Severe acute respiratory syndrome (SARS) caused by a newly identified coronavirus (SARS-CoV) is a serious emerging human infectious disease. In this report, we immunized ferrets (Mustela putorius furo) with recombinant modified vaccinia virus Ankara (rMVA) expressing the SARS-CoV spike (S) protein. Immunized ferrets developed a more rapid and vigorous neutralizing antibody response than control animals after challenge with SARS-CoV; however, they also exhibited strong inflammatory responses in liver tissue. Inflammation in control animals exposed to SARS-CoV was relatively mild. Thus, our data suggest that vaccination with rMVA expressing SARS-CoV S protein is associated with enhanced hepatitis.

Abstract. This document is the consensus of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) Subcommittee on Standardization of Immunohistochemistry on a set of guidelines for immunohistochemistry (IHC) testing in veterinary laboratories. Immunohistochemistry is a powerful ancillary methodology frequently used in many veterinary laboratories for both diagnostic and research purposes. However, neither standardization nor validation of IHC tests has been completely achieved in veterinary medicine. This document addresses both issues. Topics covered include antibody selection, fixation, antigen retrieval, antibody incubation, antibody dilutions, tissue and reagent controls, buffers, and detection systems. The validation of an IHC test is addressed for both infectious diseases and neoplastic processes. In addition, storage and handling of IHC reagents, interpretation, quality control and assurance, and troubleshooting are also discussed. Proper standardization and validation of IHC will improve the quality of diagnostics in veterinary laboratories.

Severe acute respiratory syndrome (SARS) caused by a newly identified coronavirus (SARS-CoV) remains a threat to cause epidemics as evidenced by recent sporadic cases in China. In this communication, we evaluated the efficacy and safety of two SARS vaccine candidates based on the recombinant modified vaccinia Ankara (MVA) expressing SARS-CoV spike or nucleocapsid proteins in ferrets. No clinical signs were observed in all the ferrets challenged with SARS-CoV. On the other hand, vaccination did not prevent SARS-CoV infection in ferrets. In contrast, immunized ferrets (particularly those immunized with rMVA-spike) exhibited significantly stronger inflammatory responses and focal necrosis in liver tissue after SARS-CoV challenge than control animals. Thus, our data suggest that enhanced hepatitis is linked to vaccination with rMVA expressing SARS-CoV antigens.

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In February 2004 a highly pathogenic avian influenza (HPAI) outbreak erupted in British Columbia. Investigations indicated that the responsible HPAI H7N3 virus emerged suddenly from a low pathogenic precursor. Analysis of the haemagglutinin (HA) genes of the low and high pathogenic viruses isolated from the index farm revealed the only difference to be a 21 nt insert at the HA cleavage site of the highly pathogenic avian influenza virus. It was deduced that this insert most probably arose as a result of non-homologous recombination between the HA and matrix genes of the same virus. Over the course of the outbreak, a total of 37 isolates with, and 3 isolates without inserts were characterized. The events described here appear very similar to those which occurred in Chile in 2002 where the virulence shift of another H7N3 virus was attributed to non-homologous recombination between the HA and nucleoprotein genes.

The wild mouse ecotropic retrovirus (WM-E) induces a spongiform neurodegenerative disease in mice after a variable incubation period of 2 months to as long as 1 year. We isolated a molecular clone of WM-E (15-1) which was weakly neurovirulent (incidence, 8%) but was highly leukemogenic (incidence, 45%). Both lymphoid and granulocytic leukemias were observed, and these leukemias were often neuroinvasive. A chimeric virus was constructed containing the env and 3' pol sequences of 15-1 and long terminal repeat (LTR), gag, and 5' pol sequences from a clone of Friend murine leukemia virus (FB29). FB29 has been shown previously to replicate to high levels in the central nervous system (CNS) but is not itself neurovirulent. This fnding was confirmed at the DNA level in the current study. Surprisingly, intraperitoneal inoculation of neonatal IRW mice with the chimeric virus (FrCasE) caused an accelerated neurodegenerative disease with an incubation period of only 16 days and was uniformly fatal by 23 days postinoculation. Introduction of the LTR of 15-1 into the FrCasE genome yielded a virus (FrCasEL) with a degree of neurovirulence intermediate between those of 15-1 and FrCasE. No differences were found in the levels of viremia or the relative levels of viral DNA in the spleens of mice inoculated with 15-1, FrCasE, or FrCasEL. However, the levels of viral DNA in the CNS correlated with the relative degrees of neurovirulence of the respective viruses (FrCasE > FrCasEL > 15-1). Thus, the env and 3' pol sequences of WM-E (15-1) were required for neurovirulence, but elements within the LTR and gag-pol regions of FB29 had a profound influence on the level of CNS infection and the rate of development of neurodegeneration.

Abstract. Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy of cattle, first detected in 1986 in the United Kingdom and subsequently in other countries. It is the most likely cause of variant Creutzfeldt-Jakob disease (vCJD) Sc from case 1 showed molecular features similar to typical BSE isolates, whereas PrP Sc from case 2 revealed an unusual molecular PrP Sc pattern: molecular mass of the unglycosylated and monoglycosylated isoform was higher than that of typical BSE isolates and case 2 was strongly labeled with antibody P4, which is consistent with a higher molecular mass. Sequencing of the prion protein gene of both BSE-positive animals revealed that the sequences of both animals were within the range of the prion protein gene sequence diversity previously reported for cattle.

This study examines tissues from sequential-kill, time-course pathogenesis studies to refine estimates of the age at which disease-specific PrP (PrP Sc ) can first be detected in the central nervous system (CNS) and related peripheral nervous system ganglia of cattle incubating bovine spongiform encephalopathy (BSE). Such estimates are important for risk assessments of the age at which these tissues should be removed from cattle at slaughter to prevent human and animal exposure to BSE infection. Tissues were examined from cattle dosed orally with 100 or 1 g BSE-infected brain. Incubation period data for the doses were obtained from attack rate and the sequential-kill studies. A statistical model, fitted by maximum likelihood, accounted for the differences in the lognormal incubation period and the logistic probability of infection between different dose groups. Initial detection of PrP Sc during incubation was invariably in the brainstem and the earliest was at 30 and 44 months post-exposure for the 100 g-and 1 g-dosed sequential-kill study groups, respectively. The point at which PrP Sc in 50 % of the animals would be detected by immunohistochemistry applied to medulla-obex was estimated at 9.6 and 1.7 months before clinical onset for the 100 g-and 1 g-dosed cattle, respectively, with a low probability of detection in any of the tissues examined at more than 12 months before clinical onset. PrP Sc was detected inconsistently in dorsal root ganglia, concurrent with or after detection in CNS, and not at all in certain sympathetic nervous system ganglia.