Complete genome sequence and evolution analysis of Eimeria stiedai RNA virus 1, a novel member of the family Totiviridae.

Eimeria stiedai (E. stiedai) is a coccidian that infects the liver of the domestic rabbit and may cause severe hepatic coccidiosis. Virus-like particles in E. stiedai were discovered by Revets et al. However, the complete genome sequence of the E. stiedai virus has yet to be determined. A novel virus was isolated from E. stiedai in the present study. The complete genome sequence of the E. stiedai virus was 6219 bp in length and contained two open reading frames (ORFs) with a tetranucleotide overlap (AUGA). ORF1 (2400 bp) encoded a putative coat protein of 799 amino acids (86.471 kDa) that exhibited a high level of amino acid sequence similarity to that of Eimeria tenella (E. tenella) RNA virus 1 (EtRV1; 43 % identity, NC_026140), whereas ORF2 (3303 bp) encoded a putative RNA-dependent RNA polymerase (RdRp) of 1100 amino acids (118.850 kDa) that exhibited a high level of amino acid sequence similarity to that of the E. tenella RNA virus 1 (EtRV1; 51 % identity, NC_026140). Phylogenetic analysis revealed that the E. stiedai virus was a new member of the family Totiviridae. The sequence data provided sufficient information for classification of eimeriaviruses.

Recent advances in molecular biology of parasitic viruses.

The numerous protozoa that can inhabit the human gastro-intestinal tract are known, yet little is understood of the viruses which infect these protozoa. The discovery, morphologic details, purification methods of virus-like particles, genome and proteome of the parasitic viruses, Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, and the Eimeria sp. are described in this review. The protozoan viruses share many common features: most of them are RNA or double-stranded RNA viruses, ranging between 5 and 8 kilobases, and are spherical or icosahedral in shape with an average diameter of 30-40 nm. These viruses may influence the function and pathogenicity of the protozoa which they infect, and may be important to investigate from a clinical perspective. The viruses may be used as specific genetic transfection vectors for the parasites and may represent a research tool. This review provides an overview on recent advances in the field of protozoan viruses.

Ocorrência dos principais agentes bacterianos e parasitários em fezes diarreicas de bezerros búfalos nos estados de São Paulo e Paraná / Occurrence of the main bacterial and parasite agents on diarrhetic feces of buffalo calves in the states of São Paulo and Paraná

A população de bubalinos estimada no Brasil é de aproximadamente 3 milhões de animais, encontrando-se distribuídos em todos os Estados brasileiros, com crescimento médio anual de 12%. Apesar disso, os trabalhos realizados buscando os avanços na bubalinocultura são escassos. Em função da complexidade etiológica da diarreia em bubalinos e da falta de informações recentes nesta área, o presente estudo teve como objetivo avaliar a ocorrência dos principais agentes bacterianos e parasitários envolvidos na diarreia de bezerros búfalos lactentes, de explorações leiteiras semi-intensivas e intensivas em regiões dos estados de São Paulo e Paraná. De março de 2010 a junho de 2011, foram colhidas 53 amostras para exame coproparasitológico e 46 amostras para o exame bacteriológico de animais com quadro de diarreia nos municípios paulistas de São João da Boa Vista, Dourado, Pirassununga, Registro, Pariquera Açu, Pilar do Sul e uma propriedade no estado do Paraná, município de Santana do Itararé. No exame parasitológico, 45,28% (24) foram positivos para Eimeria spp., 26,42% (14) para Strongyloidea e 1,88 (1) para Toxocara vitulorum. No exame bacteriológico, 97,83%, (45) das amostras foram positivas para E. coli, contudo, somente duas foram consideradas patogênicas (E. coli STEC). Em uma amostra (2,17%) isolou-se Klebsiella pneumoniae; já a presença de Salmonella spp. não foi constatada. Para o presente estudo, a presença de endoparasitas foi bastante relevante, principalmente os casos Eimeria spp., sendo a higiene das instalações e falhas de manejo fatores importantes na ocorrência de diarreia em bezerros búfalos no estado de São Paulo.(AU)

There are about 3 million buffalos in Brazil, spread through all of the Brazilian states; with mean annual growth of 12%. In spite of that, to the best of our knowledge few studies looking for advances in the industry have been done. Due to the etiological complexity of buffalo diarrhea and the lack of information in this area, this aimed at developing a clinical evaluation on the causes of buffalo calves bacterial and parasitical diarrhea in dairy farms of the states of São Paulo and Paraná. The survey was done in farms located in the cities of São João da Boa Vista, Dourado, Pirassununga, Registro, Pariquera Açu, Pilar do Sul (SP), and Santana do Itararé (PR). From March, 2010, to June, 2011, 53 diarrhea samples were collected and screened for endoparasite and bacteria; 45.28% (24) were positive for Eimeria spp.; 26.42% (14) had Strongyloidea; and 1.88% (1) had Toxocara vitulorum. In the bacteriological test, 97.83% (45) had E. coli, but only two were considered pathogenic (E. coli STEC); 2.17% had Klebsiella pneumonia and none presented Salmonella spp. In this study, the mainly causative agent of buffalo diarrhea was Eimeria spp., and the poor hygiene in installations and breeding failure are important factors on this diarrhea occurrence.(AU)

Eimeria necatrix virus: intracellular localisation of viral particles and proteins.

The presence of the Eimeria necatrix virus was investigated in the following life cycle stages: sporocysts, sporozoites, merozoites, and macrogametes. Electron microscopy revealed virus-like particles (VLPs) in sporozoites, which were purified from sporozoite extracts and used to raise polyclonal antibodies. Viral proteins were identified as RNA polymerase (95 kDa) and the major capsid protein (80 kDa). Polyclonal antibody was used to detect the intracellular localisation of VLPs and proteins. Immunoelectron microscopy and immunohistochemistry identified a viral protein of 95 kDa in all the E. necatrix stages studied, whereas the 80 kDa protein was found only in sporocysts and sporozoites. In addition, no VLPs were found in sporocysts. These results indicate that the synthesis of viral capsid proteins takes place during the early events of sporulation, and is then packaged into novel viruses during the late events. No VLPs were seen and no capsid proteins were found in the merozoites and macrogametes, whereas the 95 kDa RNA polymerase was present in both these stages. In addition, no VLPs or proteins were detected in chicken tissues.

Viral double-stranded RNAs of Eimeria spp. of the domestic fowl: analysis of genetic relatedness and divergence among various strains.

Genetic relatedness was examined among putative viral double-stranded RNA (dsRNA) genomes of Eimeria acervilina, E. maxima, and E. necatrix using Northern hybridization. No cross-hybridization was found among these dsRNAs, revealing little sequence homology, if any. In addition, dsRNAs from eight E. acervulina strains collected from different localities in the United States were also examined for genetic relatedness. The putative viral dsRNAs from these eight strains, including the Guelph strain, hybridized with one another to varying degrees, indicating that they are related but divergent.

RNA-dependent RNA polymerase activity associated with Eimeria necatrix virus particles containing either double-stranded or single-stranded RNA.

Single-stranded (ss) RNA containing and double-stranded (ds) RNA containing virus particles of Eimeria necatrix were isolated by centrifugation through a CsCl gradient. RNA from the gradient fractions was identified as single-stranded or double-stranded by probing northern blots with digoxigenin-labeled riboprobes. These probes were generated with SP6 and T7 RNA polymerases from a partial cDNA clone derived from 5.6-kb viral dsRNA of E. necatrix. RNA-dependent RNA polymerase (RDRP) activity was identified in these CsCl-purified virus particles. The polymerase products of the ssRNA particles consisted of dsRNA indicating replicase activity, whereas the polymerase products of the dsRNA particles consisted of ssRNA indicating transcriptase. activity. RNase treatment in high salt solution (0.3 M NaCl) of the pooled RDRP products revealed that the products consisted of both RNase-resistant dsRNA and RNase-sensitive ssRNA. These results show that both replicase and transcriptase activities were present in the purified virus. The digoxigenin-labeled products hybridized to both SP6 and T7 transcripts confirming the presence of both activities.

RNA-dependent RNA polymerase activity associated with virus-like dsRNA in Eimeria maxima and E. necatrix of the domestic fowl.

RNA-dependent RNA polymerase (RDRP) activity was identified in lysates of Eimeria maxima sporozoites and E. necatrix sporozoites and merozoites. Pretreatment of cell lysates with DNase I, RNase A, proteinase K and actinomycin D prior to RDRP assay was employed to characterize RDRP activity. DNase I and actinomycin D had little effect, while proteinase K abolished RDRP activity in both species. RNase A at a concentration of 1 mg/ml also reduced the polymerase activity in E. maxima and E. necatrix sporozoite lysates to 2% and 0%, respectively. Gel electrophoresis of RDRP products revealed that while most migrated at sizes less than 3 kb, a proportion of labelled products of E. necatrix and E. maxima also migrated to the sizes of their respective putative viral genomes. The RDRP products of E. necatrix were shown to be single-stranded by digestion with RNase in both low- and high-salt solutions and by methylmercuric hydroxide treatment. Moreover, the RDRP products of E. necatrix only hybridized to the 5.6-kb dsRNA of E. necatrix but not to the 4.5-kb dsRNAs of E. necatrix or E. maxima.

Intracellular localization of viral RNA in Eimeria necatrix of the domestic fowl.

The intracellular localization of viral RNA in three different stages of Eimeria necatrix, namely, the first- and second-generation meronts and the macrogamonts, were examined at the light microscopy level by in situ hybridization. Digoxigenin-labeled riboprobes generated from partial cDNA clones from 5.6-kb and 4.5-kb dsRNA (pzenv1 and pzenv2) were used in this study. Viral RNA was found to be confined to the cytoplasm of the eimerian host; no viral RNA was detected in chicken tissue. The intense hybridization signal observed in the cytoplasm of the immature meronts with the SP6 riboprobe of pzenv1 or the T7 riboprobe of pzenv2 was probably due to their hybridization to positive strands that had been extruded from the virus during transcription. In mature meronts the intensity of the signal is lower, signifying a decrease in transcription activity. Viral replicase activity may thus be synchronized with the growth phase of the eimerian host.

dsRNA associated with virus-like particles in Eimeria spp. of the domestic fowl.

RNA segments, identified as double-stranded, were found in sporozoites of the Guelph strains of Eimeria acervulina, E. brunetti, E. maxima and E. necatrix and in 8 of 11 strains of E. acervulina obtained from poultry houses across the United States. These RNAs were resistant to RNase A digestion in the presence of high salt concentrations (0.3 M NaCl). On agarose-gel electrophoresis, E. acervulina had one obvious band at 1.7 kb and a faint band at 3.5 kb; E. brunetti had two bands at 2.1 and 3.3 kb, respectively; E. maxima had one band at 4.5 kb; and E. necatrix had two major bands at 4.5 and 5.6 kb, respectively. No dsRNA band was seen in the three strains of E. tenella examined. Virus-like particles were purified by cesium chloride density centrifugation of homogenates of E. necatrix sporulated oocysts. The fraction at peak virus concentration had a buoyant density of 1.39 g ml-1. These virus-like particles were icosahedral, had no envelope and measured 42-44 nm in diameter. Only one RNA band at 5.6 kb was observed when nucleic acids from gradient fractions containing virus were subjected to electrophoresis. The 4.5-kb dsRNA segment of E. necatrix was not associated with a virus-like particle.

Small extrachromosomal nucleic acid segments in protozoan parasites.

Viruses have been described in the following protozoa: Babesia spp., Trichomonas vaginalis, Giardia lamblia, Leishmania braziliensis and Eimeria spp. In order to study the Babesia bovis virus, merozoites have been prepared from the blood of infected cattle. Agarose gel electrophoresis of nucleic extracts from the bovine protozoa B. bovis and Babesia bigemina were separated into genomic DNA and at least two additional nucleic acids. One molecule with a relative mobility of 5.5 kilobase pairs (kbp) was identified as a double-stranded RNA virus-like particle. Another 6.2 kbp DNA molecule had sequences related to mitochondrial genome.