Molecular evolution of influenza A/H3N2 viruses HA and NA genes circulating in Shenzhen in 2017 / 中华疾病控制杂志

Objective To investigate the molecular evolution of the Hemagglutinin (HA) and Neuraminidase (NA) genes of influenza A/H3N2 viruses in Shenzhen in the first half of 2017, so as to provide scientific basis for predicating influenza epidemic and variation. Methods A total of 40 influenza A/H3N2 viruses strains were selected and the molecular phylogenetic trees were constructed by bioinformatics software DNAStar, MEGA 7.0, etc. Then, the genetic characteristics and variation of HA and NA genes along with corresponding amino acids were analyzed. Results The homology of Shenzhen isolates reached 97.8%-100.0%, which located in the human-derived branch of Asia and North America. Compared with the vaccine strains A/Switzerland/9715293/2013(H3N2) and A/Hong Kong 14801/2014(H3N2) recommended by world Health Oraganication (WHO), there was a higher sequence similarity. Compared with the vaccine strain, HA and NA proteins had a number of amino acid sites replaced, of which HA 6 antigen sites and 2 receptor binding sites change; NA had a mutation of D151N/G located in enzyme activity sites. Potential N-glycosylation sites for HA and NA also changed. Conclusions The influenza A/H3N2 viruses in Shenzhen in the first half of 2017 has not yet formed a new subtype in the epidemic. Currently, the recommended vaccine strains still have some protective effects on the population. The replacement mutation of multiple amino acids sites of HA and NA suggests that the dynamic monitoring of molecular level of influenza A/H3N2 viruses need to be strengthened.

Applicability of a sensitive duplex real-time PCR assay for identifying B/Yamagata and B/Victoria lineages of influenza virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To develop a novel sensitive duplex real-time PCR assay for accurately identifying B/Yamagata and B/Victoria lineages of influenza virus type B.</p><p><b>METHODS</b>50 HA (hemagglutinin) gene sequences coding for B/Yamagata and B/Victoria lineage, respectively, were randomly downloaded for GenBank and analyzed by software MEGA. Primers and probes specific for HA gene of B/Yamagata and B/Victoria lineages were designed by Primer Primer and then applied in the duplex real-time RT-PCR method that was followed developed. Influenza virus B type and A type isolated in our laboratory and typing-confirmed by HAI method were used as reference strains to determine the specificity of this assay and the sensitivity of the duplex amplification was evaluated by viral load testing in terms of in vitro transcribed RNA copy number.</p><p><b>RESULTS</b>In 2006-2010, 793 influenza virus type B strains were isolated from 17 765 throat swab samples, among which 152 strains were differentiated as By lineage and 641 as Bv lineage by this assay. These results was agreement with that determined by HAI assay. This developed assay allows to accurately identify approximately 10(2) copies/microl for Bv and By lineage virus with intra- and inter-coefficient of variation (CV) < 3.5% and nearly 100% specificity.</p><p><b>CONCLUSIONS</b>This method provides sensitive and robust tool for routine diagnosis and on-time epidemiological examination of influenza virus, which could be applied in influenza surveillance laboratories for rapid molecular diagnosis.</p>

Compare real-time RT-PCR with two culture methods for influenza virus detection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Real-time RT-PCR, cell culture and embryonated eggs culture for influenza detection were compared by analyzing the data of influenza surveillance in Shenzhen in second half of 2009.</p><p><b>METHODS</b>1092 clinical samples (throat swabs) collected during second half of 2009 were tested by real-time RT-PCR, cell culture and embryonated eggs culture, and the results were analyzed by statistical methods.</p><p><b>RESULTS</b>The positive rate were 54.21%, 27.11% and 16.21% using real-time RT-PCR, cell culture and embryonated eggs culture, and the sensitive were 100%, 50% and 29.9%. The lowest dilutions of virus detected by real-time RT-PCR were 10(-2) TCID50/ml.</p><p><b>CONCLUSION</b>The sensitive of real-time RT-PCR was higher than culture and the specificity was also very high. It was more suitable for emergency detect. The sensitive of cell culture for H3N2 subtype was higher, and sensitive of embryonated eggs culture for type B was higher.</p>

Analyses of serological and genetic characteristics on novel H1N1 influenza A virus from the infected patient in Shenzhen / 病毒学报

Analysis of serological and genetic characteristics on 2009 swine-origin influenza A (H1N1) virus (S-OIV) isolated from four patients with severe disease in Shenzhen were performed. Microneutralization assay showed that the neutralizing antibody titers of the infected patients did not exceed 1 : 20 in a short term post infection, which could not neutralize the viruses efficiently. Hemagglutination inhibition (HI) tests confirmed that the antigenicity of S-OIV from the patients was distinct from the seasonal influenza A virus, but similar to the reference strains of S-OIV. Phylogenetic and molecular analysis showed that S-OIV from the patients still belonged to the classical swine lineages and did not have the genetic characteristics of highly pathogenic influenza virus. Several amino acid residue mutations on HA protein were detected, which seemed not to affect the virulence and pathogenicity of the viruses. Further, A His 275 Tyr mutation on NA protein of a virus strain was detected, which induced the oseltamivir resistance of the virus.

Association of cooking oil fumes exposure and oxidative DNA damage among occupational exposed populations / 中华劳动卫生职业病杂志

<p><b>BACKGROUND</b>Previous investigations indicate that cooks are exposed to polycyclic aromatic hydrocarbons (PAH) from cooking oil fumes (COF). However, Emission of PAH and their carcinogenic potencies from cooking oil fumes sources have not been investigated among cooks.</p><p><b>AIMS</b>To investigate the urinary excretion of a marker for oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OHdG), in different groups of cooks and different exposure groups, and to study the association between 8-OHdG and 1-hydroxypyrene (1-OHP), a biological marker for PAH exposure.</p><p><b>METHODS</b>Urine samples were collected from different groups of cooks (n = 86) and from unexposed controls (n = 36), all are male with similar age and smoking habits. The health status, occupational history, smoking, and alcohol consumption 24 hours prior to sampling was estimated from questionnaires. The urinary samples were frozen for later analyses of 8-OHdG and 1-OHP by high performance liquid chromatography.</p><p><b>RESULTS</b>Excretion in urine of 8-OHdG were similar for controls (mean 1.2 µmol/mol creatinine, n = 36), and for those who had been in the kitchen room with exhaust hood operation (mean 1.5 µmol/mol creatinine, n = 45). COF exposed cooks without exhaust hood operation had increased excretion of 8-OHdG (mean 2.3 µmol/mol creatinine, n = 18). The difference between this group and the unexposed controls was significant. The urinary levels of ln 1-OHP and ln 8-OHdG were still significantly correlated in a multiple regression analysis.</p><p><b>CONCLUSION</b>Results indicate that exposure to PAH or possibly other compounds in COF may cause oxidative DNA damage.</p>

The preparation of P particle of the norovirus strain SZ9711 from China and its affinity analysis with human histo-blood group antigens in saliva / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the binding profile of NV strain SZ9711 (GII-4) with human histo-blood group antigens (HBGAs).</p><p><b>METHODS</b>The P domain-encoding fragment was amplified by RT-PCR from the stain SZ9711 and cloned into the pGEX-4T-1 vector. The recombinant fusion protein was expressed in E. coli and purified using the column Sepharose 4B. The P protein was released by thrombin cleavage. The binding of P particles of SZ9711 and VA387 with the HBGAs were measured by saliva-based EIA method.</p><p><b>RESULTS</b>The expression of the recombinant fusion protein was shown by the SDS-PAGE, in which a 38 x 10(3)-P protein was obtained. Saliva-based EIA revealed that the P particle of SZ9711 bound to HBGAs in saliva similar to that of the strain VA387 reported previously. It bound strongly to saliva of type A, B and O(secretor) but did not interact with saliva of type O(non-secretor). Noteworthy, binding ability of SZ9711 P particle to type A saliva was lower than that of the VA387 P particle.</p><p><b>CONCLUSION</b>This is the first time that a P particle was prepared from a norovirus strain isolated in China and the binding ability of the P particle with HBGAs was analyzed. The result indicated the binding profile of the SZ9711 P particle was similar to that of VA387 reported previously. These data may be valuable in studying the relationship between noroviruses and their bindings to HGBA receptors.</p>

Epidemiological and molecular characterization of seasonal influenza A/H3N2 viruses circulating in Shenzhen, 2005 - 2007 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To determine the epidemiological characteristics of seasonal influenza in Shenzhen from 2005 to 2007 and the molecular variation of HA1 domain of influenza H3N2 viruses.</p><p><b>METHODS</b>The consultation rate for influenza-like illness (ILI) were calculated weekly for indicating the influenza activities (the Shenzhen Influenza Surveillance System mainly consisted of 16 institutions with 9 hospitals, 6 districts and one municipal centers of disease control and prevention). Pharyngeal swabs from the cases of ILI, which were collected during 2005 to 2007 from the city-wide and quality-controlled surveillance network, were used to propagate the viruses. The HA1 region of the influenza A/H3N2 viruses were detected by RT-PCR and sequenced subsequently. The analyses of pairwise amino acid variations, genetic clustering and phylogenetics was performed.</p><p><b>RESULTS</b>The activity levels of influenza showed certain changes during each year from 2005 to 2007, and there were summer peaks from May to July in 2006 and 2007. The positive rates of influenza virus were 4.78% (114/2385), 5.77% (212/3674) and 12.12% (343/2831) from 2005 to 2007 respectively. The weekly isolating rates changed accordingly with the trend of the percentages of ILI. The proportions of influenza H3N2 virus were 25.46% (28/114) and 2.83% (6/212) in 2005 and in 2006 respectively, but the proportion increased to 62.68% (215/343), which indicated that H3N2 virus became the predominant strain in 2007. Phylogenetic clustering analysis of influenza H3N2 virus revealed that there were 5 clades. The viruses which were isolated in 2005 contained in the clade I and II, the viruses in 2006 were comprised in clade III, and clade IV and V included the viruses isolated in 2007. Although the stem of cladogram developed with one accord of the time isolated viruses, the viruses which were similar to vaccine strains had circulated in Shenzhen before a given strain was determined as vaccine strain by WHO. It was also noticed that more amino acid changes at antigenic sites, especially at sites A and B in the H3N2 viruses, were found in 2007 than that in 2005 and in 2006. But the sequences at the receptor-binding sites and disulphide bond sites were conserved and no new circulating strain for genetic reassortment had been found in the period.</p><p><b>CONCLUSION</b>Shenzhen might be one of areas where the ongoing genetic drift of influenza H3N2 viruses appeared earlier in China. The changes of influenza H3N2 virus showed the active status in the population. The results suggested that monitoring seasonal influenza viruses by sequence analysis could provide important and timely information on the appearance of strains with epidemiologic significance.</p>

Study on molecular epidemiological characteristics of influenza H1N1 viruses circulating in Shenzhen,China from 2005 to 2007 / 中华流行病学杂志

Objective To study the genetic and epidemiological characteristics of HA1 of influenza H1N1 viruses circulating in Shenzhen from 2005 to 2007. Methods The HA1 region was analyzed by RT-PCR and subsequently sequenced to analyze the HA1 genetic evolution. Phylogenetic analysis was confirmed on the homology of nucleitide comparing with the reference viruses of vaccines recommended by WHO and representative virus confirmed by China CDC. Relationship between isolation rates and genetic evolutions was explored. Results The average isolation rate from 2005 to 2007 was 7.16%. Of the isolates, the proportions of influenza H1N1 viruses in 2005, 2006 and 2007 were 56.14%, 66.03%,3.61% ,respectively. Data from HA1 phylogenetic analysis showed that there were at least three clades circulated in Shenzhen. Different viruses isolated during January to April were clustered with A/New Caledonia/20/1999 viruses isolated in the latter months of 2005 clustered with A/Solomon Island/3/2006 and viruses from 2006 to 2007 were in the same clade with A/GDLH/219/2006. Results showed that most viruses had a deletion of lysine at position 130. Compared with A/New Caledonia/20/1999, the virus isolated after May of 2005 occurred T82K, Y94H, R146K, R209K, T267N amino acid substitution, while some virus isolated after May 2006 took place the amino acid substitutions of A190T, H193Y,E195D (located at antigenic site B) and R146K(antigenic site A). The sequences at the receptor-binding sites and glycosylation sites were conserved. Compared with referring viruses, A/SZ/68/2007 had 50 amino acid substitutions in the HA1 region.Of these,eleven and six were located at antigenic sites and receptor-binding sites,respectively.Four amino acid substitution resulted in the deletion of glycosylation site.Conclusion Three different genetic lineages of influenza H1N1 virus were circulated in the population in Shenzhen during 2005-2007.The special virus named A/SZ/68/2007 should be paid further attention on its antigenic and epidemiological characteristics.

Laboratory diagnosis and molecular characterization analysis of the H5N1 influenza virus isolated from the first human case in Shenzhen, China / 病毒学报

The tracheal aspirates and serum samples of a suspected human case of high-pathogenic avian influenza (firstly found in Shenzhen, China) were collected and tested by a series of assays. The results showed that the RNA extracted from the tracheal aspirate specimens of the patient was confirmed positive for H5N1 avian influenza virus by Real-time PCR. The H5N1 avian influenza virus was isolated from patient's tracheal aspirates on MDCK cell and was named A/Guangdong/2/06(H5N1). The viral load of tracheal aspirates collected at different time points were detected by Real-time PCR. The virus microneutralization and the antigenic ratio of human H5N1 isolated were also assayed. It was found that when the virus load decreased gradually after the disease onset, the serum neutralizing antibody titer in the patient increased to 1 : 160 and subsequently decreased gradually. By molecular analysis, the eight gene segments of A/Guangdong/2/06 revealed to be similar to that of H5N1 avian influenza viruses isolated from south China in 2005-2006. However, there were obvious differences in the gene sequence of the detected H5N1 viral RNA as compared with that of the strains isolated from Vietnam, Thailand and Indonesia.

Clone expression and purification of influenza A virus nucleoprotein gene / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To clone the influenza A virus NP gene into expression vector and to purify the target protein, which was used to study the preparation of monoclonal antibody.</p><p><b>METHODS</b>The RNA of influenza A virus was extracted and primers were designed according the NP gene sequence, then the NP gene of influenza A virus was expressed in E.coli DH5alpha and the NP protein was purified by affinity chromatography.</p><p><b>RESULTS</b>The recombinant expression vector-pGEX-4T-2-NP was successfully constructed and relatively pure target protein was obtained.</p><p><b>CONCLUSION</b>Through reasonably controlling the fermentation time, growth temperature and induction concentration, satisfactory soluble target product was obtained.</p>

Application of fluorescent real-time reverse transcriptase-polymerase chain reaction in detecting influenza viruses / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To apply fluorescent real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in detecting influenza viruses.</p><p><b>METHODS</b>A total of 207 oral swab samples were obtained in 16 collections from SARS patients and suspected influenza outbreak cases. They were subjected to influenza virus detection by fluorescent real-time RT-PCR, MDCK cell culture, and hemagglutinin inhibition assay.</p><p><b>RESULTS</b>Out of 207 samples, 79 (38.16%) were positive for influenza viruses when tested by fluorescent real-time PCR, and 62 (29.95%) positive when tested by MDCK cell culture. There was a statistically significant difference between them (chi square=8.64, P less than 0.005). From 104 cases in 9 collections dual serum samples were obtainable. When tested with hemagglutinin inhibition assay, 64 cases (61.54%) showed a 4-fold increase against H3N2 antigen.</p><p><b>CONCLUSION</b>This study showed that fluorescent real-time PCR is a reliable, sensitive, and fast method for detecting influenza viruses.</p>