Genetic characterization of influenza A virus and assessment of vaccine efficacy in Yantai from 2014 to 2017 / 中华预防医学杂志

In this study, the swabs were collected among patients with an influenza-like illness (ILI) admitted to 2 sentinel surveillance hospitals of Yantai from April 2014 to August 2017. All specimen were cultured and identified by hemagglutination inhibition assay. Complete sequences of Hemagglutinin (HA) of influenza A were amplified, sequenced and analyzed using molecular and phylogenetic methods. The potential vaccine efficacy were calculated using Pepitope model. The results showed that the antigenicity of A (H3N2) had changed greatly. 8 strains of influenza A (H1N1) pdm09 belonged to subclade 6B.1 and 14 strains clustered in 6B.2. 12 strains of influenza A (H3N2) fell into subgroup 3C.3a and 33 strains clustered in 3C.2a. Several residues at antigen sites and potential glycosylation sites had changed in influenza A strains. Vaccine efficacy of influenza A (H1N1) pdm09 in 2015/2016 and 2016/2017 seasons were 77.29% and 79.11% of that of a perfect match with vaccine strain, meanwhile vaccine efficacy of influenza A (H3N2) in 2014/2015, 2015/2016 and 2016/2017 were-5.18%, 16.97% and 42.05% separately. In conclusion, the influenza A virus circulated in Yantai from 2014 to 2017 presented continual genetic variation. The recommended vaccine strains still afforded protection against influenza A (H1N1) pdm09 strains and provided suboptimal protection against influenza A (H3N2) strains.

Preparation and characteristic analysis of six influenza A (H7N9) pseudovirus derived from different districts of China / 中华实验和临床病毒学杂志

Objective@#To prepare strains of influenza A (H7N9) pseudovirus derived from different districts of China for vaccine efficacy evaluation.@*Methods@#Phylogenetic tree was built based on hemagglutinin (HA) amino acid sequence analyses from 29 influenza A (H7N9) virus strains and 6 influenza A (H7N9) virus strains with HA determinants variation were selected. 293FT cells were co-transfected with plasmid pNL4-3-Luc.R-E-, pVRC-HA and pVRC-NA with codon-optimized hemagglutinin (HA) and neuraminidase (NA) derived from the six influenza A (H7N9) virus strains, respectively. Transmission electron microscopy assay and Western blot analysis were performed to demonstrate morphology and specificity of these particles, luciferase activity assay and hemagglutinin titers detection were used to determine their infectivity and hemagglutinin activity. And finally, pseudovirus-based neutralization assays were evaluated with HA immunized mice serum.@*Results@#Six influenza A (H7N9) peseudovirus particles derived from different districts of China were selected and prepared. All of the particles bearing HA and NA were characterized with classic influenza virus morphology, with TCID50 titer ranged from 104TCID50/50 μl to 105TCID50/50 μl and with hemagglutinin activity ranged from 64 to 512. Neutralization efficacies on influenza A/Shanghai/1/2013(H7N9) HA vaccine serum against 100TCID50 dose of these pseudovirus particles indicated their potential application in the vaccine cross-protective evaluation in future.@*Conclusions@#Six influenza A (H7N9) pseudovirus derived from different districts of China with potential antigenic variation on HA were constructed successfully, established foundation for their further application in vaccine cross-reactive efficacy evaluation.

Whole-genome evolution of influenza B viruses prevalent in Qingdao from 2006 to 2011 / 中华微生物学和免疫学杂志

Objective To study the whole-genome evolution of influenza B viruses prevalent in Qingdao from 2006 to 2011 .Methods RNA was extracted from influenza B viruses isolated in Qingdao from 2006 to 2011 .Each gene segment was amplified by reverse transcription polymerase chain reaction ( RT-PCR) and then sequenced .Gene sequences of each virus were determined and assembled by using Sequench -er software .A phylogenetic analysis for each gene segment was conducted by using MEGA 5.0 software pack-age.Results The phylogenetic tree of hemagglutinin ( HA) gene showed that 13 strains from 2006 to 2009 belonging to V1 clade of Victoria lineage were B/Malaysia/2506/2004-like viruses,and 12 strains from 2009 to 2011 belonging to the V2 clade of Victoria lineage were B/Brisbane/60/2008-like viruses.Moreover, strains of Yamagata lineage were all B/Florida/4/2006-like viruses including 5 strains of Y1 clade circulated from 2006 to 2008 and 7 strains of Y2 clade circulated from 2010 to 2011, respectively.The analysis of whole-genome evolution showed that 3 viruses of V2 clade presented 5+3 reassortment and 1 virus presented 1+7 reassortment.All reassortant strains matched with the vaccine strains of the present and previous season . The Yamagata and Victoria lineage strains belonged to genotype 2 and genotype 15,respectively.Compared with vaccine strains , the HA1 protein of Victoria lineage strains showed mutations at amino acid sites of H14Q, L58P, N129S, I146V, N171D and R279K, while R48K, K88R, P108A, N116K, S150I, N165Y, D196N,N202S and S229G amino acid mutations were mainly detected in Yamagata lineage strains .The sites 116 and 129,150,165,196 and 202 located in the 120,150,160 and 190 loops,respectively,which had been previously determined to be the hotspots under positive selection .Conclusion Both Yamagata and Victoria lineages of influenza B viruses were prevalent in Qingdao and evolved continuously from 2006 to 2011 .The selective pressure that a vaccine would provide was only to virus strains belonging to the same lineage ,sug-gesting a bivalent vaccine may be better for the induction of protective immunity .

Recent Trends of Antigenic Variation in Bordetella pertussis Isolates in Korea

Pertussis is a representative vaccine-preventable disease. However, there have been recent outbreaks in countries where even higher vaccination against the disease. One reason is the emergence of antigenic variants, which are different to vaccine type. In Korea, reported cases have rapidly increased since 2009. Therefore, we analyzed genotype of strains isolated in 2011-2012 by multilocus sequence typing method. As expected, the genotype profiles of tested genes dramatically changed. The major sequence type changed from ST1 to ST2, and new sequence type (ST8) appeared. In the minimum spanning tree, recent isolates belonging to the ACC-I-ST3 subgroup were detected that were composed of ST2, ST3, and ST6. In particular, the ST2 frequency increased to 81%. The novel ST8 was linked to the increased frequency of ST2. In addition, toxic strains carrying the ptxP3 promoter type were confirmed. This ptxP3 type emerged from 2009 and its frequency had increased to 100% in 2012. Based on these results, it can be inferred that the genotypic changes in the currently circulating strains are strongly associated with the recent increasing of pertussis in Korea. Therefore, the surveillance system should be strengthened, and genetic characterization of the isolates should be expanded to the whole genome sequence level.

Characterization of influenza B viruses isolated in Hunan Province after pandemic influenza A (H1N1) / 中华传染病杂志

Objective To analyze the prevalence status and the genetic characterizations of influenza B viruses isolated in Hunan Province after pandemic influenza A (H1N1) 2009,and to explore possible reasons for the prevalence.MethodsThroat swabs were collected from outpatients with influenza-like illness in 23 sentinel hospitals of Hunan Province in 2010.Influenza viruses were isolated with Madin-Darby canine kidney (MDCK) cells and identified by haemagglutination inhibition test.The genomes of 10 selected influenza B viruses were sequenced and analyzed for phylogenetic and molecular characterization.ResultsWith the reduction of isolation of pandemic influenza A (H1N1)2009 viruses,influenza B virus became the predominant isolated strain in the first half of 2010.Epidemic viruses mainly belonged to the B/Victoria lineage,and both two lineages co-circulated.Seven out of 11 influenza outbreaks caused by type B.Ten strains were filled into 2 branches of BV and BY which were classified by their lineage types in polymerase (PB2,PB1,PA),hemagglutinin (HA),neuraminidase (NA),NB,membrane protein (M1),influenza B virus membrane protein M2 (BM2),and non-structural protein (NS1,NS2) phylogenetic trees except the NP phylogenetic tree in which 10 strains were all in the BY branch.Compared with World Health Organization (WHO) vaccine strains,the amino acid identity of 11 proteins of the 10 strains was high (97.2％-100.0％).However,some amino acid point mutations were found.No mutation was found in drug resistance mutation sites.Some mutations in NA,NB,PB1,PB2 and NS2 molecules were found in 2 strains isolated from outbreaks compared with strains from sentinel surveillance.Conclusions The point mutations,insertions and genetic reassortment indicate viruses sustaining evolution,which is probably the reason for predominant influenza B viruses after pandemic influenza A (H1N1) 2009 in Hunan Province.

Antigenic variations of human influenza virus in Shiraz, Iran.

Purpose: Influenza virus is a major cause of human respiratory infections and responsible for pandemics and regional outbreaks around the world. This investigation aims to determine the prevalent influenza genotypes during 2005-2007 outbreaks in Shiraz, the capital city of Fars province, southern Iran and compare the results obtained with those of previous study. Materials and Method: Of the 300 pharyngeal swabs collected from influenza patients, 26 were found to be positive by culture and hemagglutination (HA) assays. Typing and subtyping of the isolates carried out by using multiplex RT-PCR and phylogenetic analysis performed on isolated HA genes using neighbour-joining method. Result: Out of 26 positive isolates 12 and 14 were H1N1 and H3N2 respectively. The phylogenetic and amino acid sequence analyses of our H1N1 isolates showed 99-100% genetic resemblance to A/NewCaledonia/20/99 (H1N1) vaccine strain. Most of the Iranian H3N2 isolates varied form A/California/7/2004 vaccine strain in 20 amino acids of which positions 189,226 and 227 were located in antigenic sites of HA1 molecule. These substitutions were not observed in any of the H3N2 subtypes from the same region reported previously. Conclusion: The H3N2 subtype strains prevalent during the 2005/7 influenza outbreak in southern Iran demonstrated a drastic antigenic variation and differed from A/California/7/2004 vaccine strain. The H1N1 subtypes showed a notable resemblance to A/NewCaledonia/20/99 vaccine strain and therefore were predicted to be capable of conferring sufficient immunity against H1N1 subtypes.

Evolutionary characterization of HA1 gene of influenza A H3N2 viruses from 2003 to 2008 in Hebei / 中华疾病控制杂志

Objective To understand the genetic characterization of HA1 gene of influenza A H3N2 viruses circulated in recent years in Hebei.Methods Viral RNAs of 25 H3N2 strains were extracted and amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR).The products of PCR were purified and sequenced,and the sequences were analyzed though biometic software.Results Several amino acid substitutions located in antigenic sites or receptor binding sites were found more in the isolates than the current vaccine virus or the isolates from previous year.Amino acid substitution was found in 3,140,142,144,145,158,159,189,192,193,198,204,225,226 and 227 positions in the isolates during 2003-2008,more amino acid substitutions took place in antigenic determinant A,B and receptor binding site (RBS).New phylogenetic branches appeared continuously during 2003-2008.The H3N2 strains of the same year almost clustered in the same group on the phylogenetic tree.Conclusions Amino acid substitutions continuously occurred in the HA1 genes in influenza A H3N2 viruses isolated in Hebei from 2003 to 2008,it is meaningful to pay close attention to the HA1 variation in order to prevent and control influenza.

Development of Monoclonal Antibodies to Respiratory Syncytial Virus (RSV) and Application for Study of Antigenic Variation of RSV / 감염

BACKGROUND: Strains of respiratory syncytial virus (RSV) can be classified by reactivity with monoclonal antibodies into two major subgroups A and B, then further into several antigenic variants in each subgroup. Antigenic characterization of strains prevailing in a particular community may be essential in the future development of vaccine. METHODS: We developed monoclonal antibodies (MAbs) to RSV subgroups A and B. Antigenic specificities were characterized by immunoblot assay and radioimmunoprecipitation. By the pattern of reactions with these MAbs, RSV isolated over 1990~1998 were categorized into subgroups A and B, then further into antigenic variants. RESULTS: Thirty-four monoclonal antibodies to RSV were produced; twelve were directed against nucleoprotein; seven against matrix protein; ten against fusion protein; and five against major envelope glycoprotein. During the study period, yearly epidemics of RSV infection existed. Both subgroups circulated concurrently in 6 epidemics with predominance of subgroup A, and only one of each subgroup was identified in 2. Antigenic variations were observed in matrix, fusion and major glycoprotein. Six antigenic variants were identified in subgroup A and 2 in subgroup B among 242 strains of RSV. One major variant of subgroup A dominated in all of the 7 subgroup A epidemics, while the dominant variant of subgroup B was different in each of the 7 subgroup B epidemics. CONCLUSION: During the study period, yearly epidemics of RSV infection existed. The proportion of each subgroup varied in each of the 8 epidemics. The antigenic variability of RSV should be considered in the future development of RSV vaccine.

Antigen Analysis and Restriction Fragment Length Polymorphism of the Polymerase Chain reaction Products of Varicella-Zoster Virus Wild Strains Isolated in Korea

Varicella-Zoster virus (VZV) is the causative agent of chickenpox in children. It also causes herpes zoster in old or immunocompromised people. For effective control of VZV, live attenuated vaccine (Oka strain) was developed in 1974 and has been used worldwidely. This vaccine is indicated when VZV infection can be fatal, such as leukemia, with favorable success. Recently, routine usage of VZV vaccine for general young population was proposed. However, general use of vaccine requires prior epidemiological study of wild strains prevailing in the target population. In this regard, we investigated the antigenic and genetic variations, if any, between Oka strain and wild strains isolated in Korea. Six wild strains of VZV isolated from zoster patients in Korea, Ellen (the labaratory-adapted strain) and Oka (vaccine) strains were cultivated in Vero cells. After the VZV-infected Vero cells were stained with specific monoclonal antibody panel, the immunofluorescent patterns were compared. The VZV-infected Vero cells were also extracted after metabolic labelling to be immunoprecipitated with the monoclonal antibodies. However, the patterns of immunofluorescent staining and immunoprecipitation did not show any antigenic difference among wild strains and vaccine or standard strain. When the VZV gene was amplified by polymerase chain reaction using primer VZV 115953 and 116605, and the PCR products were cleaved by restriction enzymes (Taq I, Bl II, and Hpa II), two kinds of restriction patterns were observed. These results suggested that antigenic variation was not common among wild strains of VZV isolated in Korea and between vaccine strain. But the different restriction patterns implied potential antigenic variation.