Serotype detection and phylogenetic analysis of coxsackievirus A2 causing hand, foot and mouth disease in Yunnan Province in 2021 / 中华微生物学和免疫学杂志

Objective:To analyze the non-enterovirus A71 (non-EVA71) and non-coxsackievirus A16 (non-CVA16) enteroviruses causing hand, foot and mouth disease (HFMD) in Kunming and Qujing of Yunnan Province in 2021 by sequencing the VP4/VP2 and VP1 genes and to analyze the phylogenetic characteristics of the VP1 gene of CVA2, aiming to provide reference for the prevention and control of CVA2.Methods:The samples were made and extracted strictly according to the Laboratory Manual for Hand, Foot and Mouth Disease (China Center for Disease Control and Prevention, 2018 Edition). VP4/VP2 junction regions were firstly amplified and sequenced by MD91/OL68-1 primers. These sequences were firstly edited and then "blasted" on the GenBank to determine the virus serotype. To analyze the phylogenetic characteristics of CVA2, the entire VP1 gene sequences were amplified in two segments using enterovirus species A primers. Virus serotype was again confirmed online by "Enterovirus Genotyping Tool Version 0.1". The sequences of the reference virus genotypes/sub-genotypes were downloaded according to the reference. The phylogenetic trees were constructed by Mega5.2 software and the genetic characteristics were analyzed.Results:A total of 749 non-EVA71 and non-CVA16 enteroviruses were detected in the two areas in 2021. Group A enteroviruses were the main pathogens, with CVA16 as the predominant virus, and a small number of group B enteroviruses were reported. Only five strains of CVA2 were detected with a detection rate of 0.67% (5/749), indicating that CVA2 was a rare pathogen for HFMD in the two areas. The sequencing and serotyping results were consistent using the two genomic regions of VP4/VP2 junction region and VP1 region. Phylogenetic analysis showed that three Kunming strains belonged to genotype A, while two Qujing strains belonged to genotype D.Conclusions:The detection rate of CVA2 in Kunming and Qujing was 0.67% in 2021. CVA2 was a rare pathogen for HFMD in the two regions. Phylogenetic analysis showed genotypes A and D spread in Kunming and Qujing, respectively, but had not caused epidemics. To our knowledge, this was the first report of genotype A of CVA2 in China. Strengthening the laboratory surveillance especially molecular epidemiological surveillance is valuable for the monitor and analysis of transmission source for CVA2.

Co-expression, purification and bioassay of three avian viral antigens / 生物工程学报

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.

Identification of genogroups of Infectious Bursal Disease Virus in poultry farms at Colombia / Identificación de genogrupos del virus de la enfermedad de gumboro en granjas avícolas en colombia

RESUMEN El virus de la enfermedad de Gumboro (IBDV) es un avibirnavirus con genoma dsARN que presenta altas tasas de mutación y recombinación. A pesar del efecto inmunosupresor en aves y la frecuencia con que ocurre la infección por este agente en el país son pocos los estudios que caracterizan los cuadros clínicos y se desconoce cuáles son los genogrupos circulantes. Esta investigación tuvo como objetivo determinar la frecuencia de lesiones histopatológicas en órganos del sistema inmune e identificar los genogrupos del IBDV en aves comerciales de Colombia. Para determinar la frecuencia de presentación de lesiones en órganos del sistema inmune se analizaron 381 casos clínicos de las bases de datos del Laboratorio de Patología Aviar (LPA) de la Universidad Nacional de Colombia, sede Bogotá (periodo 2016-2018). Asimismo, se secuenciaron los productos de RT-PCR del gen que codifica para la proteína viral VP2 provenientes de 35 muestras de bursas de Fabricio. Como resultado se encontró evidencia de lesiones microscópicas compatibles con procesos de inmunodepresión en órganos del sistema inmune (bursa de Fabricio, timo, bazo y médula ósea) en el 25 % (97) de los casos analizados y se identificaron los genogrupos 1, 2 y 4 en la siguiente proporción: genogrupo 1-69 % (virus clásicos), genogrupo 2-25 % (variantes) y genogrupo 4-6 % (identificado en Suramérica). Estos hallazgos demuestran la presencia de lesiones en órganos del sistema inmune y la existencia de los genogrupos 1, 2 y 3 del IBDV circulando en aves comerciales en Colombia. Esta es la primera investigación en el país con este sistema de clasificación que permite evidenciar con mayor precisión los cambios en el genoma del IBDV. Lo anterior señala la necesidad de continuar con este tipo de estudios para tener una mejor comprensión de la infección en campo y orientar el diseño e implementación de estrategias de control.

ABSTRACT Infectious Bursal Disease Virus (IBDV) is an avibirnavirus with a dsRNA genome, which has a high mutation and recombination rates. Despite the immunosuppressive effect in poultry and the frequency of infections by this agent, few studies in Colombia that characterize the clinical signs and identify the circulating genogroups have been reported. This study aimed to determine the frequency of histopathological lesions in immune organs and to identify IBDV genogroups in poultry from Colombia. In this way, the Laboratorio de Patología Aviar (LPA) databases from the Universidad Nacional de Colombia (period 2016-2018) were analyzed in order to identify the frequency of lesions present in immune organs. 35 samples of the bursa of Fabricius, positive by RT-PCR, were sequenced to the gene that codes for the VP2 protein. 97 (25 %) cases showed microscopic lesions in the immune organs. The genogroups identified and the frequencies were: genogroup 1-69 % (classical viruses), genogroup 2-25 % (antigenic variants), and genogroup 4-6 % (identified in South America). These findings demonstrate the lesions of immune organs and the presence of different genogroups circulating in commercial birds in Colombia. This indicates the need to continue with these studies in order to have a better understanding of the infection in the field and to guide the design and implementation of control strategies.

Prokaryotic expression of coxsackie virus A6 genes and preparation of polyclonal antibody against the VP1 and VP2 protein / 中华实验和临床病毒学杂志

Objective@#To construct the prokaryotic plasmid expressing the recombinant protein Coxsackie virus A6 VP1 or VP2 and prepare the antiserum of rabbit anti-CVA6 VP1 or anti-CVA6 VP2.@*Methods@#CVA6 VP1and VP2 gene fragments were amplified by reverse transcription PCR and inserted into the prokaryotic expression vectors. The recombinant plasmids were expressed in E. coli BL21 (DE3). After induction with Isopropyl β-D-Thiogalactoside (IPTG), the fusion proteins were obtained, and then purified by SDS-PAGE electrophoresis and gel extraction. The polyclonal antibodies were prepared by immunizing rabbits with the fusion proteins and analyzed with Western blot(WB) and indirect immunofluorescence assay(IFA).@*Results@#The prokaryotic expression vector of CVA6 VP1 or VP2 was confirmed by PCR, double enzyme digestion and sequencing. CVA6 VP1 and VP2 fusion proteins with high purity were obtained. WB and IFA were used to identify polyclonal antibodies.@*Conclusions@#The CVA6 VP1 and VP2 prokaryotic expression vectors were successfully constructed, and the recombinant CVA6 VP1 and VP2 proteins and their corresponding polyclonal antibodies were obtained.

Identification of epitope recognized by a monoclonal antibody against VP2 protein of bluetongue virus serotype 8 / 生物工程学报

To confirm the B cell epitope recognized by monoclonal antibody (MAb) 3G11 of bluetongue virus type 8 (BTV-8) VP2 protein prepared in our laboratory, antigen epitopes recognized by 3G11 were screened and identified by phage display technology. KLLAT sequence was found by sequencing of blue spot after four rounds panning and 283LL284 of common short peptide sequence was obtained after comparison to amino acid sequence of BTV-8 VP2 protein. The peptide sequences KLLAA, KALAT, KLAAT and KLLAT were synthesized and identified by indirect ELISA. KLLAA and KLLAT bound strongly with supernatant and as cites of 3G11 cells and reacted specifically with BTV-8 positive standard sera. Further sequence analysis showed that amino acid sequence 283LL284 was conserved among different serotypes of BTV-8 strains, and283LL284 was the key amino acids of antigen epitopes recognized by 3G11. This study laid the foundation to establish type 8 BTV specific immunological detection methods.

Establishment of two competitive ELISAs for specific detection of bluetongue virus serotype 4 / 生物工程学报

To develop a clinical diagnosis technique for bluetongue virus infection, we established serotype-specific methods to detect serotype 4 of bluetongue virus (BTV-4). Two monoclonal antibodies (mAbs) against VP2 protein of BTV-4, named 4A-1G7 and 4B-1B6, were used as competitive antibodies in the competitive enzyme-linked immunosorbent assays (C-ELISA). We detected 50 negative serum samples from sheep, goats and cattle by C-ELISA. The cut-off values of 4A-1G7 and 4B-1B6 mAbs were 49% and 40%, respectively. The results of the sensitivity, specificity and repeatability by detecting standard positive serum, were consistent with the general standard of Office International Des Epizooties. Furthermore, serum samples of BTV-4, BTV-18 and BTV-20 infection could be screened out through the combined C-ELISAs by 4A-1G7 and 4B-1B6 mAbs. Thus, this technique may diagnose BTV-4, BTV-18 and BTV-20 infections.

Immunogenicity of recombinant Lactobacillus plantarum NC8 expressing goose parvovirus VP2 gene in BALB/c mice

Goose parvovirus (GPV) continues to be a threat to goose farms and has significant economic effects on the production of geese. Current commercially available vaccines only rarely prevent GPV infection. In our study, Lactobacillus (L.) plantarum NC8 was selected as a vector to express the VP2 gene of GPV, and recombinant L. plantarum pSIP409-VP2/NC8 was successfully constructed. The molecular weight of the expressed recombinant protein was approximately 70 kDa. Mice were immunized with a 2 × 109 colony-forming unit/200 µL dose of the recombinant L. plantarum strain, and the ratios and numbers of CD11c⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, and interferon gamma- and tumor necrosis factor alpha-expressing spleen lymphocytes in the pSIP409-VP2/NC8 group were higher than those in the control groups. In addition, we assessed the capacity of L. plantarum SIP409-VP2/NC8 to induce secretory IgA production. We conclude that administered pSIP409-VP2/NC8 leads to relatively extensive cellular responses. This study provides information on GPV infection and offers a clear framework of options available for GPV control strategies.

Effect of alum on immune response in mice induced by HBoV1 VP2 VLPs / 中国免疫学杂志

Objective:To study the effect of alum on immune response in mice induced by HBoV1 VP2 VLPs.Methods:BABL/c mice were randomly divided into VLPs experimental group, alum adjuvant experimental group, PBS control group and alum adjuvant control group,the experimental group mice were intramuscular immunization with HBoV1 VP2 VLPs and HBoV1 VP2 VLPs added alum,control group mice were immunization with alum or PBS buffer,then to study the effect of alum on immune response in mice induced by HBoV1 VP2 VLPs by cellular and humoral immune strength.Results: Alum adjuvant decreased cellular immune response induced by HBoV1 VP2 VLPs(P<0.001),enhance the HBoV1 VP2 VLPs immuned serum IgG titer(P<0.05)and IgG activity(P<0.01).Conclusion:Alum adjuvant can enhance humoral immune response induced by HBoV1 VP2 VLPs,but weaken cellular immune response induced by HBoV1 VP2 VLPs,when HBoV1 VP2 VLPs used as a prophylactic vaccine it should add alum adjuvant,when used as a therapeutic vaccine,it should not add alum adjuvant.

Canarypox virus expressing infectious bursal disease VP2 protein as immunogen for chickens

Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens.

Frequência de anticorpos contra o vírus da língua azul em ovinos do estado do Ceará, Brasil / Antibodies against the bluetongue virus in sheep flocks of Ceará state, Brazil

O objetivo deste trabalho foi avaliar a ocorrência de ovinos soropositivos para o vírus da línguaazul (VLA) no Estado do Ceará, Brasil, e analisar as proteínas imunogênicas das cepas virais circulantes nesses rebanhos. O teste de imunodifusão em gel de agarose (IDGA) foi utilizado para pesquisar 271 amostras de soro oriundas de 16 rebanhos. Os resultados demonstraram que 27,3% (74/271) das amostras analisadas apresentaram anticorpos contra o agente e 68,8% (11/16) das propriedades tiveram animais positivos. O immunoblotting (IB) foi utilizado para analisar as proteínas imunogênicas do VLA a partir dos soros de animais positivos no IDGA. Os soros demonstraram forte reação contra a proteína viral VP2. Para o VLA, das sete proteínas estruturais, a VP2 é a principal a estimular a resposta imune protetora. Concluiu-se que a soropositividade para a língua azul (LA) nos rebanhos ovinos estudados no Ceará é alta, apesar dos animais não apresentarem sinais clínicos, indicativo de que o vírus ocorra de forma endêmica. Além disso, a resistência à doença apresentada pelos animais pode estar relacionada com a forte reação imunológica desses à proteína VP2. Sendo assim, outros estudos são necessários para melhor esclarecer a situação epidemiológica da LA no país, através da identificação dos vetores e sorotipos virais circulantes nas diferentes regiões.

Antibodies against the bluetongue virus in sheep flocks of Ceará state, Brazil. The objective of this work was to verify the occurrence of sheep serologically positive for bluetongue virus (BTV) in the state of Ceará, Brazil, and analyze immunogenic proteins of circulating viral strains in these flocks. The agar gel immunodifusion test (AGID) was used to examine 271 serum samples from 16 herds. The results demonstrated that 27.3% (74/271) ofthe analyzed samples presented antibodies for the agent, and that 68.8% (11/16) of the propertiespresented positive animals. Immunoblotting (IB) was used to analyze the immunogenicproteins of BTV derived from AGID positive sera. Sera showed strong reaction against viral protein VP2. Of the seven BTV structural proteins, VP2 is the major protein to elicit protective immuneresponses. It was concluded that bluetongue (BT) seropositivity in sheep flocks studied in Ceará is high, despite that the animal's do not show clinical signs, indicating that it occurs in an endemic form. The animals’ resistance to the disease may be related to the strong immune response to the protein VP2. Therefore, further studies are needed to better clarify the epidemiological situation of BT in Brazilian sheep flocks, through the identification of viral vectors and serotypes circulating in different regions.

Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens

A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry.

Seguridad viral de la Gammaglobulina anti-D / Viral safety in human anti-D immunoglobulin

Introducción. El Parvovirus B19 humano (B19) es considerado como uno de los mayores contaminantes de plasma, sangre y hemoderivados. Con el propósito de reducir el riesgo de contaminación, la Farmacopea Europea estableció el control por PCR del ADN del Parvovirus B19 en los pool es de plasma destinados a la producción de Gammaglobulina anti-D (Rh), un producto utilizado en mujeres embarazadas para pre­venir la enfermedad hemolítica del recién nacido. Además del control del ADN viral se aplican otros procesos y procedimientos adicionales para optimizar la seguridad viral y el aprovechamiento de la materia prima. Objetivos. Demostrar que el control por PCR del ADN/B19, la presencia de anticuerpos neutralizantes y la inactivación viral por pasteurización en la producción de preparados de Gammaglobulina anti-D permiten obtener un hemoderivado seguro. Materiales y Métodos. Los pooles de plasma fueron analizados para ADN/B19 mediante una técnica de PCR "in house" de adecuada sensibilidad y validada con un Standard Internacional. La extracción del ADN se realizó con columnas de sílica y la amplificación se realizó con primers específicos. Se analizaron los anticuerpos IgG contra proteína VP2 del B19 por ELISA en muestras de plasma de donaciones individuales. Se validó la pasteurización como método de inactivación viral, con virus envueltos y desnudos y se controlaron las Gammaglobulinas anti-D por PCR para descartar presencia de ADN del parvovirus B19 en producto final. Resultados. Se detectó que el 18% (n= 73) del total de pooles de plasma por PCR poseen una carga mayor a 1500 UI/ ml y el 82% con títulos menores a 1500 UI/ml. Los anticuerpos IgG anti-VP2 del B19 fueron detectados en el 75% de las muestras de plasma de donaciones individuales. La pasteurización resultó efectiva para virus envueltos y desnudos donde se detectó una pérdida de la infectividad mayor a 4.0 log de virus... (TRUNCADO)

Background. Human Parvovirus B19 (B19) is recognized as one of the majors contaminants of plasma, blood products and plasma-derived products. To reduce the risk of contamination, B19 DNA plasma pool testing by PCR during the manufacturing of anti-D Inmunoglobulin has been established by European Pharmacopoeia. This is a plasma-derived product used in pregnant women to prevent the hemolytic disease of the newborn. In addition, to optimize the viral safety and minimize the loose of plasma pools, additionals practises and procedures are implemented. Objectives. Demonstrate the B19 DNA control by PCR, the presence of antibodys with neutralizing activity and the viral inactivation by pasteurization in manufacturing of anti-D Inmunoglobulin are suitables procedures to allow to obtain a safety plasma-derived product. Materials and methods. The plasma pools were tested for B19 DNA by an "in house" PCR technique with appropriate sensitivity and validated with an Internacional Standard. The DNA was extracted with silica columns and the amplification reaction was made with specific primers. Plasma single donations were tested for VP2-specific anti-B19 IgG antibodies by ELlSA. The pasteurization was validated as a viral inactivation method, against enveloped and nude viruses. Anti-D inmunoglobulin batches were analized with PCR to ensure the absence of B19 DNA. Results. Of 73 plasma pools, 18% were found to be B19 DNA positive with a viralload > 1500 IU/ml and 82% were < 1500 IU/ml. 75% of single plasma donations were positive for VP2-specific anti-B 19 IgG antibodies. The pasteurization was effective for enveloped and nude viruses where the lost of infectivity was > 4.0 log of virus. AII anti-D inmunoglobulin batch es were negatives for B19 DNA by PCR... (TRUNCATED)

Detection of Antibodies to Infectious Bursal Disease Virus (IBDV) by Agar Gel Immunodiffusion using Recombinant VP2 Protein

Infectious bursal disease virus (IBDV) causes a highly contagious and immunosuppressive disease of chicken. Agar gel immunodiffusion using IBDV antigen extracted from bursa of Fabricius of infected chicken has been used officially for diagnosis of IBDV in Korea. In this study, in order to replace the IBDV whole virus antigen with non-infectious antigen, recombinant VP2 protein (rVP2) of IBDV was produced using recombinant baculovirus expression system. Purified baculovirus-expressed rVP2 was used as an antigen in an agar gel immunodiffusion (AGID). rVP2 antigen precipitated specifically IBDV antibodies. AGID using rVP2 antigen detected anti-IBDV antibodies from 6 dpi to 28 dpi (termination of the experiment) when specific pathogen free chickens were experimentally infected with IBDV 52/70 strain. This was consistent with result by AGID using IBDV antigen, virus neutralization test (VNT) and a commercial ELISA kit (except for one serum). The sensitivity of rVP2 was the same with that of IBDV antigen when field sera (n=324) were tested by AGID. However, AGID using rVP2 antigen detected maternal antibodies from broiler chickens (n=20) on a broiler farm up to 15 days old, although the detection rate of the AGID was relatively low compared to a commercial ELISA kit. Our results indicate that IBDV whole virus antigen from IBDV infected chickens would be replaced with recombinant VP2 protein as an antigen for AGID.

Isolation and Identification of Canine Parvovirus Serotype 2a and Its VP2 Protein Expression in Transgenic Tobacco / 中国病毒学·英文版

A strain of canine parvovirus (CPV) was isolated from feces of an ill puppy in an animal hospital in Wuhan, China. It was designated as CPV/WH02/06. This isolate was identified as serotype CPV-2a by the hemagglutination test, CPV Ag detection strip, electron microscopy, and PCR. The vp2 gene was cloned and sequenced and assigned GenBank accession number EU377537. A 1242 bp segment of the 5' region of the vp2 gene was cloned and inserted into the binary vector pBI121 and used for Agrobacterium-mediated tobacco transformation. Transgenic tobacco plants were selected on MS medium supplemented with 100 μg/mL kanamycin and 100 μg/mL timentin. Integration of the vp2 gene into the tobacco genome was confirmed by PCR using T1 progeny plants, and the expression of the VP2 protein was confirmed by Western blotting.

Co-expression of PPV VP2 and LTB on the Surface of Lactobacillus casei / 微生物学通报

Lactobacillus casei was selected as an antigen delivery vehicle for the development of oral vaccine to express recombinant LTB and porcine parvovirus (PPV) VP2 protein. The fusion protein gene encoding PPV VP2 protein and LTB, was cloned into the surface expression vector pPG, and then the recombinant expression vector pPG-VP2-LTB was electrotransformed into Lactobacillus casei 393, generating recombinant strain pPG-VP2-LTB/L. casei 393. After induced by 2% Lactose in MRS broth, an about 78 kD protein was detected in the recombinant Lactobacillus casei by SDS-PAGE. The result of Western blot indicated that the protein possessed the antigenic specificity same as the native virus protein. The result of the whole bacteria cell ELISA indicated that the LTB protein was expressed at the same time. The results of indirect immunofluorescence test and immuno-gold electron microscopy showed that the interest protein was expressed on the surface of L. casei 393. The results provide potential for the development of lactic acid bacteria oral vaccine of PPV, which used LTB as mucosal adjuvant.

Development of Peptide Antibody against Coxsackievirus B3 VP2

Coxsackievirus B3 (CVB3) is the nonenveloped virus containing a single-stranded positive-sense RNA as a genome. CVB3 infection can induce acute myocarditis and dilated cardiomypathy. CVB3 of icosahedral symmetry has four capsid proteins called VP1, VP2, VP3, and VP4. Although VP1 is a major antigenic determinant, VP2 is also an important protein for viral physiology, such as maturation cleavage and attenuation. However, VP2 study has been hampered, partly because VP2 antibody is not available. In this study, we developed peptide-based polyclonal VP2 antibody and analyzed its potency by Western blotting analysis and immunofluorescent assay. Purified B3-1 antibody (VP2 peptide antibody developed in here) showed the sensitivity and specificity, similar to VP1 monoclonal antibody which is commercially available. Moreover, this peptide antibody may be useful for double-staining with other antibodies derived from mouse. Therefore, the VP2 antibody may allow us to study CVB assembly and understand VP2 function in depth.

Efficacy of VP2 protein expressed in E. coli for protection against highly virulent infectious bursal disease virus

The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specificpathogen- free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.

Cloning and Recombinant Protein Expression of VP2 Gene of Human Parvovirus B19 / 대한임상병리학회지

BACKGROUND: Human parvovirus B19 (B19) is associated with a variety of clinical infections. As B19 antigen is not available in large quantity, studies on cloning and recombinant protein expression have been performed. We cloned and expressed VP2 protein of B19 viruses to obtain B19 antigen in large quantity. METHODS: VP2 gene was amplified by PCR. pQE vector was constructed and transformed into E. coli for the expression of recombinant VP2 protein. Fusion protein was purified by Ni-NTA protein purification system. Finally, the purified antigen was tested for ELISA to detect IgG and IgM antibodies in patients. RESULTS: Recombinant VP2 protein, produced by transformed E. coli and expressed, was confirmed to be 58 kD protein by Western blot. Antibodies were detected in sera from all of eight patients, who had been suspicous of B19 infection clinically and confirmed to harbor B19 DNA by PCR, in assays using ELISA kit coated with recombinant VP2. CONCLUSIONS: VP2 protein, purified by recombinant protein expression in this study, can be used for enzyme immunoassay for the detection of serum antibodies and can be devoted to the future study on vaccine development.

Expression of VP2 Gene of Feline Panleukopenia Virus Isolated from Tiger in Pichia pastoris Yeast / 微生物学通报

In this study,the VP2 gene of feline panleukopenia virus(FPV) isolated from tiger was successfully cloned and expressed in Pichia pastoris yeast.The VP2 gene was amplified by PCR with a pair of specific primer.Then PCR products was purified and cloned into pGEM-T for sequencing.The interesting gene fragment was recovered after the double enzyme digestion of EcoRI/NotI,then subcloned into pPICZ?A for secretory expression.The recombinant pPICZ?AVP2 was linearized with SacI and then transformed into competence yeast GS115 for expression under the induction of 1% methanol.The positive recombinants were screened by PCR method.The expression product was identified by SDS-PAGE and western-blotting.The results showed that there was a molecular weight of 32 kD,which could be specifically recognized by polyclonic antibody against FPV.It revealed that the recombinant VP2 protein had correct three-dimensional structure,which would be used as a antigen protein for the diagnosis and prevention of FPV infection in tigers.

Advances on Chlamydiophage / 微生物学通报

Bacteriophage is a kind of virus depending on bacterium,named bacterial virus,and it can multiply in bacterium.There're six types of Chlamydiophage discovered which are Chp1,Chp2,Chp3,Chp4,CPAR39 and PhiCPG1.Capsid proteins Vp1,Vp2 and Vp3 are three major structural proteins of Chlamydiophage.The study of Chlamydiophage will play great action on chlamydia infection therapy.