The propagation of a porcine epidemic diarrhea virus in swine cell lines.

A strain of porcine epidemic diarrhea virus (PEDV), P-5V, utilized as a live virus vaccine in Japan was infected to a swine cell lines, KSEK6 and IB-RS-2 cells. Clear CPE, characterized by cellular destruction, started to appear in the infected cells on 2-3 days post infection (DPI) and affected cells was completely degenerated on 4 DPI. The virus was serially passaged in the cells even without addition of trypsin. Small but clear plaques were formed under an agar overlay medium on the cells. The infective titer in the order of 10(7.00-7.50) TCID50 per ml was obtained at usual incubation temperature.

Virus propagation in a swine monocyte cell line.

The propagation of seventeen virus strains, classified into five virus families, in a swine monocyte cell line (SW/K99) was studied in the point of infective progeny production. The viruses examined were adapted to grow in a swine epithelial cell line (KSEK6) and were proved to show clear CPE in advance. These viruses were successively passaged in the monocyte cultures regardless of CPE occurrence. The cultures at 3rd passage level were titrated for their infectivity using KSEK6 cells. Out of the viruses examined, only Aujeszky's disease virus (ADV) was able to propagate in the monocytes. Four ADV strains, two virulent and two attenuated strains, were compared for their growth in two cell lines. The final amount of infective progeny measured at 72 hours incubation at 36.5 degrees C was almost in a similar order between 2 cell lines. However, the amount of infective progeny produced at 24 hours incubation was higher in SW/K99 cells than that in KSEK6 cells. The occurrence of CPE was also more evident in SW/K99 cells in an early incubation. The data indicate that viral susceptibility of the monocytes to ADV is higher than that of epithelium. Other viruses were abortive in the monocytes.

Isolation of coxsackievirus B5 from pigs.

A cytopathic virus was isolated from young pigs suffering from severe diarrhea in Okinawa, Japan in 1986. The disease was highly contagious among young pigs. The physico-chemical properties of the virus indicated an enterovirus, but, no of serological relationship was detected with reference strains of porcine enteroviruses. With the aid of Genbank for genomic sequence data, RT-PCR and hybridization method was performed. The viral isolate was identified as the Coxsackievirus (CV) B5 of human enterovirus. This is the first report of the isolation of CV B5 from pigs in Japan.

Establishment of a swine monocyte cell line.

A swine monocyte cell line was established from peripheral blood sample collected from a healthy adult male pig. The cloned cells grow actively in forming monolayers in both glass and plastic cell culture flasks with the growth medium reported previously (Kadoi, 2000) at 36.5 degrees C incubation. The plating efficiency is more than 95%. Densely grown cells in flasks show an epithelioid morphology. The fundamental properties of the cells were examined for cytological definition as monocytes. A positive property detected was guinea pig complement receptor, porcine IgG receptor, non-specific esterase, and acid phosphatase. A significant phagocytic activity proved by the inoculation of Saccharomyces cerevisiae is also one of the characteristics observed in the LPS-activated cells.

Antibody response of dogs inoculated with a large number of cultured canine monocytes adsorbed with a calicivirus in advance.

Five conventional Beagle dogs were intravenously injected with ten million canine monocyte cells (Cn/K99) cultured in vitro (Kadoi, 2000) adsorbed with a strain of calicivirus originally isolated from lions (Kadoi et al., 1997). Another two Beagle dogs were injected similarly with the virus suspension solely as control. Serum samples were collected from these dogs at intervals and specific seroneutralizing antibody production against the virus was measured in vitro. A significantly higher antibody production was demonstrated in the five dogs group. A clear booster effect was also proved in the sera of the dogs after the second virus inoculation made on day 100. A possibility of antigen presentation function of non-self monocytes is suggested.

Stability of feline caliciviruses in marine water maintained at different temperatures.

Since human caliciviruses are responsible for viral gastroenteritis transmitted by contaminated foods and the viruses barely propagate in cell culture, feline caliciviruses were employed as a model for the measurement of their stability in marine water. Survival of four strains of feline calicivirus in marine water was measured when the seed viruses were diluted 1/10 with marine water and maintained at 4 degrees C, 10 degrees C, and 20 degrees C respectively. Among the virus strains studied, a considerable amount of infective viruses remained at 10 degrees C or lower temperature conditions even for a period of 30 days.

Establishment of a canine monocyte cell line.

A canine monocyte cell line was established from the peripheral blood sample collected from a healthy young male Beagle dog. The cloned cells grew easily and were serially passaged in vitro in the medium, a slight modification further made on Dulbecco's modified Eagle medium, supplemented with 10% fetal bovine serum. The morphology of single cell was shown in triangular or round form, however, it became epithelioid in a densely grown monolayer. Non-specific esterase was detected in all cells by a cytochemical examination. The cells reacted rapidly to the addition of a small amount of LPS and differentiated to the cells of morphologically typical macrophages. Both complement receptor (CD35) and Fc gamma receptor (CD64) were demonstrated on the cell membrane.

Antibody response of cattle experimentally inoculated with parainfluenza type-3 live vaccine in a post-tracheal position.

One of the major viral respiratory diseases of cattle is parainfluenza type-3 virus infection. Attenuated live virus vaccines have been used in most countries. An experimental attempt was made to clarify the antibody response of cattle inoculated with a live vaccine in a post-tracheal position by a spray method. A significantly higher titers of antibodies in both hemagglutination inhibition and neutralization were obtained in the serum samples of animals vaccinated in this manner than those vaccinated by intramuscular injection or intranasal instillation.

Experimental infection in SPF kittens with a particular calicivirus strain originally isolated from lion.

Two SPF kittens were experimentally infected via the intranasal route with a strain of calicivirus originally isolated from sick lion suffering from vesicular disease. Fever, 40.3 degrees C and 40.7 degrees C, and vesicular formation in tongue and snout were reproduced in both kittens. The infected virus was recovered from nasal, oral and rectal swabs. A longer duration of virus recovery was proved with oral swab samples taken from 1 to 10 and 12 days post infection. This suggests that tongue and oral tissues are the main tissues for virus multiplication.

Antibody response of lions inoculated with inactivated calicivirus vaccine experimentally prepared.

The majority of lions and tigers in a Safari park in Japan were suffering from acute vesicular disease caused by a calicivirus infection. All of these animals were injected subcutaneously with an inactivated calicivirus vaccine experimentally prepared with one of the seed viruses originally isolated from sick lions. Seroneutralizing antibody of paired sera collected from ten female lions at two week intervals was measured. A significant elevation of specific antibody was detected in the serum samples and no local or systemic reactions associated with vaccination were observed.

Antigenic analysis of influenza viruses isolated in Thailand between 1991 and 1994.

We studied the epidemiology of influenza viruses in Thailand by isolating them and comparing their antigenic features with those of Japanese isolates. Between 1991 and 1994, 32 strains were isolated from 186 throat swab specimens. Twenty-one of the 32 isolates were of type A, subtype H3N2, and 11 strains were type B. It was suggested that the isolates of type A, subtype H3N2, drifted antigenically from A/Beijing/352/89-like to A/Kitakyusyu/159/93-like variants used as reference strains for comparison. The type B isolates in 1991 were suspected to be antigenically different from those of B/Bangkok/163/90, Thailand, in HI tests. These 1991 isolates were similar to B/Mie/1/93, which was isolated in the latter half of the epidemic in Japan in winter 1992/1993.

Adenovirus isolation from spleen lymphocytes of apparently healthy pigs.

A polyethylene glycol treatment was given to fuse KSEK6 cells, an established cell line derived from porcine embryo kidney, with the lymphocytes, separated from spleens of 35 apparently healthy slaughtered pigs. Eight cytopathic virus strains were isolated from the lymphocytes of these pigs. Two virus strains were isolated by inoculating the spleen tissue homogenates to KSEK6 monolayer cultures. All of viruses were identified as porcine adenoviruses according to their physicochemical, serological and immunological properties. One of these virus strains was serologically proved to be independent from six serotypes of porcine adenoviruses ever known. The electrophoretic property of viral DNA of this strain was indicated to be different from those of other reference porcine adenoviruses. This means the presence of a 7th serotype in porcine adenoviruses.

Viral susceptibility of a newly established cell line derived from the peritoneal cavity of BALB/C mouse.

Viral susceptibility of a newly established cell line, named KMP, derived from the peritoneal cavity of BALB/C mouse is described. The cells were originally cloned from the in vitro culture of ascites of the mouse injected with Ehrlich ascitic tumor cells in advance. The electrophoretic pattern of cellular DNAs, extracted from KMP, normal BALB/C mouse spleen, and Ehrlich tumor cells respectively were compared after triple digestions with restriction endonucleases. This cell line was proved to be of mouse origin, but not the sub-line of Ehrlich tumor cells. The strains of Coxsackie virus B group, swine enterovirus, influenza virus, encephalomyocarditis virus, vesicular stomatitis virus, and Aujeszky's disease virus were able to multiply well in the cell line with considerably high infectious titers in showing clear CPE and circular plaques.

A strain of calicivirus isolated from lions with vesicular lesions on tongue and snout.

In December 1992, 17 African lions and 7 Siberian tigers in a Safari park in Japan became sick with characteristic clinical symptoms of acute vesicular formations on tongue and snout. The disease was highly contagious since all of these animals showed similar symptoms within two days after the onset of the first case. Swabs were taken from affected animals in rubbing tongues, snouts and some from rectums. Cytopathic viruses were isolated on CRFK cell culture by virological tests. The physicochemical property of a representative virus strain, named Arthur/L, isolated from a male lion was identified as a member of Caliciviridae. However, seroneutralization test indicated that this virus strain was antigenically distinct from Japanese isolates of feline caliciviruses used for comparison. Viral capsid proteins of the present isolate, Arthur/L, and of a feline calicivirus, strain FC7, were compared in an electrophoresis in SDS-PAGE gel. The major viral capsid polypeptide of them were proved to be significantly different in molecular weight. The polypeptide of FC7 was estimated to be ca. 63 KDa whereas that of Arthur/L consisted of 2 components of ca. 65 and 62 KDa. The viral proteins of these two strains were also proved to be distinct by an immunoblotting test.

Beneficial use of inactivated porcine adenovirus vaccine and antibody response of young pigs.

An inactivated adenovirus vaccine experimentally prepared was inoculated into young pigs against the adenovirus infection newly appeared in Japan. Sero-neutralizing titers of the paired sera collected from 50 pigs in the interval of 3 weeks was measured. The mean of antibody titers of post-sera was 45 times higher than that of pre-sera.

Preparation of serotype specific antiserum against porcine adenoviruses in mice.

Serotyping of porcine adenoviruses needs type-specific antiserum. The injections of purified virions to SPF Balb/C mice, followed by an intraperitoneal injection of Ehrlich ascitic tumour cells, gave enough ascitic fluid containing practically applicable type-specific antibody.

Isolation of porcine adenovirus as a candidate of 5th serotype.

A strain of cytopathic virus, named strain TG/K79, was isolated from the brain of a newborn piglet, pure Hampshire breed, which died shortly after birth. The physicochemical property of virus was considered to be that of the family Adenoviridae. A significant difference between our isolated and 4 reference porcine adenoviruses was demonstrated by cross-seroneutralization test. Differences between TG/K79 and other porcine adenoviruses were also seen in electrophoretic patterns of viral DNA in agarose gel after digested by restriction endonucleases. Two SPF pigs, 2-month-old, experimentally infected via intranasal showed a fever and a hemorrhagic enteritis. A serological survey indicates that at least swine in the farm where the virus was isolated have been highly contaminated.

The propagation of a porcine hemagglutinating encephalomyelitis virus in swine kidney cell cultures.

An established cell line, KSEK6, derived from swine embryo kidney proved to be a suitable host for the propagation of a hemagglutinating encephalomyelitis virus, strain 67N. Infected cells showed clear cytopathic effect as early as 24 hours incubation at 37 degrees C. Plaques easily visible to the naked eye were also produced under agar overlay medium. The infective titer of 3rd passage level in the cells was in the order of 10(8) PFU per ml. Detecting antibodies against this virus strain in swine sera was considered to be more accurate by neutralization test than by hemagglutination inhibition.

Effect of immune serum, an antibiotic, and a corticosteroid used alone or in combination on experimental leptospirosis in Mongolian gerbils (Meriones unguiculatus).

We studied the effects of immune sera, an antibiotic, and a corticosteroid used alone or in combination on leptospira infection in Mongolian gerbils (Meriones unguiculatus). The results suggest that the combined used of immune serum and corticosteroid (pledonizoron: PZ) inhibits the effects of serum and the marked effects of antibiotic (procaine penicillin-G: PC-G) on leptospirosis. PZ had no effects and rather shortened the survival period. PZ did not affect the effects of PC-G when used in combination. These results suggest that treatment of leptospirosis with corticosteroids requires special caution.