Gerhard H. Reubel

Detection of active and latent feline herpesvirus 1 infections using the polymerase chain reaction

SummaryA polymerase chain reaction (PCR) assay was developed to detect the thymidine kinase gene of feline herpesvirus 1 (FHV-1) and to study the active and latent carrier state in a group of naturally FHV-1 infected specific pathogen free (SPF) cats. The detection limit of PCR products on ethidium bromide stained gels was 390 fg or about 3×103 copies of the FHV-1 genome. The PCR was 25% more sensitive than conventional cell culture based virus isolation techniques in detecting FHV-1 in oral/ocular swabs and 100 times more sensitive in detecting virus in cell culture supernatants. Sites of FHV-1 latency in FHV-1 carriers as determined by PCR were mainly tissues of the head, especially the trigeminal ganglia, optic nerves, olfactory bulbs and corneas. Oral fauces, salivary glands, lacrimal glands, cerebellum and conjunctiva were less consistently positive. The cerebral cortex, thymus, trachea, lung, liver, spleen, kidney, and peripheral blood mononuclear cells were consistently negative for FHV-1 genome. The distribution of FHV-1 DNA in the tissues of the head was similar whether or not corticosteroid-induced virus shedding was occurring at the time the tissues were collected. Infectious virus was never recovered from tissue homogenates regardless of the PCR status of the tissues.

Acute and Chronic Faucitis of Domestic Cats: A Feline Calicivirus–Induced Disease

The lesions of acute feline calicivirus infection are of a transient vesiculo-ulcerative nature and involve, to varying degrees, the palate, tongue, gingiva, lips, nasal philtrum, and oral fauces. Chronic ulceroproliferative faucitis is a specific but uncommon sequelae to persistent feline calicivirus oral carriage.

An experimental study of primary feline immunodeficiency virus infection in cats and a historical comparison to acute simian and human immunodeficiency virus diseases

Sixteen adolescent specific pathogen free cats were inoculated with the Petaluma strain of feline immunodeficiency virus (FIV) and two cats were then necropsied at each of 5, 10, 21, 28, 42, 56, 70, and 84 day time points following infection. Lymphadenopathy gradually increased starting at Day 10 and persisted for the duration. Gross clinical signs of fever, mild to severe malaise, anorexia, diarrhea, dehydration, and generalized soreness appeared around Day 42, peaked at Day 56, and disappeared by Days 70-84 post-infection. Leukopenia, associated initially with a mild lymphopenia and later by both a mild lymphopenia and a severe neutropenia, appeared 14-28 days following infection, troughed at Day 56, and persisted thereafter. The CD4+:CD8+ T cell ratio started to decrease around Day 28, reaching a nadir at Days 56-70. This decrease was due to a decline in the absolute numbers and percentage of CD4+ T cells and an increase in the percentage of CD8+ T cells. Significant histopathologic lesions included myeloid hyperplasia between Days 56-70 post-infection; thymitis with cortical involution and follicular hyperplasia starting at Day 42; lymphoid hyperplasia of peripheral and mesenteric nodes, spleen and tonsils beginning around Day 42; typhlitis most evident from Day 56 onward, and an interstitial nephritis and pneumonitis that was most intense after Day 42. Virus was isolated from peripheral blood mononuclear cells (PBMC) beginning 2 weeks post-infection, and plasma viremia appeared 1 week later. Plasma and PBMC-associated viremia peaked at 42-56 days following infection and decreased abruptly thereafter. Proviral DNA was detectable as early as 5 days after infection in blood leukocytes and after 10 days in other organs. The central nervous system, lungs, thymus, tonsils and mesenteric lymph nodes were the earliest sites of virus localization. Antibodies to the FIV capsid protein appeared 14 days following infection and reached peak levels by Days 42-56. Abnormalities occurring during the primary stage of FIV infection were consistent with those described for acute simian and human immunodeficiency virus-induced disease.

Effect of chronic feline immunodeficiency virus infection on experimental feline calicivirus-induced disease

Acute feline calicivirus (FCV) infection caused a more severe disease in chronically feline immunodeficiency virus (FIV) infected than in non-FIV infected cats. FIV infected cats shed significantly higher amounts of FCV through their saliva after FCV challenge than the non-FIV infected cats. However, there was no difference in the duration of FCV shedding. None of the cats exposed to FCV developed chronic FCV carriage. Both groups of cats mounted similar titers of neutralizing antibodies to FCV. Although FIV infected cats started out with significantly lower total lymphocyte and neutrophil numbers than the non-FIV infected cats, the transient lymphopenia and neutrophilia attributable to the FCV infection was of similar intensity in both groups of animals. There was no evidence that the underlying FIV-related disease or viremia was influenced by acute FCV infection. Acute FCV infection did not significantly alter the CD4+/CD8+ T lymphocyte ratio in FIV infected compared to non-FIV infected cats. The ongoing humoral IgG response to FIV was not affected by the FCV infection. There was no significant change in the proportion of FIV infected peripheral blood mononuclear cells during 8 subsequent weeks after FCV challenge as determined by polymerase chain reaction.

An epizootic of feline herpesvirus, type 1 in a large specific pathogen-free cat colony and attempts to eradicate the infection by identification and culling of carriers

This study describes the clinical course of an inadvertent feline herpesvirus, type 1 (FHV-1) outbreak in 2 specific pathogen-free (SPF) research and breeding colonies housing 690 cats and assesses a programme that was designed to eradicate the virus from the colonies. The clinical signs observed in these cats were milder, with more eye involvement than those previously described for FHV-1 infection and did not include abortion. FHV-1 eradication was based on the detection and elimination of both active and latent viral carriers. Carrier cats were detected by virus isolation from oral swabs before and after corticosteroid-induced reactivation of FHV-1 excretion. Four per cent of recovered cats were actively shedding virus prior to corticosteroid treatment; 21 % of the virus negative cats shed virus after one corticosteroid injection, and 12% of remaining culture negative cats tested positive upon a second corticosteroid treatment 6 weeks later. The colony remained virus free for 8 months after all detectable virus carriers were culled and there was no seroconversion among new kittens. A second epizootic of FHV-1 then occurred among susceptible animals. At this time, all breeding cats that had tested negative after 2 injections of corticosteroids were treated a third time; 23% of them now tested positive for FHV-1. This study demonstrates that corticosteroid treatment can be useful in improving the rate of detection, essential as a basis for decreasing the incidence of enzootic disease, but it is unlikely to detect all possible FHV-1 carriers in large populations of cats.

Interaction of acute feline herpesvirus-1 and chronic feline immunodeficiency virus infections in experimentally infected specific pathogen free cats.

Cats with or without chronic feline immunodeficiency virus (FIV) infection were exposed to feline herpesvirus, type 1 (FHV-1). FIV infected cats became sicker than non-FIV infected cats and required more supportive treatment. However, there were no differences in the length of their illness or in the levels and duration of FHV-1 shedding. FHV-1 infection caused a transient neutrophilia at Day 7 with a rapid return to preinfection levels. The neutrophilia coincided with a transient lymphopenia that was accompanied by a decline in both CD4+ and CD8+ T-lymphocytes. A brief decrease in the CD4+/CD8+ T-lymphocyte ratio occurred at Day 14 in both FIV infected and non-infected cats. This decrease was mainly the result of an absolute and transient increase in CD8+ T-lymphocytes. CD4+ and CD8+ T-lymphocyte numbers and CD4+/CD8+ T-lymphocyte ratios returned to baseline within 4-8 weeks in both FIV infected and non-infected cats. FIV infected cats produced less FHV-1 neutralizing antibodies during the first 3 weeks of infection than non-FIV infected animals. The IgM FHV-1 antibody response was depressed in FIV infected cats whereas the IgG antibody response was unaffected. FHV-1 infection evoked a comparable transient loss of lymphocyte blastogenic responses to concanavalin A and pokeweed mitogen in both FIV infected and non-infected cats. However, response to pokeweed mitogen took longer to return to normal in FIV infected animals. Lymphocytes from FIV infected cats had a greater and more sustained proliferative response to FHV-1 antigen than non-FIV infected cats. The ongoing IgG antibody response to FIV was not affected by FHV-1 infection.

FIV VACCINE STUDIES. I: IMMUNE RESPONSE TO RECOMBINANT FIV ENV GENE PRODUCTS AND OUTCOME AFTER CHALLENGE INFECTION

We have vaccinated five groups of cats (n = 25) four times with five preparations of recombinant feline immunodeficiency virus (FIV) env gene products; one group (n = 7) served as control. The vaccine formulations were as follows: (1) envelope glycoprotein of FIV Zurich 2 (FIV Z2) expressed in a Baculovirus system and isolated by gel electroelution (denatured form); (2) insect cells expressing FIV Z2 glycoprotein; (3) envelope glycoprotein of a Boston strain (FIV Bangston) expressed in insect cells and isolated by gel electroelution (denatured form); (4) glycosylated Bangston envelope protein made in insect cells and isolated in a native form; (5) non-glycosylated Bangston envelope protein made in Escherichia coli. All cats were challenged with 20 50% cat infective doses (CID50) of FIV Z2 previously titrated in cats. All vaccinated cats developed high enzyme-linked immunosorbent assay (ELISA) antibodies to the homologous antigen; crossreactivity to heterologous antigens was seen at a lower level. Virus neutralizing antibodies (tested with Petaluma virus) reached titers up to 32. After challenge, all cats seroconverted (as judged by anti gag antibodies in Western blot) and became infected (as judged by virus isolation and/or polymerase chain reaction) between 4 and 11 weeks with the exception of one cat. It is concluded that it is relatively easy to induce high ELISA antibody titers using recombinant env gene products, ELISA antibody titers do not correlate with virus neutralization or with protection.

Cloning, expression and characterization of biologically active feline tumour necrosis factor-α

Abstract We report the cloning, expression and characterization of biologically active feline tumour necrosis factor-α (fTNF-α). Messenger RNA was extracted from feline peritoneal macrophage cultures and used to synthesize cDNA for polymerase chain reaction (PCR) amplification. The PCR products were cloned into the plasmid vector pCRII and sequenced, showing 99.3% homology with a published fTNF-α gene sequence. Subcloning into the vector pGEX-2T and subsequent expression resulted in a 43 kDa fusion protein of fTNF-α and glutathione S-transferase (GST). Thrombin cleavage of the fusion protein yielded a 17 kDa protein. This protein cross-reacted with a monoclonal anti-human TNF-α antibody in Western blotting, but not with a polyclonal anti-murine TNF-α serum. Recombinant fTNF-α (rfTNF-α) and rfTNF-α-GST had a CD50 of 15 ng ml−1 and 230 ng ml−1, respectively, in the L929 cytotoxicity assay. Cats given rfTNF-α-GST intravenously manifested the typical biological effects of TNF-α, including fever, depression, and piloerection. The rfTNF-α-GST upregulated IL-2 receptor and MHC-II antigen expression on peripheral blood mononuclear cells stimulated in vitro, but had no effect on TNF-α receptor and MHC-I antigen expression.

The adenovirus that causes hemorrhagic disease of black-tailed deer is closely related to bovine adenovirus-3

SummaryDNA sequence data was obtained from an adenovirus previously shown to be the cause of a distinctive, fatal hemorrhagic disease of black-tailed deer in California. A 256 base fragment of the viral hexon gene was amplified by PCR from purified adenovirus preparations. The amplicon then was cloned and sequenced. Phylogenetic relationships with other mammalian adenoviruses were also determined. Although sequence analysis of this portion of the hexon gene indicates that the black-tailed deer adenovirus is closely related to bovine adenovirus-3, the biologic properties of the two viruses are clearly distinct.