Serological survey of bluetongue virus in sheep from Minas Gerais / Inquérito sorológico do vírus da língua azul em ovinos de Minas Gerais

Bluetongue is an infectious, non-contagious disease that affects domestic and wild ruminants, caused by a virus from the Orbivirus genus, Reoviridae family, transmitted by arthropod vectors of the Culicoides genus. This paper aims to be the first serological survey of bluetongue in sheep from the Meso-regions of Campo das Vertentes and South and Southeast of Minas Gerais. Samples were collected from sheep from different properties. The serum samples were submitted to Agar Gel Immunodiffusion (AGID) and competitive Enzyme-Linked Immunosorbent Assay (cELISA). 303 serum samples were submitted to AGID and cELISA. In these samples, 164 (54.13%) were positive in the AGID technique, and 171 (56.44%) positive in the cELISA technique, with an almost perfect agreement between the techniques (kappa index = 0.887). In all visited properties, positive animals have been found in the herd. Animals acquired from properties of the studied mesoregions were more likely to be positive in IDGA and cELISA tests than animals acquired from properties in other regions of Brazil (p<0.001). These results suggest that bluetongue virus (BTV) is widespread in the mesoregions of Campo das Vertentes and South and Southeast of Minas Gerais.(AU)

A língua azul (LA) é uma doença infecciosa, não contagiosa, que acomete ruminantes domésticos e silvestres, causada por um vírus do gênero Orbivirus da família Reoviridae, transmitida por vetores artrópodes do gênero Culicoides. O presente estudo representa o primeiro trabalho a realizar um inquérito sorológico da língua azul em rebanhos ovinos nas Mesorregiões de Campo das Vertentes e Sul e Sudoeste de Minas Gerais. Foram coletadas amostras de soro de ovinos de diferentes propriedades. As amostras de soro foram submetidas aos testes de imunodifusão em gel de ágar (IDGA) e ensaio de imunoadsorção enzimática por competição (cELISA). Ao todo 303 amostras de soro foram submetidas ao IDGA e cELISA. Dessas amostras, 164 (54,13%) foram positivas na técnica de IDGA e 171 (56,44%) positivas na técnica de cELISA, havendo concordância quase perfeita entre as técnicas (índice kappa = 0,887). Em todas as propriedades visitadas, foram encontrados animais positivos no rebanho. Animais adquiridos de propriedades das Mesorregiões estudadas, tiveram mais chances de serem positivos nos testes de IDGA e cELISA do que animais adquiridos de propriedades de outras Regiões do Brasil (p<0,001). Esses resultados sugerem que o vírus da língua azul encontra-se disseminado em ovinos nas Mesorregiões de Campo das Vertentes e Sul e Sudoeste de Minas Gerais.(AU)

Optimization of electroporation parameters for ctenopharyngodon idellus kidney cells and transient expression of grass carp reovirus NS26 protein / 病毒学报

In this study, pEGFP-N1 was chosen as the reporter plasmid and transferred into Ctenopharyngodon idellus kidney (CIK) cells by electroporation, and the optimal electroporation conditions were determined by testing the transfection efficiency with different voltages, pulse times, plasmid amounts, and numbers of shocks. The results showed that the maximum electroporation efficiency was achieved under the following conditions in a 0.2 cm electroporation cuvette containing CIK cells (1.5 x 10(7)/mL, 200 microl): electric voltage 200 V, pulse time 45 ms, plasmid 30 microg, and one electric shock. The total genomic RNA of grass carp reovirus (GCRV) was extracted in this experiment and reversely transcribed into cDNA, which was used to amplify the gene segment of GCRV non-structural protein NS26 using designed specific primers. The PCR product was recombined into pEGFP-N1 vector. The fusion protein EGFP-NS26 was successfully and efficiently expressed in the CIK cells by electroporation, which was confirmed by both fluorescent imaging and Western blot analysis. This experiment laid a foundation for further functional studies of the non-structural protein NS26 of GCRV.

Detection of avian reoviruses causing tenosynovitis in breeder flocks in Iran by reverse transcription-polymerase chain reaction [RT-PCR] and restriction enzyme fragment length polymorphism [RFLP]

Avian reoviruses [ARVs] are members of the Orthoreovirus genus; one of the 12 genera of the Reoviridae family. The ARVs are the cause of some important diseases in poultry such as reovirus-induced arthritis, tenosynovitis, chronic respiratory disease, and mal-absorption syndrome. In this study, the presence of ARVs in the Iranian breeder flocks was investigated through reverse transcriptionpolymerase chain reaction [RT-PCR] and restriction enzyme fragment length polymorphism [RFLP]. A total of 800 fecal swab samples were initially collected from breeder flocks [older than 45 weeks of age]. They were then sent to the laboratory in containers with PBS, and after that they were pooled and finally to 120 samples were obtained. The total RNA extracted from the pooled fecal samples were used to amplify the selected parts of the S1 [1023 bp] and S4 [437 bp] genes from the ARV field isolates using RT-PCR. The positive RT-PCR amplified products were further analyzed by RFLP using five restriction enzymes. Based on the findings, 5 samples were positive with the S1 primer and 6 samples were with the S4 one. The patterns observed after the digestion of PCR products revealed that the isolates of this study were identical to both the S1133 vaccine and standard strains. The findings suggested that the RT-PCR/RFLP analysis might be considered as a simple and rapid approach for the differentiation of ARV isolates. This study was the first molecular detection of the ARVs presence in the Iranian breeder flocks using the RTPCR amplification of the S1 and S4 genes and RFLP analysis

Gene sequence analysis and prokaryotic expression of sigmaB protein of Muscovy duck reovirus YB strain / 病毒学报

Muscovy ducks reovirus (DRV) is an important pathogen with a high mortality rate in Muscovy ducks, the researches in the test and the immunity were useful for the prevention and control of DRV infection. In this study, the S3 genes of the three Fujian DRVs were cloned by RT-PCR and sequencing technology. It was found that DRV-YH and YJL were close to avian reovirus (ARV) in the genetic distance, with high identities ranged from 94. 6% to 98. 9%, however, the identities of DRV-YB strain and reference ARV strains in the S3 gene were only 60.6% - 61.7%. The expression vector pET-30a-S3 harboring DRV YB strain S3 gene was constructed and transformed into E. coli BL21, and then the fusion sigmaB protein expression was induced with IPTG. The SDS-PAGE of the expressed products indicated that the fusion protein of approximately 42ku in molecular weight was expressed highly in inclusion body, and made up 67. 7% of the total proteins. The most efficient concentration of IPTG and inducing time were 0. 1 mM and 5h respectively, while the best temperature for expression was 37 degrees C. After purification with the Ni2+ affinity chromatography, the fusion sigmaB protein was 93% of the total proteins, and the purified protein amounted to 0. 86g/L. The Western blot analysis showed that the fusion aB protein was recognized specifically by the antiserum against DRV, confirming that the recombinant fusion protein had good immunoreactivity.

The isolation and identification of novel duck reovirus / 病毒学报

The virus strains were isolated from the liver and spleen of the dead young ducks characterized with symptoms of hemorrhagic-necrotic hepatitis. These isolates could cause the death of muscovy duck-embryo and chick-embryo. 1-day-old birds infected with these isolates had the same character with clinically dead birds and the virus could be isolated from artificially infected birds. These isolates could proliferate in MDEF and result in CPE. The virus could proliferate in the cytoplasm in order of crystals and arranged in the latlic-like. The viron was shown spherical, icosahedron, cubic symmetry, no-envelope, with double-layered capsid, about 70 nm in diameter by electron microscopy. The genome segments of the virus were consisted of L1-3, M1-3 and S1-4, which were similar to that of avian reovirus (ARV). Compared to 68.2%, 69.3% - 70.1%, respectively. The system evolution analysis showed that S3 gene coding sigmaB protein was placed in different branch of MDRV and ARV, indicating that S3 gene of the virus was different from ARV and MDRV. The main clinical symptoms and lesions of ducklings caused by the virus were different from the diseases caused by MDRV and ARV. It was concluded that the virus was a Novel duck reovirus belonging to Orthoreovirus genus of the Reoviridae family.

Molecular properties of grass carp reovirus in southern China and establishment of a duplex PCR detection method / 病毒学报

A strain of grass carp reovirus was isolated from sick grass carp with symptoms of haemorrhage in Guangdong province in 2009. The strain was tentatively named as GCRV-GD108 because it was isolated from grass carp and possessed 11 segments of dsRNA. Complete genome sequence analysis showed that significant differences existed between GCRV-GD108 and GCRV, as well as other known species of aquareovirus. In this study, molecular characteristics of diseased grass carp collected from different farms in Guangdong, Fujian and Hunan provinces were assayed. Based on the sequences of the 11 segments of GCRV-GD108, PCR primers corresponding to each of the segments were designed and synthesized. Total RNA of the diseased fish was extracted and used as templates of RT-PCR reaction. Specific amplification bands were obtained from all of the samples whereas no band was produced from GCRV standard strain. While using the primers specific to GCRV produced specific band in GCRV standard strain rather than in these collected samples. Sequencing of the amplification products showed that these samples displayed high similarities with each other (95.2%-99.4%), and they also shared high sequence similarities with that of GCRV-GD108 (95.0%-99.8%), suggesting that these samples shared similar molecular characteristics with those of GCRV-GD108, and were quite different from GCRV as well as the known species of genus aquareovirus. The results indicated that there are different molecular types of reovirus existed in the pond-cultured grass carp in China, and GCRV-GD108 is a representative strain in southern China, therefore great attention should be paid in order to control the disease efficiently, especially in vaccine preparation. Two pairs of primers were chosen to establish a duplex PCR assay method by combining each pair of the primers specific to GCRV-GD108 with the GCRV primer pair respectively. The duplex PCR assay method will enable the identification of GCRV-GD108 or GCRV by only a single PCR reaction.

Isolation and identification of Banna virus from mosquito for the first time in Inner Mongolia / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To identify the virus isolated from a mosquito Culex modestus collected from Tongliao city of Inner Mongolia Autonomous Region.</p><p><b>METHODS</b>A strain of virus isolated from mosquito in Tongliao city was identified by serological and molecular biological methods. The nucleotides of the virus isolate were amplified by RT-PCR, and the products were purified and sequenced. Multiple alignment, phylogenetic and amino acid (AA) analysis were carried out by software Clustal X, MEGA4 and MegAlign (DNAStar).</p><p><b>RESULTS</b>The new isolate was identified to be Banna virus by serological and molecular biological methods. Phylogenetic analysis showed that the Chinese isolates were distributed within one cluster. The homologue of nucleotide and amino acid of 12 segments between the new isolate and other strains isolated from China were 89.6%-98.4% and 90.4%-98.6%.</p><p><b>CONCLUSION</b>The virus isolated from Culex modestus in Inner Mongolia belonged to Banna virus, and it is the first time that Banna virus was isolated in this region.</p>

Isolation and identification of channel catfish (Ictalurus punctatus) hemorrhage reovirus / 病毒学报

By using cell culture and virus infection methods, a new reovirus had been isolated from channel catfish (Ictalurus punctatus) suffered with severe hemorrhage and had been identified as channel catfish reovirus (CCRV) after artificial infection in fish, electron microscopy observation, physical-chemical tests, genomic SDS-PAGE analysis and sequencing. In artificial infection test, the typical symptoms of channel catfish hemorrhage as naturally occurred could be reproduced. The isolated virus could cause typical cytopathic effect in CCO and CCK cell lines. Electron microscopy observation of ultra-thin section samples of CCRV infected CCO and CCK cells revealed that the virus replicated in cytoplasm, arrayed in crystalline, and had a non-enveloped double capsid with a diameter of 60-70 nm. Frozen-thawed, 56 degrees C 1 h, chloroform and ether had no significant effects on CCRV titer, 65 degrees C 1 h could significantly inactivated the viral infectivity. The CCRV genome SDS-PAGE analysis and nuclease sensitivity test showed that the virus genome was the same as that of viruses in Aquareovirudae and consisted of 11 segments of dsRNA assigned into three classes L1, L2, L3; M1, M2, M3 and S1, S2, S3, S4, S5 with a range of size from 0.9 to 4.4 kb. The Cloning and sequencing of the CCRV S4 segment indicated the nucleic acid number of CCRV S4 was 909 bp in length, which was exactly the same as that of GCRV S4 (AF403396) and GSRV S4 (AF403407) segments. The BLAST of CCRV S4 sequence in NCBI GenBank showed that it had a 99% and 90% similarity in sequence to the GCRV S4 and GSRV S4 segments, respectively.

Nuevas estrategias terapéuticas basadas en apoptosis y virus / Viruses and apoptosis-based therapeutic agents in human diseases

En éste trabajo se presentan los mecanismos y las moléculas que participan de la apoptosis en células de mamífero. Se discute la función de la mitocondria, se relacionan los distintos controles del sistema con patologías humanas y se presentan algunos virus neurotrópicos donde existe una importante conexión con la apoptosis. Asimismo, se indican algunos factores participantes del proceso que tienen una veta promisoria en el tratamiento de enfermedades donde la desregulación de la muerte celular programada es la causa de la patología en cuestión

Detection of human retrovirus-5 in rheumatoid arthritis patients

This study was carried out to detect the possible association of human retroviruse-5 [HRV-5] with RA and its correlation with disease activity and severity in a trial to detect its possible role as an etiologic factor. We conducted our study on 31 RA patients and 15 apparently healthy subjects serving as a control group. All patients were subjected to complete history taking, thorough clinical examination, radiological and laboratory tests. Nested PCR was done to detect the presence or absence of HRV-5 in the blood of all patients and controls as well as nine synovial fluid samples from RA patients. There was a significant presence of HRV-5 in the blood of RA patients and in four synovial fluid samples out of nine. The four patients whose synovial fluid samples were HRV-5 positive also showed HRV-5 positive blood. There was a statistically significant higher alkaline phosphatase mean in cases with HRV-5 positive blood, which may reveal Liver damage that might be caused by the virus itself. There was no statistically significant difference in the activity or severity grades in HRV-5 positive blood and synovial fluid compared to HRV-5 negative cases. The presence of HRV-5 in a significant percent in our RA patients [25.8%] should be taken into consideration since it may have a role as an etiologic factor in the pathogenesis of this disease

Preparation and storage of coltivirus antigen and its application in detection of coltivirus antibodies in serum from patients / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare purified and concentrated coltivirus high titer antigen in order to further detect antibodies against coltivirus in serum sample of patients.</p><p><b>METHODS</b>The coltivirus in C6/36 cells was cultured and harvested at different time, and the titer was titrated. The virus was purified and concentrated by polyethylene glycol (PEG), and stored at -20 degrees and 4 degrees, with and without glycerol, respectively, then the titer of coltivirus antigen was tested by indirect ELISA. By using the antigen, coltivirus antibodies in serum samples from both suspected Japanese encephalitis (JE) and viral encephalitis (VE) patients were detected.</p><p><b>RESULTS</b>The highest titer of coltivirus was found at 3-4 weeks of culturing. The antigen titer could be maintained at least for 6 months, especially antigen with glycerol either at 4 degrees or at -30 degrees even for two years. Totally 1141 serum samples from patients diagnosed clinically as JE and VE were tested. The results showed that 130 samples were coltivirus IgM antibody positive and the average positive rate was 11.4% (130/1141). Among 41 samples of paired-serum from patients in Guangzhou Children's Hospital, 9 samples were positive, the positive rate was 22.0% (9/41) in which 5 samples were diagnosed clinically as VE.</p><p><b>CONCLUSIONS</b>Stable and purified coltivirus antigen was obtained in order to test coltivirus antibodies as well as development of kits. Coltivirus probably can cause summer-autumn encephalitis in China.</p>

Susceptibility of cell lines to avian viruses

The susceptibility of the five cell lines - IB-RS-2, RK-13, Vero, BHK-21, CER - to reovirus S1133 and infectious bursal disease virus (IBDV vaccine GBV-8 strain) was studied to better define satisfactory and sensitive cell culture systems. Cultures were compared for presence of CPE, virus titers and detection of viral RNA were detected in CER and BHK-21 cells after retrovirus inoculation and in RK-13 cell line after IBDV inoculation and with high virus titers. Virus replication by production of low virus titers occurred in IB-RS-2 and Vero cells with reovirus and in BHK-21 cell line with IBDV

Rotavirus and reovirus interaction with mouse peritoneal resident phagocytic cells

Rotaviruses and reoviruses are involved in human and animal diseases. It is known that both viruses penetrate the gastrointestinal tract but their interaction with phagocytic cells is unknown. To study this interaction, peritoneal resident phagocytic cells were used and rotavirus and reovirus replication in peritoneal phagocytic cells was observed. However, rotavirus replication in these cells led to the production of defective particles since MA-104 cells inoculated with rotavirus phagocytic cell lysate did not show any evidence of virus replication. On the basis of these results, we suggest that, althought reovirus dissemination may be helped by these phagocytic cells, these cells may control rotavirus infection and probably contribute to the prevention of its dissemination.