Further evidence that the nucleic acid of avian encephalomyelitis virus consists of RNA.

The nucleic acid of the Van Roekel strain of avian encephalomyelitis virus (AEV) was determined to be RNA, according to the inability of the nucleoside analog 5-bromo 2'-deoxyuridine (BUdR) to inhibit its growth in chicken embryo kidney cell cultures. The test was carried out using known DNA and RNA viruses as controls, and the results are consistent with classification of AEV as a member of the family Picornaviridae within the genus Enterovirus.

Pathogenesis of avian encephalomyelitis viruses.

The pathogenesis of a field strain, a vaccine strain and the egg-adapted Van Roekel strain of avian encephalomyelitis virus in susceptible chicken embryos and day-old chickens was investigated using enzyme-linked immunosorbent assays for the detection of virus-specific antibody and antigen. The Van Roekel strain was shown to be highly neurotropic whereas the field and vaccine strains were enterotropic. Radioimmuno-precipitation studies using Na125I-labelled purified virus failed to detect any differences in the composition of the structural viral proteins of each strain that could account for these differences. As expected, the field and vaccine strains were more efficient than the Van Roekel strain at inducing antibody following oral administration. Primary cultures of chicken embryo brain cells supported the growth of the Van Roekel strain to a much greater extent than the field and vaccine strains.

A comparison of titration methods for live avian encephalomyelitis virus vaccines.

Factors associated with the indirect fluorescent antibody test used for the titration of avian encephalomyelitis virus (AEV) in chick embryo brain cell cultures were examined for their influence on virus replication. It was found that virus should be inoculated onto semi-confluent cell cultures and adsorbed for two hours at room temperature. The cells should then be examined for fluorescence after five days' incubation. Using these conditions, the cell culture assay was compared with the embryo and chick assays for its ability to estimate the virus content of live commercial AEV vaccines. In most cases titres obtained by the chick assay were slightly, but not significantly, higher than those obtained in the cell culture assay, although the reliability of the chick assay was, at times, questionable. In all cases titres obtained in the embryo assay were low. It is recommended that the cell culture assay be adopted as the method of choice for titrating AEV vaccines because it is rapid, reproducible, specific and greatly reduces the requirement for experimental animals.

Invasion of avian encephalomyelitis virus from the gastrointestinal tract to the central nervous system in young chickens.

Invasion sites of avian encephalomyelitis virus in the internal organs of orally infected chicks were determined by the immunofluorescent method. The invasion began when the epithelium tunica mucosae of the duodenum (together with the proventriculus, jejunum, or cecum in certain birds killed at postinoculation day 1) became test-positive. Viremia persisted for more than 5 days in the early stage of infection, then the pancreas was rapidly infected, followed by the liver, kidney, and spleen. Subsequently, the virus spread to the CNS. Rapid infection of the duodenum and pancreas was clearly observable.

An in vitro assay for quantifying the virus of avian encephalomyelitis.

Chicken embryo brain (CEB) cell cultures support the replication of embryo-adapted strains, vaccine strains, and field isolates of avian encephalomyelitis virus. A centrifugal force of 1,500 X g was applied during virus adsorption. Viral antigen was detected in the infected cells by using the indirect fluorescent-antibody technique (IFAT). Combining the infectivity of the virus in CEB cell culture with the ability to detect viral antigen by the IFAT resulted in the development of a virus-titration method. This in vitro assay proved to be more sensitive than the standard embryo-inoculation assay. It was concluded that the in vitro assay provides a satisfactory alternative to the embryo-inoculation assay.

Horizontal transmission of a pancreas-passaged avian encephalomyelitis virus in chicks.

Pancreas-passaged avian encephalomyelitis (AE) virus was transmitted horizontally in a group of 40 (1-day-old) chicks within 3 weeks after they were intermingled with two orally infected 1-day-old chicks. Viral antigen was detected in the pancreas of these contact-exposed chicks. After 5 weeks, contact-exposed chicks developed high titers against AE virus, but the chicks did not develop clinical signs of AE. The passaged virus could not be recovered from feces of six immunized chicks.

Pancreas-passaged avian encephalomyelitis virus and its immunogenicity.

A serial 34-chicken pancreas passage of avian encephalomyelitis virus by oral administration was successful. Oral inoculation test with 4 passaged viruses showed rapid infection of the duodenal wall and unchangeable infection of pancreas diminishing the viral invasiveness to other organs. The passaged virus caused neither detectable viremia nor clinical avian encephalomyelitis signs and produced neutralizing antibody of high titers.

Emergence pattern of egg-adapted avian encephalomyelitis virus by alternating passage in chickens and embryos.

The chance of inducing complete egg-adaptation was slight in alternating passage in chickens and embryos with chicken-brain-passaged viruses of wild avian encephalomyelitis viruses: egg-adaptation was accomplished by use of an incipient egg-adapting mutant, but failed with other passaged virus.

Susceptibility of chickens to avian encephalomyelitis virus. II. Behavior of the virus in day-old chicks.

The VR strain of avian encephalomyelitis virus, which had been adapted to embryonated hen's eggs, was inoculated into 2-day-old chicks by the subcutaneous route (10(2.5) approximately 10(3.0) EID50) or by the oral route (10(4.8) EID50). The chicks were examined chronologically for the distribution of the virus in the body. As a result, minute amounts of the virus were detected from the liver, spleen, pancreas, and muscle at the site of inoculation one day after inoculation and various amounts from almost all the organs 3 days and more after inoculation. The virus titer could nearly reach a maximum 7 to 9 days after inoculation. Above all, such high virus titers as ranging from 10(4.3) to 10(5.8) EID50/0.1 g were demonstrated in the brain, heart, liver, spleen, and pancreas. After that, there was a tendency for virus titer to decrease in most organs and for virus to multiply persistently in the pancreas, brain, and eyeball. Virus titer was maintained at a level of 10(2.3) approximately 10(2.8) EID50/0.1 g in these three organs even 21 days after inoculation. In the group of subcutaneous inoculation, all the chicks manifested clinical signs of infection 5 to 10 days after inoculation. On the other hand, no chicks were involved in clinical infection in the group of oral inoculation. Multiplication of the virus was delayed in the body of these chicks. Small amounts of the virus were detected from the spleen and pancreas 11 days after inoculation. Low titers (10(2.7) EID50/0.1 g at the highest) of the virus were only detected from the brain, spinal cord, spleen, pancreas, esophagus, and other organs 14 and 21 days after inoculation.

Avian encephalomyelitis virus in chicken pancreatic cell cultures.

Monolayer cell cultures consisting of epithelioid cells were made from pancreatic tissue of 10-to-13-day-old chicks. The maximum virus titer of the cell-culture fluid was obtained 8 days after inoculation with an embryo-adapted avian encephalomyelitis virus (AEV). Virus titers also increased in cell cultures inoculated with a chick-pancreas-passed AEV or a field isolant. Cell cultures inoculated with 3 strains of AEV maintained virus titers of 10(2.9)-10(3.7) 50% embryo-infective doses/ml for 15-20 days. In other cell cultures from pancreatic tissues of chicks preinfected orally with the chick-pancreas-passed AEV or the field isolant, the virus titers decreased for several days after cultivation and thereafter increased and persisted until at least the 25th or 30th day. Neither a cytopathic effect nor any inclusion body was observed in the cell cultures infected with AEV. No AEV-antigen-positive cell was detected by direct fluorescent-antibody technique.

Comparative pathogenicity of avian encephalomyelitis viruses in chicken embryos.

Multiplications of wild, various embryo-adapting and completely embryo-adapted avian encephalomyelitis (AE) viruses in chicken embryos were compared by the fluorescent-antibody technique (FAT). With a wild AE virus, viral antigens were randomly seen in the central nervous system (CNS), appearing least often in the cerebellum. Other organs seldom became test positive, except for heart and kidney. Even with 4 chicken brain-passaged viruses in the process of embryo adaptation, there was little augmentation of antigens except in the alimentary tract. However, the 2 midpassage viruses showed a peculiar localization of antigens in the white matter of the lumbosacral cord, together with the appearance of test-positive spinal ganglion cells. With 2 strains of embryo-adapted AE virus, the antigens appeared first in the spinal ganglion cells and secondly in the lumbosacral cord and then spread to the cerebrum. Subsequently, clinical signs of AE were evident. This peculiar invasion order was a prominent feature.