Disenteria de inverno é uma doença causada pelo coronavírus bovino que afeta animais adultos. Descrevem-se dois surtos de disenteria de inverno em rebanhos leiteiros nos municípios de Viamão e Vespasiano Corrêa, Rio Grande do Sul. A doença foi reproduzida experimentalmente em dois bovinos adultos. O quadro clínico se caracterizou por diarréia, inicialmente líquida esverdeada com estrias de sangue e muco, que em alguns animais, evoluiu para coloração marrom escura à sanguinolenta e que persistiu, em média, cinco dias. Na propriedade de Vespasiano Corrêa os animais apresentaram sinais respiratórios como tosse e corrimento nasal. Diminuição na produção de leite e no consumo de alimentos, além de graus variados de depressão foram também observados. Foi detectada a presença do coronavírus bovino nas fezes e secreção nasal dos animais pela técnica da nested RT-PCR. Foi realizada necropsia de um animal naturalmente infectado que morreu devido à doença. Na necropsia, observou-se mucosas pálidas, conteúdo sanguinolento com presença de grande quantidade de coágulos, principalmente no cólon espiral e petéquias na mucosa do cólon. No cólon espiral, foram observados os principais achados histológicos que incluíram criptas dilatadas sem epitélio de revestimento, ou revestidas por epitélio pavimentoso e/ou cuboidal, às vezes, com núcleos grandes e nucléolos proeminentes. Algumas criptas eram preenchidas por debris necróticos e polimorfonucleares. Amostra de fezes de um animal naturalmente infectado foi usada, através de uma suspensão administrada via oral e nasal, para tentar induzir infecção experimental em 3 bovinos de 20 meses. Foi observada diarréia em dois desses animais, no 5º dia após a inoculação. Esses animais foram eutanasiados ao 2º e 3º dias de diarréia. As lesões histológicas dos casos experimentais foram semelhantes àquelas do caso natural. Na imuno-histoquímica anti-coronavírus bovino (8F2) em cortes de tecido incluído em parafina do cólon espiral, houve marcação positiva no citoplasma de enterócitos das criptas, nos debris necróticos dessas criptas e em macrófagos na lâmina própria, tanto no caso natural como nos experimentais. Nos casos experimentais, também foi observada marcação imuno-histoquímica positiva em duodeno, jejuno, íleo, ceco, cólon descendente e epitélio de revestimento dos cornetos nasais. / Winter dysentery is caused by bovine coronavírus that affects adult animals. This report describes two outbreaks of the disease in dairy herds located in the counties of Viamão and Vespasiano Corrêa, Rio Grande do Sul. The disease was induced experimentally in two adult cattle. The most significant clinical sign was profuse and watery diarrhea, which ranged from greenish to brownish coloration with occasional blood streaks and mucus to a bloody diarrhea. In one herd, animals showed respiratory signs such as cough and nasal discharge. Most cases persisted for 5 days and also included depression, decreased milk production, and diminished food intake. The presence of bovine coronavirus was detected in feces and nasal secretions of animals by nested RTPCR. Necropsy was performed in one animal naturally affected and died due to the disease and revealed pale mucosa, sanguineous contents, and blood clots particularly within the spiral colon and pinpoint hemorrhages on the colonic mucosa. Histopathological lesions were predominant in the spiral colon and consisted of a high number of dilated crypts without epithelium or with replaced pavement epithelium with occasional immature cuboidal cells, which sometimes showed enlarged nucleus and prominent nucleolus. Some crypts were filled with epithelial desquamation and polymorphonuclear cells. Fecal samples from one naturally infected animal were used as a suspension and inoculated orally and nasal in three 20-month-old heifers. Diarrhea was observed in 2 of them and started on the 5th day post infection. These animals were euthanized at the second and third days of diarrhea. The histological lesions of experimental cases were similar to the natural case. Anti-bovine coronavirus (8F2) immunostaining was applied on paraffin embedded sections of the spiral colon and showed positive reactions in the cytoplasm of the infected crypt epithelium, sloughed necrotic cells, and within macrophages in the lamina propria of both, natural and experimental cases. Positive reactions were also seen in duodenum, jejunum, ileum, cecum, descending colon and the epithelium lining the nasal turbinate from experimental cases.

Disenteria de inverno é uma doença causada pelo coronavírus bovino que afeta animais adultos. Descrevem-se dois surtos de disenteria de inverno em rebanhos leiteiros nos municípios de Viamão e Vespasiano Corrêa, Rio Grande do Sul. A doença foi reproduzida experimentalmente em dois bovinos adultos. O quadro clínico se caracterizou por diarréia, inicialmente líquida esverdeada com estrias de sangue e muco, que em alguns animais, evoluiu para coloração marrom escura à sanguinolenta e que persistiu, em média, cinco dias. Na propriedade de Vespasiano Corrêa os animais apresentaram sinais respiratórios como tosse e corrimento nasal. Diminuição na produção de leite e no consumo de alimentos, além de graus variados de depressão foram também observados. Foi detectada a presença do coronavírus bovino nas fezes e secreção nasal dos animais pela técnica da nested RT-PCR. Foi realizada necropsia de um animal naturalmente infectado que morreu devido à doença. Na necropsia, observou-se mucosas pálidas, conteúdo sanguinolento com presença de grande quantidade de coágulos, principalmente no cólon espiral e petéquias na mucosa do cólon. No cólon espiral, foram observados os principais achados histológicos que incluíram criptas dilatadas sem epitélio de revestimento, ou revestidas por epitélio pavimentoso e/ou cuboidal, às vezes, com núcleos grandes e nucléolos proeminentes. Algumas criptas eram preenchidas por debris necróticos e polimorfonucleares. Amostra de fezes de um animal naturalmente infectado foi usada, através de uma suspensão administrada via oral e nasal, para tentar induzir infecção experimental em 3 bovinos de 20 meses. Foi observada diarréia em dois desses animais, no 5º dia após a inoculação. Esses animais foram eutanasiados ao 2º e 3º dias de diarréia. As lesões histológicas dos casos experimentais foram semelhantes àquelas do caso natural. Na imuno-histoquímica anti-coronavírus bovino (8F2) em cortes de tecido incluído em parafina do cólon espiral, houve marcação positiva no citoplasma de enterócitos das criptas, nos debris necróticos dessas criptas e em macrófagos na lâmina própria, tanto no caso natural como nos experimentais. Nos casos experimentais, também foi observada marcação imuno-histoquímica positiva em duodeno, jejuno, íleo, ceco, cólon descendente e epitélio de revestimento dos cornetos nasais. / Winter dysentery is caused by bovine coronavírus that affects adult animals. This report describes two outbreaks of the disease in dairy herds located in the counties of Viamão and Vespasiano Corrêa, Rio Grande do Sul. The disease was induced experimentally in two adult cattle. The most significant clinical sign was profuse and watery diarrhea, which ranged from greenish to brownish coloration with occasional blood streaks and mucus to a bloody diarrhea. In one herd, animals showed respiratory signs such as cough and nasal discharge. Most cases persisted for 5 days and also included depression, decreased milk production, and diminished food intake. The presence of bovine coronavirus was detected in feces and nasal secretions of animals by nested RTPCR. Necropsy was performed in one animal naturally affected and died due to the disease and revealed pale mucosa, sanguineous contents, and blood clots particularly within the spiral colon and pinpoint hemorrhages on the colonic mucosa. Histopathological lesions were predominant in the spiral colon and consisted of a high number of dilated crypts without epithelium or with replaced pavement epithelium with occasional immature cuboidal cells, which sometimes showed enlarged nucleus and prominent nucleolus. Some crypts were filled with epithelial desquamation and polymorphonuclear cells. Fecal samples from one naturally infected animal were used as a suspension and inoculated orally and nasal in three 20-month-old heifers. Diarrhea was observed in 2 of them and started on the 5th day post infection. These animals were euthanized at the second and third days of diarrhea. The histological lesions of experimental cases were similar to the natural case. Anti-bovine coronavirus (8F2) immunostaining was applied on paraffin embedded sections of the spiral colon and showed positive reactions in the cytoplasm of the infected crypt epithelium, sloughed necrotic cells, and within macrophages in the lamina propria of both, natural and experimental cases. Positive reactions were also seen in duodenum, jejunum, ileum, cecum, descending colon and the epithelium lining the nasal turbinate from experimental cases.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Richard Hesse / Bovine Coronavirus (BCoV) is known to cause enteric and respiratory diseases, such as calf diarrhea, winter dysentery, calf respiratory disease, and bovine respiratory disease complex (BRD). All of these diseases are believed to be caused by the same genotype of BCoV. BCoV exhibits tissue tropism for both the gastrointestinal and respiratory tracts. This tropism is due to 9-O-acetylated sialic acid receptor on both epithelial cells in the respiratory and enteric tract. Currently, the only vaccine available for BCoV targets the enteric form of the disease. This study addresses the hypothesis that antibodies from the enteric form of the disease can cross neutralize the respiratory form of the virus. Data from surveillance studies suggest that BCoV is one of the major contributors to BRD, for which there is no currently approved vaccine for the respiratory form of the disease. Our approach to answering this question is to sequence and analyze the complete genome of 11 respiratory and enteric coronavirus isolates using next generation sequencing (NGS). Following the NGS, viruses were selected based on phylogenetic analysis and ability to grow and be maintained in cell culture. These viruses were then be used as serum neutralization indicator viruses in SN assays. 147 bovine serums submitted to KSVDL were used to determine if there are any serological differences between the immune response to respiratory versus enteric viruses based on the antibodies produced by the animal. The overall results show that there are few differences between the enteric and respiratory isolates at the genomic level and the serological response from the animal to these viruses. The differences between enteric and respiratory virus will need to be further addressed and analyzed to conclude if there is a noteworthy difference between the viruses with different tropisms. Other factors, such as host immune response and environment, are believed to be involved in the virus tropism to certain areas of the body.

Disenteria de inverno é uma doença causada pelo coronavírus bovino que afeta animais adultos. Descrevem-se dois surtos de disenteria de inverno em rebanhos leiteiros nos municípios de Viamão e Vespasiano Corrêa, Rio Grande do Sul. A doença foi reproduzida experimentalmente em dois bovinos adultos. O quadro clínico se caracterizou por diarréia, inicialmente líquida esverdeada com estrias de sangue e muco, que em alguns animais, evoluiu para coloração marrom escura à sanguinolenta e que persistiu, em média, cinco dias. Na propriedade de Vespasiano Corrêa os animais apresentaram sinais respiratórios como tosse e corrimento nasal. Diminuição na produção de leite e no consumo de alimentos, além de graus variados de depressão foram também observados. Foi detectada a presença do coronavírus bovino nas fezes e secreção nasal dos animais pela técnica da nested RT-PCR. Foi realizada necropsia de um animal naturalmente infectado que morreu devido à doença. Na necropsia, observou-se mucosas pálidas, conteúdo sanguinolento com presença de grande quantidade de coágulos, principalmente no cólon espiral e petéquias na mucosa do cólon. No cólon espiral, foram observados os principais achados histológicos que incluíram criptas dilatadas sem epitélio de revestimento, ou revestidas por epitélio pavimentoso e/ou cuboidal, às vezes, com núcleos grandes e nucléolos proeminentes. Algumas criptas eram preenchidas por debris necróticos e polimorfonucleares. Amostra de fezes de um animal naturalmente infectado foi usada, através de uma suspensão administrada via oral e nasal, para tentar induzir infecção experimental em 3 bovinos de 20 meses. Foi observada diarréia em dois desses animais, no 5º dia após a inoculação. Esses animais foram eutanasiados ao 2º e 3º dias de diarréia. As lesões histológicas dos casos experimentais foram semelhantes àquelas do caso natural. Na imuno-histoquímica anti-coronavírus bovino (8F2) em cortes de tecido incluído em parafina do cólon espiral, houve marcação positiva no citoplasma de enterócitos das criptas, nos debris necróticos dessas criptas e em macrófagos na lâmina própria, tanto no caso natural como nos experimentais. Nos casos experimentais, também foi observada marcação imuno-histoquímica positiva em duodeno, jejuno, íleo, ceco, cólon descendente e epitélio de revestimento dos cornetos nasais. / Winter dysentery is caused by bovine coronavírus that affects adult animals. This report describes two outbreaks of the disease in dairy herds located in the counties of Viamão and Vespasiano Corrêa, Rio Grande do Sul. The disease was induced experimentally in two adult cattle. The most significant clinical sign was profuse and watery diarrhea, which ranged from greenish to brownish coloration with occasional blood streaks and mucus to a bloody diarrhea. In one herd, animals showed respiratory signs such as cough and nasal discharge. Most cases persisted for 5 days and also included depression, decreased milk production, and diminished food intake. The presence of bovine coronavirus was detected in feces and nasal secretions of animals by nested RTPCR. Necropsy was performed in one animal naturally affected and died due to the disease and revealed pale mucosa, sanguineous contents, and blood clots particularly within the spiral colon and pinpoint hemorrhages on the colonic mucosa. Histopathological lesions were predominant in the spiral colon and consisted of a high number of dilated crypts without epithelium or with replaced pavement epithelium with occasional immature cuboidal cells, which sometimes showed enlarged nucleus and prominent nucleolus. Some crypts were filled with epithelial desquamation and polymorphonuclear cells. Fecal samples from one naturally infected animal were used as a suspension and inoculated orally and nasal in three 20-month-old heifers. Diarrhea was observed in 2 of them and started on the 5th day post infection. These animals were euthanized at the second and third days of diarrhea. The histological lesions of experimental cases were similar to the natural case. Anti-bovine coronavirus (8F2) immunostaining was applied on paraffin embedded sections of the spiral colon and showed positive reactions in the cytoplasm of the infected crypt epithelium, sloughed necrotic cells, and within macrophages in the lamina propria of both, natural and experimental cases. Positive reactions were also seen in duodenum, jejunum, ileum, cecum, descending colon and the epithelium lining the nasal turbinate from experimental cases.

RNA sequences and higher-order structures in the 5’ and 3’ untranslated regions (UTRs) of positive-strand RNA viruses are known to function as cis-acting elements for translation, replication, and transcription. In coronaviruses, these are best characterized in the group 2a bovine coronavirus (BCoV) and mouse hepatitis virus (MHV), yet their precise mechanistic features are largely undefined. Here, we use a reverse genetics system in MHV to exploit the ~30% nt sequence divergence between BCoV and MHV to establish structure/function relationships of 5’ UTR cis-replication elements. It had been previously shown that a precise replacement of the 391-nt MHV 3’ UTR with the 288-nt BCoV 3’ UTR yields wt-like MHV. Our attempts to replace the 209-nt MHV 5’ UTR with the 210-nt BCoV 5’ UTR, however, yielded a non-viable chimera. Therefore, a systematic analysis of individual 5’-terminal structures was made to identify compatible elements. By placing each of four putative cis-acting domains from the BCoV 5’ UTR into the MHV genome, we learned that (i) stem-loops (SLs) I & II and SLIII are functionally compatible, (ii) SLIV is compatible if it spans parts of the 5’ UTR and the nonstructural protein 1 (nsp1) cistron, thus identifying this part of ORF 1 as a component of the cis-replication signal, (iii) a relatively unstructured 32-nt region mapping between SLIII and SLIV defines a novel virus species-specific cis-replication element, (iv) spontaneous suppressor mutations within MHV SLI and nsp1 cistron compensated for growth defects arising from the BCoV 32-nt element in the MHV genome, (v) cross talk between the 32-nt element, SLI, and the nsp1 cistron appears essential for virus replication, (vi) the BCoV 5’ UTR and nsp1 cistron function together in the MHV genome to generate a wt-like MHV phenotype, and (vii) a functional 5’ UTR-nsp1 domain in group 2a coronaviruses cannot be substituted by the corresponding genomic element from the group 2b SARS-CoV. We postulate that the interaction between the 5’ UTR and nsp1 cistron (or possibly nsp1 protein) functions as a molecular switch between genome translation and ignition of negative-strand RNA synthesis.

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