Max R. Stebbins

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.

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.

Propagation of Canine Distemper Virus in Suckling Hamsters

Summary The Lederle strain of chick embryo adapted distemper virus was propagated in suckling hamsters through 16 serial transfers. The virus elicited a lethal infection in this host in 4-7 days following inoculation. Proof of the identity of the hamster-propagated agent was derived from serum neutralization tests in hamsters and from the successful immunization of ferrets.

Plaque Formation by Infectious Canine Hepatitis (ICH) Virus

Summary Plaque formation by infectious canine hepatitis virus was demonstrated on dog kidney monolayers with both dog and swine kidney grown virus. A plaque-derived virus clone was identified as ICH by stationary tube neutralization test as well as by plaque assay. Under conditions of the study, assay by plaque plates appears to be a more sensitive titration method than by stationary tissue culture tubes.

Poliomylitis. IV. Some Cultural and other Characteristics of Chick-Embryo-Adapted Type II Strain of Poliomyelitis Virus

Summary 1. Effects of incubation time and temperature, age of embryo and route of inoculation on multiplication of a Type 2 chick-embryo propagated poliomyelitis virus were studied. The distribution of the virus in chick embryo tissues and fluids, and its action on the hatchability of infected embryos, were investigated. 2. Optimal conditions for multiplication of the virus were inoculating 6- to 9-day-old embryos via either the allantoic cavity or the yolk sac, and incubating for 4 to 6 days at 35°-37°C. 3. The best source of the virus was the embryo proper. No greater concentration of virus was found in the head than in the body. 4. Although the embryos seemed to overcome the infection by the 12 th or 13 th day, relatively few hatched out. The majority died between the 22nd and 27th day of total incubation. 5. The mouse LD50 titers of the chick-embryo-adapted virus were essentially unaffected by 9 consecutive freezings and thawings of the same virus preparation.

Bluetongue I. Propagation of Bluetongue Virus of Sheep in Suckling Hamsters

Summary The adaptation of bluetongue virus strains of different origin to the suckling hamster is reported. Details pertaining to the serial propagation of the South African Jansen strain of virus through 20 passages are given in extenso, together with results of its identification by the qualitative serum neutralization method. Mention is also made of the use of infected hamster brains for the preparation of bluetongue complement-fixing antigens.