Comparative investigations of infectious runting and stunting syndrome in vaccinated breeder chicks by inactivated reovirus and chicks from non-vaccinated breeders

Reoviruses are important pathogens responsible for poor growth performance and silent losses in the poultry industry. They are associated with many disease and syndromes such as malabsorption [runting and stunting syndrome], respiratory diseases and immunosuppression. Broiler birds are most susceptible to viral infections during the early post hatching period. Therefore, the transfer of maternal immunity to embryonated eggs is proved to be a primary means of protection from viral infections. In the present investigation, growth performance and pathology in breeder vaccinated and non-vaccinated chicks were studied after a challenge with the homologous malabsorption strain of the reovirus. Improvements in growth performance [mean live body weight, feed conversion ratio, broiler performance efficiency index, and protein efficiency index] in breeder vaccinated chicks were compared with non-vaccinated breeder chicks. The non vaccinated chicks showed various signs and lesions indicative of the reoviral malabsorption syndrome [MAS], whereas the vaccinated chicks showed very minimal alterations, demonstrating that the vaccination of breeders with homologous strains of the reovirus is profitable, and can help to increase the performance of broiler birds

Development of transgenic maize with anti-rough dwarf virus artificial miRNA vector and their disease resistance / 生物工程学报

Maize is one of the most important food crops. Rice black-streaked dwarf virus is a maize rough dwarf disease pathogen. The occurrence and transmission of maize rough dwarf disease brings great damage to maize production. The technology of using artificial miRNA to build antiviral plant has been proven effective in a variety of plants. However, such trials in maize have not been reported. We designed primers based on the sequence of maize zea-miR159a precursor and sequence of function protein genes and silencing RBSDV coding genes in RBSDV genome. We constructed amiRNA (artificial miRNA) gene for silencing RBSDV coding gene and gene silencing suppressor. We constructed pCAMBIA3301-121-amiRNA plant expression vector for transforming maize inbred lines Z31 by using agrobacterium mediated method. After molecular analysis of transgenic maize, homozygous lines with high miRNA expression were selected by molecular detection for a subsequent natural infection experiment. We studied the severity of maize rough dwarf disease according to a grading standard (grade 0 to 4). The experiment results showed that the disease resistance of transgenic homozygous maize with the anti-rough dwarf virus amiRNA vector was better than that of wild type. Among the transgenic maize, S6-miR159 transgenic maize had high disease resistance. It is feasible to create new maize variety by the use of artificial miRNA.

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.

Histopathology of cotton bollworm midgut infected with Helicoverpa armigera cytoplasmic polyhedrosis virus

This research was carried out to examine cytopathological effects of Helicoverpa armigera Cytoplasmic polyhedrosis virus (HaCPV) on infected midgut cotton bollworm (Helicoverpa armigera) using transmission and scanning electron microscope. The symptoms on infected host larvae of the host, compared with healthy ones, were getting swollen with milky-white and fragile Histopathological examinations showed infection with HaCPV small polyhedral inclusion bodies (PIB) after 1 or 2 days which were observed in columnar cells of midgut. Virions were partially or completely occupied in a polyhedral matrix to form polyhedral inclusion bodies (PIB) at periphery of virogenic stroma. PIBs were measured 0.5 to 3.5 mm and virions about 46 nm in diameter. Microvilli of infected columnar cells were affected and degenerated immediately prior to rupture of the cell. Some infected columnar cells ruptured to release PIB into the gut lumen 3 days after infection. In addition,PIB were found in goblet cells, 5 or 6 days after infection. Infected goblet cells degenerate to such an extent that only a few of the original microvillus-like cytoplasmic projections and cell organells were left. These cytopathic effects caused in the midgut by HaCPV on cotton bollworm larvae are essentially similar to those have been reported for lepidoperan and dipteran infection by CPV.

Development of real-time fluorescent quantitative PCR method for detecting Bombyx mori cytoplasmic polyhedrosis virus / 病毒学报

A pair of specific primers were designed according to the published 118 bp conserved sequence of polyhedrin gene of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) in GenBank and a serial dilutions of a recombinant plasmid were prepared and used to generate standard curves, to establish a real-time fluorescent quantitative PCR method for detection of BmCPV. The results showed that the linear relationship between virus concentration (X) and Ct (Y) was Y = -3. 582 lgX + 38.748 with the correlation coefficient R2 = 0999. The method was very sensitive, specific and reproducible. It can be applied in the rapid detection of BmCPV and the prevalence investigation of this disease.

Construction of recombinant plasmid expressing S1 gene of new type of reovirus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct the recombinant plasmid containing S1 gene of new type of reovirus, and to study the expression of protein sigma1 in Vero cells.</p><p><b>METHODS</b>The recombinant plasmid, named pC-S, was constructed by cloning S1 gene into vector pCAGGS/MCS. Then Vero cells were transfected with pC-S and collected at 24, 48, 72 h post transfection followed by SDS-PAGE and Western-Blot assay.</p><p><b>RESULTS</b>Results both SDS-PAGE and Western-Blot assay indicated that sigma1 protein could be expressed well and the highest expression level was 72 h post transfection.</p><p><b>CONCLUSIONS</b>Sigma1 protein could be expressed well in Vero cells by transfected with recombinant plasmid containing S1 gene, and could give some implications for subsequent research on virus-host interactions.</p>

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.

Comparative assessment of mammalian reoviruses versus enteroviruses as indicator for viral water pollution

Enteric viruses are important causative agents of human diseases. Among the enteric viruses, reoviruses and enteroviruses are prevalent in various aquatic environments. This study was carried out to detect and compare the presence of reoviruses and enteroviruses by ICCPCR in one wastewater and three drinking water treatment plants, studying the possibility of using reoviruses as indicator of viral water pollution and genotyping of the isolated strains. One hundred and forty four drinking water and 76 wastewater samples were collected for two years. Reoviruses and enteroviruses were detected in 12.5% [18/144] and 8.3% [12/144] of total collected drinking water samples. In the studied wastewater treatment plant [WWTP], reoviruses were detected in 26% [20/76] of total collected samples while enteroviruses were detected in 21% [16/76] of the total collected samples. Phylogenic analysis revealed that our sequences were closely related to reovirus type 1, Lang strain and Human poliovirus typel. Conclusion: The higher incidence of reoviruses than enteroviruses reflects the possibility of using reoviruses as indicator of water pollution

Highly efficient inhibition on replication of grass carp reovirus mediated by chemically synthesized small interfering RNAs / 病毒学报

Two short interfering RNAs (siRNA-RdRp1286, siRNA-RdRp1441) and one short interfering RNA (siRNA-OCP117), targeted to the RNA dependent RNA polymerase (RdRp) gene and outer capsid protein (OCP) gene of Grass carp reovirus (GCRV) respectively, were chemically synthesized and transfected into the CIK cells by lipofectamine 2000. 6 hours after transfection, the transfected CIK cells were challenged with GCRV. The culture media were collected at 48h post challenge and the virus was titrated in microculture system to evaluate the inhibition effect on GCRV replication mediated by siRNAs. Referring to the mRNA level of housekeeping gene beta-actin, RT-PCR was applied to detect the level of GCRV mRNA in transfected and challenged CIK cells. The results showed that the viral titer (lgTCID50/0. 1mL) in siRNA-RdRp1286, siRNA-RdRp1441 and siRNA-OCP117 transfected CIK cells were 4.41 +/- 0.16, 3.83 +/- 0.44 and 1.94 +/- 0.42 respectively, which were significantly lower than that in virus infection positive control (7.92 +/- 0.52) (P < 0.01). No significant change in viral titer was observed in the group transfected with siRNA negative control after challenged with GCRV (7.50 +/- 0.17, P > 0.05). Compared with the mRNA transcriptional level of beta-actin gene in virus infection positive control, the mRNA levels of GCRV in siRNA-RdRpl 286, siRNA-RdRp1 441 and siRNA-OCP117 transfected CIK cells were reduced significantly and the inhibition rate reached to (82.08 +/- 2.15)%, (89.19 +/- 1.14).% and (92.62 +/- 0.17)%, respectively. The mRNA level of GCRV in the siRNA negative control group had no noticeable change (P > 0 05).

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.

Supervivencia y transporte de los virus entéricos humanos a través de los alimentos / Survival and transport of human enteric viruses in foods

Los virus se transmiten a los seres humanos vía los alimentos como resultado de la contaminación directa o indirecta de los mismos con heces; ésta es la esencia del análisis de peligro...

The cell apoptosis induced by duck reovirus in duck embryo fibroblasts / 病毒学报

Cell apoptosis induced by duck reovirus (DRV)in duck embryo fibroblasts (DEF) was ascertained by light microscope and electron microscopy, DNA Ladder, flow cytometry and fluorescent microscopy. Typical morphological apoptotic features including cell shrinkage and condensation, margination of nuclear chromatin were observed under light microscope and the formation of apoptotic bodies by electron microscopy. DNA ladder was shown by DNA fragment analysis at 24-144h post infection. Flow cytometry showed that the cell apoptosis appeared at 24h and reached it's crest-time at 72-96h, decreased at 144h. Fluorescent microscopy showed that the apoptotic cells which showed green fluorescence appeared at 24h, the number of dead cells which showed red fluorescence increased with the time went by. The results above confirmed that the apoptosis of DEF was successfully induced by DRV.

Isolation and identification of A reovirus from masked civet cats (Paguma Larvata) / 病毒学报

192 samples of Masked Palm Civet (Paguma Larvata) from Guangdong Province were inoculated in Vero-E6 cells. One sample which came from masked palm Civet didn't cause cytopathic effects (CPE) until fourth-passage on Vero-E6 cells. Infected cells emerged granulating, shrinking, rounding and falling off. After three times freeze-thaw, cells and culture medium were harvested for electron microscopy. Virus particles were nonenveloped, double capsid and icosahedral symmetry. This virus was designated Masked Palm Civet/China/2004 (MPC/04). Hemagglutination test indicated that the virus could agglutinate healthy human type O red cells, but not the red cells of SPF chicken, experimental common bovine, rat and guinea pig. This virus was tolerant to chloroform treatment, pH 3.0 and water bath 50 degrees C 1 h. 1 M MgCl2 treatment could enhance resistance of virus to heat and increase infectivity. In order to classify the strain on the molecular level, specific primers according to mammalian reovirus were used for Reverse Transcription Polymerase Chain Reaction (RT-PCR). Appropriate specific products were amplified by RT-PCR. NCBI BLAST analysis indicated that this segment shared the highest identity to mammalian reovirus serotype 1 (T1L) virus. So we can deduce this virus is a member of the Reoviridae.

New type of cytoplasmic polyhedrosis virus isolated from mosquitoes in China / 病毒学报

0507BS3 virus was isolated from a mixed pool of Culex sp. and Anopheles sp. collected in Kashi, Xinjiang, China. 0507BS3 virus could cause cytopathic effects on C6/36 cells but not on Vero and BHK-21 cells. Viral particles had no envelope and appeared round with diameter of about 60 nm (n = 20). Viral capsid was composed of a single layer and a central core. Capsomeres on the surface of capsid were clearly visible. Electrophoresis of viral genome showed a profile of 10 double stranded RNA (dsRNA) segments. Sequencing of the tenth segment revealed the length of 964bp (GenBank ID: FJ150869). A single open reading frame (ORF) was found and encoded a protein of 275 amino acids with a molecular mass of 30.8kDa. The nucleotide sequence had no similarity with any other viral genomic sequences, but the deduced amino acid sequence significantly matched the polyhedrin genes of cytoplasmic polyhedrosis virus (CPV) in some sections. A phylogenetic tree was constructed to compare the polyhedrin gene sequences of all CPV types in GenBank. The tree demonstrated that 0507BS3 virus was only distantly related to the other CPV types and belonged to a new CPV type.

0507JS60 virus isolated in Xinjiang was identified as Liaoning virus / 病毒学报

0507JS60 virus was isolated from a pool of Culex sp. collected in Kashi, Xinjiang, which could be propagated stably on C6/36 cells and caused cytopathic effects continuously. Viral particles had no envelope and appeared round with diameter of about 55nm (n = 10). Capsomeres on the surface of capsid were clearly visible. Electrophoresis of viral genome showed a profile of 12 double stranded RNA (dsRNA) segments. Sequencing of the twelfth segment revealed the length of 760bp (GenBank ID: FJ157354). A single open reading frame (ORF) was found and encoded a protein of 174 amino acids with a molecular mass of 18.9kD. The nucleotide sequence had similarity over 89% with that of LNV, but the deduced amino acid sequence had similarity over 91% with that of LNV. A phylogenetic tree was constructed to compare the corresponding genetic sequences in Seadornavirus. The tree demonstrated that 0507JS60 virus lied in the same branch with LNV and more closely related to LNV-NE9712. 0507JS60 virus was identified as LNV, which was firstly isolated outside the Northeast of China.

Emerging a member of reoviridae family associated with acute encephalitis syndrome in Gia Lai province, 2005

Background: In recent years, some arbo viruses which causes acute encephalitis syndrome (AES) have been identified in serveral countries in the world such as Chandipura virus belonging to Rhabdoviridae family in India, Banna virus belonging to Reoviridae family in China. In Vietnam, apart from Japanese Encephalitis Virus which is considered as main cause of AES, there are a few intestinal viruses like Herpes symplex virus type 1 and 4 and Nam Dinh virus considering other causes of AES. Objective: To identify the hyppothesis that one virus strain parasitizing in mosquito in Gia Lai province causes AES in order to provide more information about virus strains which cause AES in Vietnam. Subjects and method: Aedes albopictus cell line clone C6/36 was used for the isolation of virus in 43 cerebrospinal fluid samples of patients who were treated in Gia Lai hospital, from January/2005 to July/2005. Result and Conclusion: One virus strain from a 3-year old girl in Gia Lai province was isolated in 2005. The virus coded 05VN225 has the morphology similar to other viruses belonging to Reoviridea family.The nucleic acid sequence of the virus was checked with specific primers of alphavirus and flavirus groups, Nam Dinh virus and Conti virus group B (reovirus) of the Reoviridae. The positive result was confirmed with reovirus primers. This member of the Reoviridae family was isolated from acute encephalitis syndrome in Vietnam in 2005. Further study on pathology of the virus is very necessary.

Study on the ability of mammalian reovirus BYD1 to induce apoptosis and analysis of the structure of viral major membrane penetration protein involved in proapoptosis induction / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study a newly isolated domestic mammalian reovirus, BYD1, its ability to induce apoptosis analyze the three-dimensional structure of its major membrane penetration protein to predict its function in inducing apoptosis.</p><p><b>METHODS</b>HeLa cells infected with BYD1 reovirus were metered with flow cytometer (FCM) to quantify the ratio of apoptotic cells. The data were analyzed with Student's t-test to judge the ability of BYD1 strain to induce apoptosis. The primary sequence ranged from 582 to 675 per microliter protein of BYD1, T1L, T2J and T3D were aligned and compared. The three-dimensional comparative protein structure model of microliter protein was generated by homology-modeling pipeline SWISS MODEL was applied to annotate its secondary and tertiary structure.</p><p><b>RESULTS</b>BYD1 strain was verified with the ability to induce the apoptosis of HeLa cells. The 643-675 segment composing an alpha-helix showed major difference compared with prototype T2J.</p><p><b>CONCLUSION</b>The newly isolated reovirus BYD1 is an apoptosis inducing strain. The alpha-helix (residues 643 to 675) of microliter protein of BYD1 may play a key role to induce the proapoptotic activity of infected cells.</p>

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

Lesöes articulares em frangos de corte (Gallus gallus) na infecçäo experimental pelo reovírus aviário / Articular lesion in experimental infeccion with avian reovirus in broiler (Gallus gallus)

Este trabalho descreve as lesöes articulares induzidas pela inoculaçäo oral e podal de uma amostra de reovírus aviário (S-1133) em pintos de corte de um dia de idade. Realizou-se o exame histopatológico de fragmentos da articulaçäo tibiotársica utilizando-se cinco aves dos grupos inoculados e grupo controle nos períodos de 24, 48, 72 horas e semanalmente até a oitava semana após a inoculaçäo. A primeira alteraçäo observada foi um infiltrado inflamatório misto nas bainhas tendinosas uma semana após a inoculaçäo. Na segunda semana após a inoculaçäo, houve fibroplasia, formaçäo de folículos linfóides e hiperplasia das células da membrana sinovial, alteraçöes progressivas observadas até o final do período experimental. As lesöes articulares foram similares, ocorrendo simultaneamente nos dois grupos inoculados, contudo as alteraçöes foram mais severas no grupo inoculado no coxim plantar

Lesöes viscerais induzidas experimentalmente pela inoculaçäo de uma amostra artrotrópica de reovírus em frangos de corte (Gallus gallus) / Visceral lesion induced experimentally by an artrotropic strain of reovirus in broiler (Gallus gallus)

Este trabalho descreve as lesöes viscerais induzidas pela inoculaçäo oral e podal de uma amostra artrotrópica de reovírus aviário (S-1133) em pintos de corte de um dia de idade. Realizou-se o exame histopatológico de fragmentos da Bursa de Fabricius, intestinos, fígado, baço e miocárdio utilizando-se cinco aves dos grupos inoculados e do grupo controle, nos períodos de 24, 48, 72 horas e semanalmente até a oitava semana após a inoculaçäo. No miocárdio, observou-se miocardite intersticial 48 horas pós-inoculaçäo, miocardite necrótica e pericardite na primeira semana após a inoculaçäo. Houve no pericárdio, na segunda semana após a inoculaçäo, a formaçäo de folículos linfóides e seu espessamento fibroso em períodos subseqüentes até a oitava semana. No fígado, foi observado um infiltrado inflamatório misto 72 horas após a inoculaçäo e degeneraçäo vacuolar e necrose dos hepatócitos na primeira semana pós-inoculaçäo. Observou-se, ainda, a presença de tecido linfóide ativo e de infiltrado heterofílico na segunda semana após a inoculaçäo, persistindo até o final do experimento. Verificou-se necrose progressiva do tecido esplênico de 48 horas até a primeira semana após a inoculaçäo e a partir da segunda semana observou-se hiperplasia dos folículos linfóides esplênicos. Os demais órgäos examinados näo apresentaram alteraçöes. As lesöes viscerais foram similares ocorrendo simultaneamente nos dois grupos inoculados, contudo as alteraçöes foram mais severas no grupo inoculado no coxim plantar