Serological prevalence and risk factor analysis of hepatitis G virus infection in Hubei Province of China

Hepatitis G virus (HGV),also known as GB virus C, is a recently cloned virus which may be associated with human non A-E hepatitis[1，2] It is parenterally transmitted and usually coinfected or superinfected with hepatitis B or hepatitis C virus[3-5]. Some investigations have been reported on the seroprevalence and molecular prevalence of HGV infection in different areas and different population[6-15]. Current infection of HGV is diagnosed by detection of HGV RNA, and past infection with HGV is detectable by testing anti-HGV envelope protein (E2)[16-17]. To investigate the prevalence of HGV in Hubei Province, a central area of the People's Republic of China, ELISA and RT-PCR were employed to detect serum anti-HGV and HGV RNA in 1516 patients who were divided into 16 groups.

Seroprevalence of Hepatitis E Virus in Human and Pigs in Pig Farms from Guangdong Province,Southern China

Hepatitis E is caused by hepatitis E virus （HEV）, which has been classified into four genotypes. Genotypes 3 and 4 are regarded as zoonotic pathogens. Accumulating researches indicate that genotype 4 is the main HEV strain circulating in China, and there are high levels of seropositive pigs and human in some provinces of China. In this study, serum samples from pigs and from human occupationally exposed to pigs were obtained from pig farms in Guangdong Province, in subtropical southern China, in order to investigate for the first time the prevalence of anti-HEV immunoglobulin G （IgG） in the region. Antibodies against HEV were detected by Enzyme-Linked Immunosorbent Assay （ELISA） using a commercially marketed kit. The results showed that high numbers of pigs （74/94; 78.7%） and human （50/94; 53.2%） from three pig farms in Guangdong Province were positive for anti-HEV IgG. The correlation coefficient relating the prevalence in pigs and human on different farms was 0.920. The seropositive rate in males （human） was 48.8% （20/41） and that in females was 47.7% （9/19）, which showed no statistically significant difference. These data indicated that there was a high prevalence of anti-HEV antibodies in pigs and in human with occupational exposure to pigs. The risk of infection with HEV in both human and pigs in Guangdong Province appeared to be age-dependent, to a certain extent. This study provided basic data for further researches on HEV and was a reminder that more attention should be paid to HEV infection both in pigs and workers on pig farms in the study region.

Prevalence and Spatial Distribution of Badnavirus in the Banana (Musa spp) Major Growing Areas in Burkina Faso

Banana streak virus (BSV) and Sugarcane bacilliform virus (SCBV) are two badnaviruses commonly found in all banana growing areas of the world. It is a threat to the production and improvement of Musa germplasm. In Burkina Faso, the presence of badnaviruses was reported in banana producing regions. The objective of this study was to determine the prevalence of BSV and SCBV in banana production areas of Burkina Faso. A survey followed by a symptomatologic study was conducted in banana plantations in 27 localities of the nine main banana producing regions from July to October 2018 and September to December 2020. In all, 251 leaf samples were collected and analysed for BSV and SCBV infection by Indirect Antigen Coated Plate Assay-ELISA followed by amplification of the RT/RNase H region using Polymerase chain reaction with Badna FP/RP and SCBV F/R primers, respectively. A variety of symptoms were observed on almost all plant organs which were revealed due to BSV by symptomatologic study. The results of serological and molecular diagnosis revealed a high overall prevalence of BSV in 80.48% of the samples tested. BSV was distributed in seven survey regions out of nine with prevalence ranging from 10% to 100% in North, Centre, Centre West, Hauts Bassins, Cascades, Centre East and Boucle of Mouhoun regions. Very low prevalence was recorded for SCBV in Cascades and East Centre region with 4.35 and 12.5%, respectively. Species detection using specific primers to each species revealed three main species: Banana streak Obino l’ewaï virus (BSOLV), Goldfinger virus (BSGFV) and Imové virus (BSIMV) in the samples tested, respectively in the proportions of 23%, 8% and 0.8%. Co-infection between BSV species was also detected.

Residues of 862, 921 of VP3 are associated with virulence in infectious bursal disease virus

Reverse genetics was used to study the effect of particular amino acids of infectious bursal disease virus (IBDV) on virulence. Using site-directed mutagenesis, altering of two amino acids in VP2 (Q253H, A284T) and VP3 (H783Q, V862M, I921V) in the segment A of a Chinese very virulent IBDV field strain Harbin-1, 4 virus mutants including H253/284, H783/862, H862/921, H921/783 were rescued. To evaluate the characteristics of the recovered viruses in vivo, we inoculated 4-week-old chickens with virus mutants and rescued Harbin- 1 (rHarbin-1), analyzed their bursae for pathological lesions 4 days postinfection. rHarbin-1 and H783/862, H253/284 caused severe bursal lesion, milder lesion for H862/921, mildest for H921/783. However, H253/284 caused the lowest mortality. The results showed that residue at position Q253, A284 of VP2 and V862, I921 of VP3 gene are involved with virulence, but there is difference between VP2 and VP3’s role in virulence. The ability of 862 and 921 to control virulence in VP3 is stronger than 253 and 284.

Genes transactivated by hepatitis C virus core protein, a microarray assay

AIM: To explore the new target genes transactivated by hepatitis C virus (HCV) core protein and to elucidate the pathogenesis of HCV infection. METHODS: Reverse transcribed cDNA was subjected to microarray assay. The coding gene transactivated by HCV core protein was cloned and analyzed with bioinformatics methods. RESULTS: The expressive vector of pcDNA3.1(-)-core was constructed and confirmed by restriction enzyme digestion and DNA sequencing and approved correct. mRNA was purified from HepGZ and HepG2 cells transfected with pcDNA3.1(-)-core, respectively. The cDNA derived was subjected to microarray assay. A new gene named HCTP4 was cloned with molecular biological method in combination with bioinformatics method. CONCLUSION: HCV core is a potential transactivator. Microarray is an efficient and convenient method for analysis of differentially expressed genes.

Real-time RT-PCR Assay for the detection of Tahyna Virus

A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.

Diagnostic value of antigenemia assay for cytomegalovirus gastrointestinal disease in immunocompromised patients

AIM:To investigate the utility of the cytomegalovirus(CMV)antigenemia assay for the diagnosis of CMV gastrointestinal disease(GID). METHODS:One hundred and thirty immunocompromised patients were enrolled in this study.Patients with a history of anti-CMV treatment and who had not undergone examination using the antigenemia assay were excluded.CMV-GID was defined as the detection of large cells with intranuclear inclusions alone or associated with granular cytoplasmic inclusions by biopsy.Biopsy sections were stained with hematoxylin and eosin and immunohistochemically stained with anti-CMV.We evaluated the association between CMV-GID and patient characteristics(symptoms,underlying disease,medication,leukocyte counts,and antigenemia assay).All patients were checked with an human immunodeficiency virus(HIV)antibody test before endoscopic examination.White blood cell(WBC)counts were obtained from medical records within 1 wk of endoscopy.Leukopenia was defined as a total WBC count<5000 cells/mm 3 . For HIV patients,we also checked CD4+counts from medical records. RESULTS:A total of 99 patients were retrospectively selected for analysis.Of the immunocompromised patients,19 had malignant disease,18 had autoimmune disease,19 had disorders of biochemical homeostasis, three had undergone transplantation,and 45 had HIV infection.A total of 50 patients had received immunosuppressive therapy.No patients had inflammatory bowel disease.Fifty-five patients were diagnosed as having CMV-GID.Univariate analysis indicated an association between HIV infection,leukopenia,and positive antigenemia and CMV-GID(P<0.05).Multivariate analysis using logistic regression revealed that HIV infection and positive antigenemia were the only independent factors related to CMV-GID(P<0.01).The sensitivity,specificity,positive predictive value,and negative predictive value of antigenemia for CMV-GID were 65.4%,93.6%, 91.9%,and 71.0%,respectively.In a subgroup analy-sis,patients with leukopenia displayed low sensitivity and high specificity.Minimal differences in accuracy were seen among patients with or without leukopenia. HIV-infected patients displayed low sensitivity and high specificity.Accuracy barely differed between HIV-positive and-negative patients.In HIV-infected patients, CD4 count<50 cells/μL resulted in low sensitivity and high specificity.Differences in accuracy among patients were minor,regardless of CD4 count.In patients who had undergone both quantitative real-time polymerase chain reaction(PCR)and antigenemia assay,real-time PCR was slightly more accurate in terms of sensitivity than the antigenemia assay;however,this difference was not statistically significant(P=0.312). CONCLUSION:If the antigenemia test is positive,endoscopic lesions are acceptable for the diagnosis of CMVGID without biopsy.The accuracy is not affected by HIV infection and leukopenia.Either PCR or the antigenemia assay are valid.

Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs

African swine fever(ASF),caused by the African swine fever virus(ASFV),is a devastating disease of domestic and wild pigs.There is no effective vaccine,and the control of the disease relies mainly on surveillance and early detection of infected pigs.Previously,serological assays,such as ELISA,have been developed mainly based on recombinant structural viral proteins of ASFV,including p72,p54,and p30.However,the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs.Therefore,new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required.In this study,we expressed and purified a recombinant p B602 L protein.The purified p B602 L protein was then used as an antigen to develop an indirect ELISA assay.This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China,such as classical swine fever virus,pseudorabies virus,and porcine parvovirus.This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples,including an unknown number of antiASFV sera.The coincidence of the two assays was 95%.Furthermore,the p B602 L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7 GD in pigs.The results showed that the antibody levels in all vaccinated pigs,starting from the 10 th day post-inoculation,have increased continuously during the observation period of 45 days.Our results indicate that this p B602 L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

Three sensitive and reliable serological assays for detection of potato virus A in potato plants

Vegetative propagation of seed potato often allows passaging of viruses to seed tubers,resulting in significant yield losses and reduction of potato tuber quality.Thus,virus detection approach is crucial for effective virus management programs and the production of virus-free seed potatoes.Among the reported potato-infecting viruses,potato virus A(PVA)is considered as one of the most important viruses in potato-growing regions worldwide.This study prepared four hybridoma lines secreting PVA-specific monoclonal antibodies(MAbs)(2D4,8E11,14A6 and 16H10)using purified PVA virions as an immunogen.Western blotting results indicated that all the four MAbs reacted strongly and specifically with the putative capsid protein of PVA.Using these four MAbs,this study developed antigen-coated plate enzyme-linked immunosorbent assay(ACP-ELISA),Dot-ELISA and Tissue print-ELISA for detection of PVA infection in potato plants.The results indicated that PVA can be detected in crude tissue extracts from infected potato plants diluted up to 1:327680(w/v,g m L^(-1))by ACP-ELISA or up to1:10240 by Dot-ELISA.The Tissue print-ELISA is the quickest and easiest approach among the three serological assays,and is more suitable for onsite large-scale potato screening programs.Further analyses of field-collected potato samples showed that the sensitivities and specificities of the three serological approaches were similar to those of RT-PCR in PVA detection and confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces of China.Hence,the results strongly suggest that these highly sensitive serological approaches based on PVA-specific MAbs are useful and powerful for PVA-free seed potato production programs and PVA field surveys.

Hepatitis C virus antigens enzyme immunoassay for one-step diagnosis of hepatitis C virus coinfection in human immunodeficiency virus infected individuals

BACKGROUND Current diagnosis of hepatitis C virus(HCV)infection requires two sequential steps:testing for anti-HCV followed by HCV RNA PCR to confirm viremia.We have developed a highly sensitive and specific HCV-antigens enzyme immunoassay(HCV-Ags EIA)for one-step diagnosis of viremic HCV infection.AIM To assess the clinical application of the HCV-Ags EIA in one-step diagnosis of viremic HCV infection in human immunodeficiency virus(HIV)-coinfected individuals.METHODS The study blindly tested HCV-Ags EIA for its performance in one-step diagnosing viremic HCV infection in 147 sera:10 without HCV or HIV infection;54 with viremic HCV monoinfection;38 with viremic HCV/HIV coinfection;and 45 with viremic HCV and non-viremic HIV coinfection.RESULTS Upon decoding,it was 100%accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR test.In five sera with HCV infection,HCV RNA was as low as 50-59 IU/mL,and four out of five tested positive for HCV-Ags EIA.Likewise,it was also 100%accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR in 83 sera with HCV and HIV coinfection,regardless if HIV infection was active or not.CONCLUSION The modified HCV-Ags EIA has a lower detection limit equivalent to serum HCV RNA levels of approximately 100 IU/mL.It is highly sensitive and specific in the setting of HIV coinfection,regardless of HIV infection status and CD4 count.These data support the clinical application of the HCV-Ags EIA in one-step diagnosis of HCV infection in HIV-infected individuals.

Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry

BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease.Furthermore,from natural products,active compounds against vital HCV proteins like non-structural protein 3(NS3)protease could be identified to prevent viral proliferation in the host.AIM To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODS Full-length NS3 without co-factor non-structural protein 4A(NS4A)and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli.The expressed protein was purified by metal ion affinity chromatography and gel filtration.Citrus fruit extracts were screened using fluorescence resonance energy transfer(FRET)assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry(MS)/MS technique.Among different polyphenols,highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTS NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein.Furthermore,in enzyme kinetic studies,NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3.So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease.FRET assay was developed and validated by the half maximal inhibitory concentration(IC50)values of commercially available inhibitors.Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91%of protease activity.Among the compounds identified by LCMS analysis,hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of-10.98.CONCLUSION Fused NS4A-NS3 protease is functionally more active,which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32μmol/L.

Comparative Assay of Hepatitis B and C Virus Infection Markers by Different Assay Kits^1

In order to compare sensitivity of EIA and RIA assay kits for hepatitis B and C virus (HBV and HCV, respectively) infection markers, 100 serum samples in total were collected form 50 adult women each in urban and rural areas in northeast China. The number of positive cases to the three infection markers on HBV (i.e., HBsAg +, anti HBs +, and anti HBc +) and the one on HCV (anti HCV +) were examined in two laboratories, i.e., in Laboratory A with EIA kits produced in China and in Laboratory B with RIA kits. HCV infection positivity (anti HCV +) was examined by EIA kits in both laboratories, but from different sources in and outside of China, respectively. The assay in Laboratory A gave 2 HBsAg + cases out of the 100 cases examined, whereas there were 9 positive cases in Laboratory B. In contrast, 19 cases were positive to anti HCV when examined in Laboratory A, and there were 3 cases in Laboratory B. Thus, the kits used in Laboratory A gave fewer HBsAg + and more anti HCV + cases than the kits used in Laboratory B. The prevalence of anti HBs + or anti HBc + and cases did not differ when assayed in the two laboratories with EIA and RIA kits, respectively. The agreement of positive and negative findings between the two sets of testing were 93%, 93%, 93%, 86% and 82% for HBsAg, anti HBs, anti HBc, HBV (i.e., either positive to anyone of the three markers or negative to all three markers), and anti HCV, respectively. The implication of the observation on epidemiology on HBV and HCV infection prevalence was discussed.

Feasibility of the Routine Clinical Use of a Multiplex Virus Polymerase Chain Reaction Assay Based on Blood Virus Detection in Hematopoietic Stem Cell-Transplanted Patients

Background: Multiplex virus assays are useful in immunocompromised hosts but still challenging in routine clinical settings in terms of their sensitivity, specificity, reproducibility, and time and cost performances. In recent years, we developed a qualitative multiplex virus PCR assay capable of the simultaneous detection of 13 virus species within 3 h. However, because of the multiple and concomitant nature of this virus assay, it should be validated for qualitative reliability. Materials and Methods: As a preclinical examination, this multiplex PCR was able to detect 1.25 × 103 copies/mL of 13 synthesized virus genomes and preserved same virus DNAs by the serial dilution method. Blood samples from 40 patients who underwent hematopoietic stem cell transplantation were then examined by multiplex PCR for 13 virus species, followed by quantitative real-time PCR for all 13 virus species as reference PCR when these patients developed symptoms suggestive of viral infection. Results: In 421 cumulative qualitative-quantitative tests, the multiplex PCR certainly detected 1.0 × 103 copies/mL of 5 viruses (CMV, JCV, BKV, HHV-6, ADV) that were frequently detected and thus reasonably analyzed. The positive and negative predictive values of multiplex PCR were 84.2% - 93.3% and 90.7% - 99.0%, respectively, and sensitivity and specificity were 59.0% - 83.3% and 97.2% - 99.2%, respectively, for these 5 viruses. Conclusion: From these performances, the multiplex PCR assay may be acceptable in a routine clinical laboratory setting.

Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5'UTR of Internal Ribosomal Entry Site

Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.

Development and Characterization of Monoclonal Antibody Specific to Nuclear Protein of Avian Influenza Virus Type A

Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mabs were identified by immunofluorescent assay（IFA） and enzyme linked immunosorbent assay （ELISA）. These five Mabs which were named as AIV-NP-2C3, AIV-NP-6A5, AIV-NP-3 H9, AIV-NP-7B4, AIV-NP-2H4 could react with all viruses of AIV-H9 strains in tests. The result of Western blotting showed that only the 60 ku protein antigen of AIV-H9 could be recognized by the Mabs but never recognized by New castle disease virus, REV and infectious bursa disease virus. The result of preliminary application showed that avian influenza viruses could be deetected bv Mabs in IFA and ELISA. All these Mabs will probably play important roles in preventing and monitoring avian influenza viruses.

Preparation and Preliminary Identification of Fluorescein Labeled Monoclonal Antibody against Canine Distemper Virus

[Objective] The aim of the present study was to develop a direct immunofluorescence method for the diagnosis of canine distemper （CD） with FITC-conjugated monoclonal antibodies （FITC-McAb）.[ Metbod] The McAb against CDV, designated as CE3, was purified with protein G and labeled with FITC through agitation method. After purification and identification, the optimal working concentration of FITC-labeled CE3 was determined. Then 61 clinical samples of suspected canine distemper were detected by direct immunofluorescence assay. [ Result] The absorption test, blocking test and specificity test showed that the labeled antibody had high specificity and sensitivity, but didn＇t have cross reaction with canine parvovirus （CPV）, canine parainfluenza virus （CPIV）, canine adenovirus （CAV） and rabies virus （RV）. The optimal working concentration was 1：80. The positive rate of clinical suspected samples was 48%. [ Conclusion] The direct immunofluorescence assay developed in this study was rapid, specific and convenient, and had great significance for the early diagnosis of canine distemper.

Preliminary study of a dot immunogold filtration assay for rapid detection of anti-HCV IgG

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Interaction of hepatitis C virus envelope glycoprotein E2 with the large extracellular loop of tupaia CD81

AIM: To further analyze the interaction of tupaia CD81 with hepatitis C virus (HCV) envelope protein E2. METHODS: A tupaia CD81 large extracellular loop (CD81 LEL), which binds to HCV E2 protein, was cloned and expressed as a GST-fusion protein, and interaction of HCV E2 protein with a tupaia CD81 LEL was evaluated by enzyme-linked immunosorbent assay (EIA). RESULTS: Although tupaia and human CD81 LEL differed in 6 amino acid changes, tupaia CD81 LEL was strongly recognized by anti-CD81 antibodies against human CD81 LEL conformation-dependent epitopes. Investigating LEL CD81-E2 interactions by EIA, we demonstrated that binding of tupaia CD81 LEL GST fusion protein to recombinant HCV E2 protein was markedly reduced compared to binding of human CD81 LEL GST fusion protein to recombinant HCV E2 protein. CONCLUSION: These data suggest that the structural differences in-between the tupaia and human CD81 may alter the interaction of the large extracellular loop with HCV envelope glycoprotein E2. These findings may be important for the understanding of the mechanisms of binding and entry of HCV to PTHs.

Monoclonal antibody-based serological detection of Citrus yellow vein clearing virus in citrus groves

Citrus yellow vein clearing virus （CYVCV） is considered as the causal agent of Citrus yellow vein clearing disease and belongs to the genus Mandarivirus in the family Alphaflexiviridae. Capsid protein （CP） of CYVCV Chongqing isolate （CYVCV- CQ） was produced using a prokaryotic expression system and used as the immunogen for monoclonal antibody （MAb） production. Four highly specific and sensitive murine MAbs and one polyclonal antibody were prepared in this study. Titers of the four MAbs in ascites fluids ranged from 10-6 to 10-7 as determined by indirect enzyme-linked immunosorbent assay （ELISA）. Three serological assays, including dot enzyme-linked immunosorbent assay （dot-ELISA）, tissue blot-ELISA, and double-antibody sandwich （DAS）-ELISA, were developed for quick and reliable detections of CYVCV in citrus samples. The developed dot-ELISA and DAS-ELISA methods could detect CYVCV in the infected citrus leaf crude extracts diluted at 1：2 560 and 1：10 240 （w/v, g mL^-1）, respectively. The detection result of 125 citrus leaf samples collected from citrus groves in Yunnan Province and Chongqing Municipality of China showed that approximately 36% samples were positive for CYVCV. This virus was, however, not＇detected in any sample collected from Zhejiang or Jiangxi Province, China.

Dot immunogold filtration assay for rapid detection of anti-HAV IgM in Chinese

INTRODUCTION The hepatitis A virus specific immunoglobulin M(IgM)antibody is a specific serological marker forearly diagnosis of hepatitis A..At present,themethods used at home or abroad for detecting anti-HAV IgM are RIA,ELISA and SPHAI.The dotimmunogold combination assay that has beendeveloped since 1989 is a new technique with theproperty of simple and rapid immunologicaldetection,by using the red colloidal gold particles tolabel the antibodies as indicator,and the