Roger K. Maes

Equine Multinodular Pulmonary Fibrosis: A Newly Recognized Herpesvirus-Associated Fibrotic Lung Disease:

Pulmonary fibrosis and interstitial lung disease are poorly understood in horses; the causes of such conditions are rarely identified. Equine herpesvirus 5 (EHV-5) is a γ-herpesvirus of horses that has not been associated with disease in horses. Pathologic and virologic findings from 24 horses with progressive nodular fibrotic lung disease associated with EHV-5 infection are described and compared with 23 age-matched control animals. Gross lesions consisted of multiple nodules of fibrosis throughout the lungs. Histologically, there was marked interstitial fibrosis, often with preservation of an “alveolar-like” architecture, lined by cuboidal epithelial cells. The airways contained primarily neutrophils and macrophages. Rare macrophages contained large eosinophilic intranuclear viral inclusion bodies; similar inclusion bodies were also found cytologically. The inclusions were identified as herpesviral-like particles by transmission electron microscopy in a single horse. In situ hybridization was used to detect EHV-5 nucleic acids within occasional macrophage nuclei. With polymerase chain reaction (PCR), the herpesviral DNA polymerase gene was detected in 19/24 (79.2%) of affected horses and 2/23 (8.7%) of the control horses. Virus genera–specific PCR was used to detect EHV-5 in all of the affected horses and none of the control horses. EHV-2 was detected in 8/24 (33.3%) of affected horses and 1/9 (11.1%) of the control horses. This disease has not been reported before, and the authors propose that based upon the characteristic gross and histologic findings, the disease be known as equine multinodular pulmonary fibrosis. Further, we propose that this newly described disease develops in association with infection by the equine γ-herpesvirus, EHV-5.

Diagnostic Algorithm to Differentiate Lymphoma From Inflammation in Feline Small Intestinal Biopsy Samples

Differentiating between inflammatory bowel disease (IBD) and small intestinal lymphoma in cats is often difficult, especially when only endoscopic biopsy specimens are available for evaluation. However, a correct diagnosis is imperative for proper treatment and prognosis. A retrospective study was performed using surgical and endoscopic intestinal biopsy specimens from 63 cats with a history of chronic diarrhea or vomiting or weight loss. A diagnosis of lymphoma or inflammation was based on microscopic examination of hematoxylin and eosin (HE)–stained sections alone, HE-stained sections plus results of immunohistochemical labeling (IHC) for CD3e and CD79a, and HE staining, immunophenotyping, and polymerase chain reaction (PCR) results for B and/or T cell clonality. In addition, various histomorphologic parameters were evaluated for significant differences between lymphoma and IBD using Fisher’s exact test. The sensitivity and specificity of each parameter in the diagnosis of lymphoma were also determined. Results of Bayesian statistical analysis demonstrated that combining histologic evaluation of small intestinal biopsy specimens with immunophenotyping and analysis of clonality of lymphoid infiltrates results in more accurate differentiation of neoplastic versus inflammatory lymphocytes. Important histologic features that differentiated intestinal lymphoma from IBD included lymphoid infiltration of the intestinal wall beyond the mucosa, epitheliotropism (especially intraepithelial nests and plaques), heterogeneity, and nuclear size of lymphocytes. Based on the results of this study, a stepwise diagnostic algorithm that first uses histologic assessment, followed by immunophenotyping and then PCR to determine clonality of the lymphocytes, was developed to more accurately differentiate between intestinal lymphoma and IBD.

Clinico-pathologic Features of Fatal Disease Attributed to New Variants of Endotheliotropic Herpesviruses in Two Asian Elephants (Elephas maximus)

The first herpesviruses described in association with serious elephant disease were referred to as endotheliotropic herpesviruses (EEHV) because of their ability to infect capillary endothelial cells and cause potentially fatal disease. Two related viruses, EEHV1 and EEHV2, have been described based on genetic composition. This report describes the similarities and differences in clinicopathologic features of 2 cases of fatal endotheliotropic herpesvirus infections in Asian elephants caused by a previously unrecognized virus within the betaherpesvirus subfamily. EEHV3 is markedly divergent from the 2 previously studied fatal probosciviruses, based on polymerase chain reaction sequence analysis of 2 segments of the viral genome. In addition to ascites, widespread visceral edema, petechiae, and capillary damage previously reported, important findings with EEHV3 infection were the presence of grossly visible renal medullary hemorrhage, a tropism for larger veins and arteries in various tissues, relatively high density of renal herpetic inclusions, and involvement of the retinal vessels. These findings indicate a less selective organ tropism, and this may confer a higher degree of virulence for EEHV3.

Molecular characterization of a novel coronavirus associated with epizootic catarrhal enteritis (ECE) in ferrets

Abstract A novel coronavirus, designated as ferret enteric coronavirus (FECV), was identified in feces of domestic ferrets clinically diagnosed with epizootic catarrhal enteritis (ECE). Initially, partial sequences of the polymerase, spike, membrane protein, and nucleocapsid genes were generated using coronavirus consensus PCR assays. Subsequently, the complete sequences of the nucleocapsid gene and the last two open reading frames at the 3′ terminus of the FECV genome were obtained. Phylogenetic analyses based on predicted partial amino acid sequences of the polymerase, spike, and membrane proteins, and full sequence of the nucleocapsid protein showed that FECV is genetically most closely related to group 1 coronaviruses. FECV is more similar to feline coronavirus, porcine transmissible gastroenteritis virus, and canine coronavirus than to porcine epidemic diarrhea virus and human coronavirus 229E. Molecular data presented in this study provide the first genetic evidence for a new coronavirus associated with clinical cases of ECE.

Clinicopathologic Features of a Systemic Coronavirus-Associated Disease Resembling Feline Infectious Peritonitis in the Domestic Ferret (Mustela putorius)

From 2002 to 2007, 23 ferrets from Europe and the United States were diagnosed with systemic pyogranulomatous inflammation resembling feline infectious peritonitis (FIP). The average age at the time of diagnosis was 11 months. The disease was progressive in all cases, and average duration of clinical illness was 67 days. Common clinical findings were anorexia, weight loss, diarrhea, and large, palpable intra-abdominal masses; less frequent findings included hind limb paresis, central nervous system signs, vomiting, and dyspnea. Frequent hematologic findings were mild anemia, thrombocytopenia, and hypergammaglobulinemia. Grossly, whitish nodules were found in numerous tissues, most frequently the mesenteric adipose tissue and lymph nodes, visceral peritoneum, liver, kidneys, spleen, and lungs. One ferret had a serous abdominal effusion. Microscopically, pyogranulomatous inflammation involved especially the visceral peritoneum, mesenteric adipose tissue, liver, lungs, kidneys, lymph nodes, spleen, pancreas, adrenal glands, and/or blood vessels. Immunohistochemically, all cases were positive for coronavirus antigen using monoclonal antibody FIPV3-70. Electron microscopic examination of inflammatory lesions identified particles with coronavirus morphology in the cytoplasm of macrophages. Partial sequencing of the coronavirus spike gene obtained from frozen tissue indicates that the virus is related to ferret enteric coronavirus.

Molecular characterization of noroviruses detected in diarrheic stools of Michigan and Wisconsin dairy calves: circulation of two distinct subgroups

Abstract Noroviruses have emerged as the leading worldwide cause of acute non-bacterial gastroenteritis in humans. The presence of noroviruses in diarrheic stool samples from calves on Michigan and Wisconsin dairy farms was investigated by RT-PCR. Norovirus-positive samples were found on all eight farms studied in Michigan and on 2 out of 14 farms in Wisconsin. Phylogenetic analyses of partial polymerase and capsid sequences, derived for a subset of these bovine noroviruses, showed that these strains formed a group which is genetically distinct from the human noroviruses, but more closely related to genogroup I than to genogroup II human noroviruses. Examination of 2 full and 10 additional partial capsid (ORF2) sequences of these bovine strains revealed the presence of two genetic subgroups or clusters of bovine noroviruses circulating on Michigan and Wisconsin farms. One subgroup is “Jena-like”, the other “Newbury agent-2-like”.

New hosts for equine herpesvirus 9.

Equine herpesvirus 9 was detected in a polar bear with progressive encephalitis; the source was traced to 2 members of a potential equid reservoir species, Grevy’s zebras. The virus was also found in an aborted Persian onager. Thus, the natural host range is extended to 6 species in 3 mammalian orders.

Novel calicivirus identified in rabbits, Michigan, USA.

This virus is distinct from rabbit hemorrhagic disease virus.

West Nile Virus Infection in Eastern Fox Squirrels (Sciurus niger)

Since the initial outbreak of West Nile virus (WNV) in the northeastern United States in 1999, the virus has rapidly spread westward and southward across the USA, causing high mortality in crows as well as sporadic mortality in horses, humans, and a wide variety of birds. In 2002 the epidemic widened as hundreds of equine and human cases and sporadic cases in other mammalian species were reported. This is the first report of WNV infection in three Eastern fox squirrels (Sciurus niger). Neurologic signs included head tilt, uncoordinated movement, paralysis, and tremors. Gross lesions were absent. Microscopic lesions consisted of lymphoplasmacytic inflammation involving the brain, heart, kidney, and liver. Formalin-fixed tissues from the three squirrels were tested for WNV antigen by immunohistochemical staining and for WNV-specific RNA by reverse transcriptase-polymerase chain reaction (RT-PCR). The kidneys of all three squirrels stained positive with immunohistochemistry for WNV, whereas the brain and heart were positive in only one animal. Two of the three squirrels were positive for WNV by RT-PCR.

Identification of the feline herpesvirus type 1 (FHV-1) genes encoding glycoproteins G,D,I and E: Expression of FHV-1 glycoprotein D in vaccinia and raccoon poxviruses.

The genome of feline herpesvirus type 1 (FHV-1), the major cause of viral upper respiratory disease in cats, contains several genes encoding homologues of herpes simplex virus type 1 (HSV-1) glycoproteins. Restriction mapping studies have indicated that the group D genome of FHV-1 contains a unique short region that is 9.0 kb long. The nucleotide sequence of a 6.2 kb portion of this region was determined. Analyses of this sequence have identified five open reading frames capable of encoding homologues to HSV-1 protein kinase and glycoproteins gG, gD, gI and gE. Since gD of FHV-1 is most likely an immunologically important polypeptide, vaccinia and raccoon poxvirus recombinants expressing this glycoprotein were generated. In an indirect fluorescent antibody test these recombinants reacted strongly with a rabbit anti-FHV-1 serum. High titres of virus-neutralizing antibodies were also generated in rabbits inoculated with the vaccinia virus recombinant. A 53K viral polypeptide (gD) was detected with this antiserum on Western blots containing polypeptides from potassium tartrate-purified virions.