Seropositive Reaction Rates of 9 B-Cell Epitopes of the SARS-CoV-2 Spike Protein and the Relationship between the Epitopes and Neutralizing Antibody.

OBJECTIVE: The aim of the study was to analyze the relationship between serum antibody and neutralizing antibody titers in convalescent coronavirus disease 2019 (COVID-19) patients with different disease severities, and the seropositive reaction rates of 9 reported B-cell epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Serum IgG and total antibody titers of 165 convalescent COVID-19 patients were determined by chemiluminescence, the serum neutralization antibody titers were determined by microneutralization assay, and the S/CO values of 9 peptides were detected by indirect enzyme-linked immunosorbent assay. Correlations between the aforementioned indexes were statistically analyzed, and differences in patients with different diseases severities were evaluated. RESULTS: IgG, total antibody, and neutralizing antibody titers increased with disease severity. The positive rate of the receptor-binding region (RBD) was 100%, and the average positive rate for all the 9 peptides was above 50% in 165 patients. IDf showed the highest rate of positivity (86.06%), with a rate of 95% for the (IDf + IDa) pattern. Moreover, S/CO values of RBD and mix (IDh) were significantly correlated with IgG, total antibody titers, and neutralizing antibody titers (p < 0.001), whereas the S/CO values for other 8 peptides showed no obvious correlation. CONCLUSION: In this study, a large sample was used to confirm that the peptide IDf had a high positive reaction rate for all patients (86.06%) and also had the highest detection rate in asymptomatic patients (86.67%). Only long peptide and mixed peptide showed correlation with neutralizing antibody titers, suggesting that the ability of SARS-CoV-2 antibody to neutralize virus infectivity may require the interaction of multiple sites.

A newly identified linear epitope on non-RBD region of SARS-CoV-2 spike protein improves the serological detection rate of COVID-19 patients.

BACKGROUND: Serological test is helpful in confirming and tracking infectious diseases in large population with the advantage of fast and convenience. Using the specific epitope peptides identified from the whole antigen as the detection antigen is sensitive and relatively economical. The development of epitope peptide-based detection kits for COVID-19 patients requires comprehensive information about epitope peptides. But the data on B cell epitope of SARS-CoV-2 spike protein is still limited. More importantly, there is a lack of serological data on the peptides in the population. In this study, we aimed to identify the B cell epitope peptides of spike protein and detect the reactivity in serum samples, for further providing data support for their subsequent serological applications. RESULTS: Two B cell linear epitopes, P104 and P82, located in non-RBD region of SARS-CoV-2 S protein were identified by indirect ELISA screening of an overlapping peptide library of the S protein with COVID-19 patients' convalescent serum. And the peptides were verified by testing with 165 serum samples. P104 has not been reported previously; P82 is contained in peptide S21P2 reported before. The positive reaction rates of epitope peptides S14P5 and S21P2, the two non-RBD region epitopes identified by Poh et al., and P82 and P104 were 77.0%, 73.9%, 61.2% and 30.3%, respectively, for 165 convalescent sera, including 30 asymptomatic patients. Although P104 had the lowest positive rate for total patients (30.3%), it exhibited slight advantage for detection of asymptomatic infections (36.7%). Combination of epitopes significantly improved the positive reaction rate. Among all combination patterns, (S14P5 + S21P2 + P104) pattern exhibited the highest positive reaction rate for all patients (92.7%), as well as for asymptomatic infections (86.7%), confirming the feasibility of P104 as supplementary antigen for serological detection. In addition, we analyzed the correlation between epitopes with neutralizing antibody, but only S14P5 had a medium positive correlation with neutralizing antibody titre (rs = 0.510, P < 0.01). CONCLUSION: Our research proved that epitopes on non-RBD region are of value in serological detection especially when combination more than one epitope, thus providing serological reaction information about the four epitopes, which has valuable references for their usage.

Study on the COVID-19 infection status, prevention and control strategies among people entering Shenzhen.

BACKGROUND: The novel coronavirus disease 2019 (COVID-19) confirmed cases overseas have continued to rise in the last months, and many people overseas have chosen to return to China. This increases the risk of a large number of imported cases which may cause a relapse of the COVID-19 outbreak. In order to prevent imported infection, the Shenzhen government has implemented a closed-loop management strategy using nucleic acid testing (NAT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and requiring 14 days of medical observation for individuals with an overseas tour history (Hong Kong, Macao, Taiwan province and other countries). Our study aims to describe the status of COVID-19 infection among people entering Shenzhen, and to evaluate the effect of the closed-loop management strategy. METHODS: We undertook a descriptive study and risk analysis by the entry time, time of reporting, and local confirmed cases in countries of origin. The NAT were completed in Shenzhen Center for Disease Control and Prevention (CDC), ten district-level CDCs, and fever clinics. RESULTS: A total of 86,844 people from overseas entered Shenzhen from January 1 to April 18, 2020; there were 39 imported COVID cases and 293 close contacts. The infection rate of people entering was 4.49‰ [95% Confidence interval (CI): 3.26‰-6.05‰]. Fourteen imported cases (35.9%) came from the UK, and nine (23.08%) came from the USA. People entering from the USA since March 9 or from the UK since March 13 are the high-risk population. As of July 17, there have been no new confirmed cases in Shenzhen for 153 days, and the numbers of confirmed case, close contacts, and asymptomatic cases are 0. CONCLUSIONS: The closed-loop management has been effective in preventing imported infection and controlling domestic relapse. The distribution of entry time and report time for imported cases overseas was similar. This shows that it is important to implement closed-loop management at the port of entry.

Genomic characterization of influenza A (H7N9) viruses isolated in Shenzhen, Southern China, during the second epidemic wave.

There were three epidemic waves of human infection with avian influenza A (H7N9) virus in 2013-2014. While many analyses of the genomic origin, evolution, and molecular characteristics of the influenza A (H7N9) virus have been performed using sequences from the first epidemic wave, genomic characterization of the virus from the second epidemic wave has been comparatively less reported. In this study, an in-depth analysis was performed with respect to the genomic characteristics of 11 H7N9 virus strains isolated from confirmed cases and four H7N9 virus strains isolated from environmental samples in Shenzhen during the second epidemic wave. Phylogenetic analysis demonstrated that six internal segments of the influenza A (H7N9) virus isolated from confirmed cases and environmental samples in Shenzhen were clustered into two different clades and that the origin of the influenza A (H7N9) virus isolated from confirmed cases in Shenzhen was different from that of viruses isolated during the first wave. In addition, H9N2 viruses, which were prevalent in southern China, played an important role in the reassortment of the influenza A (H7N9) virus isolated in Shenzhen. HA-R47K and -T122A, PB2-V139I, PB1-I397M, and NS1-T216P were the signature amino acids of the influenza A (H7N9) virus isolated from confirmed cases in Shenzhen. We found that the HA, NA, M, and PA genes of the A(H7N9) viruses underwent positive selection in the human population. Therefore, enhanced surveillance should be carried out to determine the origin and mode of transmission of the novel influenza A (H7N9) virus and to facilitate the formulation of effective policies for prevention and containment of a human infection epidemics.

Seroprevalence to avian influenza A(H7N9) virus among poultry workers and the general population in southern China: a longitudinal study.

BACKGROUND: Confirmed cases of avian influenza A(H7N9) virus infection in humans continue to occur in mainland China. Few confirmed cases have occurred in poultry workers despite potentially higher rates of exposure. METHODS: A serological survey was conducted in May and December 2013 in poultry market workers, and in March and September 2013 in the general population. Blood samples were collected and tested for antibodies to H7N9 and H5N1 viruses by hemagglutination inhibition (HI) assays. Multivariable analysis was employed to identify risk factors related to H7N9 infection indicated by serology among poultry workers. RESULTS: In the poultry workers, 36 of 501 (7.2%) in May and 56 of 375 (14.9%) in December had HI antibody titers ≥1:160 to H7N9. Of 96 individuals who participated in both surveys, 52 (54.2%) workers had a ≥4-fold rise in H7N9 antibody titers from May to December. In a multivariable analysis, female sex (odds ratio [OR], 2.713; 95% confidence interval [CI], 1.098-6.705) and ≥10 years of occupational exposure (OR, 3.592; 95% CI, 1.246-10.354) were identified as risk factors for infection. Seroprevalence against H5N1 at ≥1:160 was low in May (4/501 [0.8%]) and December (3/375 [0.8%]). In the general population, 0 of 417 individuals in March and 0 of 408 individuals in September had antibody titers ≥1:160 to H7N9 or to H5N1. CONCLUSIONS: Although none of the participants in our study had virologically confirmed H7N9 infection, the high proportion of poultry workers with serologic evidence of H7N9 infection between May and December 2013 suggests a substantial risk of mild H7N9 infections in this group, supporting stricter control measures in live poultry markets.

Epidemiological dynamics and phylogeography of influenza virus in southern China.

BACKGROUND: Understanding the epidemiological dynamics of influenza virus is central to surveillance and vaccine strain selection. It has been suggested that tropical and subtropical regions represent the global source of influenza epidemics. However, our understanding of the epidemiological dynamics of influenza virus in these regions is limited by a relative lack of long-term data. METHODS: We analyzed epidemiological and virological data on influenza recorded over a period of 15 years from the metropolitan city of Shenzhen in subtropical southern China. We used wavelet analysis to determine the periodicity of influenza epidemics and molecular phylogeographic analysis to investigate the role of Shenzhen and southern China in the global evolution of influenza virus. RESULTS: We show that southern China is unlikely to represent an epicenter of global influenza activity, because activity in Shenzhen is characterized by significant annual cycles, multiple viral introductions every year, limited persistence across epidemic seasons, and viruses that generally are not positioned on the trunk of the global influenza virus phylogeny. CONCLUSIONS: We propose that novel influenza viruses emerge and evolve in multiple geographic localities and that the global evolution of influenza virus is complex and does not simply originate from a southern Chinese epicenter.

Insight into the practical performance of RT-PCR testing for SARS-CoV-2 using serological data: a cohort study.

BACKGROUND: Virological detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through RT-PCR has limitations for surveillance. Serological tests can be an important complementary approach. We aimed to assess the practical performance of RT-PCR-based surveillance protocols and determine the extent of undetected SARS-CoV-2 infection in Shenzhen, China. METHODS: We did a cohort study in Shenzhen, China and attempted to recruit by telephone all RT-PCR-negative close contacts (defined as those who lived in the same residence as, or shared a meal, travelled, or socially interacted with, an index case within 2 days before symptom onset) of all RT-PCR-confirmed cases of SARS-CoV-2 detected since January, 2020, via contact tracing. We measured anti-SARS-CoV-2 antibodies in serum samples from RT-PCR-negative close contacts 2-15 weeks after initial virological testing by RT-PCR, using total antibody, IgG, and IgM ELISAs. In addition, we did a serosurvey of volunteers from neighbourhoods with no reported cases, and from neighbourhoods with reported cases. We assessed rates of infection undetected by RT-PCR, performance of RT-PCR over the course of infection, and characteristics of individuals who were seropositive on total antibody ELISA but RT-PCR negative. FINDINGS: Between April 12 and May 4, 2020, we enrolled and collected serological samples from 2345 (53·0%) of 4422 RT-PCR-negative close contacts of cases of RT-PCR-confirmed SARS-CoV-2. 1175 (50·1%) of 2345 were close contacts of cases diagnosed in Shenzhen with contact tracing details, and of these, 880 (74·9%) had serum samples collected more than 2 weeks after exposure to an index case and were included in our analysis. 40 (4·5%) of 880 RT-PCR-negative close contacts were positive on total antibody ELISA. The seropositivity rate with total antibody ELISA among RT-PCR-negative close contacts, adjusted for assay performance, was 4·1% (95% CI 2·9-5·7), which was significantly higher than among individuals residing in neighbourhoods with no reported cases (0·0% [95% CI 0·0-1·1]). RT-PCR-positive individuals were 8·0 times (95% CI 5·3-12·7) more likely to report symptoms than those who were RT-PCR-negative but seropositive, but both groups had a similar distribution of sex, age, contact frequency, and mode of contact. RT-PCR did not detect 48 (36% [95% CI 28-44]) of 134 infected close contacts, and false-negative rates appeared to be associated with stage of infection. INTERPRETATION: Even rigorous RT-PCR testing protocols might miss a substantial proportion of SARS-CoV-2 infections, perhaps in part due to difficulties in determining the timing of testing in asymptomatic individuals for optimal sensitivity. RT-PCR-based surveillance and control protocols that include rapid contact tracing, universal RT-PCR testing, and mandatory 2-week quarantine were, nevertheless, able to contain community spread in Shenzhen, China. FUNDING: The Bill & Melinda Gates Foundation, Special Foundation of Science and Technology Innovation Strategy of Guangdong Province, and Key Project of Shenzhen Science and Technology Innovation Commission.

Incidence of novel coronavirus (2019-nCoV) infection among people under home quarantine in Shenzhen, China.

BACKGROUND: Since the outbreak of 2019-nCoV in December, Chinese government has implemented various measures including travel bans, centralized treatments, and home quarantines to slowing the transmission across the country. In this study, we aimed to estimate the incidence of 2019-nCoV infection among people under home quarantine in Shenzhen, China. METHODS: We used a stratified multistage random sampling method to recruit participants and collected demographic information and laboratory results of people under home quarantine. We conducted descriptive analysis to estimate the basic characteristics and to calculate the incidence in out study population. RESULTS: A total of 2004 people under home quarantine participated in this study, of which 1637 participants finished the questionnaire with a response rate of 81.7%. Mean age of the participants was 33.7 years, ranging from 0.3 to 80.2 years. Of people who provided clear travel history, 129 people have traveled to Wuhan city and 1,046 people have traveled to other cities in Hubei province within 14 days before the home quarantine. Few (less than 1%) participants reported contact history with confirmed or suspected cases during their trip and most of these arrived at Shenzhen between Jan 24, 2020 to Jan 27, 2020. The incidence of COVID-19 in the sample was 1.5‰ (95% CI: 0.31‰-4.37‰). CONCLUSION: Home quarantine has been effective in preventing the early transmission of COVID-19, but that more needs to be done to improve early detection of COVID-19 infection.

[Identification and sequence analysis of E gene of Dengue virus type 2 strain isolated from patient serum in Shenzhen].

OBJECTIVE: To isolate and identify the pathogen of Dengue fever from Shenzhen city in 2005 - 2006, and to analyze the molecular characteristics of the isolated Dengue virus strain as well as to explore its possible origin. METHODS: IgM and IgG of serum samples taken from 60 suspected Dengue fever patients were detected by ELISA and immunochromatography, and 9 specimens were positive. Nine samples from patients with early stage Dengue fever were used to isolate virus with C6/36 cell line and the positive cell cultures were identified by MGB fluorescent PCR. The type of isolated virus strain was determined by RT-semi-nested-PCR and fluorescent PCR. E gene of isolated virus strain was amplified by RT-PCR and sequenced. Homology and phylogenetic tree of E gene of Shenzhen Dengue virus with the strains isolated from other areas were constructed. RESULTS: Of nine antibody-positive serum samples, one strain of Dengue virus was successfully isolated. The isolated virus strain was confirmed as Dengue virus type 2 and designated as DEN2-SZ0521. The homology of nucleotide sequence and the deduced amino acid sequence of E gene of SZ0521 with standard type 2 Dengue virus NGC strain was 94.2% and 98.2%, but the homology with standard Dengue virus 1, 3, 4 in the same fragment were 59.1%, 57.2%, 58.5% and 68.1%, 66.7%, 63.2%, respectively. The phylogenetic tree indicated that SZ0521 had the greatest similarity with the Malay0412a/Tw strain and they lied in the same branch of the phylogenetic tree. The corresponding homology of nucleotide sequence and amino acid sequence was 99.8% and 100%, respectively. The isolated Dengue virus type 2 belonged to genotype IV with Indonesia-76, Somalia-84 and Sri Lanka-90. CONCLUSION: Dengue virus was isolated from Shenzhen for the first time, and it was classified as type 2. It was confirmed that the type 2 Dengue virus may come from the epidemic area in Malaysia.

[Epidemiological and molecular characterization of seasonal influenza A/H3N2 viruses circulating in Shenzhen, 2005 - 2007].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Rapid detection of influenza virus N2 subtype by fluorescence quantitative RT-PCR].

OBJECTIVE: Developing a rapid method to detect influenza virus N2 subtype by fluorescence real-time quantitative RT-PCR. METHODS: According to conservative sequences of NA gene of influenza virus N2 subtype, a pair primers and Taq-man probe were designed, respectively. Using one step RT-PCR kit to set up real-time RT-PCR system for detection of influenza virus N2 subtype, after the standard quantitative curve of the assay was established using 10-fold serial dilution of TCID50, the sensitivity was determined. The specificity and test for influenza virus illness (ILI) samples were also determined using this RT-PCR system. RESULTS: The sensitivity of detection influenza virus N2 subtype was 2.56 x 10(-6) and the regression coefficient of the quantitative curve was 0.997. The amplification efficiency and specificity of this assay was 99.9% and 100%, respectively. The real-time RT-PCR for N2 subtype results of ILI samples was in accordance with HAI method completely. CONCLUSION: The detection system based on real-time RT-PCR could be utilized to rapidly and sensitively detect influenza virus N2 subtype.

A multiplex real-time RT-PCR for detection and identification of influenza virus types A and B and subtypes H5 and N1.

A multiplex real-time RT-PCR method for the simultaneous detection of influenza virus types A and B and identification of subtypes H5 and N1 in a single tube is described. The method was developed with four sets of primers and probes which were specific to influenza virus (sub)types A, B, H5, and N1, and evaluated by using a total of 40 influenza virus reference strains, including 17 avian influenza A (12 H5N1, 1 H1N1, 1 H3N2, 1 H4N6, 1 H7N3, and 1 H9N2), 18 human influenza A (11 H3N2, 6 H1N1 and 1 H5N1) and 5 influenza B viruses. The method exhibited a high specificity and sensitivity of approximately 10(1)-10(2)copies/microl for each (sub)type and a high reproducibility with intra- and inter-assay CV from 0.13 to 4.24%. In an analysis of 189 clinical samples from patients during the year 2004 and 2005, the method identified 81 positive samples (42.9%) and identified simultaneously 14 type B samples and 11 subtype N1 samples, in comparison only 46 positive samples (24.3%) identified by the conventional culturing method. The method would be a useful molecular diagnostic tool for large-scale screening of clinical samples for influenza virus.

[Study on the molecular characteristic of natural infection of rodents with Hantaviruses in Shenzhen city].

OBJECTIVE: In order to investigate Hantavirus (HV) infection of captured rodents and to understand the genotypes and the molecular characteristic of Hantaviruses in Shenzhen. METHODS: The captured rodents were classified and the density of distribution was calculated. A total of 472 animals were captured, among which Rattus norvegicus was the dominant group. The total viral RNA was extracted from the lung tissues positive with HV antigens by immunofluorescent assay and gene sequence of M fragment was amplified with RT-nested-PCR by using the Hantavirus genotype specific primers. The amplified genes were then sequenced, and subjected to genotyping and homology analysis. RESULTS: The results of genotype analysis showed that the Hantaviruses taken from twenty-one lung specimens in Rattus norvegicus in Shenzhen city belonged to the Hantavirus type II (SEOV). Results in homology analysis suggested that the homology among twenty-one samples should be rather high with 95.4% of nucleotide sequence identity and they belonged to the same subtype. Phylogenetic tree analysis showed that they were branched into at least six different lineages, and were highly homologized with SZ2083. We also found that these virus strains had not shown more highly homology of nucleotide sequence in nearest district, whereas revealed consistency in farther district. CONCLUSION: The major hosts of Hantaviruses in Shenzhen city were Rattus norvegicus and the epidemic strains were genotyped as SEO-type. Nucleotide sequence and deduced amino acid sequence from different rodents were highly homologous, while nucleotide mutation had also been observed. Further studies are required to explore the possible viruses' sequence mutation.

[Study on characterization of the hemagglutinin gene of avian influenza virus H5N1 subtype originated from the pan Pacific regions].

OBJECTIVE: To understand the gene characterization of hamagglutinin (HA) of AIV H5N1 subtype originated from the pan Pacific regions in order to find out its mutation possibility. METHODS: 50 HA gene sequences originated from pan Pacific regions were downloaded, including Thailand, Vietnam, Hong Kong, Indonesia, mainland of China, Mongolia and Russia, the alignments of nucleic acid and amino acid sequence of HA gene were analyzed, and the phylogenetic relationships among 50 H5N1 high pathogenic avian influenza (HPAI) virus were evaluated by bio-software DNASTAR 5.0 and MEGA 3.1. RESULTS: 50 HPAI viruses were separated into 3 clusters, including Thailand and Vietnam cluster, China-Indonesia-Northern Asia cluster and the other cluster primary composed by isolates from 1996 to 2001 similar with Gs/Gd like or the other in the respect of genetype. The analysis of HA molecular characterization shows that the isolates from Thailand and Vietnam could have the similar antigenic profiles, while the others could have different molecular characterizations especially in the respect of epitope determinant sites, besides the similarity in a certain zones. CONCLUSION: Hamagglutinin characterization of HPAI virus originated from the pan Pacific regions could show zone specificity, as well as with the similarity in a certain range.

[Rapid detection of influenza virus A by fluorescence quantitative RT-PCR].

OBJECTIVE: To develop a method to rapidly detect influenza virus type A by fluorescence real-time quantitative RT-PCR. METHODS: According to conservative sequence of influenza virus type A M2 gene, a pair primers and Taqman probe were designed, respectively. After the quantitative curve of the assay were established using tenfold serial dilution of TCID50, the sensitivity and the specificity of clinic samples were determined. RESULTS: The detection sensitivity of the assay was 2.56 x 10(-5) TCID50 and the regression coefficient of the quantitative curve was 0.999. The specificity was determined by testing five other specimens, all of which yielded negative results. CONCLUSION: The detection system based on real-time RT-PCR was rapid, sensitive and steady, which could be used to detect the clinic samples.

[Study on the rapid detection of Dengue viruses by Taqman MGB real-time fluorescent PCR].

OBJECTIVE: To develop the Taqman MGB real-time fluorescent PCR assay for rapid detection of Dengue viruses. METHODS: Using Taqman MGB technique, a pair of universal primers and Taqman MGB probe were designed according to a highly reserved sequence of the 3'-noncoding region of dengue viruses type 1-4. Dengue virus strains were used as standard and Japanese encephalitis virus strains were used as control, the real-time PCR assay for specific and sensitive detection of the dengue viruses was established. While 8 serum specimens of ELISA positive were detected by the RT-PCR and fluorescent PCR. RESULTS: The sensitivity of real-time PCR was 0.17pg/microl (cDNA)or 10(-5)TCID50. There was no cross-reaction with Japanese encephalitis virus. Of 8 specimens, 2 were positive by RT-PCR and 5 were positive by real-time PCR. The test could be completed in 4 hours. CONCLUSION: The Taqman MGB real-time PCR assay was fast, sensitive and specific. It could be applied to the quick early diagnosis of dengue viruses.

[Expression and assay in vitro of phage single chain antibody against nucleoprotein of influenza virus A type].

OBJECTIVE: To expression the phage single chain antibody against nucleoprotein of influenza virus A type in E. coli strain HB2151 by screening the positive clone from the phage antibody library against nucleoprotein, which will prepare for the construction of fast leak kit of Influenza virus A type. METHODS: The positive clone ratio in the phage antibody library was enriched by three-round continual solid panning, ELISA, SDS-PAGE and Western-blot methods were used to analyze the protein secreted into the supemants and periplasmic. Affinity chromatography was used to purified the protein expressed in periplasmic and the titer was also analyzed using ELISA method. RESULTS: 10 clones were screened from96 clones and among the 10, there were 3 stronger positive with OD450nm value of 0.469, 0.582 and 0.507, respectively. The phage single chain antibody against Influenza virus A type was primary expressed in periplasmic. The ELISA results were positive even if the single chain antibody purified by affinity chromatography was diluted 4096 folds. CONCLUSION: Using phage antibody library technique, phage single antibody against nucleoprotein of influenza virus A type was preliminary expressed in E. coli.

[Evaluation on construction and expression in vitro of nucleic acid vaccine of nucleoprotein of influenza virus A].

OBJECTIVE: Constructing nucleic acid vaccine of influenza virus A to study the protective effects. METHODS: NP gene was reverse transcripted from influenza virus A and linked with pSECTAG 2/Hygro A to constructed nucleic acid vaccine of influenza virus A. The constructed nucleic acid vaccine was transinfected into VERO cell resorting to lipofectamineTM2000. NP gene of influenza virus A was expressed in VERO cell by the method of ELISA. RESULTS: The results showed that the NP gene was expressed to the highest level at 36h after transinfection with the A40 value of 0.382. From 36 h to 60 h after transinfection, the expression of NP gene was stable (the A45, value at 48h and 60h was 0.385 and 0.387, respectively) . CONCLUSION: The nucleic acid vaccine of influenza virus A was successfully constructed, which sets up groudworks for the research of effection of nucleic acid vaccine in vivo.

[Detection of SARS-coronavirus in both human and animals by RT-PCR].

OBJECTIVE: To find the most suitable RT-PCR detection method for SARS-Coronavirus (SARS-CoV) detection in both human and animals, and to study the source of SARS virus by investigating the condition of virus carried by wild, domestic animals and animals sold in market. METHODS: 350 throat washes of confirmed, suspected and observed SARS cases were tested by TaqMan and molecular beacon fluorescence RT-PCR methods. 386 animals with 442 nasal-throat swabs, fecal swabs of animals and cell cultured were detected by TaqMan method and conventional RT-PCR method. RESULTS: 10 positive were detected from 41 SARS clinical confirmed cases (24.39%). The results of TaqMan and molecular beacon are basically coincident with the coincident rate of 88.89%. 18 cell cultures with CPE were detected and find 16 positive, among which, 14 were civet cats (87.5%). The detecting results of animal samples from Dongmen Market (Shenzhen) and other markets and farms in peripheral areas show that: the total positive rate of 4 kinds of wild animal (civet cat, raccoon dog, hog-badge and Chinese ferret-badge) is 39.02% , which has an significant difference (P < 0.01) compared with the positive rate of other animals as 0. The positive rate of PCR for nasal and fecal swabs from 109 major wild animals is 44.04%, but the positive rate of 145 wild live-animals is 0. CONCLUSION: TaqMan and molecular beacon PCR methods both can be used in SARS detection.The results could support the hypothesis that animals (especially civet cat) could carry SARS virus.