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1、 Braz. Arch. Biol. Technol. v.52 n. special: pp.45-49, Nov. 2009 45 Vol. 52, Special Number: pp. 45-49, November 2009 ISSN 1516-8913 Printed in Brazil BRAZILIAN ARCHIVES OF BIOLOGY AND TECHNOLOGY A N I N T E R N A T I O N A L J O U R N A L Bovine Coronavirus Detection in a Collection of Diarrheic St
2、ool Samples Positive for Group A Bovine Rotavirus Aline Fernandes Barry, Alice Fernandes Alfieri, Danilo Tancler Stipp and Amauri Alcindo Alfieri * Laboratrio de Virologia Animal; Departamento de Medicina Veterinria Preventiva; Universidade Estadual de Londrina; C. P.: 6001; 86051-990; Londrina - PR
3、 - Brasil ABSTRACT Neonatal diarrhea is an important cause of economic losses for cattle farmers. The main viral etiologies of enteric diseases are group A rotaviruses (GARV) and the bovine coronavirus (BCoV). Although both viruses infect calves of the same age, the occurrence of mixed infections is
4、 still under studied. The present study describes the co-infection of BCoV and GARV in stool samples. Forty-four diarrheic fecal samples from calves up to 60 days old that had previously tested positive for GARV by SS-PAGE were analyzed using semi-nested PCR for BCoV. A product with 251 bp of the BC
5、oV nucleoprotein gene was amplified in 15.9% (7/44) of the samples, demonstrating that co- infection is not an unusual event. These results reinforce the need for testing for both GARV and BCoV, even in fecal samples that previously tested positive for one virus. Key words: calf, diarrhea, BCoV, rot
6、avirus, SN-PCR, co-infection *Author for correspondence: alfieriuel.br INTRODUCTION Neonatal diarrhea is one of the main causes of calf mortality worldwide. The disease results in major economic losses in many dairy and beef cattle herds that result from treatment costs and calf deaths. Calf diarrhe
7、a is considered to be a multi- factorial disease. Several environmental, management-related, nutritional, and physiological factors may occur either alone or in synergy with different infectious agents such as protozoans, bacteria, and/or viruses. In calves, bovine group A rotavirus (GARV) and bovin
8、e coronavirus (BCoV) are the viruses most commonly associated with neonatal diarrhea and it is not unusual that both viruses can concomitantly infect calves (Snodgrass et al., 1986; Holland, 1990; Alfieri et al., 2006; Oliveira Filho et al., 2007). Rotaviruses belong to the Reoviridae family. They h
9、ave icosahedral symmetry and a non-enveloped capsid formed by three concentric layers of protein that is 70-90 nm in diameter. The rotavirus genome is composed of 11 double-stranded RNA segments (dsRNA). Based on the common group antigen (VP6 protein), rotaviruses are classified antigenically into s
10、even serogroups (A-G). The co-migration of genomic segments 7, 8, and 9 in polyacrylamide gel electrophoresis (PAGE) is an important characteristic of group A rotaviruses (Estes, 1996). Several techniques have been developed to diagnose bovine GARV by detecting the viral Barry, A. F. et al. Braz. Ar
11、ch. Biol. Technol. v.52 n. special: pp.45-49, Nov. 2009 46 particle (cell culture, electron microscopy, immune electron microscopy), viral protein (ELISA, latex agglutination, direct immunofluorescence), and viral genome (SS-PAGE, RT-PCR). All of these methods vary in specificity and sensitivity and
12、 present both advantages and disadvantages (Alfieri et al., 1996; Markowska-Daniel et al., 1996). PAGE followed by silver staining (SS-PAGE) is a well-established technique that is often employed for rotavirus detection (Herring et al., 1982; Pereira et al., 1983). It enables the identification of r
13、otavirus groups by analyzing the migration pattern of the 11 segments of dsRNA, and it has all of the characteristics required for large scale use, such as low cost and ease of execution (Markowska-Daniel et al., 1996). The Coronavirus genus belongs to the Coronaviridae family and is divided into th
14、ree genetically distinct groups. BCoV is classified in group 2 and is a pneumoenteric virus that causes diarrhea and respiratory disease in calves and winter dysentery in adult cattle (Takiuchi et al., 2009). The virus has an envelope 120-160 nm in diameter and a helical nucleocapsid. The genome is
15、a single-stranded, positive-sense RNA molecule of 27-32 kb. The 13 open reading frames (ORFs) of the genome encode five major structural proteins (ICTV dB 2009). The frequency of BCoV infection is quite variable between different geographical regions, the production type (dairy or beef herds), and t
16、he diagnostic techniques used. In Brazil, only a few studies have evaluated the BCoV occurrence in stool samples from calves. The rates of infection range from 14 to 39% (Jerez et al., 2002; Takiuchi et al., 2006; Oliveira Filho et al., 2007; Stipp et al., 2009). A few different methods exist to show the presence of BCoV in stool samples, including electron microscopy (EM), isolation in tissue culture, hemagglutination (HA) / inhibition
