RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens.

OBJECTIVE: To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases. METHODS: A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID 50 per µL and copies per µL, and the newly established methods were tested in clinical specimens collected in recent years. RESULTS: The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID 50 per µL and 10 virus copies per µL, and for the complete VP1 gene was 1 CCID 50 per µL and 100 virus copies per µL, using serially-diluted virus stocks of five serotypes. As a proof-of-concept, 25 serotypes were identified and complete VP1 sequences of 23 serotypes were obtained by this system among 858 clinical specimens positive for HEVs during the past eight surveillance seasons. CONCLUSION: This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

Incidence of human rabies and characterization of rabies virus nucleoprotein gene in dogs in Fujian Province, Southeast China, 2002-2012.

BACKGROUND: Rabies is a global fatal infectious viral disease that is characterized by a high mortality after onset of clinical symptoms. Recently, there has been an increase in the incidence of rabies in China. The aim of this study was to investigate the incidence of human rabies and characterize the rabies virus nucleoprotein gene in dogs sampled from Fujian Province, Southeast China from 2002 to 2012. METHODS: Data pertaining to human rabies cases in Fujian Province during the period from 2002 through 2012 were collected, and the epidemiological profiles were described. The saliva and brain specimens were collected from dogs in Quanzhou, Longyan and Sanming cities of the province, and the rabies virus antigen was determined in the canine saliva specimens using an ELISA assay. Rabies virus RNA was extracted from canine brain specimens, and rabies virus nucleoprotein gene was amplified using a nested RT-PCR assay, followed by sequencing and genotyping. RESULTS: A total of 226 human rabies cases were reported in Fujian Province from 2002 to 2012, in which 197 cases were detected in three cities of Quanzhou, Longyan and Sanming. ELISA assay revealed positive rabies virus antigen in six of eight rabid dogs and 165 of 3492 seemingly healthy dogs. The full-length gene fragment of the rabies virus nucleoprotein gene was amplified from the brain specimens of seven rabid dogs and 12 seemingly healthy dogs. Sequence alignment and phylogenetic analysis revealed that these 19 rabies virus nucleoprotein genes all belonged to genotype I, and were classified into three genetic groups. Sequencing analysis showed a 99.7% to 100% intra-group and an 86.4% to 89.3% inter-group homology. CONCLUSIONS: This study is the first description pertaining to the epidemiological characteristics of human rabies cases and characterization of the rabies virus nucleoprotein gene in dogs in Fujian Province, Southeast China. Our findings may provide valuable knowledge for the development of strategies targeting the prevention and control of rabies.

Neutralizing antibodies respond to a bivalent dengue DNA vaccine or/and a recombinant bivalent antigen.

There is currently no effective vaccine to prevent dengue infection, despite the existence of multiple studies on potential methods of immunization. The aim of the present study was to explore the effect of DNA and/or recombinant protein on levels of neutralizing antibodies. For this purpose, envelope domain IIIs of dengue serotypes 1 and 2 (DEN-1/2)were spliced by a linker (Gly­Gly­Ser­Gly­Ser)3 and cloned into the prokaryotic expression plasmid pET30a (+) and eukaryotic vector pcDNA3.1 (+). The chimeric bivalent protein was expressed in Escherichia coli, and one­step purification by high­performance liquid chromatography was conducted. Protein expression levels of the DNA plasmid were tested in BHK­21 cells by indirect immunofluorescent assay. In order to explore a more effective immunization strategy and to develop neutralizing antibodies against the two serotypes, mice were inoculated with recombinant bivalent protein, the DNA vaccine, or the two given simultaneously. Presence of the specific antibodies was tested by ELISA and the presence of the neutralizing antibodies was determined by plaque reduction neutralization test. Results of the analysis indicated that the use of a combination of DNA and protein induced significantly higher titers of neutralizing antibodies against either DEN­1 or DEN­2 (1:64.0 and 1:76.1, respectively) compared with the DNA (1:24.7 and 1:26.9, DEN­1 and DEN­2, respectively) or the recombinant protein (1:34.9 and 1:45.3 in DEN­1 and DEN­2, respectively). The present study demonstrated that the combination of recombinant protein and DNA as an immunization strategy may be an effective method for the development of a vaccine to prevent dengue virus infection.

The effect of temperature on the extrinsic incubation period and infection rate of dengue virus serotype 2 infection in Aedes albopictus.

Dengue fever is an acute mosquito-borne viral disease caused by dengue virus (DENV). Temperature may affect the efficiency of the mosquito vectors in spreading DENV. Aedes albopictus mosquitoes were infected orally with a DENV2 suspension and incubated at different temperatures. Subsequently, DENV2 antigen was collected from salivary gland and thorax-abdomen samples on different days postinfection and tested using an immunofluorescence assay to determine the extrinsic incubation period and infection rate. As the temperature increased, the extrinsic DENV2 incubation period in Ae. albopictus gradually shortened, and infection rates showed a tendency to initially increase, followed by a subsequent decrease.

[Characteristics of complete genome of pandemic A/H1N1/2009 influenza virus isolated in Fujian Province, China].

This study aims to investigate the characteristics of genomic variation of pandemic A/H1N1/2009 influenza virus isolated in Fujian Province, China. Complete genome sequence analysis was performed on 14 strains of pandemic A/H1N1/2009 influenza virus isolated from Fujian during 2009-2012. All virus strains were typical low-pathogenic influenza viruses, with resistance to amantadine and sensitivity to neuraminidase inhibitors. Eight genome fragments of all strains were closely related to those of A/California/07/2009 (H1N1) vaccine strain, with > or = 98.2% homology. Compared with the vaccine strain, the influenza strains from Fujian had relatively large variation, and variation was identified at 11 amino acid sites of the HA gene of A/Fujiangulou/SWL1155/2012 strain, including 4 sites (H138R, L161I, S185T, and S203T) involved inthree antigen determinants (Ca, Sa, and Sb). In conclusion, the influenza vaccine has a satisfactory protective effect on Fujian population, but the influenza strains from Fujian in 2012 has antigenic drift compared with the vaccine strain, more attention should therefore be paid to the surveillance of mutations of pandemic A/H1N1/2009 influenza virus.

[Molecular epidemiology of HFMD-associated pathogen coxsackievirus A6 in Fujian Province, 2011-2013].

In order to characterize the molecular epidemiology of HFMD-associated Coxsackievirus A6 (CVA6) in Fujian Province, a total of 1340 specimens from non-EV71 non-CVA16 HFMD patients were collected during 2011-2013. Isolated virus strains were identified and subtyped. Full-length coding regions for the VP1 gene of the predominant serotype CVA6 isolates were amplified and sequenced. Among the 375 non-EV71 non-CVA16 HFMD cases confirmed by virus isolation and molecular subtyping, 182 (48.5%) were found to be caused by CVA6, accounting for 7.9%, 16.2% and 39.6% HFMD-associated enteroviruses in FujianProvince during 2011, 2012, and 2013, respectively. Compared with general features observed in the HFMD epidemic, no difference in CVA6-specificity or severity rates was observed between geographical origins, gender, or age groups. Nucleotide sequence analyses of VP1 genes revealed high diversity levels of 16.2%-18.6% among CVA6 strains from Fujian Province, in contrast to the prototype CVA6 strain, and showed low levels of diversity in the amino acid sequences (4.3%-6.2%). Phylogenetic analysis also indicated that CVA6 isolates from Fujian Province were distinct from the prototype strain and other isolates from abroad; however, it was homologous to domestic strains, although the Fujian isolates clustered into multiple branches. These results suggested that significant changes in the pathogenic spectrum of HFMD in Fujian Province occurred during 2011-2013, as CVA6 was one of the predominant serotypes of HFMD. CVA6 isolates from Fujian Province were co-circulating and co-evolving with other domestic strains as multiple closely related CVA6 transmission chains were observed in Fujian Province overall and within each prefecture.

Molecular epidemiology of Echovirus 30 in Fujian, China between 2001 and 2011.

Echovirus 30 (E-30) was responsible for an outbreak of aseptic meningitis between April 1 and June 2, 2011 in Fujian Province, China. A molecular epidemiology study of 115 E-30 strains was performed to characterize the genetic features of the etiologic agent of the 2011 aseptic meningitis outbreak. The phylogenetic trees of the complete VP1 gene (876 bp) from 74 of 115 isolates and 50 reference sequences were analyzed. Three lineages (E-30_h, i, and j) were detected that had co-circulated in Fujian in the last decade, of which E-30_j was new. The other 72 Fujian strains and 16 representative strains from other provinces of China all belong to E-30_h and E-30_i. Two distinct E-30 clusters including virus isolates obtained during adult surveillance were associated with the 2011 outbreak and differed from Fujian isolates prior to 2011, suggesting that the viruses may vary and adult infections play an important role in viral transmission. Thus, the multiple lineages of E-30 in Fujian and variant viruses enhanced transmissibility, which may be related to the epidemic activity of E-30.

Molecular epidemiology of hepatitis C virus among different groups of people in the province of Fujian, China.

Genotyping of hepatitis C virus (HCV) can provide valuable information for prognosis and treatment duration prediction. To explore the genetic diversity of HCV in Fujian Province, China, 112, 104 and 48 anti-HCV-positive serum samples were collected from volunteer blood donors, IDUs and patients, respectively, from Jan 2008 to Dec 2008 and were genotyped through sequence analysis, followed by phylogenetic analysis in the C/E1 and NS5B regions. Genotypes could be determined for 85.61 and 84.85 % of samples in the C/E1 and NS5B region, respectively. 6a was the most prevalent subtype, which accounted for 42.04 and 43.75 % in the C/E1 and NS5B region, respectively. Mixed infection and potential recombination were detected in this study. Kappa tests indicated that similar results were obtained by two genotyping methods targeting the C/E1 and NS5B regions. The differences in the main prevalent subtype between the three target groups suggest diversity of HCV prevalence in different populations.

Identification of origins of marker chromosomes using fluorescence in situ hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the value of fluorescence in situ hybridization (FISH) and bacterial artificial chromosome FISH (BAC-FISH) for the diagnosis for patients with marker chromosomes.</p><p><b>METHODS</b>Sixteen patients with marker chromosomes were analyzed with technologies including GTG-banding, Q-banding, multiplex FISH and BAC-FISH.</p><p><b>RESULTS</b>The marker chromosomes in the 16 patients were verified as der(Y) (2 cases), psu dic(Y) (1 case), psu dic(15) (1 case), dic(15) (1 case), del(Y) (1 case), r(X) (5 cases), i(14 or 22) (2 cases), i(18) (1 case).</p><p><b>CONCLUSION</b>FISH and BAC-FISH can both verify the origin of marker chromosomes and provide accurate information for the diagnosis and treatment of patients.</p>

[Genotype I Japanese encephalitis virus is the main genotype in mosquito in Fujian province].

OBJECTIVE: To grasp the infection rate and genotypes of Japanese encephalitis virus (JEV) in mosquito in Fujian province. METHODS: Mosquito specimens in Sanming city, Jianyang city and Fuzhou city in Fujian province were collected in 2010. RT-PCR was used to detect the JEV sequence from the mosquitoes by specific primers. The sequence splicing and the differentiation analysis for nucleotides, deduced amino acid sequence and phylogenetic tree were performed by the software of ATGC, Clustal X (1.83), MegAlign, GeneDoc 3.2 and Mega (4.0). RESULTS: Totally 6987 mosquitoes were collected and main species was Culex tritaeniorhynchus and Anopheles sinensis. The infection rate of JEV in mosquitoes in Sanming, Jianyang and Fuzhou were 1.25%, 1.76% and 0.65%, respectively. One full genome in the positive specimens was sequenced. And further study showed that the positive JEV sequences belonged to genotype I. CONCLUSION: Genotype I Japanese encephalitis virus is the main genotype in mosquitos in Fujian province.

[Sequencing and analysis of the complete genome sequence of WU polyomavirus in Fuzhou, China].

WU polyomavirus (WUPyV), a new member of the genus Polyomavirus in the family Polyomaviridae, is recently found in patients with respiratory tract infections. In our study, the complete genome of the two WUPyV isolates (FZ18, FZTF) were sequenced and deposited in GenBank (accession nos. FJ890981, FJ890982). The two sequences of the WUPyV isolates in this study varied little from each other. Compared with other complete genome sequences of WUPyV in GenBank (strain B0, S1-S4, CLFF, accession nos. EF444549, EF444550, EF444551, EF444552, EF444553, EU296475 respectively), the sequence length in nucleotides is 5228bp, 1bp shorter than the known sequences. The deleted base pair was at nucleotide position 4536 in the non-coding region of large T antigen (LTAg). The genome of the WUPyV encoded for five proteins. They were three capsid proteins: VP2, VP1, VP3 and LTAg, small T antigen (STAg), respectively. To investigate whether these nucleotide sequences had any unique features, we compared the genome sequence of the 2 WUPyV isolates in Fuzhou, China to those documented in the GenBank database by using PHYLIP software version 3.65 and the neighbor-joining method. The 2 WUPyV strains in our study were clustered together. Strain FZTF was more closed to the reference strain B0 of Australian than strain FZ18.

Construction and characterization of a full-length infectious clone from a fast-replicating, X4-tropic HIV-1 subtype B' isolate.

In HIV-1 epidemics in China, HIV-1 subtype B' is the most predominant subtype circulating in intravenous drug users. In this study, we constructed an HIV-1 full-length infectious molecular clone based on the primary virus LWJ, which was isolated from an HIV-infected patient in Fujian Province, China. Phylogenetic and bootscanning analysis of the viral sequence revealed that the isolate LWJ belonged to HIV-1 subtype B'. The infectious clone was designated as "pLWJ". The virus (LWJ-c) produced from this infectious clone by in vitro transfection of 293T cells could infect both human peripheral blood mononuclear cells (PBMCs) and human the T cell line MT4. Interestingly, the cloned LWJ-c virus utilized CXCR4 as its co-receptor and could replicate in vitro with similar efficiency and kinetics compared to its parental primary isolate LWJ as well as the clade B reference virus NL4-3. The LWJ-c virus could also cause cytopathic effects in both PBMCs and MT cells. Sequence analysis of the envelope glycoprotein of pLWJ showed that a conserved GPGR motif and an arginine at position 11 were present in the V3 loop, which was consistent with previous reports regarding CXCR4 co-receptor usage and syncytium-inducing (SI) phenotype. Thus, the infectious clone represents a fast-replicating, high-producing, CXCR4-tropic and syncytium-inducing isolate. Given the prevalence of HIV-1 subtype B' in China, this infectious clone can be a very useful tool to provide a versatile molecular model for research focusing on the biological properties of this subtype.

[Construction of sub-unit dengue vaccines and analysis of its immunogenicity].

OBJECTIVE: To construct sub-unit vaccines of dengue virus type 1 to 4 and to analyze its immunogenicity. METHODS: Envelope domain III s of dengue serotypes 1 and 2, as well as 3 and 4, were spliced by a linker (Gly-Gly-Ser-Gly-Ser)3 and cloned into vector pET-30a, then transformed into E. coli to express recombinant fusion proteins. The recombinant proteins were purified by high-performance liquid chromatography and mixed to immunize BALB/c mice. The neutralizing antibodies were tested by neutralizing assay, as well as in newborn mice challenged intracranially with dengue virus type 1 to 4. RESULTS: Mice immunized with proteins could produce neutralizing antibodies, with titers of 1:34. 9, 1: 45.3, 1: 24.7 and 1:38.4 for DEN-1 to 4 respectively. 100% newborn mice challenged with DEN-1 or 2 in combination with sera from mice immunized with recombinant proteins were protected, whereas 83% protection was obtained when challenged with DEN-3 or 4. CONCLUSION: The recombinant proteins possess excellent immunogenicity to induce neutralizing antibodies and would be valuable for development of a tetravalent sub-unit vaccine.

[A duplex nested PCR assay detecting of Vibrio cholerae and its application on environmental specimens].

OBJECTIVE: To establish a duplex nested PCR assay system which is capable for detecting O1 and O139 groups of Vibrio cholerae simultaneously, and is applicable to environmental specimens from routine cholera surveillance. METHODS: Based on nucleic acid sequences available in GenBank, six sets of primers were designed by PrimerSelect program of DNAStar, targeting the rfb gene that encodes the O antigens of O1 and O139 V. cholerae, respectively. The specificity of several primer combinations was tested. A duplex nested PCR assay system for simultaneously detecting O1 and O139 V. cholerae was established, subsequently, its sensitivity, specificity, reproducibility and field evaluation were tested. The sensitivity of this assay was evaluated by comparing detection limits of nested PCR and conventional PCR. Its reproducibility was tested by 32 positive samples (11 samples positive for O1, 21 samples positive for O139) from environmental surveillance. In addition, the selected amplicons from positive samples were sequenced and analyzed with relevant sequences. RESULTS: This newly-established duplex nested PCR assay might distinguish O1 V. cholerae from O139 V. cholerae, based on fragment lengths of amplicons, with reliable reproducibility, and no specific amplification was observed as compared with other vibrio species. The sensitivity of this nested PCR was (15 000) higher than conventional PCR, and there was no interference observed with multiple primers and complicated templates in the same vial. In its field evaluation, 32 positive DNA samples were detected and be further confirmed with double or triple tests, implying reliable reproducibility and consistency of this system. These results indicated that this assay had reliable reproducibility. No amplification was observed in all negative specimens and also suggested the acceptable specificity of this assay. Sequence analysis of the selected amplification products revealed 100% homogeneous with relevant genes from V.cholerae, indicating that these amplicons were originated from V. cholerae. CONCLUSION: This duplex nested PCR assay system should be rapid, sensitive and especially applicable to small laboratories, and be suitable for dynamic environmental surveillance.

[RtL164V, a mutation possibly associated with lamivudine resistant HBV].

OBJECTIVE: To study the efficacy of lamivudine treatment in chronic hepatitis B patients affected by structures of HBV P-genes. METHODS: P genes of HBV isolated from sera were amplified by means of one-step PCR and then sequenced. The sequences of the P-genes from responders, primary non-responders and rebounders were compared before and after their lamivudine treatments. RESULTS: (1) Among the primary non-responders and rebounders, commixture genotype B and C was found in 2 patients with genotype B and in 1 patient with genotype C; genotype shift from B into C was also observed in one patient after lamivudine therapy. (2) During the course of the therapy YMDD mutation emerged in all 8 primary non-responders and rebounders, which existed in some patients of the 3 groups before their lamivudine treatment. (3) An rtL164V mutation in the reverse transcriptase region was observed in all primary non-responders before and after lamivudine therapy and also in rebounders when viral breakthrough occurred, which was not seen in the responders. (4) Four amino acid substitutions at rt91, rt168, rt234 and rt256 in the reverse transcriptase region were seen in the rebounders and primary non-responders. CONCLUSION: YMDD mutation was not the only key point closely linked to HBV resistant to lamivudine therapy. RtL164V may be a novel mutation correlated with lamivudine-resistance.

[Study on the public health emergent events in Fujian province in 2004--2007].

OBJECTIVE: To study the public health emergent events (PHEE) in Fujian province, from 2004 to 2007. METHODS: Descriptive and analytic methods were used to analyze the PHEE in Fujian province according to the internet-based surveillance reports. RESULTS: From 2004 to 2007, there were 304 emergency events being surveyed. Of all the events, there were 7 (2.30%) belonged to serious-degree of grade II, 57(18.75%) to grade III and 240 (78.95%) to grade IV, but with no grade I. Results showed that the attack rate in affected population was 25.82 per thousand, the mortality rate was 0.08 per thousand and the fatality rate was 0.32%. The numbers of emergency events decreased 2.82% on average, each year. A total number of 169 (55.60%) events occurred in schools with 71 (23.36%) in the countryside. Numbers due to infectious disease-born was 233 (76.64%) including avian flu, cholera and dengue fever were predominant pathogens of the grade II and grade III emergency events. 57 (18.75%) of the events was due to food poisoning. The epi-garph showed that there were two peaks. i.e. in Mar-Apr and Sep, contributed 43.1% to the total number of events. CONCLUSION: Emergency events showed a stable decrease in Fujian province with communicable disease and food poisoning the two major sources and more commonly seen in schools and countryside. We suggest that the government and community pay more attention to the emergency events of avian flu, cholera and dengue fever.

[Application of purified recombinant outer membrane lipoprotein LipL32 in detecting antibodies among leptospirosis cases].

OBJECTIVE: To establish recombinant outer membrane lipoprotein LipL32-based antibody detection assays in identifying leptospirosis. METHODS: Recombinant leptospiral outer membrane protein LipL32 was obtained by genetic engineering method. This purified protein was used in the indirect and sandwich ELISA assays to test the antibodies in sera of human beings and rats, and the results were compared with those obtained by microscopy agglutination test (MAT) and imported ELISA kit. RESULTS: When the acute and convalescent phase specimens from 9 leptospiral patients were tested, the detected rates of three ELISAs were similar to the MAT. Among the 45 probable cases which MAT showed positive, 32 (71.11%) samples were positive by r32-I-ELISA, 36 (80.00%) by r32-S-ELISA, while 28.89% (13/45) samples were positive and 55.56% (25/45) were suspicious by D.A.I-ELISA. The specificity of r32-I-ELISA and r32-S-ELISA were 97.10% (67/69) for 69 specimens. 43 healthy specimens were negative by both r32-I-ELISA and r32-S-ELISA, 14 healthy specimens were negative by D.A.I-ELISA. Among 16 non-leptospirosis patients, two specimens were positive by r32-I-ELISA and r32-S-ELISA, D.A.I-ELISA and identified one positive specimen, while 12 specimens were suspicious by D.A.I-ELISA. For 10 syphilis specimens, data obtained through three ELISAs were in consistent with that by MAT. A sandwiched ELISA, using rLipL32 protein as the antigen was developed to detect rat sera. Considering MAT as standard test, the sensitivity and specificity were 86.75% (131/151), 99.19% (122/123) respectively with coincidence rate as 92.34% (253/274). CONCLUSION: The recombinant protein LipL32 had high immunoreactivity and could be used as an antigen for the detection of pathogenic leptospirosis. In summary, the novel sandwiched ELISA with rLipL32 showed similar sensitivity and specificity to that of MAT in the antibody detection of rat leptospirosis. It was suitable for large scales field sero-epidemiological studies.

[A sero-epidemiology study on hepatitis E virus infection in Fujian province].

OBJECTIVE: To understand the sero-prevalence of hepatitis E virus (HEV) infection among different populations and animals in Fujian province. METHODS: One thousand one hundred and fifty-one serum samples were collected from 5 species of animals including swine, dog, cow, sheep and rat. A total of 2209 and 1722 serum samples from the general population and from the exposed population were collected. Anti-HEV IgG was detected by ELISA. The general population was composed of healthy blood donors and the individuals who had attended physical examination including farmers, handlers, veterinarians, cooks who worked with pigs or chickens while the poultry wholesale suppliers made up the exposure population. RESULTS: The infection rates of HEV in animals were different between species (chi2 = 406.25, P < 0.01) with the highest seen in the pig group. With pigs being kept at home, the rates were between 70.00% and 94.12% but the rate was 39.77% for those families that keeping the pigs at farms. The infection rate of HEV was 23.3% in the general population and 33.3% in the exposed populations, respectively. A significantly higher infection rate for anti-HEV was found in the exposed population when comparing with general population. The positive rate of anti-HEV IgG was significantly higher in the exposed population that closely having had contact with chickens than those who had contact with pigs. The increasing trend of HEV infection rate with age had been found but there was no significant difference between males and females in the general population. In the exposed population, the infection rate in males was significantly higher than that in females. CONCLUSION: The infection ratse of HEV in pigs and in the exposure population were much higher, especially for those persons in close contact with chickens or pigs, suggesting that the sub-clinical infection for HEV might exist. These data further supported the hypothesis that HEV might have been an zoonotic disease.