R. A. Williams

Demonstration of sites of latency of infectious laryngotracheitis virus using the polymerase chain reaction.

Mature laying chickens were inoculated intratracheally with a field strain of infectious laryngotracheitis (ILT) virus. Tracheal swabs were collected regularly from all birds for virus culture. At various times post-inoculation, pairs of birds were killed and tissues removed for detection of virus products using conventional tissue homogenization and culture, organ culture, indirect immunofluorescence (IF) and also the polymerase chain reaction (PCR). The latter was used to detect a DNA sequence from the ILT virus thymidine kinase gene. Following inoculation the birds developed mild respiratory disease with clinical signs characteristic of ILT from 3 to 10 days post-inoculation. Trachea and turbinate tissues were virus-positive as determined by virus isolation, organ culture, IF and PCR on day 4 post-inoculation. After recovery from the acute phase, virus shedding initially ceased, then intermittent, low level shedding was recorded for five of the six remaining birds. In an attempt to locate sites of latency, pairs of birds were sampled at 31, 46 and 61 days post-inoculation. Virus was not detected in upper respiratory tract or ocular tissues by conventional techniques, or in the trigeminal, proximal and distal ganglia. All tissues were also negative by PCR, except for the trigeminal ganglia of five of the six birds. All PCR-positive birds had previously shed ILT virus intermittently between days 19 and 59 post-inoculation. As we did not detect viral DNA in any of the other tissues sampled from clinically recovered birds, we conclude that the trigeminal ganglion is the main site of latency of ILT virus.

Latency and reactivation of infectious laryngotracheitis vaccine virus.

SummaryLatency and reactivation of a commercial infectious laryngotracheitis virus vaccine were demonstrated in live chickens. Virus was re-isolated at intervals between seven and fourteen weeks post-vaccination and this may be of epizootiological significance.

Quasispecies evolution of a hypervariable region of the feline calicivirus capsid gene in cell culture and in persistently infected cats.

Feline calicivirus (FCV) is a respiratory pathogen of cats that is capable of causing persistent infections. This study examined the evolution of a hypervariable region of the FCV capsid gene both during 90 passages in cell culture and during replication in persistently infected cats. This region of the capsid protein is known to contain neutralization epitopes and may be a target for immune evasion during virus persistence in the host. Sequence analysis showed that FCV exists as a quasispecies which evolved both in cell culture and in persistently infected cats. Changes involved both loss of sequence present in the infecting isolate and a gain of both synonymous and non-synonymous nucleotide substitutions to generate sequences not detected within earlier isolates. Overall, these changes led to a reduction in population heterogeneity over time. Where virus populations were highly homogeneous allowing a consensus sequence to be determined, evolution rates for the consensus sequence ranged from 0.10-1.07 substitutions per nucleotide per year. Marked changes in virus neutralization profiles were seen in isolates obtained sequentially from a persistently infected cat. This was not the case with cell culture passaged virus, suggesting that the individual amino acid changes found only in virus from persistently infected cats may significantly alter the antigenic profile of FCV, and may be the result of immune selection.

Experimental infection of laying turkeys with Rhinotracheitis virus: Distribution of virus in the tissues and serological response

Twenty-four laying turkey hens shown to be free of antibodies to turkey rhinotracheitis virus were inoculated intranasally with an isolate of the virus. A mild respiratory disease developed between 5 and 9 days post infection (pi). Two birds were selected at random at intervals between days 1 and 20 pi, killed and tissues examined for the presence of virus. At autopsy between days 2 and 12 abnormalities were found in the oviducts including the deposition of inspissated albumen. Yolk material was occasionally found in the abdominal cavity and there was one instance of egg peritonitis. Eggs with abnormal shells were found in the uterus on days 3 and 9. By direct immunofluorescence (IF) staining, virus was detected in the trachea between days 1 and 7 pi and in the turbinates between days 2 and 5 pi. Virus could also be isolated from these sites using turkey embryo tracheal organ cultures but this method was slightly less sensitive than IF for these tissues. No virus was demonstrated in the lungs or air sacs. Viral antigens were detected by IF in the epithelium of the uterus on day 7 pi and in this and all other regions of the oviduct on day 9 pi. Virus was isolated only from middle magnum and vagina on day 9 pi. On other occasions up to 20 days pi the above tissues and spleen, ovary, liver, kidney and hypothalamus were all negative for virus. Antibodies detected by ELISA and serum neutralisation both reached, high titre by 12 days pi and were maintained at a high level (Iog2 12-15) throughout the period of observation (89 days).

Development of a live attenuated vaccine against turkey rhinotracheitis.

Three preparations of a strain of turkey rhinotracheitis (TRT) virus were tested for their ability to protect turkey poults against challenge with virulent virus given 3 weeks later. The preparations were as follows: one had been passaged in turkey embryo tracheal organ culture (TOC) 98 times, another had been passaged in primary chick embryo fibroblast (CEF) monolayers 28 times and the third had undergone 17 passages in Vero cell monolayers. Each was administered by the eyedrop route to groups of 21-day-old TRT-seronegative turkey poults. The TOC preparation caused clinical signs consistent with TRT infection, indicating the virus had not been attenuated. The CEF and Vero preparations produced no clinical effects. Following challenge with virulent TRT virus at 21 days post-inoculation, the CEF group developed clinical signs consistent with TRT but the TOC and Vero virus groups showed none. All other parameters correlated with these findings. All groups showed an increase in specific SN and ELISA antibodies following challenge. These results indicated that after relatively few passages in Vero cells, this strain of TRT virus became satisfactorily attenuated and was able to offer protection against clinical disease following experimental challenge. Two of the three virus preparations (TOC and Vero) were also shown to spread from the inoculated birds to uninoculated contact birds, introduced into the groups at 5 days post-inoculation, and they induced protection in these contacts against virulent virus challenge.

Further studies on the development of a live attenuated vaccine against turkey rhinotracheitis.

Turkey rhinotracheitis (TRT) virus attenuated by passaging in Vero cells was tested at two different passage levels (15 or 25 passages) and two dose levels [10(3) or 10(4) TCID50 (50% tissue culture infectious doses) per bird] to determine the efficacy in protecting turkey poults against experimental challenge with virulent TRT virus. Following administration by the eyedrop route at 10 days of age, all four preparations proved successful in providing protection against clinical disease and establishment of challenge virus in the trachea when challenged with virulent virus 3 weeks later. Twelve-day-old poults given the 25th Vero passage TRT virus at a dose of 10(3.5) TCID50 per bird were protected against experimental challenge with virulent virus for at least 22 weeks post-primary inoculation. The 25th passage virus was tested for safety by administering ten times the dose (10(4.5) TCID50 per bird) used in the previous trial to a group of 10-day-old turkey poults. None of the birds showed any clinical signs during 21 days post-inoculation. Attempts to back-passage the virus from bird to bird were unsuccessful.

A comparison of direct electron microscopy, virus isolation and a DNA amplification method for the detection of avian infectious laryngotracheitis virus in field material

Seventy-two post-mortem samples of mainly tracheal tissue from commercial chickens from 25 commercial chicken flocks with suspected infectious laryngotracheitis (ILT) were examined for the presence of the virus using direct electron microscopy (EM), virus isolation (VI) in primary chick embryo liver cell culture and a DNA amplification method (polymerase chain reaction; PCR). ILT virus was identified in 22 outbreaks, and in 58 of the 72 specimens. PCR detected virus in 52 of the 72 specimens and VI was positive in 48. In five instances, VI was positive where the other methods were negative and in three, PCR was the only test positive. Direct EM examination detected virus in only 19 of the 58 positive samples and in no case was EM the only method positive. An advantage of PCR was that it could sometimes detect virus in samples that were too heavily contaminated with bacteria for virus to be isolated and on other occasions it was positive for ILT virus when the only virus that could be detected by growth in tissue culture was adenovirus.

Quasispecies evolution of a hypervariable region of the feline calicivirus capsid gene in cell culture and persistently infected cats

The study determines the sequence evolution of feline calicivirus both in cell culture and in persistently infected cats and relates this to changes in virus neutralisation.

Isolation of infectious laryngotracheitis virus from proximal femora of lame broiler chickens

Two herpesviruses previously isolated from seven of 19 affected joint/bone samples in an earlier survey of lameness in broilers were identified. They were characterised as infectious laryngotracheitis (ILT) virus using serum neutralisation, immunofluorescence, restriction enzyme analysis and polymerase chain reaction techniques. In experimentally infected chicks, one of the isolates caused mild ILT and intranuclear inclusion bodies were present in the tracheal epithelium after four days. It is considered unlikely that these viruses were involved in the pathological changes in the affected legs. The possibility that ILT pathogenesis and epidemiology are more complex than currently understood is discussed.

Pathogenicity of latent infectious laryngotracheitis virus in chickens

Groups of specific pathogen-free chickens were inoculated with the same dose of a field strain of infectious laryngotracheitis virus that had either been isolated from tracheal swabs taken from infected birds during acute phase shedding, or that had been isolated during an episode of virus shedding after a latent period of 12 to 17 weeks. Birds in both groups developed characteristic clinical signs of ILT including difficulty in breathing, rales and sneezing. Thus, ILT virus shed after a latent period is capable of causing disease in susceptible birds similar to that seen in the field.