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>

Construction and identification of nemo-like kinase gene recombinant adenovirus vector / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct the nemo-like kinase (NLK) gene recombinant adenovirus vector.</p><p><b>METHODS</b>The AdEasy system was used to construct the recombinant adenovirus vector. Using reverse transcriptase polymerase chain reaction (RT-PCR), the full-length gene of NLK and its mutants (K155M, T286V, and C425Y) were amplified from HEK293 cells. The FLAG tag was appended at the C-terminal of NLK. After ligation and transformation, the NLK gene and its mutants were cloned into the pAdTrack-CMV vector. It was detected by PCR, sequencing, and Western blot analysis. Using DNA recombination and homogenous recombination, the normally expressed plasmids were linearized by the restriction enzyme-PmeI and PacI, then the enzyme-digested products were recycled by using ethanol precipitation. The purified product was transfected to HEK293A packaging cells with FuGENE HD transfection reagent. After amplification of the recombinant adenovirus, Western blot analysis was performed to detect the expression of NLK gene and its mutants.</p><p><b>RESULTS</b>The successful construction of pAdtrack-CMV-NLK (and mutants) was confirmed by PCR and sequencing. Western blot analysis showed that the target genes and the recombinant adenovirus were obtained. This recombinant virus was able to express NLK protein and its mutants correctly in HCT 116 cells.</p><p><b>CONCLUSION</b>The NLK gene recombinant adenovirus vector was successfully constructed and identified.</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.

Baculovirus Host-Range / 中国病毒学·英文版

Baculoviruses are used as microbial insecticides, protein expression vectors, epitope display platforms, and most recently as vectors for gene therapy. Understanding the mechanisms that control baculovirus host-range and tissue tropisms are important for assessing their safety and for improving their properties for these biotechnology applications. In the past two decades some progress has been made and several baculovirus genes that influence host-range have been identified.Despite this progress, our understanding of the underlying mechanisms that restrict baculovirus host-range is still limited. Here we review what is currently known about baculovirus genes that influence virus host-range.

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>

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.

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.

A case of human highly pathogenic avian influenza in Shenzhen, China: application of field epidemiological study / 中华流行病学杂志

<p><b>OBJECTIVE</b>Based on analyzing the characteristics of a case with human avian influenza and the effects of field epidemiological study.</p><p><b>METHODS</b>An emergency-response-system was started up to follow the probable human Highly Pathogenic Avian Influenza case initially detected by the "Undefined Pneumonia Surveillance System of Shenzhen". Public health professionals administered several epidemiologic investigations and giving all the contacts of the patient with a 7-day-long medical observation for temporally related influenza-like illness. Reverse transcriptase-polymerase chain reaction (RT-PCR) with primers for H5 and N1 was applied to test respiratory tract samples and/or throat swabs of the patient and all his contacts specific for the hemagglutinin gene of influenza A H5N1. Activities and strategies such as media response,notification in the public, communications with multiple related sectors, social participation and information exchange with Hong Kong were involved in field control and management.</p><p><b>RESULTS</b>The patient was a male, 31 years old,with an occupation as a truck driver in a factory,and had been residing in Shenzhen for 7 years. Started with an influenza-like syndrome, the patient received treatment on the 4th day of the onset, from a clinic and on the 6th day from a regular hospital. On the 8th day of the disease course, he was confirmed by Shenzhen Center for Disease Control and Prevention as human avian flu case and was then transferred to Intensive Care Unit (ICU). On the 83rd day of commence, the patients was healed and released from the hospital. The patient had no significant exposure to sick poultry or poultry that died from the illness before the onset of the disease. The patient and five family members lived together, but no family member was affected and no contact showed positive results for H5N1. A small food market with live poultry, which was under formal supervision and before illness the patient once visited, located near his apartment. Totally, 35 swabs from live birds and bird's coops in the market for H5 nucleic acid were tested and all were negative. The influenza H5N1 virus isolated for the case was named as A/Guangdong/02/2006 (H5N1) or GD/2/06. Phylogenetic relationships and molecular characterization analysis revealed that all the segments of the H5N1 virus named GD/2/06 still belonged to avian segments. Investigation process and control measures were released to the general public through the media. Soon after the laboratory confirmation, information was released to the society, as well as Hong Kong Center for Health Protection. Local Departments of Agriculture, Industries & Business, and Entry-Exit Inspection & Quarantine Bureau together with the Public Health Department put up combined actions. A computer-based telephone survey was initiated to investigate attitudes and knowledge of residents in town, revealing that positive atmosphere dominated and no panic existed.</p><p><b>CONCLUSION</b>Rapid laboratory diagnosis of the virus was the key for successful treatment and survival result of the case. Still, the pathogen was from birds resources. No human-to-human transmission was observed, however, source of infection was unclear. Field epidemiological study could offer special methods for the responses of emergency public health problems.</p>

Ascovirus and its Evolution / 中国病毒学

Ascoviruses, iridoviruses, asfarviruses and poxviruses are all cytoplasmic DNA viruses. The evolutionary origins of cytoplasmic DNA viruses have never been fully addressed. Morphological, genetic and molecular data were used to test if all four cytoplasmic virus families (Ascoviridae, Iridoviridae, Asfarviridae, and Poxvirirdae) evolved from nuclear replicating baculoviruses and how the four virus groups are related. Molecular phylogenetic analyses using DNA polymerase predicted that cytoplasmic DNA viruses might have evolved from nuclear replicating baculoviruses, and that poxviruses and asfarviruses share a common ancestor with iridoviruses. These three cytoplasmic viruses again shared a common ancestor with ascoviruses. Morphological and genetic data predicted the same evolutionary trend as molecular data predicted. A genome sequence comparison showed that ascoviruses have more baculovirus protein homologues than do iridoviruses, which suggested that ascoviruses have evolved from baculoviruses and iridoviruses evolved from ascoviruses. Poxviruses showed genetic and morphological similarity to other cytoplamic viruses, such as ascoviruses, suggesting it has undergone reticulate evolution via hybridization, recombination and lateral gene transfer with other viruses. Within the ascovirus family, we tested if molecular phylogenetic analyses agree with biological inference; that is, ascovirus had an evolutionary trend of increasing genome size, expanding host range and widening tissue tropism for these viruses. Both molecular and biological data predicted this evolutionary trend. The phylogenetic relationship among the four species of ascovirus was predicted to be that TnAV-2 and HvAV-3 shared a common ancestor with SfAV-1 and the three virus species again shared a common ancestor with DpAV-4.

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>