To verify the presence of CPV in the fecal samples of a dog with clinical symptoms of severe enteritis, we performed PCR with primer sets for the conserved region in various strains of CPV (sense, 5'-AAAGAGAGCCAGGAGAGGTA-3'; anti-sense, 5'-TTCTGACAGCAGGTTGACCA-3') at 94℃ for 1 minute, 55℃ for 1 minute, and 72℃ for 1 minute (30 cycles) [24]. DNA for PCR analysis was obtained from the feces and isolated using QIAamp DNA stool mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

The fecal sample was homogenized (10% w/v) in phosphate-buffered saline (PBS) and subsequently clarified by centrifuging the solution at 2,800 rpm for 10 minutes. The supernatant was filtered through a 0.45-µm filter and then a 0.22-µm filter (Schleicher & Schuell, Keene, NH, USA) to obtain the viral fraction. The filtered supernatant was overlaid on a 70% monolayer of A72 canine fibroma cells in alpha-minimum essential medium supplemented with 2% fetal bovine serum, non-essential amino acids, and antibiotics (at a final concentration of 100 units/mL of penicillin G sodium, 0.1 mg/mL of streptomycin sulfate, and 29 µg/mL of amphotericin B). The cytopathic effect (CPE) of the isolate on A72 cells was observed at 3 days post-infection (dpi). The presence of CPV on infected cells was confirmed by indirect fluorescent assay (IFA) by using an anti-CPV monoclonal antibody (Genobiotech, Daejeon, Korea) and fluorescein isothiocyanate-conjugated anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA). Then, the isolated virus was passaged 4 times on A72 cells. CPE-positive A72 cell samples in their fourth passage were alternatively frozen and thawed 3 times, and then centrifuged at 3,000 rpm for 10 minutes at 4℃. The presence of CPV in the supernatant was confirmed by PCR analysis with type 2b-specific primers and CPE generation on the A72 cell monolayer before performing the in vivo infection tests.

To determine the type of the isolated CPV, PCR was performed using CPV-2b-specific primer pairs (sense, 5'-CTTTAACCTTCCTGTAACAG-3'; anti-sense, 5'-CATAGTTAAATTGGTTATCTAC-3') at 94℃ for 30 seconds, 55℃ for 2 minutes, and 72℃ for 40 seconds (30 cycles).

The VP2 gene of the clinical isolate was amplified using PCR with primer pairs (sense, 5'-ATGAGTGATGGAGCAGTTCAACC-3'; anti-sense, 5'-TTAGTATAATTTTCTAGGTGCTAG-3') at 94℃ for 30 seconds, 55℃ for 2 minutes, and 72℃ for 40 seconds (30 cycles). The PCR product was isolated by electrophoresis on a 1% agarose gel and purified using QIAquick gel extraction kit (Qiagen) according to the manufacturer's instructions. The purified sample was cloned into apCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA) and used to transform competent JM109 cells. The colonies were grown for 18 hours at 37℃ on Luria broth agar plates containing ampicillin. The plasmids were extracted by using GENE ALL Plasmid SV mini kit (General Biosystem, Seoul, Korea), and the inserts were subjected to dideoxy chain-termination sequencing (Applied Biosystems, Foster City, CA, USA). Sequence homology was estimated using BLASTn of the National Center for Biotechnology Information.

Seven male beagle dogs, aged 6 to 8 weeks, were used as test animals. The absence of the parvoviral antigen and the anti-parvoviral antibody in these animals was confirmed by the parvoviral antigen detection kit (Isu Abxis, Seoul, Korea) and parvoviral antibody detection kit (ImmunoComb, Biogal-Galed Labs., Galed, Israel), respectively. These dogs were allocated to 3 groups: group I, group II, and group III, which comprised 2, 2, and 3 dogs, respectively. Group I served as the control group and were orally administered with 1 mL of PBS without any infective agents. The dogs in group II were orally administered with the same volume ofthe clinical isolate, and those in group III were orally administered with the attenuated virus 2 weeks before the oral challenge with the clinical isolate. The dosage of the virus was 10, 50% tissue culture infectious dose (TCID₅₀) of CPV [16,25]. The clinical condition of each animal was monitored daily. Blood samples were obtained at 2-day intervals to analyze viremia and antibody levels each condition was detected using PCR and ImmunoComb (Biogal-Galed Labs.), respectively. Feces of each animal were also taken at 2-day intervals for PCR investigation of fecal shedding of CPV. On the day of death or 11 days after inoculation, 10 different anatomical samples were taken (from the spleen, mesenteric lymph node, thymus, cardiac muscle, liver, lung, kidney, duodenum, jejunum, and ileum) and analyzed by PCR. Histological changes in the jejunum and ileum were evaluated after hematoxylin and eosin staining.

We studied CPV infection in a dog that had severe enteritis, which was later confirmed by using a parvoviral antigen detection kit (Isu Abxis). PCR analysis of the DNA isolated from the fecal samples with the universal primers also showed positive results, as expected (Fig. 1). To isolate the virus from the fecal samples, we inoculated the filtered viral fraction on the A72 cells. The infected A72 cells showed cytoplasmic atrophy and elongation, nuclear polymorphism, and degradation, as well as detachment from the bottom of the plate in their final stage. On average, these changes occurred at 3 dpi and peaked at 5 dpi. CPVs in CPE-positive A72 cells were successfully visualized using IFA. A significantly higher increase in fluorescence was observed in the CPV-infected A72 cells than in the negative control cells (Fig. 2). These results clearly show that the isolate was a CPV, and we characterized this isolated virus.

To identify the type of clinical isolate, we performed PCR with primer sets specific to CPV-2b. As shown in Fig. 1, the genomic size was found to be 472 bp, as expected, indicating that this clinical isolate belongs to the CPV-2b type. We then determined the nucleotide sequence of the virus and found that the isolate was a novel type since its VP2 region was not identical to that of any of the CPVs listed on GenBank. As shown in Table 1, this isolate has Asn at amino acid position 426 in VP2, while CPV-39 and CPV-133, which are classical CPV-2b strains, have Asp at that position.

In order to develop a new vaccine against CPV infection, we passaged the clinical isolate 4 times on A72 cells and performed the tests described in Materials and Methods with the attenuated virus to determine its protection abilities. The dogs in group II that were inoculated with the fecal viral sample alone experienced diarrhea at 3 dpi, and their symptoms immediately progressed to severe enteritis with anorexia, vomiting, diarrhea, dehydration, and dyspnea; they finally died at 6 dpi. In contrast, the 2 dogs in group III that were administered with attenuated virus prior to the oral challenge with the clinical isolate remained healthy until the termination of the experiment and did not show any clinical symptoms. The 2 dogs in group I, the negative control group without any artificial infection, were healthy for the first 6 days since the start of the experiment but experienced anorexia and diarrhea at 7 dpi, which progressed to severe diarrhea and dehydration by around 10 dpi. These pathological conditions of the dogs in group I might be attributed to the transmission of CPV from the dogs in group II.

The presence of CPV in the blood and feces was examined by PCR. Consistent with the clinical symptoms was the observation that the dogs in group II exhibited positive bands at 3-5 dpi; this indicated viremia and fecal shedding of CPV. The dogs in group I showed the presence of CPV at 9-11 dpi. However, the dogs in group III did not show viremia at all, although 2 of them (dog #5 and #7) showed fecal shedding of CPV at 5-9 dpi (Fig. 3).

To examine the development of antibody against CPV, the antibody levels in the blood were determined by using ImmunoComb; the results showed that antibody titers against CPV arose significantly at 7-11 dpi in the dogs of group III (Fig. 4). The dogs in the other groups, except dog #3 in group II, did not show such elevated antibody titers throughout the experiment. The antibody titer of dog #3 increased to 7 arbitrary units at 5 dpi, although this dog died at 6 dpi.

An important factor that should be studied to understand the virulence of this virus is its tropism. To examine this factor, we performed PCR to detect CPV in 10 different anatomical samples from the test animals. Most of the samples from each dog in groups I and II had CPVs, but in the group III dogs, CPVs were present in only 1or 2 different samples, including the ileum, mesenteric lymph nodes, spleen, and kidneys (Fig. 5).

Histological examination of the jejunum and ileum of the dogs in groups I and II showed severe destruction of the villi and mucosal layers, including loss of surface epithelium, dilated crypts, shortening and blunting of villi, and inflammatory and hemorrhagic infiltration into the lamina propria (Fig. 6). No such significant histologic lesions were found in the intestines of the dogs in group III.