (Goose/Guangdong lineage) H5 highly pathogenic avian influenza (HPAI) viruses belonging to clade 2.3.4, such as H5N2, H5N5, H5N6, and H5N8, have been identified in China since 2008 and have since evolved into four genetically distinct clade 2.3.4.4 groups (A–D). Since 2014, HPAI clade 2.3.4.4 viruses have spread rapidly via migratory wild aquatic birds and have evolved through reassortment with prevailing local low pathogenicity avian influenza viruses. Group A H5N8 viruses and its reassortant viruses caused outbreaks in wide geographic regions (Asia, Europe, and North America) during 2014–2015. Novel reassortant Group B H5N8 viruses caused outbreaks in Asia, Europe, and Africa during 2016–2017. Novel reassortant Group C H5N6 viruses caused outbreaks in Korea and Japan during the 2016–2017 winter season. Group D H5N6 viruses caused outbreaks in China and Vietnam. A wide range of avian species, including wild and domestic waterfowl, domestic poultry, and even zoo birds, seem to be permissive for infection by and/or transmission of clade 2.3.4.4 HPAI viruses. Further, compared to previous H5N1 HPAI viruses, these reassortant viruses show altered pathogenicity in birds. In this review, we discuss the evolution, global spread, and pathogenicity of H5 clade 2.3.4.4 HPAI viruses.

Genomic Characterization of an Unusual Human G3P3 Rotavirus with Multiple Cross-species Reassortment / 病毒学报

One unusual human G3P[3] group A rotavirus (RVA) strain M2-102 was identified in stool sample collected from a child with diarrhea in Guangxi Province, China in 2014. It is well known that G3P[3] is a genotype commonly identified in feline and canine RVAs. However, the preliminary phylogenetic analyses of the VP7 and VP4 genes of strain M2-102 indicated that these two genes were closely related to bat RVA strain MYAS33 and simian strain RRV, respectively, whereas both clustered distantly to feline/canine-like RVA strains. In this study, full genome sequencing and molecular analyses were conducted to obtain the true origin of strain M2-102. It was revealed that strain RVA/Human-wt/CHN/M2-102/2014/G3P[3] exhibited a G3-P[3]-I3-R3-C3-M3-A9-N3-T3-E3-H6 genotype constellation for VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes. Phylogenetic analyses revealed that 5 genes (VP7, VP1, VP2, NSP2 and NSP3) from strain M2-102 were closely related to those of bat strain MYAS33 from Yunnan Province which was thought a true bat RVA strain rather than a virus transmitted between species, while another 5 genes (VP4, VP3, NSP1, NSP4 and NSP5) clustered closely with those of simian strain RRV, yet the VP6 gene was closely related to that of human G3P[9] strain AU-1 and AU-1-like RVAs. The epidemiological data indicated that the child infected with M2-102 came from a countryside village, located in Dong Autonomous County of Sanjiang (subtropical hilly wooded area), Liuzhou city in Guangxi Province which might provide natural environment for reassortment events occurring among animal and human RVAs. Therefore, the data suggest that human strain M2-102 might originate from multiple reassortment events among bat, simian and human AU-1-like RVAs, yet it is not clear whether the genomic backbone based on bat MYAS33 (5 genes) and simian RRV (5 genes) like rotaviruses had been obtained through reassortment before being transmitted to the human. This is the first report on whole genome analysis of human G3P[3] RVA from China.

Preparation and Identification of High Immunogenic A/PR/8/34 Maternal Strain HA Protein for Influenza Virus Classical Reassortment / 病毒学报

Preparation of maternal strain A/PR/8/34 HA antiserum for influenza virus classical reassortment. A/PR/8/34 virus was digested by bromelain after inactivation and purification. 5%-20% sucrose continuous density gradient centrifugation method was used to purify HA protein. SIRD method was used to select the target protein. SDS-PAGE method was used to identified HA protein. High Immunogenic A/PR/8/34 HA protein was successfully prepared and HI titer reached 10240. High purity HA antiserum was identified by SIRD method. The key reagent in the classical reassortment of influenza virus was prepared, and the complete set of technical methods were explored, which laid the foundation for the independent research and development of seasonal influenza vaccine strains of China.

Protective efficacy of a high-growth reassortant swine H3N2 inactivated vaccine constructed by reverse genetic manipulation

Novel reassortant H3N2 swine influenza viruses (SwIV) with the matrix gene from the 2009 H1N1 pandemic virus have been isolated in many countries as well as during outbreaks in multiple states in the United States, indicating that H3N2 SwIV might be a potential threat to public health. Since southern China is the world's largest producer of pigs, efficient vaccines should be developed to prevent pigs from acquiring H3N2 subtype SwIV infections, and thus limit the possibility of SwIV infection at agricultural fairs. In this study, a high-growth reassortant virus (GD/PR8) was generated by plasmid-based reverse genetics and tested as a candidate inactivated vaccine. The protective efficacy of this vaccine was evaluated in mice by challenging them with another H3N2 SwIV isolate [A/Swine/Heilongjiang/1/05 (H3N2) (HLJ/05)]. Prime and booster inoculation with GD/PR8 vaccine yielded high-titer serum hemagglutination inhibiting antibodies and IgG antibodies. Complete protection of mice against H3N2 SwIV was observed, with significantly reduced lung lesion and viral loads in vaccine-inoculated mice relative to mock-vaccinated controls. These results suggest that the GD/PR8 vaccine may serve as a promising candidate for rapid intervention of H3N2 SwIV outbreaks in China.

Biosafety issues and public concerns on recombinant influenza viruses generated in the laboratories / 生物工程学报

Understanding inter-species transmission of influenza viruses is an important research topic. Scientists try to identify and evaluate the functional factors determining the host range of influenza viruses by generating the recombinant viruses through reverse genetics in laboratories, which reveals the viruses' molecular mechanisms of infection and transmission in different species. Therefore, the reverse genetic method is a very important tool for further understanding the biology of influenza viruses and will provide the insight for the prevention and treatment of infections and transmission. However, these recombinant influenza viruses generated in laboratories will become the potential threat to the public health and the environment. In this paper, we discussed the biological safety issues of recombinant influenza viruses and suggested we should set up protocols for risk management on research activities related to recombinant highly pathogenic influenza viruses.

Origin and the recombinant model of H7N9 virus prevailing in China / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the origin and the recombinant model of H7N9 virus prevailing in China by sequence analysis.</p><p><b>METHODS</b>The sequences of H7N9 virus were collected and analyzed with the software BLAST and MEGA 5.0. The phylogenetic trees were constructed after multiple sequences alignment. The homologous sequences of H7N9 segments were determined and the model was inferred according to the origin of H7N9 segments.</p><p><b>RESULTS</b>The most relevant sequences of HA, NA, NS and PB2 segments were located at one branch of the phylogenetic tree, while the closest relevant sequences of PB1, PA, NP and MP contained two H9N2 virus origins. According to the analysis of sequence origin, H7N9 viruses might be divided into 5 genotypes: namely A, B, A/Shanghai/1/2013-H7N9, A/Pigeon/Shanghai/S1069-H7N9 and A/Zhejiang/HZ1/2013-H7N9, and the genotype A consisted of A1 and A2 subtypes.</p><p><b>CONCLUSION</b>The prevailing H7N9 virus might be derived from 5 different viruses after 4 times of recombination, which resulted in the two major types of A and B. The subtypes of A1 and A2 were two different derivatives from one reassortant. The A/Pigeon/Shanghai/S1069-H7N9 strain might be the recombinant of type A H7N9 virus with a local H9N2 virus during the H7N9 epidemics. The A/Zhejiang/HZ1/2013-H7N9 strain could be the re-arrangement of subtype A2 with type B H7N9 virus.</p>

Analysis of evolution features of whole genome of influenza virus H3N2 in Qingdao between year 2007 and 2011 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate the evolution features of whole-genome of influenza virus H3N2 prevalent in Qingdao from year 2007 to 2011.</p><p><b>METHODS</b>The RNA of 58 strains of influenza virus H3N2 prevalent in Qingdao between 2007 and 2011 was extracted and all segments amplified by RT-PCR. The sequence was then detected and assembled by software Sequencer. A total of 589 strains of influenza virus H3N2 with more than 300 amino acid recorded by GenBank were selected. The phylogeny and molecular features of all gene segments were analyzed by software Mega 5.0, referred by the heavy chain of hemagglutinin (HA1).</p><p><b>RESULTS</b>Hemagglutinin (HA) genes of influenza virus H3N2 prevalent in Qingdao between year 2007 and 2011 formed a single trunk of phylogenetic tree. Every prevalent strain originated in last season. The analysis of the evolution of whole genome found that reassortment virus strains were prevalent between year 2009 and 2010, but between 2010 and 2011 there were two series of prevalent strains, which showed complicated reassortment. Compared with the vaccine strains, the variant amino acids of protein of virus HA1 between year 2007 and 2011 were 8, 6, 6, 8 and 11, involving 13 antigenic sites. The sequence analysis of M2 protein showed that the isolated influenza virus H3N2 mutated in amino acid site 31, from serine to asparagine (S31N). HA1 gene of influenza virus H3N2 isolated in Qingdao between 2007 and 2011 shared the similar phylogenetic tree with the globally prevalent strain. The comparison of the sequence and the analysis of the antigenicity found co-infection between H3N2 and A/H1N1 in the strain A/Qingdao/F521/2011.</p><p><b>CONCLUSION</b>The evolution features of all segments of influenza virus H3N2 prevalent in Qingdao between year 2007 and 2011 were complicated.</p>

Analysis of genetic variation of hemagglutinin and three internal genes of influenza B virus isolated in Zhejiang province from 1999 to 2012 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To explore the characteristics of the genetic variation of hemagglutinin( HA) and three internal genes coding for the nucleoprotein ( NP) , matrix protein ( M) and nonstructural protein ( NS) of influenza B virus.</p><p><b>METHODS</b>A total of 31 strains of influenza B virus were isolated in Zhejiang province from 1999 to 2012, and then were amplified and sequenced the genes of HAl , NP, M and NS. The phylogenetic tree was constructed, the nucleotide substitution rate of the above individual gene was estimated and the variation sites of amino acids were analyzed.</p><p><b>RESULTS</b>The 31 isolated strains of influenza B virus were divided into two distinct lineages Victoria and Yamagata in the phylogenetic tree of HAl gene,represented by B/Victoria/2/87 and B/Yamagata/16/88. Phylogenetic analysis of the NP gene showed that the NP gene of Victoria-like influenza B strains which were isolated after 2010 was highly homologous with Yamagata-like isolates, and thereby they were found to be on the same branch of the phylogenetic tree of the NP gene. Nucleotide substitution rates of HAl , NP, M and NS genes were estimated to be 2. 29 x 10 -3 ,1. 39 X 10-3 ,1. 78 X 10-3 ,1. 30 X 10-3 /site per year, respectively. Variations of amino acid of HAl domain of Victoria-like isolates mainly included K48E ,L58P ,N75K,K80R,K129N/S,N165K,S172P ,Sl97N/D and A202V; while those in Yamagata-like isolates were R48K, S1501, N166Y, N203S, G230D and D233N. Determined amino acid sequences of NP of Victoria-like influenza B isolates were similar to Yamagata-like isolates after 2010 and variations happened on four characteristic amino acid sites, naming A60D, I233V, N513S and V5341, compared with previous Victoria-like influenza B isolates.</p><p><b>CONCLUSION</b>Significant variation was found among influenza B strains isolated in Zhejiang province from 1999 to 2012. The surface HAl gene evolved more rapidly than internal genes. Gene reassortment and gene mutation were the main evolutionary mechanism of influenza B virus.</p>

Hacia la erradicación de la poliomielitis: logros y retos en México

Desde la implementación de las estrategias globales de erradicación, la incidencia de parálisis poliomielítica ha disminuido dramáticamente. Cuatro estrategias han contribuido notablemente: a) Altas coberturas de inmunización con vacuna oral de polio (VOP), b) Inmunización suplementaria durante los Días Nacionales de Vacunación, c) Vigilancia epidemiológica efectiva de casos de parálisis flácida aguda (PFA), y d) Bloqueos vacunales en zonas de alto riesgo. Sólo quedan tres países polioendémicos, no obstante, cualquier país corre el riesgo potencial de importación del virus de algunas de estas áreas, de la liberación accidental del virus resguardado en laboratorios de diagnóstico clínico o investigación, o de la presencia de virus circulantes derivados de vacuna en el medio ambiente. Este documento pretende exponer los antecedentes históricos que hicieron posible la eliminación de la enfermedad en México, así como los retos para lograr un mundo libre de poliomielitis.

Since the strategies to eradicate polio were implemented, the incidence of paralytic polio has dropped dramatically. Four main strategies have greatly contributed: a) High immunization coverage rate with oral polio vaccine (OPV), b) Supplementary immunization activities during the National Immunizations Days c) An effective epidemiological surveillance system for acute flaccid paralysis (AFP) and d) Intensified immunization activities in high risk areas. Three countries remain polio endemic, nevertheless, any country has a potential risk of the virus importation from one of these endemic areas; an accidental release of poliovirus from a research or clinical laboratory, or from having a circulating vaccine-derived poliovirus in the environment. The present document aims to provide an historical background that made possible the disease elimination in Mexico. Moreover, we discuss the challenges that every country needs to face in order to achieve a polio-free world.

Genome sequencing and genetic analysis of a natural reassortant H5N1 subtype avian influenza virus possessing H9N2 internal genes / 病毒学报

Abstract:One H5N1 subtype avian influenza virus, A/duck/Shandong/009/2008 (Dk/SD/009/08), was isolated from apparently healthy domestic ducks in some live bird market in East China during our epidemiological surveillance. To investigate the genetic composition, Dk/SD/009/08 was subjected to genome sequencing. The amino acid motif of cleavage site was "PLRERRRK-R/GL", which was consistent with the characterization of the HPAIV. According to the newest unified nomenclature system of H5N1, Dk/SD/ 009/08 was classified into Clade 2.3.4. The BLAST results showed that four gene segments (HA, NA, NP and NS) had the highest nucleotide identities with H5N1 subtype AIVs whereas the remaining four (PB2, PB1, PA and M) displayed the closest relationship with H9N2 subtype. Therefore, Dk/SD/009/08 might be a natural reassortant virus. The phylogenetic analysis further indicated that G1-like H9N2 subtype AIVs which was prevalent mainly in quails of Southern China might provide the internal genes for Dk/ SD/009/08.

Phylogenetic analysis of 2009 H1N1 (A) influenza virus based on genomic sequence features / 生物医学工程学杂志

From April 2009 onward, a new strain of human H1N1 influenza virus has swept over the world. The genome of influenza virus consists of 8 segments, encoding 10 proteins, respectively. The reassortments among the 8 segments cause the variation of influenza virus. Therefore, phylogenetic analysis of the 8 genes is very important. In this paper, we choose neighboring word frequency as the genomic features, using VC++ programming to analyze evolution of the 8 segments of H1N1 virus. As a result, we found that PB2 genes and PA genes of these three isolated virus were originated from North American avian influenza virus, that PB1 genes were originated from the seasonal influenza virus of human, and that HA genes, NS genes and NP genes came from the North American classical swine influenza A virus. The NA segments and M segments were originated from the European swine influenza virus.

A novel packaging system of recombinant AAV5/5 vector / 生物工程学报

We developed a scalable AAV5/5 vector packaging system by using replication competent recombinant herpes simplex type 1 virus as helper virus. The fragment containing rep and cap genes of AAV5 was inserted into the non-necessary gene (UL2) of HSV1 genome, resulting in the helper virus rHSV1-rep5cap5. An AAV5/5 vector pAAV5neo carrying two AAV5 ITRs was constructed by inserting a neo gene expression cassette into the plasmid backbone of pAV5CMV-GFP. pAAV5neo-EGFP was constructed by inserting EGFP gene into pAAV5neo. BHK21 cell was transfected with pAAV5neo-EGFP and cultured in the presence of G418. EGFP expression positive monoclonal cells were picked up, and one that produced rAAV5/5-EGFP with the highest efficiency under the help of rHSV1-rep5cap5 was chosen as the production cell line named as C020. rAAV5/5-EGFP was produced by infecting C020 cells with rHSV1-rep5cap5, and crudely purified by our previous method of 'chloroform treatment-PEG8000/NaCl precipitation- chloroform extract'. rAAV5/5-EGFP preparation with high purity was obtained by ultrafiltration with molecular weight cut-off value of 100 kDa. SDS-PAGE stained with Coomassie brilliant blue R250 showed clearly specific pattern of three bands of AAV capsid proteins. rAAV5/5-EGFP was also assayed using negative stain transmission electron microscopy and the majority of the virus particles were found solid. About 30% green fluorescent cells could be seen after infecting HEK293 cells with rAAV5/5-EGFP 24 h at 1 x 10(5) vg/cell. In conclusion, we have established an efficient AAV5/5 vector production system and could produce recombinant AAV5/5 virus in large amounts for gene therapy research.

Influenza humana a H1N1: pandemia esperada / H1N1 human influenza: pandemic expected

La influenza es responsable de incremento en la morbilidad y mortalidad, del costo de las hospitalizaciones y de ausentismo escolar y laboral. Es producida por el virus de la influenza perteneciente a la familia Orthomixoviridae y es un virus ARN envuelto. El período de incubación va de 1 a 4 días. Las manifestaciones clínicas incluyen fiebre y varían desde conjuntivitis leve a neumonía grave con falla multiorgánica, hemorragia pulmonar, náuseas, vómito y diarrea. Se ha reportado pandemias importantes con cifras de defunciones alarmantes. La influencia AH1N1, actualmente circulante desde marzo del 2009, es el producto de la recombinación genética del virus de la influenza porcina euroasiática, influenza porcina de Norteamérica, influenza aviar no H5, e influenza humana. Hasta el 13 de septiembre del presente año la OMS ha notificado la apararición de más de 296471 casos y al menos 3486 defunciones. En la regtión de América se informa de más de 124126 casos con 2625 defunciones. Según el Ministerio de Salud, en Venezuela se informa de 5171 casos sospechosos, 1316 confirmados y 67 defunciones por influenza AH1N1, hasta el 17 de septiembre de 2009. El lavado frecuente de manos, aislamiento de los sospechosos, tratamiento con oseltamivir o zanamivir y la inmunización al personal susceptible al tener disponible la vacuna son medidas indispensables en la prevención de la diseminación de la pandemia.

Influenza impacts morbidity, mortality and health care costs. It causes school and work absenteeism. The responsible microorganisms are RNA viruses belonging to Orthomixoviridae family. Uncomplicated influenza begins after an incubation period of 1 to 4 days. Symptoms include fever, and in some cases mild conjunctivitis, but other patients have severe pneumonia with multiorgan failure, pulmonary bleeding, nausea, vomiting, and diarrhea. New influenza AH1N1 is a genetic recombination of Euro-Asian swine influenza virus, seasonal influenza virus, and H3N2 virus as the one isolated in Australia in 2007 (A/Brisbane/10/2007). As of September 13, 2009, the World Health Organization reported more than 296471 confirmed cases worldwide with at least 3486 deaths. In the Americas the figure reaches more than 124126 cases, and 2625 dealths. Venezuelan Ministry of Health has confirmed 1316 cases with 67 deaths. Regular hand hygiene measures, isolation of cases, oseltamivir or zanamivir therapy to suspected or confirmed cases, and vaccination of susceptible people once the new vaccines become available are all important prevention measures.

Isolation and primary identification of viruses in mosquitoes in the south of Xinjiang / 中华预防医学杂志

<p><b>OBJECTIVE</b>To isolate viruses from mosquitoes in the south of Xinjiang and identify these viruses primarily.</p><p><b>METHODS</b>A total of 13 491 mosquitoes were collected in the south of Xinjiang from Jul to Aug, 2005. These mosquitoes were divided into 130 groups and grinded respectively. The supernates were inoculated in C6/36 and Vero cells. Viruses isolated were detected, the genomic nucleic types by electrophoresis of viral genomes and the morphologies observed under electronmicroscope.</p><p><b>RESULTS</b>All 42 viruses were isolated, which caused CPEs on C6/36 but not on Vero cells. 27 viruses showed similar genomic profiles with 12 dsRNA segments. 1 virus displayed genomic profile with 10 dsRNA segments. 5 viruses took on similar genomic profiles with about 4 kbp DNA band. 9 viruses did not get any taxonomy information. Electromicroscopic pictures of these viruses revealed that above four types of viruses had distinguished morphologies indicating different virus species.</p><p><b>CONCLUSION</b>There should be several virus species in the mosquitoes in the south of Xinjiang. dsRNA virus with 12 genomic segments should play analysis a predominant role in the south of Xinjiang.</p>

Construcion of a chimeric Japanese encephalits virus/dengue virus-2 / 病毒学报

The prM/E gene of DV2 was cloned into the JEV (SA14-14-2 strain) replicon vector which had been constructed previously, and the resulting recombinant plasmid was named pPartialdeltaprM/E. The constructed chimeric clone was linearized and then was transcripted into RNA in vitro. The produced RNA was transfected into BHK-21 cells. Five to seven days later, CPE could be observed on the transfected BHK-21cells, and then the supernatant containing the chimeric virus was collected. The Supernatant was inoculated to BHK-1 cells and C6/36 cells, respectively. CPE could be observed about 4 days post the infection of C6/36cell with the chimeric virus. The results from RT-PCR, IFA, Western blot showed that the virus contained the chimeric RNA and the envelop protein of DV2. However, the chimeric virus could not be passaged in BHK-21 cell. The successful construction of the infectious clone JE/DEN-2 laid the basis for the further research of the DV vaccine.

Sequencing and analysis of the full coding sequence of Batai virus isolated in China / 病毒学报

By RT-PCR and TAIL-PCR, the full coding region of Batai virus isolated in China (YN92-4 strain)was sequenced for the first time. According to the results, the genome of the virus contained three segments S, M and L of 947, 4,371 and 6,860 nucleotides, respectively. The S segment coded a nucleoprotein of 234 amino acids and a nonstructural protein of 102 amino acids, the M and L segments coded a precursor protein of 1 ,435 amino acids and RNA polymerase of 2,239 amino acids, respectively. Compared with the full coding sequence of Batai viruses isolated out of China, the S and M segments of YN92-4 and ON-7/B/01 showed the highest homology in nucleotide and amino acid sequenes with similarity of 97.7% (100%) and 95.7% (98%), respectively. Since there was no full coding sequence information on the L segment in GenBank for the reference, the L segment of YN92-4 was compared with that of Bunyamwera virus and the homology of nucleotide and amino acid was 73.5%and 81.6%, respectively. Phylogenetic analysis showed YN92-4 strain was clustered into one group with the prototype of Batai virus (MM2222). The results suggested that the YN92-4 strain had no occurrance of genetic reassortment (like Ngari virus) and was close to the Batai virus (ON-7/B/01 strain) isolated from cattle serum in Japan.

Generation and characterization of a cold-adapted attenuated live H3N2 subtype influenza virus vaccine candidate / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>H3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. The aim of this study was to establish a system for rescuing of a cold-adapted high-yielding H3N2 subtype human influenza virus by reverse genetics.</p><p><b>METHODS</b>In order to generate better and safer vaccine candidate viruses, a cold-adapted high yielding reassortant H3N2 influenza A virus was genetically constructed by reverse genetics and was designated as rgAA-H3N2. The rgAA-H3N2 virus contained HA and NA genes from an epidemic strain A/Wisconsin/67/2005 (H3N2) in a background of internal genes derived from the master donor viruses (MDV), cold-adapted (ca), temperature sensitive (ts), live attenuated influenza virus strain A/Ann Arbor/6/60 (MDV-A).</p><p><b>RESULTS</b>In this presentation, the virus HA titer of rgAA-H3N2 in the allantoic fluid from infected embryonated eggs was as high as 1:1024. A fluorescent focus assay (FFU) was performed 24-36 hours post-infection using a specific antibody and bright staining was used for determining the virus titer. The allantoic fluid containing the recovered influenza virus was analyzed in a hemagglutination inhibition (HI) test and the specific inhibition was found.</p><p><b>CONCLUSION</b>The results mentioned above demonstrated that cold-adapted, attenuated reassortant H3N2 subtype influenza A virus was successfully generated, which laid a good foundation for the further related research.</p>