Isolation And Characterization Of A Calicivirus From Mink

The paper describes the isolation, identification, and biological and molecualr analysis of a virus isolated from the sick minks. Results indicate that the isolate is a mink calicivirus (MCV). In June 2007, an infectious disease was outbreak at a mink(Mustela vison)farm in Dalian, China. The observed clinical symptoms of the sick minks included mucopurulent ocular discharges, dry muzzle and others. Motality occurred to some cases, and autopsy of the dead minks showed hemorrhagic pneumonia. Samples of nasal swabs, ocular swabs, blood and organs such as lung, heart, liver, kidney, spleen and brain were collected and inoculated on Vero-E6 cell cultures after grinding. With continuous passages on cell culture, a typical MCV cytopathic effect (CPE) appeared in the cells inoculated with the grinded lung samples. The cells manifested an increase of granules in cytoplasma, shrinked and became rounding, and finally fall off from the monolayers in the flask, suggesting the infection of the cells by viruses. The viruses were harvested by 3-times freezing-and-thawing, and were then used for the virus identification and studies for its biological characteristics, using different relevant tests. Cell infection spectrum study showed that the virus grew well in Vero E6, Marc145 and BHK21 cells and produced cytopathic effect (CPE), but it could not grow in chicken embryo. The results of chloroform-sensitive test, ether-sensitive test and deoxidized salt-sensitive test demonstrated that the virus is not sensitive to the fat-soluble reagent. Under electron microscopy, the virus particle appeared as an unenveloped, with a goblet-like capsid and positive icosahedron symmetry. The virus is not sensitive to 2'-deoxy-5-fluorouridine (FUDR), indicating that the nucleic acid type of the virus is RNA. The pH toleration tests showed that the virus tolerates to the acidic environment as low as pH 3.0, but not to pH >10.0. The virus is still active after 1 hour period at 50?C. Hemagglutinin test revealed that the virus agglutinates red blood cells of human "O" type, but not agglutinate any red blood cells from chicken, geese, rabbits, pigs, cattle, goats, sheep, dogs, rats and guinea pigs. UV-inactivation test showed that the virus can be completely inactivated in 1 hour under UV light. All the results together from the above tests suggest that the virus is a member of Caliciviridae. Using a pair of calicivirus specific primers (DL1, DL2) in a reverse transcription polymerase chain reaction (RT-PCR), a 331 bp sequence fragment was expectly amplified and obtained. A BLAST comparison of the sequencing result of the virus to the 11 nucleotide sequences of calicivirus in GenBank was conducted and homology of the sequencies was performed using the DNAStar and BioEdit sequence analysis softwares. The results showed that the sequence homology of the current isolated virus with the 3 strains isolateded in United States in 1980 ( AF338405,AF338406,AF338407)is 93.5%～96.8% for nucleotides,and 91.3 ~ 97.1% for amino acids. In conclusion, the isolated virus is a mink calicivirus, and it is named as MCV strain DL-07.