Samia Metwally

Discovery of Swine as a Host for the Reston ebolavirus

Not Reston at All Reston ebolavirus is named, mistakenly perhaps, for Reston, Virginia, where it was discovered in the 1970s in imported macaques. After some alarm it was found not to be virulent in humans, uniquely among the ebola viruses, which are characteristically fatal causing a horrific spectrum of symptoms. Using a panviral detection assay, Reston ebolavirus has been rediscovered by Barrette et al. (p. 204) in domesticated pigs in the Philippines in association with other viruses that cause respiratory illness. The strains involved are closely related to the original macaque strain and, given how little variance there is among the viruses, it appears that it is freely circulating between these species possibly, like several other zoonotic viruses, having a reservoir in bats. Serological assays indicated that farm workers have become infected, although no obvious symptoms of human disease have been reported. Respiratory infections in pigs in the Philippines are associated with a cocktail of viruses, including a monkeys filovirus. Since the discovery of the Marburg and Ebola species of filovirus, seemingly random, sporadic fatal outbreaks of disease in humans and nonhuman primates have given impetus to identification of host tropisms and potential reservoirs. Domestic swine in the Philippines, experiencing unusually severe outbreaks of porcine reproductive and respiratory disease syndrome, have now been discovered to host Reston ebolavirus (REBOV). Although REBOV is the only member of Filoviridae that has not been associated with disease in humans, its emergence in the human food chain is of concern. REBOV isolates were found to be more divergent from each other than from the original virus isolated in 1989, indicating polyphyletic origins and that REBOV has been circulating since, and possibly before, the initial discovery of REBOV in monkeys.

A pandemic strain of calicivirus threatens rabbit industries in the Americas

Rabbit Hemorrhagic Disease (RHD) is a severe acute viral disease specifically affecting the European rabbit Oryctolagus cuniculus. As the European rabbit is the predominant species of domestic rabbit throughout the world, RHD contributes towards significant losses to rabbit farming industries and endangers wild populations of rabbits in Europe and other predatory animals in Europe that depend upon rabbits as a food source. Rabbit Hemorrhagic Disease virus (RHDV) – a Lagovirus belonging to the family Caliciviridae is the etiological agent of RHD. Typically, RHD presents with sudden death in 70% to 95% of infected animals. There have been four separate incursions of RHDV in the USA, the most recent of which occurred in the state of Indiana in June of 2005. Animal inoculation studies confirmed the pathogenicity of the Indiana 2005 isolate, which caused acute death and pathological changes characterized by acute diffuse severe liver necrosis and pulmonary hemorrhages. Complete viral genome sequences of all USA outbreak isolates were determined and comparative genomics revealed that each outbreak was the result of a separate introduction of virus rather than from a single virus lineage. All of the USA isolates clustered with RHDV genomes from China, and phylogenetic analysis of the major capsid protein (VP60) revealed that they were related to a pandemic antigenic variant strain known as RHDVa. Rapid spread of the RHDVa pandemic suggests a selective advantage for this new subtype. Given its rapid spread, pathogenic nature, and potential to further evolve, possibly broadening its host range to include other genera native to the Americas, RHDVa should be regarded as a threat.

Foot-and-mouth disease in feral swine: susceptibility and transmission.

Experimental studies of foot-and-mouth disease (FMD) in feral swine are limited, and data for clinical manifestations and disease transmissibility are lacking. In this report, feral and domestic swine were experimentally infected with FMDV (A24-Cruzeiro), and susceptibility and virus transmission were studied. Feral swine were proved to be highly susceptible to A-24 Cruzeiro FMD virus by intradermal inoculation and by contact with infected domestic and feral swine. Typical clinical signs in feral swine included transient fever, lameness and vesicular lesions in the coronary bands, heel bulbs, tip of the tongue and snout. Domestic swine exhibited clinical signs of the disease within 24 h after contact with feral swine, whereas feral swine did not show clinical signs of FMD until 48 h after contact with infected domestic and feral swine. Clinical scores of feral and domestic swine were comparable. However, feral swine exhibited a higher tolerance for the disease, and their thicker, darker skin made vesicular lesions difficult to detect. Virus titration of oral swabs showed that both feral and domestic swine shed similar amounts of virus, with levels peaking between 2 to 4 dpi/dpc (days post-inoculation/days post-contact). FMDV RNA was intermittently detectable in the oral swabs by real-time RT-PCR of both feral and domestic swine between 1 and 8 dpi/dpc and in some instances until 14 dpi/12 dpc. Both feral and domestic swine seroconverted 6-8 dpi/dpc as measured by 3ABC antibody ELISA and VIAA assays. FMDV RNA levels in animal room air filters were similar in feral and domestic swine animal rooms, and were last detected at 22 dpi, while none were detectable at 28 or 35 dpi. The FMDV RNA persisted in domestic and feral swine tonsils up to 33-36 dpi/dpc, whereas virus isolation was negative. Results from this study will help understand the role feral swine may play in sustaining an FMD outbreak, and may be utilized in guiding surveillance, epidemiologic and economic models.

FOOT-AND-MOUTH DISEASE IN NORTH AMERICAN BISON (BISON BISON) AND ELK (CERVUS ELAPHUS NELSONI): SUSCEPTIBILITY, INTRA- AND INTERSPECIES TRANSMISSION, CLINICAL SIGNS, AND LESIONS

There is limited information about the pathogenesis and epidemiology of foot-and-mouth disease (FMD) in North American bison (Bison bison) or elk (Cervus elaphus nelsoni). In these two experimental infection studies, we compared the susceptibilities of bison and elk to FMD virus (FMDV), respectively, with that of cattle; determined whether intra- and interspecies transmission could occur in bison and cattle, and elk and cattle; determined suitability of conventional available laboratory tests to detect FMDV infection in bison and elk; and investigated whether bison or elk are efficient long-term carriers of FMDV. In both studies, after a period of acclimation to the containment at Plum Island Animal Disease Center, animals were infected by intraepithelial tongue inoculation with 10,000 bovine tongue infective doses of FMDV, strain O1 Manisa. Inoculated animals were kept with contact animals; subsequently, inoculated and/or exposed contact animals were placed in rooms with unexposed animals. All bison developed oral mucosal and foot lesions similar to those of cattle. Bison developed fever, lameness, inappetence, and ptyalism. Physical examinations on bison revealed numerous small vesicles and erosions affecting tongue, gingiva, muzzle, hard and soft palates, coronary bands, and interdigital skin. Inoculated elk developed transient fever and mild focal tongue and foot lesions. Contact elk developed neither clinical signs nor gross pathologic lesions of FMD. At necropsy, lesions in bison included numerous extensive vesicles, erosions, and/or ulcers in the oral cavities, feet, and rumen pillars depending on the stage of disease. Less extensive oral, foot, and rumen lesions were present in the inoculated elk. All bison and inoculated elk developed antibodies to FMDV and were positive for FMDV by reverse transcription-polymerase chain reaction (RT-PCR). Transmission occurred between cattle and bison, and bison and bison. It did not occur between elk and cattle. Elk-to-elk transmission studies resulted in only one contact elk developing serologic evidence of a subclinical infection. Other exposed elk developed neither clinical, pathologic, virologic, nor serologic evidence of disease. FMDV was not isolated from animals past 28 days postinfection.

Diagnosis of Porcine teschovirus encephalomyelitis in the Republic of Haiti

In February and March 2009, approximately 1,500 backyard pigs of variable age became sick, and approximately 700 of them died or were euthanized in the Lower Artibonite Valley and the Lower Plateau of the Republic of Haiti. The main clinical sign was posterior ataxia followed by paresis and/or paralysis on the second or third day of illness. No gross lesions were observed at postmortem examinations. The morbidity and mortality were approximately 60% and 40%, respectively. Diagnostic samples (whole blood, brain, tonsil, lymph nodes, spleen, and lung) were negative for Classical swine fever virus and African swine fever virus. Porcine teschovirus type 1 was detected by reverse transcription polymerase chain reactions in brain samples. Results of virus isolation, electron microscopy of virus particles, histopathological analysis on brain tissues, nucleic acid sequencing, and phylogenetic analysis of the viral isolate supported the diagnosis of teschovirus encephalomyelitis. The outbreak of the disease in Haiti is the first appearance of the severe form of teschovirus encephalomyelitis in the Americas. This disease poses a potential threat to the swine industries in other Caribbean countries, as well as to Central and North American countries.

Analysis of clinical samples for early detection of classical swine fever during infection with low, moderate, and highly virulent strains in relation to the onset of clinical signs.

Classical swine fever (CSF) is a transboundary viral disease affecting swine. The clinical course of disease and the best diagnostic samples for early detection were examined using low, moderate, and highly virulent strains of CSFV inoculated into 8-12 week old domestic pigs. Clinical signs were monitored and recorded. Nasal swabs, tonsil scrapings, blood and tonsils were tested using virus isolation, immunohistochemistry, and real-time reverse transcriptase PCR (rRT-PCR).Severe clinical signs appear 3 days post infection (dpi) with the highly virulent strain, correlating with positive tonsil scrapings, tonsil and blood by virus isolation and rRT-PCR (83-100%), whereas nasal swabs become comparable by 5dpi (89-100%). The moderate strain caused less severe clinical signs between 5 and 7dpi, with tonsil scrapings, tonsil and blood positive by 7dpi (83-100%), and nasal swabs were comparable at 10dpi (67-90%). The low virulent strain showed mild clinical signs at 7dpi, with blood, tonsil and tonsil scrapings positive by virus isolation and rRT-PCR. Except for one sample at 10dpi, nasal swabs remained negative throughout the course of infection. This study indicates that irrespective of virulence, whole blood and tonsil scrapings are the sample of choice for early detection of CSFV in live pigs.

Identification and characterization of mimotopes of classical swine fever virus E2 glycoprotein using specific anti-E2 monoclonal antibodies.

Classical swine fever virus (CSFV) shares high nucleic acid and amino acid sequence homology with the other members of the pestivirus genus, namely bovine viral diarrhea virus and border disease virus. All three viruses are able to infect swine and generate cross reactive antibodies, which is problematic during differential diagnosis for classical swine fever (CSF). Toward the development of a new generation of CSF specific diagnostic tools, monoclonal antibodies specific for CSFV were mapped using phage display technology. Six mimotopes were identified, some of which were found to be antigenic and/or specific for CSF when used as coating antigens in an ELISA for the detection of CSF antibodies in swine serum. Two mimotopes in particular termed V2-2 and V7-1 recognized numerous strains of CSF antisera and bound fewer BVD and BD antisera compared to a commercial CSF antibody ELISA. These two mimotopes may be useful to the pestivirus field in the development of a highly specific CSF antibody ELISA as well as in the development of other potential diagnostic technologies.

FOOT-AND-MOUTH DISEASE IN A SMALL SAMPLE OF EXPERIMENTALLY INFECTED PRONGHORN (ANTILOCAPRA AMERICANA).

Abstract There is limited information on the pathogenesis and epidemiology of foot-and-mouth disease (FMD) in North American wildlife and none concerning pronghorn (Antilocapra americana). In an experimental study of 13 pronghorn and six steers (Bos taurus), we compared the susceptibility of pronghorn to FMD virus (FMDV) strain O, with that of cattle (Bos taurus). We also determined the potential for intra- and interspecies transmission of FMDV strain O in pronghorn and cattle, assessed the application of conventional laboratory tests in their suitability to detect FMDV infection in pronghorn, and evaluated the potential role of pronghorn as efficient long-term carriers of FMDV. After acclimation to containment at Plum Island Animal Disease Center, two pronghorn and one steer were each infected by intraepithelial tongue inoculation with 10,000 bovine tongue infective doses of FMDV, strain O1 Manisa. Inoculated animals were housed with contact animals. When contact-exposed animals developed fever they were placed in rooms with previously unexposed animals. All inoculated and exposed cattle and pronghorn developed clinical disease typical of FMD. Pronghorn developed severe foot lesions and mild to moderate oral lesions, primarily on the tongue. Duration of clinical signs in both species was 2–3 wk with foot abnormalities evident to the end of the study (51 d postexposure). Other lesions included pancreatitis, myositis of the tongue, and secondary lesions including pleuritis, pneumonia, decubital ulcers, and tenosynovitis. Virus transmission occurred between pronghorn, from cattle to pronghorn, and from pronghorn to cattle. Conventional laboratory tests detected virus and antibodies against nonstructural and structural FMDV proteins in pronghorn and cattle. Virus was present in some animals for 1 wk but was not detectable by virus isolation or PCR at 3 wk postinfection or afterward.