A multiplex liquid-chip assay based on Luminex xMAP technology for simultaneous detection of six common respiratory viruses.

We utilized one-step multiplex reverse transcription-PCR (RT-PCR) and Luminex xMAP technology to develop a respiratory multiplex liquid-chip assay (rMLA) for simultaneous detection of 6 common respiratory viruses, including influenza virus type A (FluA) and type B (FluB), para-influenza virus type 3 (PIV-3), respiratory syncytial virus (RSV), human metapneumovirus (MPV) and a threatening virus to China, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Performance of rMLA was evaluated by comparing with real-time RT-PCR. Detection data from clinical specimens showed that the rMLA had diagnostic sensitivities of 97.10% for FluA, 94.59% for FluB, 98.68% for PIV-3, 94.87% for RSV and 95.92% for MPV (No Data for MERS-CoV due to the lack of positive specimens). Data of analytical sensitivities showed that the detection limits of the rMLA assay were 5-25 viral RNA copies per µl for FluA, FluB, PIV-3 and MERS-CoV, approximate to the real-time RT-PCR assay; while the values were 8 and 22copies/µl for MPV and RSV, lower than the real-time RT-PCR(78 and 114 copies/µl respectively). The results indicated that the rMLA is a sensitive, specific detection tool and comparable to real-time RT-PCR, especially suitable for high-throughput detection of respiratory specimens.

[Tracing investigation and diagnosis of one transfusion-transmitted malaria case in Zhejiang Province].

OBJECTIVE: To identify the sources of infection and the mode of transmission of a malaria case with unknown origin. METHODS: Clinical data of the case were collected and the epidemiological investigation was conducted. The blood samples of the patient and the suspected infection source (blood donor) were detected by microscopy, rapid diagnostic test strip (RDT) and nested PCR. RESULTS: The patient did not visited malaria endemic areas. After a blood transfusion, the patient had chills and fever, and was confirmed as falciparum malaria by microscopy with bone marrow and peripheral blood smears and RDT. The blood donor was a worker returned from Africa. Before blood donation she was sick like malaria carrier, and took anti-malarial drug. She was then confirmed as falciparum malaria by RDT and microscopy. The blood samples from the patient and the blood donor were diagnosed as falciparum malaria by nested PCR, and the similarity of the small subunit rRNA (SSU rRNA) sequence was 100%, showing they were mix-infected with K1 and MAD20 genotypes of Plasmodium falciparum. CONCLUSION: This patient is confirmed P. falciparum infection via blood transfusion from a donor who returned from Africa.

[Secretory expression of salivary ATP diphosphohydrolase (apyrase) from Aedes albopictus in Pichia pastoris].

The gene-coding mature apyrase protein from Aedes albopictus was amplified by RT-PCR and cloned in frame with the a-factor secretion signal peptide into Pichia pastoris secreting expression vector pGAPZalpha-A resulting in the pGAPZa-A-apyrase. After being linearized by Bln I restriction enzyme, the recombinant pGAPZalpha-A-apyrase was trans-formed into Pichia pastoris GS115 by electroporation. Recombinant strains pGAPZalpha-A-apyrase/GS115 were screened on YPDS plates containing Zeocin and identified by PCR. The recombinant protein of apyrase (M(r) 60000) has been expressed in the supernatant of Pichia pastoris.

[Laboratory testing for a case of imported Plasmodium ovale infection in Zhejiang Province].

Blood sample obtained from a patient, which returned from Equatorial Guinea, with clinical diagnosis of Plasmodium infection was confirmed as imported P. ovale infection by etiology and molecular biological methods. 50 microl blood was obtained before taking anti-malarial drugs to make thin and thick blood smears, Giemsa stained, and observed by microscopy. Genomic DNA was extracted from the blood sample, and detected for DNA fragment of P. ovale, P. vivax, P. falciparum or P. malariae by real-time fluorescent quantitative PCR. P. ovale parasites were found in both thin and thick blood smears, and confirmed by quantitative PCR. With the results of laboratory testing, epidemiological history and clinical manifestations, the patient was diagnosed as imported P. ovale infection.

[Pathogen identification and clinical diagnosis for one case infected with Babesia].

OBJECTIVE: To identify the pathogen and make diagnosis on a case who was misdiagnosed as malaria. METHODS: Clinical and epidemiological information of the suspected case was collected. Blood samples during hospitalization were collected and examined microscopically. Genomic DNA from the blood samples was amplified by Babesia 18S RNA genus- and species- specific primers, respectively, and the amplified products were used in sequencing and BLAST sequence analysis. RESULTS: The case had a fever over 20 days repeatedly with anaemia (RBC 2.59 x 10(12), HB 5.5 g/L) and hepatosplenomegaly. The unidentified parasites were found in the bone marrow and blood smear after Giemsa staining. Epidemiological information revealed that this case had a history of blood transfusion and tick bites. 1 625 bp and 449 bp band generated by PCR amplification from blood sample using Babesia genus- and species-specific primers, and the sequence homology was 99% in comparison to Babesia microti (AB241632) with BLAST analysis. CONCLUSION: The clinical information, epidemiological history, and the PCR identification confirm the diagnosis of Babesia microti infection.

[Temporal and spatial population dynamics of rabies virus isolates in China].

In order to study phylogeography, population dynamics and molecular evolution of rabies viruses (RABVs) isolates from China, especially spatio-temporal dynamics, the timescale of RABVs evolution and its pattern of migration, we performed an extensive comparative analysis of RABV N gene sequence data, representing 167 isolates sampled from 20 provinces in a 78-year period (from 1931 through 2009). The available Chinese isolates could be divided into two distinct clades:Phylogroup clades I comprised Chinese group 1-4; Phylogroup clades II contained Chinese group 5-8. We found no evidence for positive selection (dN/dS>1) acting at any codon and found strong selective constraints for N gene. Bayesian Markov Chain Monte Carlo (MCMC) analysis suggested that the Chinese rabies viruses originated within the last 2000 years and the mean rates of nucleotide substitution for the N gene were approximately 4 x 10(-4) substitutions per site per year. The analyses of the spatial and spatio-temporal evolution indicated that RABV isolates from China migrated among different Provinces.

[Molecular epidemiology investigation of rabies virus nucleoprotein genes in China].

OBJECTIVE: To study the relationship between the distribution of rabies virus and genetic variation, the genetic characterization and variation of rabies virus strains in China were analyzed. METHODS: The downstream 720 nucleotides of Nucleoprotein (N) gene coding region of the rabies specimens from different areas and host animals were sequenced, and then homology and phylogenesis were analyzed. RESULTS: Nucleotide similarities of 34 N gene sequences were 87.5%-100%, and the deduced amino acid similarities were 93.3%-99.6%. Most of the nucleotide variations were synonymous mutations. CONCLUSION: The 34 rabies specimens all belong to genotype I and are of regional characteristic. The rabies viruses in high-incidence areas in China are of various origins and present the transmission tendency from high-incidence areas to surrounding regions. There may be cross-infection and mutual spread of rabies virus between wildlife and domestic animals as well as native and foreign animals.

[Sequencing and analyses on glycoprotein gene of rabies viruses isolated in Zhejiang province, China].

OBJECTIVE: Based on sequencing the genomes of glycoprotein (GP) gene of rabies viruses isolated in Zhejiang, we analyzed the properties of rabies viruses genetic variation in molecular level, and to compare with those of other representative vaccine strains and street virus strains, get the information about rabies viruses variation. METHODS: Suckling mice against rabies virus were selected. Overlapped fragments were amplified by RT-PCR and full-length genomes were assembled to analyze the nucleotide and deduced protein similarities and phylogenetic analyses of the GP genes. RESULTS: The fourteen full-length genomes were completely sequenced and they had the same genetic structure with 1575 nts and deduced protein with 524 aa. Genetic analysis revealed that the nucleotide and amino acid homologies of GP gene from Zhejiang strains and other vaccine strains or street virus strains were 82.3% - 99.9% and 85.1% - 99.8%. The fourteen strains were genotype 1 according to the phylogenetic analyses. The GP amino acids of Zhejiang strains rabies virus strains without any recombination occurred in GP and no larger variation appeared in the major antigenic sites. CONCLUSION: The comprehensive analysis based on the first-level structure of GP demonstrated that it was possible that some advantageous antigenic epitopes existed in certain areas and potential antigenic determinants. It was evident that the GP gene of Zhejiang strains appear to be stable and their sequence similarity with the representative strains of street virus in China were higher than those of other vaccine strains. Some differences showed in the genetic structure and evolution relationship among Zhejiang strains, other street strains in other regions and vaccine strains.

[Sequencing and analysis of complete genome of rabies viruses isolated from Chinese Ferret-Badger and dog in Zhejiang province].

Based on sequencing the full-length genomes of four Chinese Ferret-Badger and dog, we analyze the properties of rabies viruses genetic variation in molecular level, get the information about rabies viruses prevalence and variation in Zhejiang, and enrich the genome database of rabies viruses street strains isolated from China. Rabies viruses in suckling mice were isolated, overlapped fragments were amplified by RT-PCR and full-length genomes were assembled to analyze the nucleotide and deduced protein similarities and phylogenetic analyses from Chinese Ferret-Badger, dog, sika deer, vole, used vaccine strain were determined. The four full-length genomes were sequenced completely and had the same genetic structure with the length of 11, 923 nts or 11, 925 nts including 58 nts-Leader, 1353 nts-NP, 894 nts-PP, 609 nts-MP, 1575 nts-GP, 6386 nts-LP, and 2, 5, 5 nts- intergenic regions(IGRs), 423 nts-Pseudogene-like sequence (psi), 70 nts-Trailer. The four full-length genomes were in accordance with the properties of Rhabdoviridae Lyssa virus by BLAST and multi-sequence alignment. The nucleotide and amino acid sequences among Chinese strains had the highest similarity, especially among animals of the same species. Of the four full-length genomes, the similarity in amino acid level was dramatically higher than that in nucleotide level, so the nucleotide mutations happened in these four genomes were most synonymous mutations. Compared with the reference rabies viruses, the lengths of the five protein coding regions had no change, no recombination, only with a few point mutations. It was evident that the five proteins appeared to be stable. The variation sites and types of the four genomes were similar to the reference vaccine or street strains. And the four strains were genotype 1 according to the multi-sequence and phylogenetic analyses, which possessed the distinct district characteristics of China. Therefore, these four rabies viruses are likely to be street viruses already existing in the natural world.

[New animal hosts of rabies virus in mountain areas in Zhejiang province].

OBJECTIVE: To understand the prevalence of rabies among wild animals and the animal species in rabies epidemic areas of Zhejiang province. METHODS: One hundred and sixty samples were collected from the brain tissues of cats, stoats, Apodemus agrarius, Moschus chinensis, and Sus scrofa in Lishui and Chunan cities of Zhejiang province. Each sample was divided into four parts: cerebrum, mesencephali, cerebellum and gyms hippocampi which were used to determine the positive samples by detection of rabies virus specific antigens and nucleotides, using DFA and RT-PCR methods. RESULTS: Positive slides in the tests contained a glaring, apple green brilliance fluorescence using rabies virus specific monoclonal antibody against nucleoprotein. Using Nested-PCR method targeted at part of N gene, five positive samples were identified which consisting of four positive samples from stoats with positive ratio as 8.33% (4/48) and one positive sample from Apodemus agrarius with positive ratio as 1.75% (1/57). However, no positive result was found from cats, Moschus chinensis, and Sus scrofa samples. CONCLUSION: Rabies virus positive samples were identified from stoats and Apodemus agrarius in the mountain areas, with biological diversity in Lishui and Chunan cities of Zhejiang province, indicating that stoats and Apodemus agrarius might have played a role in human rabies and acted as host of rabies virus. In order to effectively prevent and control rabies virus under these complicated geographical and ecological environment, we must understand and evaluate the infection situation among animals in these regions.

[Study on the difference of genes and the type identification of hantavirus from Lishui, Zhejiang province].

OBJECTIVE: To isolate hantavirus from Lishui county--one of the epidemic regions for hemorrhagic fever with renal syndrome (HFRS), in Zhejiang province, and to identify the serotype and molecular/biological characteristics of a new HTN subtype hantavirus (HV) strains, hopefully to provide evidence for HFRS prevention and therapy. METHODS: Data on the host animals was collected from Lishui, Zhejiang province in 2007. Direct immunofluorscece assay was adopted to determine HFRS antigens and the lung tissues from HV infected Vero-E6 cells for HV isolation, then total RNA was extracted from Hantavirus Lishui strains and amplified by RT-PCR M, S segments of strains genome were also cloned and sequenced and compared with those of other strains of HV. RESULTS: 2 strains virus (ZLS6-11 and ZLS-12) were successfully isolated from 7 positive lung samples of mice and were identified as HTNV by anti-McAb and phylogenetic analysis. With sequence compation,we found that 2 strains with complete M and S segment had higher homology with HTN-type strains than with other types of HV, but 13.4%-20.7% and 10.3%-16.1% of the genes were found which were different from HTNV. The phylogenetic trees constructed by complete S and M segment showed that ZLS6-11 and ZLS-12 strains were located in HTNV group,and structured independent embranchment. CONCLUSION: ZLS6-11 and ZLS-12 Strains were believed to belong to HTN-type and phylogenetically different from the HTNV.

[Complete genome sequencing and analyses of rabies viruses isolated from wild animals (Chinese Ferret-Badger) in Zhejiang province].

OBJECTIVE: Based on sequencing the full-length genomes of two Chinese Ferret-Badger, we analyzed the properties of rabies viruses genetic variation in molecular level to get information on prevalence and variation of rabies viruses in Zhejiang, and to enrich the genome database of rabies viruses street strains isolated from Chinese wildlife. METHODS: Overlapped fragments were amplified by RT-PCR and full-length genomes were assembled to analyze the nucleotide and deduced protein similarities and phylogenetic analyses of the N genes from Chinese Ferret-Badger, sika deer, vole, dog. Vaccine strains were then determined. RESULTS: The two full-length genomes were completely sequenced to find out that they had the same genetic structure with 11 923 nts including 58 nts-Leader, 1353 nts-NP, 894 nts-PP, 609 nts-MP, 1575 nts-GP, 6386 nts-LP, and 2, 5, 5 nts- intergenic regions (IGRs), 423 nts-Pseudogene-like sequence (Psi), 70 nts-Trailer. CONCLUSION: The two full-length genomes were in accordance with the properties of Rhabdoviridae Lyssa virus by blast and multi-sequence alignment. The nucleotide and amino acid sequences among Chinese strains had the highest similarity, especially among animals of the same species. Of the two full-length genomes, the similarity in amino acid level was dramatically higher than that in nucleotide level, so that the nucleotide mutations happened in these two genomes were most probably as synonymous mutations. Compared to the referenced rabies viruses, the lengths of the five protein coding regions did not show any changes or recombination, but only with a few-point mutations. It was evident that the five proteins appeared to be stable. The variation sites and types of the two ferret badgers genomes were similar to the referenced vaccine or street strains. The two strains were genotype 1 according to the multi-sequence and phylogenetic analyses, which possessing the distinct geographyphic characteristics of China. All the evidence suggested a cue that these two ferret badgers rabies viruses were likely to be street virus that already circulating in wildlife.

Effects of H pylori infection on gap-junctional intercellular communication and proliferation of gastric epithelial cells in vitro.

AIM: To explore the effects of H pylori infection on gap-junctional intercellular communication (GJIC) and proliferation of gastric epithelial cells in vitro. METHODS: A human gastric epithelial cell line (SGC-7901) cultured on coverslips was exposed overnight to intact H pylori (CagA(+) or CagA(-) strains) and sonicated extracts, respectively. GJIC between the cells was detected by fluorescence redistribution after photobleaching (FRAP) technique. Proliferation of SGC cells was determined by methylthiazolyl tetrazolium (MTT) assay. RESULTS: When compared with control in which cells were cultured with simple medium alone, both CagA(+) and CagA(-) H pylori isolates could inhibit GJIC (CagA(+): F = 57.98, P < 0.01; CagA(-): F = 29.59, P < 0.01) and proliferation (CagA(+): F = 42.65, P < 0.01; CagA(-): F = 58.14, P < 0.01) of SGC-7901 cells. Compared with CagA(-) strains, CagA(+) H pylori more significantly down-regulated GJIC of gastric cells (intact H pylori: t = 13.86, P < 0.01; sonicated extracts: t = 11.87, P < 0.01) and inhibited proliferation gastric cells to a lesser extent in vitro (intact H pylori: t = 3.06, P < 0.05; sonicated extracts: t = 3.94, P < 0.01). CONCLUSION: Compared with CagA(-) H pylori strains, CagA(+) strains down-regulate GJIC of gastric epithelial cells more significantly and inhibit proliferation of gastric cells to a lesser extent in vitro. H pylori, especially CagA(+) strains, may play an important role in gastric carcinogenesis.