Susan K. Schommer

Detection by Reverse Transcription-PCR and Genetic Characterization of Field Isolates of Swine Hepatitis E Virus from Pigs in Different Geographic Regions of the United States

ABSTRACT Hepatitis E virus (HEV) is an important public health concern in many developing countries. HEV is also endemic in some industrialized counties, including the United States. With our recent discovery of swine HEV in pigs that is genetically closely related to human HEV, hepatitis E is now considered a zoonotic disease. Human strains of HEV are genetically heterogenic. So far in the United States, only one strain of swine HEV has been identified and characterized from a pig. To determine the extent of genetic variations and the nature of swine HEV infections in U.S. pigs, we developed a universal reverse transcription-PCR (RT-PCR) assay that is capable of detecting genetically divergent strains of HEV. By using this universal RT-PCR assay, we tested fecal and serum samples of pigs of 2 to 4 months of age from 37 different U.S. swine farms for the presence of swine HEV RNA. Thirty-four of the 96 pigs (35%) and 20 of the 37 swine herds (54%) tested were positive for swine HEV RNA. The sequences of a 348-bp region within the ORF2 gene of 27 swine HEV isolates from different geographic regions were determined. Sequence analyses revealed that the 27 U.S. swine HEV isolates shared 88 to 100% nucleotide sequence identities with each other and 89 to 98% identities with the prototype U.S. strain of swine HEV. These U.S. swine HEV isolates are only distantly related to the Taiwanese strains of swine HEV, with about 74 to 78% nucleotide sequence identities; to most known human strains of HEV worldwide, with <79% sequence identities; and to avian HEV, with 54 to 56% sequence identities. Phylogenetic analysis showed that all the U.S. swine HEV isolates identified in this study clustered in the same genotype with the prototype U.S. swine HEV and the two U.S. strains of human HEV. The data from this study indicated that swine HEV is widespread and enzoonotic in U.S. swine herds and that, as is with human HEV, swine HEV isolates from different geographic regions of the world are also genetically heterogenic. These data further raise potential concerns for zoonosis, xenozoonosis, and food safety.

Porcine reproductive and respiratory syndrome virus field isolates differ in in vitro interferon phenotypes.

Abstract Type I interferons (IFN-α and -β) play an important role in the innate host defense against viral infection by inducing antiviral responses. In addition to direct antiviral activities, type I IFN serves as an important link between the innate and adaptive immune response through multiple mechanisms. Therefore, the outcome of a viral infection can be affected by IFN induction and the IFN sensitivity of a virus. North American porcine reproductive and respiratory syndrome virus (PRRSV) field isolates were studied with regard to IFN-α sensitivity and induction in order to understand the role of type I IFN in PRRSV pathogenesis. PRRSV isolates were differentially sensitive to porcine recombinant IFN-α (rIFN-α) and varied in their ability to induce IFN-α in porcine alveolar macrophages (PAM) cultures as measured by a porcine IFN-α specific ELISA on cell culture supernatants. Fifty-two plaques were purified from three PRRSV isolates (numbers 3, 7, and 12) and tested for IFN sensitivity and IFN induction. Plaque-derived populations were composed of heterogeneous populations in terms of IFN-inducing capacity and sensitivity to rIFN-α. When macrophages infected with isolates 3, 7, or 12 were treated with polycytidylic acid (polyI:C), IFN-α production was enhanced. Cells infected with isolate 3 and treated with polyI:C showed the most consistent and strongest enhancement of IFN-α production. It was demonstrated that the relatively low concentrations of IFN-α produced by isolate 3 contributed to the enhanced IFN-α synthesis in response to polyI:C. Isolates 7 and 12 significantly suppressed the enhanced IFN-α production by isolate 3 in polyI:C treated cells. To determine if suppression was at the level of IFN-α transcription, quantitative RT-PCR was performed for IFN-α mRNA and compared to GAPDH and cyclophilin mRNA quantification. However, the relative number of IFN-α transcript copies did not correlate with IFN-α protein levels, suggesting a post-transcriptional mechanism of suppression. In summary, these results demonstrate that PRRSV field isolates differ both in IFN-α sensitivity and induction. Furthermore, a PRRSV field isolate strongly enhance polyI:C-induced IFN-α production in PAM cultures and this priming effect was suppressed by other PRRSV isolates.

Association of two newly recognized herpesviruses with interstitial pneumonia in donkeys (Equus asinus)

Over a period of 6 years, antemortem and postmortem examinations were performed on a number of donkeys suffering from respiratory disease. For many cases, initial diagnostic efforts failed to identify an etiology consistent with the pathologic findings. However, retrospective examination of these cases using consensus primer polymerase chain reaction, designed to recognize herpesviruses from all 3 subfamilies of the Herpesviridae, amplified a fragment of the highly conserved herpesvirus DNA polymerase gene from a number of these animals. Two novel herpesviruses, herein designated asinine herpesvirus 4 (AHV4) and asinine herpesvirus 5 (AHV5), were consistently detected in lung tissue from donkeys in which the histopathology was characterized by interstitial pneumonia and marked syncytial cell formation but not in lung tissue from donkeys with evidence of bacterial or verminous pneumonia. Nucleotide sequence and phylogenetic analysis places these new viruses within the Gammaherpesvirinae subfamily and indicates that they are most closely related to the recently identified zebra herpesvirus and wildass herpesvirus as well as equine herpesviruses 2 and 5.

Detection and Multigenic Characterization of a Herpesvirus Associated with Malignant Catarrhal Fever in White-Tailed Deer (Odocoileus virginianus) from Missouri

ABSTRACT Between 1998 and 2001, tissues from four captive white-tailed deer were observed to have histologic lesions of systemic lymphocytic vasculitis. These lesions suggested malignant catarrhal fever, although epizootic hemorrhagic disease and bluetongue were included in the differential diagnosis. Initial diagnostic efforts, including virus isolation and reverse transcription-PCR for epizootic hemorrhagic disease virus and bluetongue virus, failed to identify an etiologic agent. However, consensus primer PCR targeted to the herpesvirus DNA polymerase gene detected viral genomic DNA in each of these four cases. Nucleotide sequence analysis of the amplified product demonstrated that the detected virus was identical over the compared region to the recently described malignant catarrhal fever virus of white-tailed deer (H. Li, N. Dyer, J. Keller, and T. B. Crawford, J. Clin. Microbiol. 38:1313-1318, 2000). Additional nucleotide sequencing of both the DNA polymerase gene and DNA packaging gene followed by phylogenetic analysis solidified this newly recognized herpesvirus as a member of the Gammaherpesvirinae and suggests that this virus, along with ovine herpesvirus 2, alcelaphine herpesvirus 1, alcelaphine herpesvirus 2 and caprine herpesvirus 2, may be part of a separate clade within this subfamily.

Detection of Leptospira Interrogans DNA and Antigen in Fixed Equine Eyes Affected with End-Stage Equine Recurrent Uveitis

Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide. Leptospira has been implicated as an etiologic agent in some cases of ERU and has been detected in fresh ocular tissues of affected horses. The objective of this study was to determine the presence of Leptospira antigen and DNA in fixed equine ocular tissues affected with end-stage ERU. Sections of eyes from 30 horses were obtained. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes diagnosed with ERU based on clinical signs and histologic lesions. Sections were subjected to immunohistochemical staining with an array of rabbit anti-Leptospira polyclonal antibodies. DNA extractions were performed by using a commercial kit designed for fixed tissue. Real-time PCR analysis was completed on extracted DNA. The target sequence for PCR was designed from alignments of available Leptospira 16S rDNA partial sequences obtained from GenBank. Two of 10 test samples were positive for Leptospira antigen by immunohistochemical assay. Zero of 20 controls were positive for Leptospira antigen. All test samples and controls were negative for Leptospira DNA by real-time PCR analysis. Leptospira was detected at a lower frequency than that previously reported for fresh ERU-affected aqueous humor and vitreous samples. Leptospira is not frequently detectable in fixed ocular tissues of horses affected with ERU when using traditional immunohistochemical and real-time PCR techniques.

Use of a PRRSV Infectious Clone to Evaluate in Vitro Quasispecies Evolution

hallmark of RNA viruses. Virus populations in vivo, referred to as quasispecies, are comprised of a heterogeneous mix of related variants that are randomly generated as a result of errors by the viral RNA-dependent RNA polymerase. This diversity is considered to be an important mechanism of virus persistence and pathogenesis in many virus systems and provides a mechanism to rapidly respond to changes in the host environment. Initially PRRSV genetic variation was studied using consensus genome sequences. There have been several papers published on PRRSV variation during experimental infection of pigs, however only one started with a biologically cloned virus. Our laboratory is currently using a North American infectious clone to investigate the mechanisms of PRRSV persistence and pathogenesis. One purpose of this study was to evaluate the stability of the infectious clone for its manipulation and subsequent use. Use of the infectious clone also allows us to begin with a single DNA sequence, providing a well-defined starting point for studying PRRSV evolution. The other goal of this study was to investigate PRRSV quasispecies evolution in an environment that excludes the immunologic pressure that is present in the previous studies which involved experimental infection in swine. Four regions of the genome were selected for analysis in this study: Nsp2, ORF3, ORF5, and ORF6. The Nsp2 protein is the most variable region among arteriviruses, 7 and has been implicated in having a role in the humoral immune response. The ORF3 protein has the greatest percentage of amino acid changes between the modified live vaccine (Ingelvac) and its parent strain, VR-2332, the isolate from which the infectious clone used in this study was derived. ORF5 has been the focus of previous quasispecies investigations and its corresponding protein has been associated with virus neutralization. The most conserved region of the PRRSV genome across all North

Hepatitis and enteritis caused by a novel herpesvirus in two monitor lizards (Varanus spp.)

Reported cases of herpesvirus-induced disease are uncommon in most species of reptiles, with the majority of reports in chelonians. Two monitor lizards (Varanus spp.) presented for postmortem examination at the Veterinary Medical Diagnostic Laboratory at the University of Missouri. Tan, 1–2-mm foci were grossly visible on the mucosal surface of the intestine and in the liver. Microscopically, there was multifocal necrosis in the lamina propria of the small intestine and in the liver. Many of the degenerate cells contained large, eosinophilic intranuclear inclusions. Enveloped icosahedral virions consistent with herpesvirus were detected by electron microscopy. A 180-bp DNA fragment was amplified by polymerase chain reaction from samples of small intestine and liver using primers that targeted a portion of the herpesvirus DNA polymerase gene. The sequence of the fragment was determined to be most closely related to Varanid herpesvirus 2 (80% nucleotide identity, 82% amino acid identity). Based on histological and molecular findings, a novel pathogenic herpesvirus of lizards in the family Varanidae is proposed.

Genetic engineering alveolar macrophages for host resistance to PRRSV

Standard strategies for control of porcine reproductive and respiratory syndrome virus (PRRSV) have not been effective, as vaccines have not reduced the prevalence of disease and many producers depopulate after an outbreak. Another method of control would be to prevent the virus from infecting the pig. The virus was thought to infect alveolar macrophages by interaction with a variety of cell surface molecules. One popular model had PRRSV first interacting with heparin sulfate followed by binding to sialoadhesin and then being internalized into an endosome. Within the endosome, PRRSV was thought to interact with CD163 to uncoat the virus so the viral genome could be released into the cytosol and infect the cell. Other candidate receptors have included vimentin, CD151 and CD209. By using genetic engineering, it is possible to test the importance of individual entry mediators by knocking them out. Pigs engineered by knockout of sialoadhesin were still susceptible to infection, while CD163 knockout resulted in pigs that were resistant to infection. Genetic engineering is not only a valuable tool to determine the role of specific proteins in infection by PRRSV (in this case), but also provides a means to create animals resistant to disease. Genetic engineering of alveolar macrophages can also illuminate the role of other proteins in response to infection. We suggest that strategies to prevent infection be pursued to reduce the reservoir of virus.

Swine models, genomic tools and services to enhance our understanding of human health and diseases

The pig is becoming increasingly important as a biomedical model. Given the similarities between pigs and humans, a greater understanding of the underlying biology of human health and diseases may come from the pig rather than from classical rodent models. With an increasing need for swine models, it is essential that the genomic tools, models and services be readily available to the scientific community. Many of these are available through the National Swine Resource and Research Center (NSRRC), a facility funded by the US National Institutes of Health at the University of Missouri. The goal of the NSRRC is to provide high-quality biomedical swine models to the scientific community.

Use of heteroduplex mobility assays (HMA) for pre-sequencing screening and identification of variant strains of swine and avian hepatitis E viruses.

Hepatitis E virus (HEV), the causative agent of human hepatitis E, is an important public health problem in many developing countries and is also endemic in many industrialized countries including the US. The discoveries of avian and swine HEVs by our group from chickens and pigs, respectively, suggest that hepatitis E may be a zoonosis. Current methods for molecular epidemiological studies of HEV require PCR amplification of field strains of HEV followed by DNA sequencing and sequence analyses, which are laborious and expensive. As novel or variant strains of HEV continue to evolve rapidly both in humans and other animals, it is important to develop a rapid pre-sequencing screening method to select field isolates for further molecular characterization. In this study, we developed two heteroduplex mobility assays (HMA) (one for swine HEV based on the ORF2 region, and the other for avian HEV based on the ORF1 region) to genetically differentiate field strains of avian and swine HEVs from known reference strains. The ORF2 regions of 22 swine HEV isolates and the ORF1 regions of 13 avian HEV isolates were amplified by PCR, sequenced and analyzed by HMA against reference prototype swine HEV strain and reference prototype avian HEV strain, respectively. We showed that, in general, the HMA profiles correlate well with nucleotide sequence identities and with phylogenetic clustering between field strains and the reference swine HEV or avian HEV strains. Field isolates with similar HMA patterns generally showed similar sequence identities with the reference strains and clustered together in the phylogenetic trees. Therefore, by using different HEV isolates as references, the HMA developed in this study can be used as a pre-sequencing screening tool to identify variant HEV isolates for further molecular epidemiological studies.