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.

Research progress on invasion-related virulence factors in Porphyromonas gingivalis / 中华口腔医学杂志

Porphyromonas gingivalis (Pg) is a kind of gram-negative obligate anaerobes. It can invade and internalize within host cells. The invasion ability of Pg is very important for the occurrence and development of diseases and has been a hot topic for a long time. Remaining pathogenic characteristics in cell is one of its pathogenesis. In the process of invading host, the specific bacterial adhesin combine with the ligand of host cells, which activate various signal transduction pathways and trigger bacterial internalization. Virulence factors in Pg, such as fimbriae, gingival protease, hemagglutinins and outer membrane vesicles play significant roles in the process. This review summarized the research progress of the virulence factors which relate to Pg′s invasion, which provided a serious of new ideas on exploring Pg′s pathogenesis and the prevention and treatment of related diseases.

ABO Incompatible Living Donor Liver Transplantation: A Single Center Experience / 대한이식학회지

BACKGROUND: This study examined the outcomes of ABO incompatible living donor liver transplantation (LDLT). The changes in the immunologic factors that might help predict the long term outcomes were also studied. METHODS: Twenty-three patients, who underwent ABO incompatible LDLT from 2010 to 2015, were reviewed retrospectively. The protocol was the same as for ABO compatible LDLT except for the administration of rituximab and plasma exchange. The clinical outcomes and immunologic factors, such as isoagglutinin titer and cluster of differentiation 20+ (CD20+) lymphocyte levels were reviewed. RESULTS: The center showed a 3-year survival of 64% with no case of antibody-mediated rejection. When transplantation-unrelated mortalities (for example, traffic accidents and myocardial infarction) were removed from statistical analysis, the 3-year survival was 77.8%. Although isoagglutinin titers continued to remain at low levels, the CD20+ lymphocyte levels recovered to the pre-Rituximab levels at postoperative one year. CONCLUSIONS: As donor shortages continue, ABO incompatible liver transplantation is a feasible method to expand the donor pool. On the other hand, caution is still needed until more long-term outcomes are reported. Because CD20+ lymphocytes are recovered with time, more immunologic studies will be needed in the future.

Effect of evolution and mutations of H3N2 virus hemagglutinin genes on the antigenic epitopes in Guangdong / 中华传染病杂志

Objective To reveal evolution characteristics and antigenic epitope variabilities of hemagglutinin (HA) gene of H3N2 viruses in Guangdong during 2014-2015.Methods The HA gene nucleotide sequence of influenza H3N2 virus isolated during 2014-2015 in Guangdong Province was selected by spatial-temporal distribution.The HA gene nucleotide sequences were compared with global HA genes downloaded from GenBank and GISAID and the gene nucleotide mutations were analyzed.The phylogenetic tree and the entropy chart were conducted.Results Compared with HA gene of vaccine strain A/Texas/50/2012,the substitutions of 25 amino acid sites occurred in the HA gene of the 17 Guangdong strains during 2014 to 2015.The epitope A,B,D and E of the HA1 gene developed mutations,which involved 11 amino acid sites.One glycosylation site deletion occurred in A/Guangdong/ 55/2015 in virtue of the N38K site mutation.The high mutation site was at 175 with entropy value of 1.16.The 363 site might be the positive selection while the 368 and 425 sites might be the negative selection sites by comprehensive evaluation of the screening results of the single likelihood ancestor countincy (SLAC),fixed effects likelihood (FEL) and internal fixed-effects likelihood (IFEL) model.Conclusions The mutations emerge in the epitopes A,B,D and E of HA gene of the Guangdong H3N2 stains.Evolution at 363 site is positive selection while those at 368 and 425 sites are negative selection.The mutation accumulation might lead to the epidemic of influenza H3N2.

Genetic characteristic analysis of haemagglutinin of two episodes of influenza B virus outbreaks in Guizhou ;Province in 2016 / 中华传染病杂志

Objective To understand the genetic variations of influenza B virus outbreaks in Guizhou province in 2016,and to compare the matching situation of outbreak epidemic strains with the vaccine strains recommended by WHO and representative strains in China.Methods The haemagglutinin HA1 gene of 8 strains isolated from two episodes of influenza B virus outbreaks in Tongren area was amplified and sequenced.The sequencing products were analyzed by bioinformatics software DNAStar. Results The two episodes of influenza outbreaks were both caused by influenza B Victoria lineage virus (BV).The homologies of the isolated strains were 99.8%—100.0% in nucleotide and 99.5 %—100.0%in amino acid.Mutation was only detected at 274 site in some strains.Compared with reference strain B/Victoria/2/87,the homologies were 91 .8%—92.0% and 91 .5 %—92.0%,respectively.Mutations developed at 17 amino acid sites,among which,I143V,V163I and V201I site were associated with the main antigenic determinant area B,C and D.Compared with previous vaccine strain B/Brisbane/60/2008, the homologies were 98.2%—98.3% and 98.5 %—99.0%,respectively,and mutations were detected at 3 sites.Mutations at I143V and N155D were detected in all 8 strains and at T247I in some strains.The mutation of I143V was associated with antigenic determinant area B.Compared with the representative strain B/Chongqing-Yuzhong/1384/2010,the homologies were 96.7%—96.8% and 97.0%—97.5 %, respectively.A total of 6 sites developed mutations,among which,5 sites were P84L,I143V,N155D, V172I and T223N mutations.The mutation of T247I was detected in some strains,and I143V was associated with area B.Compared with the epidemic strain in Guizhou in 2016,the homologies were 99.8%—100.0% and 99.5 %—100.0%,respectively.Mutation was only detected at site 247 in some strains and was not associated with the main antigenic determinant area.Conclusions The two episodes of influenza outbreaks in Guizhou are caused by the same BV lineage epidemic virus strain.Haemagglutinin gene of BV lineage virus is constantly changing.However,there is no new mutation emerged at important site.Compared with previous influenza vaccine strain B/Brisbane/60/2008 recommended by WHO,BV lineage virus is well matched and could provide a positive protection effect.

Immunogenicity of hemagglutinin of H7 N9 expressed in yeast Pichia pastoris / 军事医学

Objective To express the hemagglutinin of H7N9 in Pichia pastoris and analyze its immunogenicity. Methods The HA [1 -525 amino acids(aa)] of H7N9 [A/Hangzhou/1/2013(H7N9)] lacking the C-terminal transmembrane anchor-coding sequence was amplified by PCR and cloned into the expression vector pPICZαA.The plasmid pPICZ-HA7-S-was transformed into P.pastoris and the HA1-525 was detected by ELISA.Then the HA1-525 was precipitated by PEG20000.After immunization with the HA1-525 , the anti-HA7 antibody in mouse serum was detected by ELISA and hemagglutinin inhibition ( HI) test.Results The HA1-525 was expressed in P.pastoris after being induced with methanol. Western blotting confirmed that there were specific dispersion bands and the molecular weight of HA1-525 decreased to 58 × 103 after being digested by endo H.Anti-HA7 antibody was found in serum of HA1-525 immunized mice and the hemaggluti-nation-inhibition titers reached 1∶700 after the third doses.Conclusion This study shows the HA1-525 expressed in P.pastoris can induce the neutralizing antibody in mice.

Research on preparation of influenza virus vaccine without thimerosal for children dose and its stability / 国际检验医学杂志

Objective To investigate the preparation of influenza virus vaccine without thimerosal for children dose and its sta-bility .Methods H1N1 ,H3N2 ,B-type influenza virus were inoculated into allantoic fluid to prepare three batches of influenza virus vaccine without thimerosal for children dose and the vaccine stability test was performed .Single radial immunodiffusion(SRID) and enzyme-linked immunosorbent assay(ELISA) were used to detect the concentration of hemagglutinin and egg albumin .Total protein concentration ,appearance ,sterility test ,endotoxin ,free formaldehyde and the pH value of vaccine were also measured .Results The pH value of vaccine was 7 .2 ,with total protein concentration of 182-189 mg/mL .Hemagglutinin concentrations of H1N1 ,H3N2 and B-type influenza virus decreased when they had been placed in 2-8 ℃ for 3 ,6 ,9 ,12 ,18 months or (37 .0 ± 2 .0) ℃ for 7 ,14 days ,however ,they maintained at 6 .0 μg/0 .25 mL or more at last .Conclusion Influenza virus vaccine without thimerosal for chil-dren dose shows improved safty and is accord with the standard of Chinese Pharmacopoeia(2010 edition) .

Lectins: Proteins with Diverse Applications.

Lectins are proteins that bind to carbohydrates and sugar containing substances in a specific and reversible way or precipitate glycoconjugates. These heterogeneous class of carbohydrate-binding proteins or glycoproteins of non-immune origin are capable of specific recognition of, and reversible binding to, carbohydrates without altering their covalent structure. Lectins are found in a diversity of organisms and possess the ability to agglutinate erythrocytes with known carbohydrate specificity since they have at least one non-catalytic domain that binds reversibly to specific monosaccharides or oligosaccharides. This review aims to highlight the applications of lectins in various fields of biology. Lectins are isolated from their natural sources by chromatographic procedures with various modulations to increase their production. The yields of animal lectins are usually low compared with the yields of plant lectins such as legume lectins, which form a major source of these proteins. Lectins manifest a diversity of activities including anti-insect activities, antitumor, immunomodulatory, antimicrobial and HIV-1 reverse transcriptase inhibitory, which may find applications in many therapeutic areas. A small number of lectins demonstrate anti-parasitic activities.

Phylogenetic analysis of hemagglutinin (HA) gene of the novel avian influenza virus A/H7n9 / 第二军医大学学报

Objective To investigate the evolution and variations in coding amino acids of hemagglutinin (HA) gene of the novel avian influenza virus H7N9 in 2013 epidemic. Methods The HA gene sequences of influenza virus H7N9, H7N2, H7N3 and H7N7 subtypeswere downloaded from the database of The National Center for Biotechnology Information (NCBI) and The Global Initiative on Sharing All Influenza Data (GISAID). MEGA 5. 05 software was used for sequence analysis and N- J method was used for constructing the phylogenetic trees. The amino acid sequences at the receptor binding sites, glycosylation sites, and cleavage siteswas aligned and analyzed. Results The HA genes this novel A/H7N9 virus in 2013 shared a 95. 3%- 95. 6% similarity with JQ906573. 1| Zhejiang (H7N3 virus) isolated in 2011. The most important variation in this novel H7N9 isolates was found at the receptor binding site: Q226L. The 5 glycosylation sites were highly conservative. One basic amino acid (R) at the HA cleavage sites, located between aa339 and aa340, was also found in this novel isolate. Conclusion The HA gene of this novel H7N9 isolate might originate from H7 subtypes carried by birds in China. Thebinding site change caused by Q226L variation might be responsible for human infection of this novel H7N9 isolate.

Construction and identification of a eukaryotic expression vector of fusion protein containing avian H5N1 influenza virus hemagglutinin antigen and the cholera toxin B subunit / 中华传染病杂志

Objective To construct a eukaryotic vector which contains avian H5N1 influenza virus hemagglutinin (HA) antigen and the cholera toxin B subunit (CTB) and to investigate its expression in COS7 cells,and the ability to induce specific immune responses in vivo in different periods.Methods After cloned by polymerase chain reaction (PCR),CTB and HA genes were digested with BamH Ⅰ and connected into CTB-HA gene with T4 ligase.The connected gene was referred to as CH.After double digestion,CH gene was inserted into a eukaryotic recombinant plasmid pCI-neo.The pCI-CH plasmid was then transfected into COS7 cells.Western blot was used to detect the expression of HA antigen.After New Zealand white rabbits were immunized,the titer of HA antigen-specific antibody in serum and its specificity with other strains such as H1N1,H9N1,H3N2 and influenza B virus were determined by indirect enzyme-linked immunosorbent assay.Results The pCI-CH vector (DNA vaccine) was successfully constructed,which could be efficiently expressed in COS7 cells and induce specific antibodies against pCI-CH in rabbits.Cross reactions indicated that DNA vaccine pCI-CH specific antisera could not only react with H5N1 strain (P/N＞2.1),but also H1N1,H9N1 and H3N2 strains,but did not cross react with influenza B virus.Conclusion The newly constructed avian H5N1 influenza virus nucleic acid vaccine has good immunogenicity.

Haemagglutinin gene mutations and sequences of influenza viruses A (H1N1) / 中华传染病杂志

ObjectiveTo understand the detections of influenza A (H1N1) in 2009,and haemagglutinin (HA) gene mutations and the comparisons with standard strains.MethodsThe nasopharyngeal swabs from patients with influenza-like illness (ILI) in National Influenza Sentinel Surveillance Hospital and the outbreak epidemic area were collected.The virus typing and A (H1N1) viruses were tested by real time-polymerase chain reaction (RT-PCR).Then the pathogens were isolated with MDCK cells,the virus titer was determined with hemagglutination test and the virus typing was identified with hemagglutination inhibition test (HA1).The RT-PCR products of HA1 gene of virulent strains were sequenced and then analyzed through bioinformatics.Results A total of 996 pharyngeal swab specimens were tested,and nucleic acid positive cases included 337 A (H1N1) subtype,1 seasonal A (H1N1) subtype,67 A (H3N2) subtype,and 12 B type.The positive rate of nucleic acid detection of influenza was 41.87％ and that of A (H1N1) was 33.84％.Thirty-six influenza A (H1N1) virus strains were isolated,and 10 of them were successfully sequenced and several amino acid mutations were identified.There were 6 amino acid mutations found compared with vaccine strain A/California/07/2009 (H1N1),and 1 site was in area B of epitope.Conclusions A (H1N1) is absolute predominant among isolated strains in 2009.HA gene of virulent strains is mutated compared with vaccine strain provided by World Health Organization,which shows that the area B of epitope changes,while the key amino acid position 222 doesn't change.

Sequence analysis of the hemagglutinin gene of isolates viruses from 3 novel influenza A ( H1N1 )deaths / 中华检验医学杂志

Objective To understand the origin and variation of the hemagglutinin gene of isolates viruses from 3 novel influenza A( H1N1 ) deaths in Changsha ( A/Hunan Kaifu/SWL4142/2009 ( H1N1 ) , A/Hunan Changsha/SWL4346/2009 ( H1 N1 ) and A/Hunan Furong/SWL4224/2009( H1N1 )). Methods The nasopharyngeal swab specimens from the 3 novel influenza A( H1N1 ) deaths in Changsha were tested by RT-PCR and influenza viruses were isolated simultaneously. With the sequencing primers recommended by World Health Organization (WHO), the HA gene of sequences of 3 novel influenza A( H1N1 ) deaths were tested by CEQTM 8000 Genetic Analysis System, through dye terminator cycle sequencing. The sequencing results were submitted to GenBank, then the results were analyzed for amino acid alignment and phylogenetic tree analysis with ClustalX and Mega4.1 software. Results All the nucleotide homologies of HA gene sequences in A/Hunan Kaifu/SWL4142/2009 ( H1N1 ), A/Hunan Changsha/SWL4346/2009 ( H1N1 ) and A/Hunan Furong/SWL4224/2009( H1N1 ) are 99% as compared with the novel influenza A( H1N1 ) virus strains of A/NewYork/3502/2009 ( H1N1 ), A/Shanghai/71T/2009 ( H1N1 ) and A/Chita/01/2009 ( H1N1 )The nucleotide homology of the 3 HA gene sequences are more than 99. 5% the same compared with the novel influenza A( H1N1 ) virus strain ( A/Sichuan/1/2009( H1N1 ) ) in China. Phylogenetic tree analysis reveals that 2009 novel influenza A(H1N1 ) viruses including 3 HA gene sequences of A/Hunan Kaifu/SWL4142/2009 ( H1 N1 ), A/Hunan Changsha/SWL4346/2009 ( H1N1 ), A/Hunan Furong/SWL4224/2009( H1N1 ) had a close evolutionary relationship with the swine H1 virus isolates in North America ( A/Swine/Indiana/P12439/00), but a distant evolutionary relationship with those human seasonal A( H1 N1 ) influenza virus and avian. After comparing with genes of A/Swine/Indiana/P12439/00, we found that the HA gene sequences of the 3 viruses isolated had 28,30 and 27 amino acids with mutation respectively, but only one (R53K) amino acids mutation at 21 important antigenic sites in the 3 viruses isolated. Multiple alignment of 364 HA genes sequences of novel influenza A ( H1N1 ) viruses in the world showed they had 119 nonconserved amino acids, 5 non-conserved position at important antigenic sites. Conclusions The HA gene sequences from 3 viruses isolated in this study and other influenza A ( H1N1 ) viruses might originate from swine A( H1N1 ) in North America by variation. The 3 HA gene sequences of viruses isolated have high homology as compared with the novel influenza A ( H1N1 ) virus strains worldwide, and the 3 HA gene sequences of viruses isolated are in stable condition as the vast majority of novel influenza A( H1N1 ) virus strains in the world.

Variations in surface protein genes of avian influenza virus before and after infecting mouse / 中华临床感染病杂志

Objective To investigate the variations of surface protein genes of avian influenza virus (AIV)before and after infecting mouse.Methods Mouse lung tissue was infected with A/Goose/Guangaong/NH/2003(H5N1)and the virus was isolated 12 hours and 9 days after replication in lung tissue of mouse.The isolated strains were amplified in embryonated chicken eggs,anti the virion RNA was transcribed into cDNA by reverse transeriptase.After amplification and purification,dideoxy-mediated chain termination was performed to detect synthetic oligonucleotide primers and DNA sequence was analyzed.Results The homology of nucleotide sequence for HA gene of three isolated strains was 99.6%-99.8%.and that of amino acid sequences was 99.3%-99.6%.The homology of nucleotide sequence for NA gene of three strains was 99.8%-99.9%.all of them were synonymous mutatinns.No variation was found in M gene.Conclusion After replication in mouse lung tissue,no significant mutation was found in the surface protein genes of AIV except some point mutations in HA genes.

Surveillance of hemagglutinin genetic variation of influenza viruses in Shanghai and Wuxi / 中华传染病杂志

Objectives To explore the type and subtype distribution of influenza viruses in influenza-like patients and the hemagglutinin(HA)genetic variation of influenza A viruses in Shang- hai and Wuxi during the influenza prevalent season from 2004 to 2006.Methods Throat swabs were collected from the influenza-like patients in the sentinel hospitals and during the outbreaks,and then inoculated into MDCK cells to isolate influenza viruses,which were subsequently identified by direct immunofluorescence(DIF)and reverse transcription-polymerase chain reaction(RT-PCR).HA seg- ments of influenza A viruses were sequenced to analyze the genetic variation of HA.Results One hundred and twenty-six strains of influenza viruses,including 53 H3N2,43 H1N1 and 30 influenza B viruses were isolated from August 2004 to September 2006,and 7 outbreaks.All these outbreaks oc- curred in February or March The pathogens were identified as H1N1 in one outbreak,H3N2 in two outbreaks,B in two outbreaks and mix infections in two outbreaks(1 H1N1 and B,1 H3N2 and B, respectively).By sequencing the HA segment,the H3 and H1 segments were all homologous to the isolates from different countries in the same period.Conclusion H3N2 and H1N1 are the major strains prevalent in Shanghai and Wuxi,which reach the peak from January to March No HA and NA recom- binant strains and new HA and NA subtypes are found in these areas.The variations of H1 and H3 are similar to those found in other countries.

Construction of Newcastle disease virus hemagglutinin-neuraminidase (HN) gene and its antitumor immune response effects / 第二军医大学学报

Objective: To construct an eukaryotic expression plasmid containing gene coding for the hemagglutinin neuraminidase(HN) of Newcastle disease virus (NDV), and to study its mechanism and value in antitumor therapy. Methods: The HN cDNA was abtained from NDV with RT PCR and an eukaryotic expression vector of HN gene ( pcDNA3 HN ) was constructed. The antitumor effect was evaluated after injecting pcDNA3 HN into mice bearing B16 melanoma. Results: The HN cDNA of NDV was successfully cloned and pcDNA3 HN had a good expression in COS 7 cells. Animal experiments suggested that the pcDNA3 HN could significantly increase CTL and NK activity of tumor bearing mice. Conclusion:The eukaryotic expression plasmid containing the gene coding for the (HN) has the function of increasing CTL and NK activity of tumor bearing mice.