Construction of recombinant adenovirus expression vector of avian reovirus σC protein and its effect on proliferation of hepatocellular carcinoma cells / 中国生物制品学杂志

@#Objective To construct the recombinant adenovirus vector pAd-σC for the expression of avian reovirus(ARV)aC protein and to detect its effects on the proliferation of hepatocellular carcinoma cells,in order to build up a basis for the development of novel anti-tumor vaccines.Methods The recombinant shuttle vector pShuttle-σC was constructed by PCR amplification of ARV σC gene,and then transformed into competent BJ5183 cells containing the adenovirus vector pAdessy-1.The recombi-nant adenovirus vector pAd-σC was obtained by homologous recombination,and the virus was packaged in HEK293 cells.The virus titer was measured by TCID_(50),the expression of σC protein was determined by Western blot and ELISA,and the effect of virus on the proliferation of human hepatocellular carcinoma cells SMMC7721 was detected by CCK-8 assay.Results The recombinant shuttle vector pShuttle-σC was confirmed to be constructed correctly by double enzyme digestion and sequen-cing,and the recombinant adenovirus vector pAd-σC was constructed correctly as identified by colony PCR.σC protein was successfully expressed in hepatocellular carcinoma cells SMMC7721.The recombinant adenovirus Ad-σC had a titer of 10~(7.5)/0.1 mL,which inhibited the proliferation of hepatocellular carcinoma cells SMMC7721.Conclusion The recombinant adenovirus vector pAd-trC containing ARV σC gene was successfully constructed,and its inhibitory effect on tumor cell proliferation was preliminarily analyzed,which lays a foundation for revealing the molecular mechanism of ARV oncolytic effect and further developing novel anti-tumor biological preparation.

Cytotoxic effect of in vitro expanded NK cell-carrying oncolytic reovirus on colorectal cancer cells / 中国肿瘤生物治疗杂志

@# Objective: To evaluate whether human NK cells expanded in vitro can be used as carrier cells of reovirus and to investigate its clinical application value. Methods: Expansion of human NK cells in vitro, and flow cytometry was used to analyse the purity of CD3-CD56+ cells. Expanded NK cells were loaded with reovirus and observed by confocal microscopy, to determining the location of reovirus on NK cells. CCK-8 assay was used to detect reovirus-induced oncolysis of expanded NK cells carrying reovirus (Reo-NK) to tumor cells in the presence of neutralizing antibodies; Real-time fluorescence quantitative PCR was used to assess the relative expression of viral RNA in tumor cells. Cytotoxicity assay were performed to detect Reo-NK cells against KRAS mutant (DLD-1) and KRAS wild type (CaCo-2, HT29) colorectal cancer cell lines, ELISA matched paired antibodies assay was performed to measure the perforin level released by NK cells. Results: Confocal microscopy demonstrated that NK cells retained reovirus on the surface. Expanded NK cells could delivery reovirus to tumor cells in the presence of neutralizing antibodies, and the reovirus after delivery still had significant oncolytic activity (P<0.01); Corresponding qPCR result displayed that the expression of viral RNA in tumor cells significantly increased over time (P<0.01). Compared with NK group, Reo-NK group evidently enhanced the cytotoxicity on colorectal cancer cell lines with both KRAS gene mutant and wild (all P<0.05), and significantly increased the release of perforin (all P<0.05). Conclusion: In vitro expanded NK cells provide a convincing cell carrier for reovirus, while reovirus enhances the cytotoxicity of NK cells, and the combination of the two show a stronger killing effect on colorectal cancer cells，that has important clinical application value.

Progress of anti-tumor mechanisms of reovirus / 肿瘤研究与临床

Oncolytic viruses have made great breakthroughs in cancer treatment, especially for reovirus, which can effectively induce the death of tumor cells without harming the normal tissues. More than 80％ tumor cells are sensitive to reovirus infection. Reovirus induces the apoptosis of tumor cells and exerts anti-tumor immunity to achieve anti-tumor activity, and the curative effect of combination therapy with reovirus and chemotherapeutic drugs exceeds the effect of monotherapy. Reovirus can exert anti-tumor effects through different mechanisms, which is of great significance for the new and effective treatment of tumors in the future.

Effects of transport and storage conditions on the nucleic acid detection for three kinds of mouse RNA viral samples / 中国比较医学杂志

Objective To analyze the effect of transport and storage conditions on the detection of pathogenic nucleic acid MHV, Reo-3, MNV in laboratory mouse cecal contents samples. Methods MHV, Reo-3 and MNV were mixed with mouse cecal contents and used as reference samples,respectively. They were placed in the lysis buffer of RNA extraction reagent(buffer AVL)or normal saline, and stored at 4℃ and room temperature(22℃-25℃). RNA of these samples was extracted at 1,2,3,7,and 14 days. Then the amount of nucleic acid in samples was detected by real-time fluorescence quantitative PCR. Results A greater decrease of the amount of nucleic acid was observed when the samples were placed in normal saline than that kept in buffer AVL. The amount of nucleic acid in samples stored at 4℃ was found to be higher than that stored at 25℃ room temperature. The amount of nucleic acid in the samples which were kept in buffer AVL at 4℃ for 3 days was higher than 50%,still detectable in the samples kept for 7 days,and undetectable at 14 days. Conclusions Mouse cecal content samples are preferably stored in the lysis buffer of RNA extraction reagent and transported at 4℃ for the detection of MHV, Reo-3, and MNV nucleic acid. It is better to complete the detection test within 3 days.

Reoviral Oncotropism Against c-Myc Overexpressing HS 68 Cells

Naturally occurring reoviruses are live replication-proficient viruses specifically infecting human cancer cell while sparing normal counterpart. Since the discovery of reoviruses in 1950s, reoviruses have shown various degrees of safety and efficacy in pre-clinical or clinical application for human anti-cancer therapeutics. I have recently shown that cellular tumor suppressor genes, such as p53, ATM (Ataxia telangiectasia mutated), and RB (Retinoblastoma associated), are important in determining reoviral oncotropism. Thus, it is interesting to examine whether the aberrancy of c-Myc expression, whose normal function also plays an important role in the maintenance of genomic integrity, could affect reoviral oncolytic tropism. Hs68 cells are non-tumorigenic normal cells and resistant to reoviral cytopathic effects. Importantly, I found that c-Myc overexpression in human HS68 cells effectively induced reovirus cytophatic effects compared to mock expressed cells as shown by the typical reoviral cytophathology and an increased level of caspase-3 activity. Taken together, overexpression of c-Myc could play an important role in determining reoviral oncolytic tropism.

Immunogenicity of a vaccine of S fragments from a new type reovirus generates humoral and cellular immune responses in mice / 中国实验动物学报

Objective To explore the humoral and cellular immune responses of a vaccine of S fragments from a new type reovirus R4 strain in mice.Methods Four recombinant plasmids were constructed by respectively cloning S 1, S2, S3,S4 genes into pcDNA3.1+, and mice were intramuscularly immunized with the recombinant plasmids in a dose of 100 μg/mouse.Control vector pcDNA3.1 +and phosphate buffered saline (PBS) were used as negative controls.The spe-cific antibody level and IgG subclass (IgG1, IgG2a, and IgG2b) were detected by ELISA, and cellular immune responses to R4 were assessed using an interferon ( IFN)-γELISpot assay .Results All recombinant plasmids induced significantly higher levels of anti-R4 IgG compared with that of the controls (pcDNA3.1 +and PBS), and the titers were highest in the mice immunized with S1 and S3.On the other hand, S1 gene induced highest IgG2a antibody and the cellular immune re-sponse was best .Conclusions After the mice immunized with S 1 gene recombinant plasmid , this plasmid can initiate both cellular and humoral immune responses in mice .S1 gene recombinant plasmid is a promising vaccine candidate .

In vitro antiviral activity of seeds from Guettarda angelica against avian viruses.

The aim of this study was to evaluate the inhibition of the aqueous extract of seeds (AEs) from Guettarda angelica on cell infection by two avian RNA viruses: avian reovirus (ARV) and metapneumovirus (AMPV). The cytotoxic and antiviral activities were evaluated by MTT assay to determine the 50% cytotoxic (CC50) and inhibitory concentrations (IC50). The selectivity index (SI=CC50/IC50) also carried out. AEs exhibited antiviral activity only against ARV presenting IC50 of 23.59μg/mL. This inhibition was not due to any cytotoxic effect of AEs since the CC50 on Vero cells was of 400.60μg/mL and its SI was of 17.00; this extract also showed a virucidal effect on ARV. Previous studies also demonstrated antiviral activity of AEs extract against three animal herpesviruses. Thus, the seeds from G. angelica showing antiviral activity against DNA and RNA viruses, enveloped and non-enveloped, could be promising source of new antiviral agents encouraging its fractionation to isolate the active compound.

The occurrence of orthoreovirus, rotavirus and chicken anemia virus in chickens of the poultry industry in Minas Gerais, Brazil / Ocorrência de orthoreovirus, rotavirus e vírus da anemia das galinhas em frangos de corte da indústria avícola de Minas Gerais

Fifty-four fecal samples taken from broiler chickens from 1 to 45 days of age, and of pullets from 10 to 13 weeks of age, original from eight different poultry regions in the state of Minas Gerais, Brazil, were collected from March 2008 to January 2010 for avian Orthoreovirus (ARV) and avian Rotavirus (AvRV) analyses. For the assay of ARV, RNA was immediately extracted (Trizolâ) and transcribed into cDNA for assaying in a nested-PCR with ARV-specific primers. For AvRV, polyacrylamide gel electrophoresis (PAGE) was performed with RNA extracts obtained by phenol-chloroform extraction. CAV was additionally investigated through a nested-PCR of thymus and spleen. Results found 5.55% positive for ARV and 9.25% for AvRV. Also, CAV and ARV genomes were detected in co-infection, in a highly prostrated and claudicating chicken flock. No ARV or AvRV infections were detected in pullets. Material of a clinically affected flock was inoculated into SPF embryos, resulting in embryonic hemorrhage, whitish foci in the chorio-allantoic membrane and death. Sequencing of ARV amplicons and isolate cDNA grouped local strains with the ARV S1133 strain, historically used in live vaccines, suggesting the continued circulation of this vaccine virus strain in intensive poultry regions. Detection rates for ARV and AvRV, as well as the presence of CAV, were additionally indicative of failing biosecurity strategies for the intensive poultry regions examined.

Avaliou-se a ocorrência de Orthoreovirus (ARV) e Rotavirus (AvRV) aviários na avicultura industrial de Minas Gerais. Foram colhidas cinquenta e quatro amostras de fezes de frangos de corte entre um e 45 dias e de frangas de postura de 10 a 13 semanas de idade. Para análise de ARV, o RNA foi imediatamente extraído (Trizol), transcrito em cDNA e avaliado em uma PCR com oligonucleotídeos iniciadores específicos para ARV. Para a investigação de AvRV, os extratos de RNA foram obtidos por fenol-clorofórmio e submetidos à eletroforese em gel de poliacrilamida. Todas as amostras foram também avaliadas para o DNA do vírus da anemia das galinhas (CAV) em uma nested-PCR específica. Em frangos de corte, a positividade encontrada para ARV foi de 5,55% e para AvRV de 9,25%. CAV foi detectado em coinfecção em um plantel com refugagem, claudicação e prostração. Nenhuma amostra de poedeiras foi positiva para ARV ou AvRV. Material de plantel com sinais clínicos foi purificado e inoculado em ovos SPF embrionados, sendo obtidas lesões hemorrágicas e focos brancos na membrana cório-alantóide. O sequenciamento dos produtos de PCR e de embrião agrupou os isolados de ARV com a estirpe S1133, historicamente usada como vacina viva. Os resultados sugerem a continuada circulação da infecção por estirpes assemelhadas a ARV S1133 nas regiões de avicultura industrial. Os índices de detecção de ARV, AvRV e CAV indicam que a intensificação nas regiões produtoras tem resultado em falhas de biosseguridade.

Suppression of RNA Interference Pathway in vitro by Grass Carp Reovirus / 中国病毒学·英文版

The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined.The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK).The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay.GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells,no GCRV-specific siRNA could be detected.Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene.These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway,unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.

Molecular Characterization of Nonstructural Protein NS38 of Grass Carp Reovirus / 中国病毒学·英文版

Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, consistent with other reoviruses, is considered to cooperate with the NS80 protein in viral particle assembly. To investigate the molecular basis of the role of NS38, a complete protein was expressed in E.coli for the first time. It was found that there is a better expression of NS38 induced with IPTG at 28 ℃ rather than 37 ℃. In addition, the antiserum of NS38 prepared with purified fusion protein and injected into rabbit could be used for detecting NS38 protein expression in GCRV infected cell lysate, while there is not any reaction crossed with purified virus particle, confirming NS38 is not a component of the viral structural protein. The result reported in this study will provide evidence for further viral protein-protein and protein-RNA interaction in dsRNA viruses replication.

Anticorpos contra vírus de galinha em cracídeos / Detection of antibodies against commercial poultry viruses in cracids

Os cracídeos são Galliformes silvestres das Américas. Com o objetivo de investigar a presença de anticorpos contra vírus de galinhas em cracídeos, foram coletadas 51 amostras de soro de 10 diferentes espécies dessas aves. Esses animais eram mantidos em criatórios conservacionistas e zoológicos nos Municípios de Santa Maria, Soledade, Passo Fundo, Sapucaia, Gravataí, Viamão e Três Coroas, Estado do Rio Grande do Sul, Brasil. Anticorpos neutralizantes foram detectados em 5,9 por cento (3/51) do total de amostras testadas contra o vírus da bronquite infecciosa das galinhas, 15,7 por cento (8/51) contra o reovírus aviário e 35,3 por cento (18/51) contra o vírus da doença infecciosa da bolsa. Todas as amostras foram negativas para o vírus da bouba aviária no teste de IDGA. A detecção de anticorpos para vírus de aves comerciais sugere que os cracídeos podem ser susceptíveis à infecção por esses vírus.

The cracids are wild Galliformes native from the Americas. Fifty one serum samples were collected from individuals of 10 different species of cracids in order to obtain information regarding to the antibody status of different viruses. These birds were kept in shelters and zoos localized in Santa Maria, Soledade, Passo Fundo, Sapucaia, Gravataí, Viamão and Três Coroas counties, in the Rio Grande do Sul State, Brazil. Neutralizing antibodies were detected in the individuals serum from different species specific referring to infectious bronchitis virus in 5.9 percent (3/51) of the samples, to avian reovirus in 15.7 percent (8/51) and, to infectious bursal disease virus in 35.3 percent (18/51). All samples were negative for fowlpox virus, as measured by IDGA test. The detection of commercial poultry viruses antibodies suggests that cracids could be susceptible to infection by those viruses.

Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus / 中国病毒学·英文版

Grass carp reovirus (GCRV), a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstructural protein NS80, encoded by GCRV segment 4, has a high similarity with uNS in MRV(Mammalian orthoreoviruses), which may be associated with viral factory formation. To understand the function of the uNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region (335.742) was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28oC. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80(335.742) protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody (mouse) and NS80(335-742) specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.

Cloning and expression of the σC gene of avian reovirus / 中国兽医学报

The σC gene of ARV S1133 was designed to amplify by reverse transcription chain reaction(RTPCR).The σC gene was inserted into the vector pMD19-T,identified by PCR method and restriction enzyme,and sequenced.It showed that the insert cloned gene fragment was the σC gene of ARV.Then the gene was inserted intc the pET32a(+) and indicated that fusion expression vector pET32a-σC was constructed.The recombinant fusion protein was highly expressed in E.coli BL21 induced by 1.0 mmol/L IPTG for 5 hours in the form of inclusion bodies.The weight of recombinant fusion protein molecular is 54 000.Western-blot with ARV antibodies against the fusion protein showed the recombinant protein has a favourable reactivity.

High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells / 中国病毒学·英文版

Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.

Detección de títulos de anticuerpos contra anemia infecciosa aviar y su relación con otros virus inmunosupresores en pollos de engorde: estado Zulia Venezuela / Detection of chicken infectious Anemia antibodies and their relationship with other immunosuppressor virus in broilers chicken: Zulia state Venezuela

En Venezuela, la incidencia de enfermedades respiratorias virales e inmunosupresoras son dos de los mayores problemas en la industria avícola nacional, y hasta el momento no se cuenta con suficiente información epidemiológica al respecto que ayude a establecer medidas de control, por lo que el objetivo de esta investigación fue determinar serológicamente la presencia de anemia infecciosa aviar, reovirus y gumboro, y su relación entre ellas, así como determinar el nivel de anemia en las aves evaluadas. Se tomaron muestras de aproximadamente 14 a 15 aves, de forma semanal a diferentes edades (1, 7, 14, 21, 28, 35 y 42 días), en tres granjas comerciales, tomándose un total de 295 aves. Los títulos de anticuerpos se midieron a través de la prueba ELISA, y el nivel de anemia, por la técnica de microhematocrito. Se detectaron porcentajes de anticuerpos séricos: 90,8 por ciento (268/295) para anemia infecciosa aviar; 82,4 por ciento (244/295) para reovirus y 97 por ciento (286/295) para la enfermedad infecciosa de la bursa. En cuanto a los valores de hematocrito se encontró en forma general que, el 19,6 por ciento (58/295) de las aves evaluadas presentaron anemia, mostrando valores de hematocrito entre 20 y 27 por ciento. Se observó una correlación positiva altamente significativa entre la anemia infecciosa aviar y los otros virus inmunosupresores estudiados, con gumboro (r=0,437; P<0,0001) y reovirus (r=0,312; P<0,0001). Los resultados obtenidos en este trabajo permiten demostrar la presencia del virus de la anemia infecciosa aviar en pollos de engorde en la región, de manera aislada o asociada con reovirus y gumboro, que pudiesen estar afectando en forma subclínica o clínica las granjas avícolas zulianas.

The incidence of viral respiratory and immunosuppressant diseases are two of the biggest problems in the Venezuelan poultry industry, however in the country, there is not enough epidemiological information that helps to establish control measures. The aim of this research was to determine the presence of serological chicken anemia virus, reovirus and gumboro, and it´s correlation between them as well as to determine the level of anemia in the evaluated birds. In this research, approximately fourteen or fifteen (14 or 15) birds were tested weekly and at different ages (1; 7; 14; 21; 28; 35 y 42 days), in three commercial farms. The samples were taken from a total of 295 birds. The viral antibodies were determined by ELISA test and the anemia levels by micro-hematocrit. The presence of seropositivity was 90.8% (268/295) for the chicken anemia virus, 82.4% (244/295) for reovirus and 97% (286/295) for gumboro. Of the total, 9.6% (58/295) of the evaluated birds presented anemia, showing values of hematocrits between 20% and 27%. A positive correlation was found between chicken anemia virus and the other immunosuppressor viruses studied, gumboro (r=0.437; P<0.0001) and reovirus (r=0.312; P<0.0001). The obtained results in this research demonstrated the presence of viral anemia, in broilers, in the Zulia Region, with or without the presence of reovirus and/or gumboro. That could have an effect, in either sub-clinical or clinical forms.

Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells / 中国病毒学

Grass carp reovirus (GCRV), a disaster agent to aquatic animals, belongs to Genus Aquareovirus of family Reoviridea. Sequence analysis revealed GCRV genome segment 8 (s8) was 1296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa. To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter, the recombinant baculovirus, which contained the GCRVs8 and eGFP (enhanced green fluorescence protein)genes, was constructed by using the Bac-to-Bac insect expression system. In this study, the whole GCRVs8 and eGFP genes, amplified by PCR, were constructed into a pFastBacDual vector under polyhedron (PH) and p10 promoters, respectively. The constructed dual recombinant plasmid (pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid (AcGCRVs8/eGFP) by transposition. Finally, the recombinant bacluovirus (vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells. The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection, and gradually enhanced and extended around 5days culture in P1(Passage1) stock. The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus (BV) stock. Additionally, PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus. Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.

Characterization and Detection of Enteric Viruses in Surface Water, Finished Water, Tap Water by Total Culturable Virus Assay (TCVA) Method

The information of species and quantity of enteric viruses in surface water, finished water, and tap water is important in helping understand the pathogenesis of viruses, providing information about health and hygiene, improving handling technique of drinking water, and establishing the standards of water quality. Using standard total culturable virus assay-most probable number (TCVA-MPN) method, we tried to detect infectious enteric viruses in surface water, finished water, and tap water samples that were collected and evaluated according to the information collection rule (ICR). The results obtained with TCVA method were compared to the results from both reverse transcription-polymerase chain reaction (RT-PCR) and integrated cell culture-RT-PCR (ICC-RT-PCR) method. Five of 86 samples (5.8%) were positive as determined by the TCVA-MPN method. Two of 86 samples (2.3%) were positive for reovirus as determined by the RT-PCR and ICC-RT-PCR, and contained infectious reovirus. One of 86 samples (1.7%) was positive for coxsackievirus type B3 as determined by the RT-PCR and ICC-RT-PCR.

Detection of Enteroviruses and Mammalian Reoviruses by RT-PCR and Integrated Cell Culture-PCR in CPE-positive Surface Water Samples

The environmental water samples assayed by total culturable virus assay (TCVA) were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and integrated cell culture-PCR (ICC-PCR) method for enteroviruses and reovirus since they are the usually detected virus groups by culture assays. The detection sensitivities of the TCVA, RT-PCR, and ICC-PCR were compared and the overall reliability of the detection was analyzed to confirm environmental samples for enteric viruses. A total of eight samples from different areas was analyzed by performing TCVA, RT-PCR, and ICC-PCR. Virus concentrations in surface water samples ranged from 1.03 to 47.3 most probable numbers of infectious units (MPN) per 100 liters. When primers specific for both enteroviruses and reoviruses were used in both RT-PCR and ICC-PCR, all the samples (100%) were positive for the viruses. Reoviruses were the most frequently detected ones among the samples. According to the sequence results of enteroviruses, five of the samples were contaminated by coxsackievirus type B3, and the rest by coxsackievirus type B6, echovirus type 30, or vaccine strain poliovirus type 3. It was observed that both enteroviruses and reoviruses were detected concurrently in all CPE-positive environmental water samples.

Detection of Enteroviruses and Mammalian Reoviruses by RT-PCR and Integrated Cell Culture-PCR in CPE-positive Surface Water Samples

The environmental water samples assayed by total culturable virus assay (TCVA) were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and integrated cell culture-PCR (ICC-PCR) method for enteroviruses and reovirus since they are the usually detected virus groups by culture assays. The detection sensitivities of the TCVA, RT-PCR, and ICC-PCR were compared and the overall reliability of the detection was analyzed to confirm environmental samples for enteric viruses. A total of eight samples from different areas was analyzed by performing TCVA, RT-PCR, and ICC-PCR. Virus concentrations in surface water samples ranged from 1.03 to 47.3 most probable numbers of infectious units (MPN) per 100 liters. When primers specific for both enteroviruses and reoviruses were used in both RT-PCR and ICC-PCR, all the samples (100%) were positive for the viruses. Reoviruses were the most frequently detected ones among the samples. According to the sequence results of enteroviruses, five of the samples were contaminated by coxsackievirus type B3, and the rest by coxsackievirus type B6, echovirus type 30, or vaccine strain poliovirus type 3. It was observed that both enteroviruses and reoviruses were detected concurrently in all CPE-positive environmental water samples.

Isolation and Characterization of Reovirus in Korea

Reovirus was found to inhabit both the respiratory and the enteric tract of human find animals. The genome of reovirus comprises 10 segments of double-stranded RNA, total size 24 kbp. Nine strains of reovirus were isolated from human and field mice in Korea. Aseptically collected sera from human and lung tissues from field mice were used for virus isolation For serotype determination, hemagglutination inhibition test was used, and three strains were confirmed to type 2 and six strains to type 3. To determine the genomic diversity and molecular phylogeny of reoviruses isolated in Korea, part of 54 genomic segment of reovirus was enzymatically amplified and directly sequenced. In nucleotide level, Apo98-35 strain showed 15.4%, 19.3%, and 14.4% differences compared to type 1 (T1L, Lang), type 2 (T2J), and type 3 reference strains, respectively. In amino acid level, Apo98-35 strain showed 10.5%, 13.7%, and 9.5% differences compared to type 1, type 2, and type 3 reference strains, respectively. Using the maximum parsimony method based on 285 bp spaning region of the S4 genomic segment, phylogenetic analysis indicated that Apo98-35 from Korea formed different phylogenetic branch. Our data obtained by sequence and phylogenetic analyses of reoviruses are consistent with the distinct geographically dependent evolution of reoviruses in Korea.