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The oral cavities of snakes are replete with various types of bacterial flora. Culture-dependent studies suggest that some of the bacterial species are responsible for secondary bacterial infection associated with snakebite. A complete profile of the ophidian oral bacterial community has been unreported until now. Therefore, in the present study, we determined the complete bacterial compositions in the oral cavity of some snakes from India. Methods: Total DNA was isolated from oral swabs collected from three wild snake species (Indian Cobra, King Cobra and Indian Python). Next, the DNA was subjected to PCR amplification of microbial 16S rRNA gene using V3-region-specific primers. The amplicons were used for preparation of DNA libraries that were sequenced on an Illumina MiSeq platform. Results: The cluster-based taxonomy analysis revealed that Proteobacteria and Actinobacteria were the most predominant phyla present in the oral cavities of snakes. This result indicates that snakes show more similarities to birds than mammals as to their oral bacterial communities. Furthermore, our study reports all the unique and common bacterial species (total: 147) found among the oral microbes of snakes studied, while the majority of commonly abundant species were pathogens or opportunistic pathogens to humans. A wide difference in ophidian oral bacterial flora suggests variation by individual, species and geographical region. Conclusion: The present study would provide a foundation for further research on snakes to recognize the potential drugs/antibiotics for the different infectious diseases.(AU)
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Snakes , Bacterial Infections , Actinobacteria , Proteobacteria , High-Throughput Nucleotide Sequencing , Anti-Bacterial Agents , Polymerase Chain ReactionABSTRACT
The helicase genes and hypervariable regions (HVRs) of three avian hepatitis E viruses (HEVs) detected at three different farms were sequenced and characterized. Two isolates (DW-L and GI-B2) were classified as genotype 2 and one isolate (GR-B) was classified as genotype 1. A phylogenetic tree, based on the helicase gene and HVR nucleotide sequences, revealed the newly detected viruses and other avian HEVs were classified similarly. Unlike previously reported avian HEVs, the DW-L isolate detected in broiler breeders with characteristic lesions of avian HEV had no prolinerich motif in its HVR, suggesting that the proline-rich motif is non-essential for viral replication and infection.
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Agriculture , Base Sequence , Genotype , Hepevirus , Korea , TreesABSTRACT
Hepatitis C virus ( HCV) infection is one of the main reasons causing liver cirrhosis and hepatocellular carcinoma. The quasispecies composition and the evolution of HCV are very complicated. An in-depth analysis of the quasispecies composition of HCV is critical for elucidating the mechanisms of HCV transmission. The regions encoding the envelope glycoproteins (E1 and E2) and the nonstructural protein 2 (NS2) are hyper variable regions (HVR). Analyzing the quasispecies of HCV HVR with advanced sequen-cing and bio-information technologies would be beneficial for understanding the sources of HCV infection, the routes of transmission and the evolution of HCV. Currently, three generations of sequencing methods and some bio-information soft-wares are available for analyzing HCV HVR quasispecies. When an outbreak of HCV infection happens, using the new generation of sequencing and bio-information methods to seek the first case and the routes of transmission would provide scientific basis for the prevention and treatment of HCV in-fection.
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Objective To construct a recombinant human adenovirus type 3 ( HAd3 ) vector ex-pressing one major epitope of dengue virus type 1.Methods The gene encoding the envelope protein (304-314 aa) of dengue virus type 1 was inserted into the hypervariable region 1 ( HVR1 ) of HAd3 hexon by using overlap PCR.The recombinant gene was cloned into the shuttle plasmid, then linearized with AsisⅠrestriction enzyme and co-transformed into Escherichia coli BJ5183 strains with the digested backbone plas-mid for homologous recombination.The recombinant plasmid pBRAdΔE3GFP-DENV1 was transfected into AD293 cells to rescue recombinant adenovirus strains (rAdΔE3GFP-DENV1).ELISA and Western blot as-say were performed to evaluate the humoral responses induced in BALB/c mice after the immunization with rAdΔE3GFP-DENV1 strains.Results The recombinant adenovirus strains were successfully rescued. ELISA and Western blot assay showed that the antibodies in serum sample could recognize dengue virus type 1 strains.Conclusion The recombinant adenovirus strains expressing the epitope of dengue virus type 1 were successfully constructed.This study provided evidence for the development of multivalent vaccines against dengue virus.
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Objective To assess the correlation between polymorphisms in the coagulation factor Ⅶ (F Ⅶ) gene hypervariable region 4 (HVR4) site and risk related to coronary heart disease (CHD) in different ethnic populations,especially the Asian populations.Methods Publications up to April 2013,from CBM,CNKI,Wanfang Database,VIP,PubMed,Cochrane Library and Embase were searched to collect data from case-control studies related to F Ⅶ gene HVR4 site and CHD in populations from different ethnicities.Quality of studies was evaluated,available data extracted and both RevMan 5.1 and Stata 11.0 softwares were used for Meta-analysis.Results Fifteen case-control studies were included,involving 3167 cases with CHD group and 3168 cases in the control group.Results on this Meta-analysis showed that:a) polymorphism of the FⅦ gene HVR4 site H7/H6 +H5and CHD,b) H7H7/H6H6 + H7H6 and CHD were both slightly correlated between people with different ethnic backgrounds.However,the H6 allele versus H7 + H5 allele and CHD showed different results-a high correlation seen in different ethnic groups.H5 allele versus H6 + H7 allele and CHD did not appear significant difference(OR=l.20,95％CI:0.76-1.90,P=0.43).Conclusion Both FⅦgene HVR4 polymorphisms H7 allele and the H7H7 genotype might have served as protective factors for CHD in different ethnic groups,H6 allele might serve as a risk factor for CHD,but H5 allele was likely not to be associated with CHD in different ethnic groups.
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ObjectiveTo discuss the relationship between the cross-reactivity of antibody against the hypervariable region 1 ( HVR1 ) of hepatitis C virus and early viral response ( EVR ) in patients undergoing antiviral therapy.MethodsBy ELISA and HCV HVR1 antibody cross-reactivity matrix reagent,the differences of anti-HCV hypervariable region antibody were tested in baseline serum from 46 patients with chronic hepatitis C before antiviral therapy.HCV genotyping and HCV RNA were analyzed by RT-PCR method.The HCV RNA assay was done at three time points:before treatment,pegylated interferon in combination with ribavirin therapy for 12 and 48 weeks.ResultsIn 46 cases of chronic hepatitis C patients,there were 16 cases with HCV 2a type,30 cases with l b and 33 patients obtained EVR.The EVR incidence of type 2a[ 93.8％ ( 15/16 ) ] was higher than that of type 1 b[ 60.0％ (18/30),x2 =4.316,P ＜ 0.05 ].In the EVR group of type 1b chronic hepatitis C patients,the positive number of average multi-target HVR1 antigen was ( 12 ± 4),which was significantly higher than that in the Non-EVR patients [ (7 ± 5 ),t =2.797,P ＜0.01 ].Bv the Fisher exact test,it was showed that patients'serum response to HVR1 antigens numbered 001,003,009,013,016 were higher in EVR group than those in non-EVR group,with statistically significant (P ＜ 0.05 ).ConclusionThe cross-reactivity of HVR1 antibody may play an important role in predicting the effectiveness of antiviral therapy.
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Objective To detect antibodies against the hypervariable region 1(HVR1) of hepatitis C virus in blood screening.Methods The HVR1 antibodies were detected by F4HVR1 antigen,and then were compared with other antibodies of HCV.Results Among HCV-RNA positive samples,HVR1 antibody was 96.8% positive.The positive rate of HVR1 antibodies in 90 suspected HCV-infected samples was 61.1%,which was close to those against C and NS3,and higher than NS4 and NS5(P
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Objective To investigate the relationship between hypervariable regionsⅠ(HVRⅠ) mutation of mitochondrial DNA (mtDNA) and lung squamous carcinoma and to explore its significance in carcinogenesis. Methods White blood cells and carcinoma tissues were obtained from 13 cases of lung squamous carcinoma patients and mtDNA were extracted by one step method. HVRⅠfragments were amplified by PCR. Mutations were determined by DNA sequencing. Results In 13 lung squamous carcinoma patients, 8 cases showed mutation in HVR Ⅰ, and 30 mutations were found in 25 different nucleotide sites, in which 17 were point mutations and 13 were insertions and deletions, including a 10-bp deletion in one patient. Conclusion These results suggest that the mutation rate of HVRⅠsequence in lung squamous carcinoma tissue was relatively high, and point mutation might play an important role in lung carcinogenesis.
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Objective: To compare the sequence diversity of HVR1 in the putative envelope protein E2 of the genotypeⅡand genotype Ⅲ HCV in Chinese. Methods: The cDNAs[nucleotide(nt)1449-1586(HCV-J) or nt1460-1582(HCV-J6)] derived from plasma of 55 patients infected with genotype Ⅱ HCV and 38 patients infected with genotype Ⅲ HCV were amplified,purified and directly sequenced by RT-nested polymerase chain reaction(PCR) and dideoxynucleotide chain termination method. Results: The HVR1 was found in amino acid(aa) 384-408 positions of both types HCV E2 protein. There were 5 similar conserved amino acids in 2 types HCV HVR1:aa385(Thr), aa389, 390, 406(Gly)and aa403(Phe).Besides, 401(Ser) was also highly conserved in genotype Ⅱ HCV HVR1. Although the variation characteristic of 2 types was similar, but the sequence diversity(SD),the kinds and frequency of some amino acids in some HVR1 positions and the conserved region near the HVR1 had some differences between 2 genotypes. Conclusion: Further study on the diversity of HVR1 and its biological significance will be helpful to understand the mechanism of HCV persistent infection and the development of HCV vaccine.
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Purpose To understand the clinical significance of sequence variations in the hypervariable region(HVR) of hepatitis C virus during infections. Methods 8 cases of acute hepatitis C and 20 of chronic hepatitis C were followed for two years.Blood samples were taken at intervals of six months for analysis of HCV?HVR sequences by reverse transcription-polymerase chain reaction(RT?PCR) and direct sequencing methods. Results Results showed that HCV?HVR sequences of the 28 patients changed in various degrees.92% of these nucleotide substitutions led to changes of corresponding amino-acid sequences.Only 8% of changed nucleotide were synonymous substitutions.Variation of amino acid ranged from 1 to 20(mean 8,30%).The most common nucleotide substitution(62%) occurred in the first position of codon,31% in the second and the rest in the third.HVR variation rate was 0.89×10-1 per genome site per year in acute hepatitis C,compared with 2.31×10-1 per genome site per year in chronic hepatitis C (P<0.05),but variations had no relation to HCV subtype.Variation of HVR in the flare up type (ALT>150 u/L) was much more than that in the quiescent type (ALT<100 u/L). Conclusions Our results suggested that sequence variation of HVR during HCV chronic infection seems to be an adaptive response to HCV to evade the host immune pressure and might play a major role in the establishment of persistent infection as well as in the flare-up of hepatitis.
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BACKGROUND: Meicillin-resistant Staphylococcus aureus(MRSA) is a common cause of nosocomial infections worldwide. Identification of strains by molecular typing facilitates epidemiological studies and improves disease control This study was performed to determine the usefulness of mecA-associated hypervariable region(HVR) polymerase chain reaction (PCR) and random amplified polymorphic DNA(RAPD) analysis in the investigation of a nosocomial MRSA infections. METHODS: Methicillin-resistance was identified by NCCLS disk diffusion method using the oxacillin disk. And PCR was done for detection of mecA gene. Antimicrobial susceptibility test, HVR-PCR and RAPD using 3 primers were performed for epidemiological analysis on isolates of MRSA. RESULTS: During the period from 1997 Dec. to 1998 May, 120 strains of S. aureus were isolated from clinical specimens. Among them, 78 strains were MRSA, and 72 strains were mecA positive. The strains of mecA positive MRSA were classified into four types by antibiogram, six genotypes by HVR-PCR, and 29 groups by RAPD using three primers. The combination of HVR genotypes and RAPD analysis showed 43 different types in 72 mecA positive MRSA isolates The five strains which were repeatedly isolated from the same patients showed the same HVR genotypes and RAPD analysis. CONCLUSIONS: Antibiogram, HVR-PCR, and RAPD could classify MRSA isolates into only 4-6 types, respectively, but combination of these methods could improve the typability. And combination of results of RAPD analysis using three primers were better than that using one primer in epidemiological studies of MRSA because of same reasons. It can be concluded that molecular typing of MRSA using HVR-PCR and RAPD assay is useful in epidemiolgical investigation of nosocomial infections caused by MRSA, because of its simplicity and reproducibility.
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Humans , Cross Infection , Diffusion , Epidemiologic Studies , Genotype , Methicillin Resistance , Methicillin-Resistant Staphylococcus aureus , Microbial Sensitivity Tests , Molecular Typing , Oxacillin , Polymerase Chain Reaction , StaphylococcusABSTRACT
BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) are becoming increasingly responsible for the outbreaks of nosocomial infections around the world. Because MRSA are often resistant to antimicrobial agents and because of the current necessity to use non-beta-lactam antimicrobial therapy, infections with these organisms are difficult to treat. The purpose of this study was to explain how the genetic background contributes to the phenotypic expression of MRSA and to enable immediate and proper identification of MRSA in nosocomial infections. METHODS: A total of 240 staphylococci strains were tested for epidemiological research. The molecular genetic methods, such as dot blot hybridization, polymerase chain reaction, and southern blot hybridization, were compared in terms of the immediate and proper identification of the pathogens. The arrangement of dru sequence, the repeat unit of genes, was clarified particularly through mecA probe to compare and distinguish the isolated MRSA strains. RESULTS: By the dot blot hybridization, the mecA gene was detected in 120 MRSA strains and in 8 of 50 methicillin-suscpetible S. aureus (MSSA) in Group II (resistance to methicillin is induced in vitro). Among isolates included in Group I (resistance to methicillin is not induced in vitro) mecA genes were not detected. By PCR, the amplified DNA band of 533 bp was confirmed in 120 MRSA but not in MSSA. By the southern blot hybridization, the signal of the amplified electrophoresis band in PCR was similarly observed. The amplified band of mec related hypervariable region was observed in all methicilin resistant (MR) staphylococci but not in MSSA. The size of PCR products was between 194 bp and 1,353 bp. mec-related HVR was classified into seven genotypes. The nucleotide sequence of genotype E was similar to that of mec related HVR and eight units were directly repeated. CONCLUSION: Both methods allowed rapid detection of mecA gene. Further differentiation of MRSA and other staphylococci strains was possible by PCR detection of polymorphism of HVR sequence which would be useful in tracing back the source of resistant clones.
Subject(s)
Anti-Infective Agents , Base Sequence , Blotting, Southern , Clone Cells , Cross Infection , Disease Outbreaks , DNA , Electrophoresis , Genotype , Methicillin , Methicillin Resistance , Methicillin-Resistant Staphylococcus aureus , Molecular Biology , Polymerase Chain ReactionABSTRACT
Interferon-alpha (IFN-alpha) has been used to treat hepatitis C virus (HCV)-induced hepatitis, but it has been effective in only about half of the treated patients, with recurrence appearing in the other half. As a consequence of the possible complications associated with IFN-alpha and the high cost of treatment, it has become extremely important to select the proper patients for IFN-alpha treatment. In our previous study, we found that the quasispecies in the hypervariable region (HVR) 1 of HCV were various and that a new quasispecies can appear in non-responders and/or lead to deterioration in the patients' condition. The preliminary data we obtained in the process of our previous research led us to believe that the quasispecies of HVR 1 has something to do with the effect of IFN-alpha. Thus, in this investigation, we tried to determine the predictive factors of IFN-alpha therapy. Thirty patients with HCV infection were treated with IFN-alpha. Among them, 15 patients recovered after six months IFN-alpha treatment, but the remaining 15 patients showed no response after six months IFN-alpha treatment. We cloned HVR 1 DNA by reverse transcription-polymerase chain reaction (RT-PCR) and examined the quasispecies of HVR 1. As the quasispecies of HVR 1 in non-responders varied more than in the complete remission group, we concluded that the sequence variation in HVR 1 of HCV can be used to predict the effect of IFN-alpha.