Kazuhiro Uwatoko

Rapid method utilizing the polymerase chain reaction for detection of canine parvovirus in feces of diarrheic dogs

By using primers based on the sequence of the VP2 gene of canine parovirus (CPV), we established a rapid and specific assay for identification of the virus from fecal specimens based on the polymerase chain reaction (PCR). By use of a pair of primers, a specific 226-bp sequence was amplified by the PCR. All strains of CPV tested gave a specific amplification product by the PCR, while neither porcine parovirus nor host cell did so. The PCR assay can detect fewer particles of CPV than the conventional methods, being able to detect CPV from fecal specimens in a rapid manner, provided that gel filtration of the samples through a spun column was done to remove inhibitory substances from the fecal specimens. These results suggest that the PCR assay can detect the presence of CPV in dogs early enough to prevent secondary infection by CPV in veterinary hospitals.

Rapid and efficient method to eliminate substances inhibitory to the polymerase chain reaction from animal fecal samples

To detect pathogenic viruses in animal fecal specimens by polymerase chain reaction (PCR) assays, it is important to remove or inactivate PCR-inhibitory substances. Recently, it was reported that such inhibitory substances in human feces could be efficiently eliminated by a cationic surfactant, Catrimox-14 (Iowa Biotechnology, Iowa) during extraction of viral RNA. In the present report, Catrimox-14 was successfully applied to detect pathogenic viruses in fecal specimens from a variety of animals. By extraction of viral DNA in the presence of this cationic surfactant, the PCR assay could detect canine parvovirus (CPV) in all fecal specimens prepared from 13 kinds of animals, i.e., cat, chicken, cow, dog, gerbil, goat, golden hamster, horse, mouse, pig, rat, rabbit, or sheep. Pretreatment by gel-filtration or boiling failed to remove or inactivate the PCR-inhibitory substances in fecal specimens from mouse, goat, rat, and sheep.

Effect of heparin on a haemagglutinin of porcine reproductive and respiratory syndrome virus

Heparin inhibited haemagglutination by porcine reproductive and respiratory syndrome virus (PRRSV) and by Aujeskys disease virus, but failed to inhibit haemagglutination by parainfluenza virus type 3. The minimal inhibitory concentration of heparin required to inhibit 8 HA U of PRRSV haemagglutinin ranged from 0.1 to 1 U ml-1. Mouse erythrocytes failed to combine with the haemagglutination inhibitory factor of heparin. However, mouse erythrocytes treated with heparinase had greatly reduced agglutinability by PRRSV. The formation of a haemagglutinin-heparin complex could be observed by sedimenting heparin with the haemagglutinin. All these findings suggest that a heparin-like molecule on the surface of mouse erythrocytes serves as the virus-cell receptor.

Canine parvovirus binds to multiple cellular membrane proteins from both permissive and nonpermissive cell lines

For identification of canine parvovirus (CPV) binding protein, the SDS-solubilized cell membrane fraction from a permissive cell line. CRPK, was subjected to the virus overlay protein blot assay (VOPBA). Competitive inhibition experiments showed the presence of multiple CPV-binding proteins with molecular masses of 36, 35, 33, 31, 29, 27, 25, and 23 kDa. CPV-binding proteins of same molecular masses were also detected in membrane fractions from nonpermissive, as well as other permissive, cell lines. We confirm that the mechanism of nonpermissiveness to CPV is not operative at the cellular attachment level.