The present review describes the current status of multiplex quantitative real time polymerase chain reaction(q PCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. ...The present review describes the current status of multiplex quantitative real time polymerase chain reaction(q PCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex q PCR for the detection of hepatitis viruses, including hepatitis A virus(HAV), hepatitis B virus(HBV), hepatitis C virus(HCV), hepatitis D virus(HDV), and hepatitis E virus(HEV). In addition, multiplex q PCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex q PCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex q PCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process.展开更多
Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequence...Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.展开更多
BACKGROUND Extended-spectrumβ-lactamase(ESBL)-producing Escherichia coli(E.coli)are among the main pathogens in urinary tract infections(UTIs)among kidney transplant patients(KTPs).AIM To estimate the prevalence of E...BACKGROUND Extended-spectrumβ-lactamase(ESBL)-producing Escherichia coli(E.coli)are among the main pathogens in urinary tract infections(UTIs)among kidney transplant patients(KTPs).AIM To estimate the prevalence of ESBL-producing E.coli in KTPs and to evaluate the most prevalent serotypes and antibacterial susceptibility patterns of isolated bacteria in Tehran,Iran.METHODS A total of 60 clinical isolates of uropathogenic E.coli were collected from 3 kidney transplant centers from April to May 2019.Antimicrobial susceptibility testing was performed by the disk diffusion method as recommended by the Clinical Laboratory and Standards Institute.The serotyping of E.coli isolates was performed by the slide agglutination method.The presence of blaTEM,blaSHV,and bla CTX-M genes was evaluated by polymerase chain reaction.RESULTS The frequency of ESBL-producing E.coli in KTPs was found to be 33.4%.All of the 60 E.coli isolates were found to be susceptible to doripenem(100%)and ertapenem(100%).High resistance rates to ampicillin(86%),cefotaxime(80%),and cefazolin(77%)were also documented.The most frequent serotypes were serotype I(50%),serotype II(15%),serotype III(25%),and serotype VI(10%).The gene most frequently found was blaTEM(55%),followed by blaCTX-M(51%)and blaSHV(41%).CONCLUSION Molecular analysis showed that blaTEM was the most common ESBL-encoding gene.The high resistance toβ-lactams antibiotics(i.e.,ampicillin,cefotaxime,and cefazolin)found in E.coli from KTPs with UTIs remains a serious clinical challenge.Further efforts to control ESBL-producing E.coli should include the careful use of all antibiotics as well as barrier precautions to reduce spread.展开更多
Bovine Respiratory Disease (BRD) causes a severe form of pneumonia in all age of cattle. This study was designed to investigate the distribution of capsular types, serotypes, and virulence-associated genes of the majo...Bovine Respiratory Disease (BRD) causes a severe form of pneumonia in all age of cattle. This study was designed to investigate the distribution of capsular types, serotypes, and virulence-associated genes of the major bacterial pathogens from BRD outbreak samples in Ethiopia. In this study 166 samples were collected from clinically sick (<i>n</i> = 107) and pneumonic lung tissue (<i>n</i> = 59). Laboratory assay confirmed isolation of <i>M. haemolytica</i> 37 (22.29%), <i>P. multocida</i> 25 (15.06%), <i>B. trehalosi</i> 12 (7.23%), and <i>H. somni</i> 15 (9.04%). PCR assay of <i>P. multocida</i> capsular typing revealed 21 (84.0%) cap A (<i>hyaD-hyaC</i>) and 4 (16.0%) cap D (<i>dcbF</i>) strains. <i>M. haemolytica</i> serotypes belonged to A: 1, A: 2, and A: 6 from 26 (70.27%), 4 (10.81%), and 7 (18.92%) isolates, respectively. <i>P. multocida</i> biotyping showed isolation of A: 1, A: 2, and A: 3 from 3 (14.29%), 2 (9.52%), and 16 (76.19%) isolates, respectively. <i>M. haemolytica</i> harbored more than 60% <i>ssa</i> gene, and 90.91% <i>sodA</i> while <i>FbpA</i>, <i>TbpA</i>, and <i>lktC</i> genes were found in all isolates. Likewise, all <i>P. multocida</i> exhibited <i>toxA</i>, <i>FbpA</i>, <i>TbpA</i>, and <i>pmSLP</i> genes. The current finding showed that <i>M. haemolytica</i> serotype A: 1 is frequently associated with BRD followed by <i>P. multocida</i> biotype A: 3. These two isolates harbored diverse virulence-associated genes and presented the pathogenic potential of the current isolates. Thus, investigation of pathogenic strains of BRD, virulence genes distribution, and molecular epidemiology of the disease from wider areas of the country are essential. Hence, continuous outbreak surveillance and molecular approaches are indispensable in designing efficient prevention strategies.展开更多
Objective:To analyse the prevalence of serotypes,antibiotic resistance,and virulence genes of Group B Streptococcus(GBS)strains isolated from pregnant women at 35-37 weeks of gestation in Ho Chi Minh City,Vietnam,from...Objective:To analyse the prevalence of serotypes,antibiotic resistance,and virulence genes of Group B Streptococcus(GBS)strains isolated from pregnant women at 35-37 weeks of gestation in Ho Chi Minh City,Vietnam,from January 2022 to January 2023.Methods:GBS strains were isolated through selective culture methods and confirmed by PCR.Serotyping,virulence gene detection,and antibiotic susceptibility testing were performed using PCR,gel electrophoresis techniques and Kirby-Bauer test.Results:Totally,61 GBS isolated from 300 participants have been identified including seven GBS serotypes(Ⅰa,Ⅰb,Ⅱ,Ⅲ,Ⅳ,Ⅴ,andⅥ).SerotypesⅦ,Ⅷ,andⅨwere not detected in the study population.Antibiotic resistance patterns varied:13.1%of isolates were fully susceptible,while the majority showed multi-drug resistance,with 34.4%resistant to three antibiotics.SerotypeⅠa demonstrated high susceptibility(35.7%),while serotypeⅢshowed extensive resistance,with 87.5%being resistant to at least three antibiotics.All strains are susceptible to vancomycin andβ-lactams susceptibility also remained high,but resistance to clindamycin,erythromycin,and tetracycline was high(>65%).The virulence genes scpB,cylB,fbsB,and cfb were highly prevalent(90%-100%),indicating their potential for vaccine and diagnostic development.Conclusions:Our findings provide valuable insights into GBS serotypes,resistance,and virulence factors,contributing to community monitoring,preventive measures,diagnostics,and vaccine development.However,the limited sample size necessitates further research.展开更多
[Objective]The aim was to provide a theoretical basis for preparation of polyclonal antibodies and monoclonal antibody that of type specificity, as well as Foot-and-mouth Disease Virus （FMDV） typing.[Method] The rec...[Objective]The aim was to provide a theoretical basis for preparation of polyclonal antibodies and monoclonal antibody that of type specificity, as well as Foot-and-mouth Disease Virus （FMDV） typing.[Method] The recombinant plasmid pGEM-CP1 that contained VPI gene of FMDV of serotype C was used as template for the VP1 and its C terminus coding fragments of FMDV of serotypes C amplification. The coding fragments of VP1 and its C terminus were respectively cloned into prokaryotic expression vector for prokaryotic expression and the reactionogenicity was detected. The purified fusion protein of FMDV VP1 and its C terminus of serotype C were used to construct the indirect ELISA method to detect positive sera of four serotypes A, O, C and Asia 1 of guinea pig and determine the cross reactivity of FMDV antibody of VP1 and its C terminus of serotype C with other three serotypes. [ Result] The recombinant prokaryotic expression plasmids of PPRO-CVP1 and pPRO-CVPlc were constructed, FMDV VP1 and its C terminus of serotype C were expressed in high level, and the molecular weight of target proteins was 33 kD and 20 kD respectively. Western blot result showed that the fusion protein of VP1 and its C terminus could react with the positive sera of guinea pig of the same serotype. ELISA results revealed that VP1 and its C terminus of serotype C are type-specific and no cross-reactivities were shown between guinea pig positive sera of FMDV of serotype C with the other three serotypes, and the C terminus showed better type-specificity. [ Conclusion] FMDV specific antigen of serotype C was obtained.展开更多
Dear Editor,Dengue virus(DENV)is a positive-sense single-stranded RNA virus belonging to the Flaviviridae family,which causes dengue—a disease affecting over 400 million people annually worldwide.DENV is transmitted ...Dear Editor,Dengue virus(DENV)is a positive-sense single-stranded RNA virus belonging to the Flaviviridae family,which causes dengue—a disease affecting over 400 million people annually worldwide.DENV is transmitted through the bite of mosquitoes from the Aedes genus,primarily Aedes aegypti,and has a wide distribution in tropical and subtropical areas(de Souza et al.,2022).展开更多
Streptococcus suis serotype 2(SS2)is a zoonotic pathogen that can cause acute infection,such as septicemia in pigs and streptococcal toxic shock-like syndrome(STSLS)in humans,indicating that SS2 can evade innate immun...Streptococcus suis serotype 2(SS2)is a zoonotic pathogen that can cause acute infection,such as septicemia in pigs and streptococcal toxic shock-like syndrome(STSLS)in humans,indicating that SS2 can evade innate immunity.Macrophages perform essential antimicrobial functions in the innate immune system by engulfing and killing pathogens.Previously,a dna K mutant strain that showed impaired phagocytosis resistance ability was screened from the transposon mutant library of SS2,but the specific mechanism is unclear.In this study,we further demonstrated that DnaK was required for SS2 to be antiphagocytosed by macrophages and survive in adverse environments.A mouse challenge experiment indicated that DnaK promoted bacteremia and systemic dissemination of SS2,enhancing bacterial pathogenicity.Western blot and immunofluorescence results indicated that DnaK could be secreted by SS2 and was able to enter RAW264.7 macrophages.Then,the endocytic receptor LRP1 regulated by DnaK was identified through RNA sequencing(RNA-Seq).We found that DnaK decreased both the mRNA and protein levels of LRP1.Knockdown of the LRP1β-chain(LRP1β)significantly decreased the phagocytosis rate of the SS2 strain ZY05719,suggesting that LRP1 is a phagocytic receptor of SS2.Furthermore,inhibitor treatment assays revealed that DnaK decreased LRP1 protein levels through the transcription factor PPARγand the ubiquitin-proteasome system.In summary,DnaK contributes to the phagocytosis resistance of SS2 by decreasing LRP1 protein levels in macrophages,providing new insights into the antiphagocytosis mechanisms of SS2 and helping to understand its pathogenesis.展开更多
Streptococcus suis serotype 2(SS2)is an emerging zoonotic pathogen that causes meningitis in humans and pigs.This pathogen generates substantial economic losses in the swine industry while posing a significant threat ...Streptococcus suis serotype 2(SS2)is an emerging zoonotic pathogen that causes meningitis in humans and pigs.This pathogen generates substantial economic losses in the swine industry while posing a significant threat to public health security.The mechanisms through which SS2 penetrates the brain and induces meningitis remain incompletely understood.This study examines the role and mechanism of SS2 collagenase-like protease(Clp)in facilitating bacterial passage across the blood-brain barrier(BBB).The research demonstrates that SS2 Clp enhanced virulence and tissue colonization while promoting BBB degradation in mice.The Δclp mutant exhibited reduced ability to traverse human brain microvascular endothelial(hCMEC/D3)cell monolayers compared to wild-type SS2,while the addition of recombinant protein rClp increased permeability.Furthermore,rClp significantly enhanced SS2 adhesion to hCMEC/D3,suppressed the expression of intercellular tight junction proteins ZO-1,Occludin,and Claudin-5 independent of its enzyme activity,and triggered hCMEC/D3 apoptosis through cell receptor ligand apoptosis and mitochondrial apoptosis pathways,partially dependent on its enzyme activity,leading to BBB disruption and enhanced permeability.Additionally,Clp enhanced the infiltration of macrophages(F4/80+),monocytes(F4/80-Ly6C+),and neutrophils(Ly6G+)into the brain following SS2 infection.These findings establish that SS2 Clp is essential for bacterial passage across the BBB,offering a theoretical foundation for improved prevention and treatment strategies for SS2-induced meningitis.展开更多
Objective:To assess the burden of Group B Streptococcus(GBS)and analyze the distribution of scrotypes in relation to their source.The review highlights data gaps in transmission dynamics and regional food consumption ...Objective:To assess the burden of Group B Streptococcus(GBS)and analyze the distribution of scrotypes in relation to their source.The review highlights data gaps in transmission dynamics and regional food consumption practices,which are esscntial for designing cffective public health strategies and advancing vaccine development.Methods:Scarches were conducted in Web of Science,MEDLINE,Science Direct,PubMcd,and Scopus databases to find studies related to GBS during 1990-2025.Eligible studies were those that described prevalence,scrotype distribution or scquence type(ST)of GBS in Southeast Asian countries.Random-cffects meta-analysis was used to pool data.Results:A total of 26 studies met the inclusion criteria from cight countries.The pooled estimate of maternal GBS colonization was 15.1%,with scrotypesII,v,ⅡI,V,and I a accounting for the majority of cases(91.24%)in the Southcast Asia studies.Data on ST was limited;however,ST1 was found to be predominant in Malaysia and Thailand,while ST283 was notably linked to the consumption of raw fish.Conclusions:The pooled estimate of the maternal colonization with GBS was 15.1%which is cquivalent to many other primary and review reports worldwide.Distribution of scrotype and ST is needed to be studied in Southcast Asian countries to devise cffective preventive measures.These findings underscore thc importance of surveillance and tailored prevention strategies to combat GBS infections in Southcast Asia.展开更多
Dengue virus(DENV)is a positive-sense single-stranded RNA virus belonging to the genus Flavivirus within the Flaviviridae family.Four serotypes,DENV 1-4,are distributed globally[1].Hanoi metropolitan city is an endemi...Dengue virus(DENV)is a positive-sense single-stranded RNA virus belonging to the genus Flavivirus within the Flaviviridae family.Four serotypes,DENV 1-4,are distributed globally[1].Hanoi metropolitan city is an endemic hotspot for DENV transmission in Vietnam[2,3].The largest outbreak occurred in 2017,with more than 36000 cases and 7 deaths reported,causing by all four serotypes with the predominance of DENV1,following by DENV2[4,5].During the following dengue season,we collected 390 blood and serum samples from 197 hospitalized patients in a national hospital in Hanoi city,Northern Vietnam to identify the circulating DENV serotypes responsible for the 2018-2019 outbreak.展开更多
[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []V...[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []Vlethod] The gene fragments en- coding SS2 adhesive related-factors MRP, FBPS and CPS2J and a housekeeping gene aroA were amplified by reverse transcription PCR from the total RNA of SS2, cloned, and sequenced. The recombinant plasmids containing the target genes were constructed, and used as templates in Real-time PCR. [Result] Dynamic curves, stan- dard curves and melting curves of the adhesive related-factors and aroA were ob- tained by the optimized Real-time PCR system. The standard curves showed a good linear relationship between template copy number and circulation number, and the correlation coefficients （FF） of the standard curves were over 0.995. Also, these as- says were highly specific a^d there was single specific melting peak for every gene. Moreover, the assays were highly sensitive and had a detection limit of 1.0×102 copies in 1 μl of initial templates. Finally, it was highly repeatable and had a coeffi- cient of variation less than 2% for intra-assay. [Conclusion] This study will provide a way to reveal the adhesion mechanism of SS2 to different host cells at molecular level.展开更多
Nutritional value of vegetables and high prices of meat and meat originated food compel common people to consume plant originated food particularly salad vegetables. Microbial population of vegetable surfaces contains...Nutritional value of vegetables and high prices of meat and meat originated food compel common people to consume plant originated food particularly salad vegetables. Microbial population of vegetable surfaces contains a large number of pathogenic bacteria including members of Enterobactereace like Escherichia coli (E. coli). A survey was conducted in three major markets of Rawalpindi, Pakistan. Tomato, lettuce, cabbage and cucumber samples were collected from three shops of each market. Each vegetable was analysed as unwashed and washed for total coliforms, faecal coliforms and E. coli by FAO (Food Quality Manual). About two hundred and fifty E. coli isolates were preserved, serotyped for presence of O157 serotype. Total coliforms, faecal coliforms and E. coli count exceeded the permissible limits in most samples. The highest Total coliforms were associated with cabbage (3.78 log10 cfu/g). Cucumber was the least contaminated by Total coliforms (2.15 log10 cfu/g). E. coli was detected in tomato, lettuce, cucumber and cabbage. Washed samples showed reduced bacterial population. Seventy six isolates of E. coli were biochemically characterized and serotyped for O157 antigen. A majority of strains could not be identified by serotyping. These findings conclude with high potentially pathogenic microbial load on salad vegetables and urge for preventive action on priority basis.展开更多
Campylobacter is one of the most common bacterial enteropathogens of food borne origin in industrialized countries with C. jejuni being the most common species followed by C. coli. The prevalence of Campylobacters in ...Campylobacter is one of the most common bacterial enteropathogens of food borne origin in industrialized countries with C. jejuni being the most common species followed by C. coli. The prevalence of Campylobacters in and around Chandigarh, India was studied by phenotypic and genotypic methods. Fecal samples from 1145 diarrheal patients and 102 healthy subjects from hospital and community were cultured on Campylobacter media and identified by Gram stain, biochemical investigations and serotyping. Molecular identification of Campylobacter isolates was done using specific primers to unique regions of 16S rRNA, Campylobacter jejuni (hipO), Campylobacter coli (aspK), Campylobacter lari (glyA) and Campylobacter upsaliensis (lpxA) genes. Identification of specific genes to look for resistance to nalidixic acid, ciprofloxacin, tetracyclin and streptomycin was also done. Campylobacters were isolated from 2.6% of patients with diarrhea. Campylobacteriosis was more prevalent in children ≤5 years old and during summer season. The most frequent serotypes were S:27, B:2, Z5:52 and V:32. All the Campylobacters isolated by culture were confirmed genotypically by identification of 16S rRNA, hipO and aspK genes. Of the 30 isolates, 27 were C. jejuni and 3 were C. coli. No C. lari or C. upsaliensis were detected. Antibiotic resistance was 40% for nalidixic acid, 23.3% for ciprofloxacin, 50% for tetracyclin and 20% for streptomycin. Campylobacter prevalence is low in the region with C. jejuni being the most common species. A high degree of resistance was found for nalidixic acid and tetracyclin but moderate for ciprofloxacin and streptomycin.展开更多
Campylobacter is one of the most common food-borne bacterial enteropathogens. We planned to investigate the prevalence and antibiotic resistogram of Campylobacter in poultry in and around Chandigarh. Poultry samples (...Campylobacter is one of the most common food-borne bacterial enteropathogens. We planned to investigate the prevalence and antibiotic resistogram of Campylobacter in poultry in and around Chandigarh. Poultry samples (n = 127) were obtained from slaughter houses/retail outlets and cultured microaerophilically on Campylobacter media. The isolates were identified phenotypically and by molecular investigation. Identification of specific genes to look for resistance to nalidixic acid, ciprofloxacin, tetracyclin and streptomycin was also done. Campylobacter was isolated from 57/127 (44.9%) of the samples. The most frequent serotypes identified were B: 2, S: 27, Z5: 52 and Z7: 57. All culture isolates (100%) were reconfirmed as Campylobacter by 16S rRNA polymerase chain reaction. Molecular identification of isolates revealed the presence of C. jejuni in 45 (79.0%), C. coli in 1 (1.8%) and co-infection of C. coli and C. jejuni in 11 (19.3%). No C. lari and C. upsaliensis were detected. Antibiogram typing showed nalidixic acid resistance in 36.8%, ciprofloxacin resistance in 35.0% and 31.5% resistance for both streptomycin and tetracyclin. A high level of Campylobacter prevalence was found among the poultry with C. jejuni being the most commonly isolated species. Resistance to major antibiotics among Campylobacter isolates from poultry was also very high. The study of prevalence of Campylobacter in poultry and its resistance to major antibiotics will help to plan risk burden strategies throughout the food chain.展开更多
A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test,affinity purified polyclonal antibodies from Guinea pigs which were immunized...A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test,affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody,and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip,the capture antibody was laid on a sample pad,the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C,Swine vesicular disease (SVD),Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically,the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple,easy and fast for clinical testing on field sites; no special instruments and skills are required,and the result can be obtained within 15 min. To our knowledge,this is the first rapid immunochromatogarpic assay for serotype A of FMDV.展开更多
Fowl adenovirus serotype 4(FAdV-4)strain SD1511 was isolated from chickens with severe inclusion body hepatitis and hydropericardium syndrome in Shandong Province,China.The isolate was cultured in primary chicken embr...Fowl adenovirus serotype 4(FAdV-4)strain SD1511 was isolated from chickens with severe inclusion body hepatitis and hydropericardium syndrome in Shandong Province,China.The isolate was cultured in primary chicken embryo kidney cells.A study of pathogenicity indicated that SD1511 readily infected 7–35-d-old chickens by intramuscular injection and intranasal and oral routes,causing 50%–100%mortality.The 35-d-old chickens suffered more severe infection than 7-and 21-d-old chickens with mortality highest in the intramuscular injection group.The serum from surviving chickens showed potent viral neutralizing capability.The complete genome of SD1511 was sequenced and analyzed.The strain was found to belong to the FAdV-4 cluster with more than 99%identity with the virulent FAdV-4 strains isolated in China in recent years except for some distinct variations,including deletions of open reading frame 27(ORF27),ORF48,and part of ORF19.Our findings suggest that SD1511 might be used as a prototype strain for the study of pathogenesis and vaccine development.展开更多
The pathogenesis of HBsAg （＋）/HBsAb （＋） double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Euk...The pathogenesis of HBsAg （＋）/HBsAb （＋） double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Eukaryotic expression plasmids PCI-SY, PCI-adw, PCI-adr, PCI-ayw, which expressed S gene product of different serotypes, were constructed and transfected into HepG2 cells. Recombinant proteins were purified from the transfected cells. At the same time, HBsAg mouse antiserum was obtained by immunizing mice with PCI-SY plasmid. HBsAg/HBsAb coexistence was simulated using these antigens and antiserum. Furthermore, the expression plasmids expressing different serotypes of S gene product including PCI-adw, PCI-adr, and PCI-ayw were injected into mice via tail vein. HBsAg and HBsAb in mice sera were tested at the first and 7th day respectively after antigen plasmids injection. Both in vitro simulation and in vivo animal models demonstrated that HBsAg antigen and HBsAb of the same serotypes Could not coexist, but HBsAg antigen and HBsAb of different serotype could coexist. HBsAg/HBsAb double positive hepatitis B virus infection could be due to infection of viruses of different serotypes.展开更多
Since 2012,the clinical cases of inclusion body hepatitis showed an increasing trend in China,causing considerable economic losses to the poultry industry.In this study,a fowl adenovirus strain CH/GDLZ/201801 was isol...Since 2012,the clinical cases of inclusion body hepatitis showed an increasing trend in China,causing considerable economic losses to the poultry industry.In this study,a fowl adenovirus strain CH/GDLZ/201801 was isolated from a chicken flock experiencing inclusion body hepatitis and analyzed by complete genome sequencing.The pathogenicity of the new virus strain was examined by experimental infection of specific pathogen free chickens.The isolate was identified by immunofluorescence and the virions presented typical icosahedral particles under transmission electron microscopy.The full genome of the isolate was 44,329 nucleotides in length with 58%G+C content.Phylogenetic analysis,based on the whole genome,revealed that the new isolate was closest to serotype 8a from the species Fowl aviadenovirus E(FAdVE).Recombination analysis and phylogenetic analysis showed that the new isolate is a recombinant strain between FAdV-8a and FAdV-8b.In infection experiments,three infected chickens showed clinical signs and one chicken died on day 7 post infection,corresponding to 5%mortality.Macroscopic and microscopic lesions in the liver were observed,and viral antigen could be detected in the livers by immunohistochemical staining and TEM.Taken together,our study describes the genomic characteristics and pathogenicity of a FAdV-8a strain in China.It would lay a solid foundation for further study of the pathogenic mechanism and vaccine development of the virus.展开更多
文摘The present review describes the current status of multiplex quantitative real time polymerase chain reaction(q PCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex q PCR for the detection of hepatitis viruses, including hepatitis A virus(HAV), hepatitis B virus(HBV), hepatitis C virus(HCV), hepatitis D virus(HDV), and hepatitis E virus(HEV). In addition, multiplex q PCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex q PCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex q PCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process.
基金supported by a grant from Beijing Municipal Natural Science Foundation [L212011]National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention [131031102000210003&102393230020020000002]。
文摘Objective To establish and modify quantitative real-time polymerase chain reaction(qPCR)-based serotyping assays to distinguish 97 pneumococcal serotypes.Methods A database of capsular polysaccharide(cps)loci sequences was generated,covering 97 pneumococcal serotypes.Bioinformatics analyses were performed to identify the cps loci structure and target genes related to different pneumococcal serotypes with specific SNPs.A total of 27 novel qPCR serotyping assay primers and probes were established based on qPCR,while 27 recombinant plasmids containing serotype-specific DNA sequence fragments were constructed as reference target sequences to examine the specificity and sensitivity of the qPCR assay.A panel of pneumococcal reference strains was employed to evaluate the capability of pneumococcal serotyping.Results A total of 97 pneumococcal serotyping assays based on qPCR were established and modified,which included 64 serotypes previously reported as well as an additional 33 serotypes.Twenty-seven novel qPCR serotyping target sequences were implemented in the pneumococcal qPCR serotyping system.A total of 97 pneumococcal serotypes,which included 52 individual serotypes and 45 serotypes belonging to 20 serogroups,could not be identified as individual serotypes.The sensitivity of qPCR assays based on 27 target sequences was 1–100 copies/μL.The specificity of the qPCR assays was 100%,which were tested by a panel of 90 serotypes of the pneumococcal reference strains.Conclusion A total of 27 novel qPCR assays were established and modified to analyze 97pneumococcal serotypes.
基金Supported by Research Department of School of Medicine Shahid Beheshti University of Medical Sciences,No.17920,and accepted by the ethic committee,Code.IR.SBMU.MSP.REC.1398.349.
文摘BACKGROUND Extended-spectrumβ-lactamase(ESBL)-producing Escherichia coli(E.coli)are among the main pathogens in urinary tract infections(UTIs)among kidney transplant patients(KTPs).AIM To estimate the prevalence of ESBL-producing E.coli in KTPs and to evaluate the most prevalent serotypes and antibacterial susceptibility patterns of isolated bacteria in Tehran,Iran.METHODS A total of 60 clinical isolates of uropathogenic E.coli were collected from 3 kidney transplant centers from April to May 2019.Antimicrobial susceptibility testing was performed by the disk diffusion method as recommended by the Clinical Laboratory and Standards Institute.The serotyping of E.coli isolates was performed by the slide agglutination method.The presence of blaTEM,blaSHV,and bla CTX-M genes was evaluated by polymerase chain reaction.RESULTS The frequency of ESBL-producing E.coli in KTPs was found to be 33.4%.All of the 60 E.coli isolates were found to be susceptible to doripenem(100%)and ertapenem(100%).High resistance rates to ampicillin(86%),cefotaxime(80%),and cefazolin(77%)were also documented.The most frequent serotypes were serotype I(50%),serotype II(15%),serotype III(25%),and serotype VI(10%).The gene most frequently found was blaTEM(55%),followed by blaCTX-M(51%)and blaSHV(41%).CONCLUSION Molecular analysis showed that blaTEM was the most common ESBL-encoding gene.The high resistance toβ-lactams antibiotics(i.e.,ampicillin,cefotaxime,and cefazolin)found in E.coli from KTPs with UTIs remains a serious clinical challenge.Further efforts to control ESBL-producing E.coli should include the careful use of all antibiotics as well as barrier precautions to reduce spread.
文摘Bovine Respiratory Disease (BRD) causes a severe form of pneumonia in all age of cattle. This study was designed to investigate the distribution of capsular types, serotypes, and virulence-associated genes of the major bacterial pathogens from BRD outbreak samples in Ethiopia. In this study 166 samples were collected from clinically sick (<i>n</i> = 107) and pneumonic lung tissue (<i>n</i> = 59). Laboratory assay confirmed isolation of <i>M. haemolytica</i> 37 (22.29%), <i>P. multocida</i> 25 (15.06%), <i>B. trehalosi</i> 12 (7.23%), and <i>H. somni</i> 15 (9.04%). PCR assay of <i>P. multocida</i> capsular typing revealed 21 (84.0%) cap A (<i>hyaD-hyaC</i>) and 4 (16.0%) cap D (<i>dcbF</i>) strains. <i>M. haemolytica</i> serotypes belonged to A: 1, A: 2, and A: 6 from 26 (70.27%), 4 (10.81%), and 7 (18.92%) isolates, respectively. <i>P. multocida</i> biotyping showed isolation of A: 1, A: 2, and A: 3 from 3 (14.29%), 2 (9.52%), and 16 (76.19%) isolates, respectively. <i>M. haemolytica</i> harbored more than 60% <i>ssa</i> gene, and 90.91% <i>sodA</i> while <i>FbpA</i>, <i>TbpA</i>, and <i>lktC</i> genes were found in all isolates. Likewise, all <i>P. multocida</i> exhibited <i>toxA</i>, <i>FbpA</i>, <i>TbpA</i>, and <i>pmSLP</i> genes. The current finding showed that <i>M. haemolytica</i> serotype A: 1 is frequently associated with BRD followed by <i>P. multocida</i> biotype A: 3. These two isolates harbored diverse virulence-associated genes and presented the pathogenic potential of the current isolates. Thus, investigation of pathogenic strains of BRD, virulence genes distribution, and molecular epidemiology of the disease from wider areas of the country are essential. Hence, continuous outbreak surveillance and molecular approaches are indispensable in designing efficient prevention strategies.
文摘Objective:To analyse the prevalence of serotypes,antibiotic resistance,and virulence genes of Group B Streptococcus(GBS)strains isolated from pregnant women at 35-37 weeks of gestation in Ho Chi Minh City,Vietnam,from January 2022 to January 2023.Methods:GBS strains were isolated through selective culture methods and confirmed by PCR.Serotyping,virulence gene detection,and antibiotic susceptibility testing were performed using PCR,gel electrophoresis techniques and Kirby-Bauer test.Results:Totally,61 GBS isolated from 300 participants have been identified including seven GBS serotypes(Ⅰa,Ⅰb,Ⅱ,Ⅲ,Ⅳ,Ⅴ,andⅥ).SerotypesⅦ,Ⅷ,andⅨwere not detected in the study population.Antibiotic resistance patterns varied:13.1%of isolates were fully susceptible,while the majority showed multi-drug resistance,with 34.4%resistant to three antibiotics.SerotypeⅠa demonstrated high susceptibility(35.7%),while serotypeⅢshowed extensive resistance,with 87.5%being resistant to at least three antibiotics.All strains are susceptible to vancomycin andβ-lactams susceptibility also remained high,but resistance to clindamycin,erythromycin,and tetracycline was high(>65%).The virulence genes scpB,cylB,fbsB,and cfb were highly prevalent(90%-100%),indicating their potential for vaccine and diagnostic development.Conclusions:Our findings provide valuable insights into GBS serotypes,resistance,and virulence factors,contributing to community monitoring,preventive measures,diagnostics,and vaccine development.However,the limited sample size necessitates further research.
基金Supported by Key Projects in the National Science &Technology Pillar Program during the Eleventh Five-year Plan Period (2006BAD06A14)~~
文摘[Objective]The aim was to provide a theoretical basis for preparation of polyclonal antibodies and monoclonal antibody that of type specificity, as well as Foot-and-mouth Disease Virus （FMDV） typing.[Method] The recombinant plasmid pGEM-CP1 that contained VPI gene of FMDV of serotype C was used as template for the VP1 and its C terminus coding fragments of FMDV of serotypes C amplification. The coding fragments of VP1 and its C terminus were respectively cloned into prokaryotic expression vector for prokaryotic expression and the reactionogenicity was detected. The purified fusion protein of FMDV VP1 and its C terminus of serotype C were used to construct the indirect ELISA method to detect positive sera of four serotypes A, O, C and Asia 1 of guinea pig and determine the cross reactivity of FMDV antibody of VP1 and its C terminus of serotype C with other three serotypes. [ Result] The recombinant prokaryotic expression plasmids of PPRO-CVP1 and pPRO-CVPlc were constructed, FMDV VP1 and its C terminus of serotype C were expressed in high level, and the molecular weight of target proteins was 33 kD and 20 kD respectively. Western blot result showed that the fusion protein of VP1 and its C terminus could react with the positive sera of guinea pig of the same serotype. ELISA results revealed that VP1 and its C terminus of serotype C are type-specific and no cross-reactivities were shown between guinea pig positive sera of FMDV of serotype C with the other three serotypes, and the C terminus showed better type-specificity. [ Conclusion] FMDV specific antigen of serotype C was obtained.
文摘Dear Editor,Dengue virus(DENV)is a positive-sense single-stranded RNA virus belonging to the Flaviviridae family,which causes dengue—a disease affecting over 400 million people annually worldwide.DENV is transmitted through the bite of mosquitoes from the Aedes genus,primarily Aedes aegypti,and has a wide distribution in tropical and subtropical areas(de Souza et al.,2022).
基金funded by the National Key Research and Development Program of China(2021YFD1800400)the National Natural Science Foundation of China(32373018)+2 种基金Jiangsu Agriculture Science and Technology Innovation Fund,China(CX(23)1029)the Excellent Research Innovation Team in Universities in Anhui Province,China(2022AH010088)the Shennong Scholar Project of Anhui Agricultural University,China(rc392101)。
文摘Streptococcus suis serotype 2(SS2)is a zoonotic pathogen that can cause acute infection,such as septicemia in pigs and streptococcal toxic shock-like syndrome(STSLS)in humans,indicating that SS2 can evade innate immunity.Macrophages perform essential antimicrobial functions in the innate immune system by engulfing and killing pathogens.Previously,a dna K mutant strain that showed impaired phagocytosis resistance ability was screened from the transposon mutant library of SS2,but the specific mechanism is unclear.In this study,we further demonstrated that DnaK was required for SS2 to be antiphagocytosed by macrophages and survive in adverse environments.A mouse challenge experiment indicated that DnaK promoted bacteremia and systemic dissemination of SS2,enhancing bacterial pathogenicity.Western blot and immunofluorescence results indicated that DnaK could be secreted by SS2 and was able to enter RAW264.7 macrophages.Then,the endocytic receptor LRP1 regulated by DnaK was identified through RNA sequencing(RNA-Seq).We found that DnaK decreased both the mRNA and protein levels of LRP1.Knockdown of the LRP1β-chain(LRP1β)significantly decreased the phagocytosis rate of the SS2 strain ZY05719,suggesting that LRP1 is a phagocytic receptor of SS2.Furthermore,inhibitor treatment assays revealed that DnaK decreased LRP1 protein levels through the transcription factor PPARγand the ubiquitin-proteasome system.In summary,DnaK contributes to the phagocytosis resistance of SS2 by decreasing LRP1 protein levels in macrophages,providing new insights into the antiphagocytosis mechanisms of SS2 and helping to understand its pathogenesis.
基金supported by the National Key Research and Development Program of China(2021FYD1800405)the National Natural Science Foundation of China(32072823).
文摘Streptococcus suis serotype 2(SS2)is an emerging zoonotic pathogen that causes meningitis in humans and pigs.This pathogen generates substantial economic losses in the swine industry while posing a significant threat to public health security.The mechanisms through which SS2 penetrates the brain and induces meningitis remain incompletely understood.This study examines the role and mechanism of SS2 collagenase-like protease(Clp)in facilitating bacterial passage across the blood-brain barrier(BBB).The research demonstrates that SS2 Clp enhanced virulence and tissue colonization while promoting BBB degradation in mice.The Δclp mutant exhibited reduced ability to traverse human brain microvascular endothelial(hCMEC/D3)cell monolayers compared to wild-type SS2,while the addition of recombinant protein rClp increased permeability.Furthermore,rClp significantly enhanced SS2 adhesion to hCMEC/D3,suppressed the expression of intercellular tight junction proteins ZO-1,Occludin,and Claudin-5 independent of its enzyme activity,and triggered hCMEC/D3 apoptosis through cell receptor ligand apoptosis and mitochondrial apoptosis pathways,partially dependent on its enzyme activity,leading to BBB disruption and enhanced permeability.Additionally,Clp enhanced the infiltration of macrophages(F4/80+),monocytes(F4/80-Ly6C+),and neutrophils(Ly6G+)into the brain following SS2 infection.These findings establish that SS2 Clp is essential for bacterial passage across the BBB,offering a theoretical foundation for improved prevention and treatment strategies for SS2-induced meningitis.
文摘Objective:To assess the burden of Group B Streptococcus(GBS)and analyze the distribution of scrotypes in relation to their source.The review highlights data gaps in transmission dynamics and regional food consumption practices,which are esscntial for designing cffective public health strategies and advancing vaccine development.Methods:Scarches were conducted in Web of Science,MEDLINE,Science Direct,PubMcd,and Scopus databases to find studies related to GBS during 1990-2025.Eligible studies were those that described prevalence,scrotype distribution or scquence type(ST)of GBS in Southeast Asian countries.Random-cffects meta-analysis was used to pool data.Results:A total of 26 studies met the inclusion criteria from cight countries.The pooled estimate of maternal GBS colonization was 15.1%,with scrotypesII,v,ⅡI,V,and I a accounting for the majority of cases(91.24%)in the Southcast Asia studies.Data on ST was limited;however,ST1 was found to be predominant in Malaysia and Thailand,while ST283 was notably linked to the consumption of raw fish.Conclusions:The pooled estimate of the maternal colonization with GBS was 15.1%which is cquivalent to many other primary and review reports worldwide.Distribution of scrotype and ST is needed to be studied in Southcast Asian countries to devise cffective preventive measures.These findings underscore thc importance of surveillance and tailored prevention strategies to combat GBS infections in Southcast Asia.
基金the“Metropolitan Mosquitoes Project”funded by the Swedish Research Council for Environment,Agricultural Sciences and Spatial Planning(Formas,grant number 2016-00364).
文摘Dengue virus(DENV)is a positive-sense single-stranded RNA virus belonging to the genus Flavivirus within the Flaviviridae family.Four serotypes,DENV 1-4,are distributed globally[1].Hanoi metropolitan city is an endemic hotspot for DENV transmission in Vietnam[2,3].The largest outbreak occurred in 2017,with more than 36000 cases and 7 deaths reported,causing by all four serotypes with the predominance of DENV1,following by DENV2[4,5].During the following dengue season,we collected 390 blood and serum samples from 197 hospitalized patients in a national hospital in Hanoi city,Northern Vietnam to identify the circulating DENV serotypes responsible for the 2018-2019 outbreak.
基金Supported by National Natural Science Foundation of China(31072155)Natural Science Foundation of Jiangsu Province(BK2010068)+1 种基金Fund for Independent Innovation of Agricultural Science in Jiangsu Province[CX(11)2060]Special Fund for Agroscientific Research in the Public Interest(201303041)~~
文摘[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []Vlethod] The gene fragments en- coding SS2 adhesive related-factors MRP, FBPS and CPS2J and a housekeeping gene aroA were amplified by reverse transcription PCR from the total RNA of SS2, cloned, and sequenced. The recombinant plasmids containing the target genes were constructed, and used as templates in Real-time PCR. [Result] Dynamic curves, stan- dard curves and melting curves of the adhesive related-factors and aroA were ob- tained by the optimized Real-time PCR system. The standard curves showed a good linear relationship between template copy number and circulation number, and the correlation coefficients （FF） of the standard curves were over 0.995. Also, these as- says were highly specific a^d there was single specific melting peak for every gene. Moreover, the assays were highly sensitive and had a detection limit of 1.0×102 copies in 1 μl of initial templates. Finally, it was highly repeatable and had a coeffi- cient of variation less than 2% for intra-assay. [Conclusion] This study will provide a way to reveal the adhesion mechanism of SS2 to different host cells at molecular level.
文摘Nutritional value of vegetables and high prices of meat and meat originated food compel common people to consume plant originated food particularly salad vegetables. Microbial population of vegetable surfaces contains a large number of pathogenic bacteria including members of Enterobactereace like Escherichia coli (E. coli). A survey was conducted in three major markets of Rawalpindi, Pakistan. Tomato, lettuce, cabbage and cucumber samples were collected from three shops of each market. Each vegetable was analysed as unwashed and washed for total coliforms, faecal coliforms and E. coli by FAO (Food Quality Manual). About two hundred and fifty E. coli isolates were preserved, serotyped for presence of O157 serotype. Total coliforms, faecal coliforms and E. coli count exceeded the permissible limits in most samples. The highest Total coliforms were associated with cabbage (3.78 log10 cfu/g). Cucumber was the least contaminated by Total coliforms (2.15 log10 cfu/g). E. coli was detected in tomato, lettuce, cucumber and cabbage. Washed samples showed reduced bacterial population. Seventy six isolates of E. coli were biochemically characterized and serotyped for O157 antigen. A majority of strains could not be identified by serotyping. These findings conclude with high potentially pathogenic microbial load on salad vegetables and urge for preventive action on priority basis.
文摘Campylobacter is one of the most common bacterial enteropathogens of food borne origin in industrialized countries with C. jejuni being the most common species followed by C. coli. The prevalence of Campylobacters in and around Chandigarh, India was studied by phenotypic and genotypic methods. Fecal samples from 1145 diarrheal patients and 102 healthy subjects from hospital and community were cultured on Campylobacter media and identified by Gram stain, biochemical investigations and serotyping. Molecular identification of Campylobacter isolates was done using specific primers to unique regions of 16S rRNA, Campylobacter jejuni (hipO), Campylobacter coli (aspK), Campylobacter lari (glyA) and Campylobacter upsaliensis (lpxA) genes. Identification of specific genes to look for resistance to nalidixic acid, ciprofloxacin, tetracyclin and streptomycin was also done. Campylobacters were isolated from 2.6% of patients with diarrhea. Campylobacteriosis was more prevalent in children ≤5 years old and during summer season. The most frequent serotypes were S:27, B:2, Z5:52 and V:32. All the Campylobacters isolated by culture were confirmed genotypically by identification of 16S rRNA, hipO and aspK genes. Of the 30 isolates, 27 were C. jejuni and 3 were C. coli. No C. lari or C. upsaliensis were detected. Antibiotic resistance was 40% for nalidixic acid, 23.3% for ciprofloxacin, 50% for tetracyclin and 20% for streptomycin. Campylobacter prevalence is low in the region with C. jejuni being the most common species. A high degree of resistance was found for nalidixic acid and tetracyclin but moderate for ciprofloxacin and streptomycin.
文摘Campylobacter is one of the most common food-borne bacterial enteropathogens. We planned to investigate the prevalence and antibiotic resistogram of Campylobacter in poultry in and around Chandigarh. Poultry samples (n = 127) were obtained from slaughter houses/retail outlets and cultured microaerophilically on Campylobacter media. The isolates were identified phenotypically and by molecular investigation. Identification of specific genes to look for resistance to nalidixic acid, ciprofloxacin, tetracyclin and streptomycin was also done. Campylobacter was isolated from 57/127 (44.9%) of the samples. The most frequent serotypes identified were B: 2, S: 27, Z5: 52 and Z7: 57. All culture isolates (100%) were reconfirmed as Campylobacter by 16S rRNA polymerase chain reaction. Molecular identification of isolates revealed the presence of C. jejuni in 45 (79.0%), C. coli in 1 (1.8%) and co-infection of C. coli and C. jejuni in 11 (19.3%). No C. lari and C. upsaliensis were detected. Antibiogram typing showed nalidixic acid resistance in 36.8%, ciprofloxacin resistance in 35.0% and 31.5% resistance for both streptomycin and tetracyclin. A high level of Campylobacter prevalence was found among the poultry with C. jejuni being the most commonly isolated species. Resistance to major antibiotics among Campylobacter isolates from poultry was also very high. The study of prevalence of Campylobacter in poultry and its resistance to major antibiotics will help to plan risk burden strategies throughout the food chain.
基金Financial supported by the Gansu ProvincialSci. & Tech. Department (1002NKDA037)
文摘A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test,affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody,and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip,the capture antibody was laid on a sample pad,the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C,Swine vesicular disease (SVD),Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically,the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple,easy and fast for clinical testing on field sites; no special instruments and skills are required,and the result can be obtained within 15 min. To our knowledge,this is the first rapid immunochromatogarpic assay for serotype A of FMDV.
基金the National Key Technology Research and Development Program of China(No.2015BAD12B01)the China Agriculture Research System(No.CARS-40-K13)
文摘Fowl adenovirus serotype 4(FAdV-4)strain SD1511 was isolated from chickens with severe inclusion body hepatitis and hydropericardium syndrome in Shandong Province,China.The isolate was cultured in primary chicken embryo kidney cells.A study of pathogenicity indicated that SD1511 readily infected 7–35-d-old chickens by intramuscular injection and intranasal and oral routes,causing 50%–100%mortality.The 35-d-old chickens suffered more severe infection than 7-and 21-d-old chickens with mortality highest in the intramuscular injection group.The serum from surviving chickens showed potent viral neutralizing capability.The complete genome of SD1511 was sequenced and analyzed.The strain was found to belong to the FAdV-4 cluster with more than 99%identity with the virulent FAdV-4 strains isolated in China in recent years except for some distinct variations,including deletions of open reading frame 27(ORF27),ORF48,and part of ORF19.Our findings suggest that SD1511 might be used as a prototype strain for the study of pathogenesis and vaccine development.
基金supported by grants from National Key Basic Research Program of China (No.2005CB522900)College Science and Technology Research Program of Anhui Province (No.KJ2008B300)
文摘The pathogenesis of HBsAg （＋）/HBsAb （＋） double positive hepatitis B virus infection was investigated by simulating HBsAg/HBsAb coexistence in vitro and establishing HBsAg/HBsAb double positive model in vivo. Eukaryotic expression plasmids PCI-SY, PCI-adw, PCI-adr, PCI-ayw, which expressed S gene product of different serotypes, were constructed and transfected into HepG2 cells. Recombinant proteins were purified from the transfected cells. At the same time, HBsAg mouse antiserum was obtained by immunizing mice with PCI-SY plasmid. HBsAg/HBsAb coexistence was simulated using these antigens and antiserum. Furthermore, the expression plasmids expressing different serotypes of S gene product including PCI-adw, PCI-adr, and PCI-ayw were injected into mice via tail vein. HBsAg and HBsAb in mice sera were tested at the first and 7th day respectively after antigen plasmids injection. Both in vitro simulation and in vivo animal models demonstrated that HBsAg antigen and HBsAb of the same serotypes Could not coexist, but HBsAg antigen and HBsAb of different serotype could coexist. HBsAg/HBsAb double positive hepatitis B virus infection could be due to infection of viruses of different serotypes.
文摘Since 2012,the clinical cases of inclusion body hepatitis showed an increasing trend in China,causing considerable economic losses to the poultry industry.In this study,a fowl adenovirus strain CH/GDLZ/201801 was isolated from a chicken flock experiencing inclusion body hepatitis and analyzed by complete genome sequencing.The pathogenicity of the new virus strain was examined by experimental infection of specific pathogen free chickens.The isolate was identified by immunofluorescence and the virions presented typical icosahedral particles under transmission electron microscopy.The full genome of the isolate was 44,329 nucleotides in length with 58%G+C content.Phylogenetic analysis,based on the whole genome,revealed that the new isolate was closest to serotype 8a from the species Fowl aviadenovirus E(FAdVE).Recombination analysis and phylogenetic analysis showed that the new isolate is a recombinant strain between FAdV-8a and FAdV-8b.In infection experiments,three infected chickens showed clinical signs and one chicken died on day 7 post infection,corresponding to 5%mortality.Macroscopic and microscopic lesions in the liver were observed,and viral antigen could be detected in the livers by immunohistochemical staining and TEM.Taken together,our study describes the genomic characteristics and pathogenicity of a FAdV-8a strain in China.It would lay a solid foundation for further study of the pathogenic mechanism and vaccine development of the virus.
