Victor J. Cabasso

Propagation of Infectious Canine Hepatitis Virus in Tissue Culture

Summary The serial propagation of ICH virus by the roller tube method in dog kidney tissue culture through 15 consecutive passages is reported. The growth of the virus under these conditions causes a specific cytopatho-genic degeneration of the tissue growth, similar to that reported for poliomyelitis virus in the presence of human or monkey tissues. Identification of the tissue culture virus was made by both the complement-fixation and serum neutralization methods using a known infectious canine hepatitis fox immune serum.

Propagation of Canine Distemper Virus on the Chorio-Allantolc Membrane of Embryonated Hen Eggs

Summary Studies are reported in which the Lederle stock strain of canine distemper virus was adapted and maintained through 74 serial passages on the chorio-allantoic membranes of developing chick embryos. The chick embryo adapted strain retained its capacity to produce lethal infection in ferrets through the 24th egg passage level but apparently lost its ability to do so between the 24th and 28th egg passages. Ferrets inoculated with chick embryo adapted virus of passage levels higher than the 28th transfer failed to show any signs of disease, but were found immune on rechallenge with fully virulent ferret spleen virus. Cross neutralization tests carried out in chick embryos and in ferrets indicate that the chick embryo adapted strain and the parent strain of ferret spleen virus are immunologically related.

INFECTIOUS CANISE HEPATITIS VIRUS

.4mong the recognized viral denizens of the dog, infectious canine hepatitis (ICH) made its appearance fairly late, but now more is known about it than about the earlier denizens, distemper and rabies. The main reason for the rapid accumulation of knowledge concerning ICH was its prompt propagation in tissue culture monolayers. ICH attracted attention a5 a disease of dogs in 1947, when Rubarth‘ published his classical report on almost 20 years of research, and soon thereafter was identified in the U.S2z3 and in Great Britain.4 Before long it was recognized in most parts of the world. However, the existence of such a disease had been suspected much earlier. Cowdry and Scott: reported in 1930 their discovery of intranuclear inclusions in the hepatic and endothelial cells of two dogs. While the presence of these inclusions could not be explained a t the time, it was predicted that a filtrable agent would eventually be found to be responsible for them. A few years later, Green and Shillinger,6 who had experimentally infected dogs with fox encephalitis virus, demonstrated the presence of intranuclear inclusions in their t iswes . The immunological identity or’ ICH virus and the virus responsible for fox encephalitis was established in 19-49.’ For a time this led to confusion regarding the name of the canine disease, which was referred to as “fox encephalitis in the dog” by some authorsR and many veterinary practitioners, although in dogs the disease has no encephalitic symptoms. The fox was soon relegated to second place, however, and gradually the virus came to be known as infectious canine hepatitis, an agent also responsible for encephalitis in foxes.

Poliovirus antibody three years after oral trivalent vaccine (Sabin strains)

A sample group of children who had no poliovirus antibody before vaccination was tested three years after vaccination. For poliovirus Types II and III, 100 per cent and 94.5 per cent of the children, respectively, retained antibody titers of at least 1:16. Only for Type I was the antibody-positive rate appreciably lower than at two years after vaccination, 81.3 per cent compared with 94.5 per cent; eight more children had a titer below 1:16. However, all but two children had a Type I titer of at least 1:8.

Immunizing properties of live attenuated measles virus

Summary Early clinical trials indicate that it is possible to immunize children with live attenuated measles virus. The susceptible subjects develop either no symptoms at all or a modified measles with fever, leukopenia, and a faint eruption. Koplik spots are uncommon. These children have no catarrhal symptoms and do not appear toxic. All of the susceptible children developed a significant titer of neutralizing antibodies. No complications occurred in any of our cases. The urgent need of a measles vaccine is stressed, and there are indications that measles immunization as a routine procedure may not be too distant.

Poliomyelitis. III. Propagation of MEFl Strain of Poliomyelitis Virus in Developing Chick Embryo by Allantoic Cavity Inoculation.

Summary and conclusions Data describing the cultivation of the MEFl Lansing type poliomyelitis virus in the developing chick embryo are reported. This strain of poliomyelitis virus was derived from the original MEFl strain following its adaptation to suckling hamsters. Neutralization tests performed at three passage levels with bona fide immune hamster and monkey sera prepared against three homotypic strains of the Lansing type virus point to the identity of the chick embryo adapted and propagated virus with this type of poliomyelitis agents. A moderate neutralization has also been shown to take place between the chick embryo adapted virus and immune monkey Brunhilde and Leon sera, but, as indicated, it is difficult to attribute much significance to this finding at the present time. Some experimental evidence was obtained indicating that the chick embryo adapted virus does not induce paralysis or death in Rhesus monkeys even following intracerebral inoculation with comparatively massive doses of virus. The effect of this virus on the immune response of Rhesus monkeys and chimpanzees is being further investigated.

Propagation of Canine Distemper (CD) Virus in Tissue Culture

Summary A chick-embryo-adapted canine distemper virus was propagated successfully for 64 passages in cultures of total chick-embryo tissues. As measured on the chorioallantoic membrane of live chick embryos, average virus titers of these passages ranged between 103.0 and 104.5/ml infected culture suspension. These titers are of the same order as those obtained with live-chick-embryo-grown virus. Identity of tissue-culture-grown agent with original CD virus employed was established by virus neutralization method and by ferret immunization. Effective freeze-dried vaccines were produced with different passage levels of tissue-culture-grown virus.

Further evidence of immunologic dissimilarity of distemper (CD) and measles (M) viruses.

Summary Additional experimental evidence of dissimilarity of canine distemper (CDV) and measles (MV) viruses are presented: 1. Ferrets inoculated with repeated doses of MV-adjuvant mixture and which developed M antibodies proved as susceptible as control animals to challenge with CDV. 2. Paired convalescent sera from measles patients showed significant measles antibody rises, while CD antibodies remained essentially absent. 3. Growth of MV in HeLa cells was not inhibited by presence of CD antiserum or normal serum, while MV antiserum apparently reduced virus multiplication. Presence of measles antiserum did not interfere with propagation of CDV. 4. By 14th day after MV infection, serologically negative monkeys developed high antibody titers for MV but none for CDV. It remains to be determined whether results obtained are attributable to particular virus strains used, or apply also to other isolates of MV and CDV. Authors thank Marie Murrin and Hubert Klaver for technical assistance and E. Manoogian for help in preparation of manuscript.

Distemper and measles viruses. I. lack of immunogenic crossing in dogs and chickens.

Summary Puppies, vaccinated against canine distemper (CD) or immunized and challenged with CDV, developed high levels of homologous antibodies but failed to develop CF or serum neutralizing antibodies for measles virus (MV). Similarly, chickens hyperimmunized with CD virus did not develop measles neutralizing antibodies, although CD antibodies in appreciable levels appeared in 6 of 6 birds. Hyperimmunization of another group of chickens with MV was followed by no neutralizing CD antibodies in any of 6, whereas 5 of 6 birds gave a substantial measles response.

A bivalent live virus vaccine against canine distemper (CD) and infectious canine hepatitis (ICH).

Summary The virulence of ICH virus has been effectively modified by serial propagation in dog-kidney cultures followed by adaptation to and serial cultivation in swine-embryo tissue. The modified virus is an effective immunizing agent, and produces in the vaccinated animal no adverse reaction except for an occasional case of light and transient opacification of the eye following massive peripheral injection of the virus. The modified ICH virus may spread through the urine of vaccinated animals to intimate contact animals, but although these develop antibodies they show no signs of disease. A combined vaccine prepared by mixing modified CD and ICH viruses elicited good protection against both diseases in doubly susceptible animals, and in puppies already immune to ICH did not interfere with the production of immunity to CD.