Carlos H. Romero

Protection of goats against peste des petits ruminants with recombinant capripoxviruses expressing the fusion and haemagglutinin protein genes of rinderpest virus

Goats were protected against a lethal challenge of peste des petits ruminants (PPR) virus following vaccination with a recombinant capripoxvirus containing either the fusion (F) gene of rinderpest virus or the haemagglutinin (H) gene of rinderpest virus. The H gene recombinant produced high titres of neutralizing antibody to rinderpest virus in the vaccinated goats, whereas the F gene recombinant failed to stimulate detectable levels of neutralizing antibody. A similar response to the two recombinant vaccines has previously been reported for cattle. Neither recombinant produced detectable levels of specific antibodies to PPR virus.

VENEREAL TRANSMISSION OF PSEUDORABIES VIRUSES INDIGENOUS TO FERAL SWINE

Between 1995 and 1998, we designed a series of studies in which we attempted to determine the main routes of transmission involved in the natural infection of pseudorabies virus (PRV) indigenous to free-ranging feral swine (Sus scrofa). Naturally infected feral sows transmitted the infection to uninfected feral boars, with which they had been commingled for a 6-wk period. Pseudorabies virus was isolated from boar preputial swabs, but not from nasal swabs. Three of the same PRV-infected feral sows did not transmit the infection to domestic boars during a 16 wk commingling period, despite the fact that they became pregnant. Feral boars, naturally infected with PRV, transmitted the virus to domestic gilts while penned together during 6 wk. Pseudorabies virus was isolated from vaginal swabs, but not from nasal swabs of gilts, after 2 and 3 wk of commingling. When the same infected boars were commingled with either feral or domestic boars for 13 wk, PRV transmission did not occur. None of the exposed boars developed neutralizing antibodies or yielded virus from their preputial or nasal swabs. Our results indicate that PRV indigenous to feral swine is preferentially transmitted to feral or domestic swine of the opposite sex by the venereal route. This mode of transmission differs from that seen in the natural transmission of PRV prevalent in domestic swine, where contaminated secretions, excretions and aerosols are responsible for the spread of the virus. Based on these results, we feel that as long as feral swine do not come into direct contact with domestic swine, PRV-infected feral swine probably pose only a limited risk to the success of the National Pseudorabies Eradication Program. The fact that PRV is usually transmitted from feral to domestic swine at the time of mating would indicate that the isolation of domestic herds by the use of a “double fence,” should be adequate protection against reinfection with PRV.

Mechanisms of transmission of Aujeszky's disease virus originating from feral swine in the USA.

To understand the possible mechanisms of transmission of Aujeszkys disease virus (pseudorabies or PRV) from a feral pig reservoir, intranasal infections were initiated in domestic pigs and in pigs from a herd derived from captured feral pigs. Virus strains originating from feral pigs and from domestic pigs were compared. Similar shedding patterns were obtained in both feral-derived and domestic pigs, however, virus strains from feral pigs were markedly attenuated. Virus could be isolated after acute infection from nasal secretions, tonsils and occasionally from genital organs. In studies of transmission of PRV by cannibalism, either latently infected or acutely infected tissue was fed to both domestic and feral-derived pigs. In two similar experiments, latently infected tissue did not transmit virus, but tissues from acutely infected pigs did transmit infection. Cannibalism was observed typically in both types of pigs older than 6 weeks of age. It was concluded that transmission of PRV originating from feral pigs can occur by several mechanisms including the respiratory route and by cannibalism of pigs that die of acute infection. Transmission of PRV from feral swine may, however, result in sub-clinical infection.

Genetic identification of novel poxviruses of cetaceans and pinnipeds

Summary.Novel poxviruses were identified in skin lesions of several species of cetaceans and pinnipeds using polymerase chain reaction targeting DNA polymerase and DNA topoisomerase I genes of members of the subfamily Chordopoxvirinae. With the exception of parapoxviruses, no molecular data of marine mammal poxviruses were available to infer genetic and evolutionary relatedness to terrestrial vertebrate poxviruses. Viruses were assigned to a cetacean poxvirus 1 (CPV-1) group based on nucleotide and amino acid identities of gene fragments amplified from skin lesions of Asian bottlenose (Tursiops aduncus), Atlantic bottlenose (Tursiops truncatus), rough-toothed (Steno bredanensis), and striped (Stenella coeruleoalba) dolphins. A different poxvirus was detected in skin lesions of a bowhead whale (Balaena mysticetus) and provisionally assigned to a CPV-2 group. These viruses showed highest identity to terrestrial poxviruses of the genera Orthopoxvirus and Suipoxvirus. A novel species-specific poxvirus was also identified in skin lesions of Steller sea lions (Eumetopias jubatus). None of these poxviruses were found to have amplifiable hemagglutinin gene sequences. Novel parapoxviruses were also identified in skin lesions of Steller sea lions and spotted seals (Phoca largha). A significant degree of divergence was observed in sequences of Steller sea lion parapoxviruses, while those of spotted seals and harbor seals (Phoca vitulina) were highly conserved.

A novel porcine gammaherpesvirus.

A novel porcine gammaherpesvirus was detected in the blood of domestic pigs by PCR. With degenerate-primer PCR and subsequent long-distance PCR approaches a 60-kbp genome stretch was amplified. Sequence analysis revealed the presence of the gammaherpesvirus ORFs 03 to 46 as well as a putative chemokine receptor and a v-bcl-2 gene. The 60-kbp sequence was compared with the corresponding sequence of the porcine lymphotropic herpesvirus 1 (PLHV-1) published recently and the sequence of PLHV-2, which was amplified from porcine tonsil. Considerable sequence differences (amino acid identities: 49-89%) were found between the novel virus and PLHV-1 as well as PLHV-2, which were very closely related to each other (amino acid identities: 85-98%). The novel virus had essentially the same genome organization as PLHV-1 and -2 and was therefore designated PLHV-3. Like PLHV-1 and -2, PLHV-3 was frequently found in the blood and in lymphoid organs of domestic and feral pigs from different geographic locations. In the blood, the PLHVs were detected predominantly in B-cells. Indication for latent as well as productive PLHV-3 infection was found in the porcine B-cell line L23. It can be concluded that the PLHVs are widespread and are likely to cause a persistent B-lymphotropic infection. Since PLHV-1 has been implicated in the development of porcine posttransplantation lymphoproliferative disease, all porcine lymphotropic gammaherpesviruses are of concern when pigs are used as donors in xenotransplantation.

Genital infection and transmission of pseudorabies virus in feral swine in Florida, USA

Seventeen feral swine (FS) naturally infected with pseudorabies virus (PRV) and treated with dexamethasone (4 mg/kg body wt) on five consecutive days shed virus primarily from the genital tract and less frequently from the upper respiratory tract. The FS isolates were identified as PRV by virus neutralization with specific polyclonal antiserum and by direct immunofluorescence. Restriction endonuclease analysis with BamHI showed that representative samples from a total of 62 isolates were identical to each other, but differed in at least 5 DNA bands from the PRV Shope reference strain profile. DNA purified from FS isolates propagated in Vero cells or DNA extracted directly from genital swabs were amplified in the polymerase chain reaction using primers specific for the gpII (gB) gene of PRV. This amplification yielded a product of the expected size (200 bp), which specifically hybridized to a digoxigenin-labelled 30-mer probe complementary to an area within the region defined by the primers. In a transmission experiment, PRV was recovered from the vagina at 1 and 6 weeks after uninfected feral gilts were mixed with infected feral boars. PRV was not isolated from the upper respiratory tract of either gilts or boars. At eight weeks, 4 of the 5 gilts had developed low titer neutralizing antibodies to PRV. Our results indicate that PRV in FS is transmitted through sexual contact.

Potential sites of virus latency associated with indigenous pseudorabies viruses in feral swine

Free-ranging feral swine (Sus scrofa) are known to be present in at least 32 states of the USA and are continuously expanding their range. Infection with pseudorabies virus (PRV) occurs in feral swine and the primary route of transmission in free-living conditions seems to be venereal. Between 1995 and 1999, naturally infected feral swine and experimentally infected hybrid progeny of feral and domestic swine, were kept in isolation and evaluated for occurrence of latent PRV indigenous to feral swine in sacral and trigeminal ganglia and tonsil. Sacral ganglia were shown, by polymerase chain reaction (PCR) amplification of the thymidine kinase (TK) gene of PRV, to be the most frequent sites of latency of PRV. Nine (56%) of 16 sacral ganglia, seven (44%) of 16 trigeminal ganglia, and five (39%) of 13 tonsils from naturally infected feral swine were positive for PCR amplification of TK sequences of PRV. These tissues were negative for PRV when viral isolation was attempted in Vero cells. DNA sequencing of cloned TK fragments from the sacral ganglia of two feral swine, showed only one nucleotide difference between the two fragments and extensive sequence homology to fragment sequences from various domestic swine PRV strains from China, Northern Ireland, and the USA. The hybrid feral domestic swine, experimentally inoculated with an indigenous feral swine PRV isolate by either the genital or respiratory route, acquired the infection but showed no clinical signs of pseudorabies. Virus inoculated into either the genital or respiratory tract could, at times, be isolated from both these sites. The most common latency sites were the sacral ganglia, regardless of the route and dose of infection in these experimentally infected hybrids. Nine of 10 sacral ganglia, six of 10 trigeminal ganglia, and three of 10 tonsils were positive for PCR amplification of TK sequences. No virus was isolated from these tissues in Vero cells. The demonstration of the sacral ganglia as the most common sites of latency of pseudorabies viruses indigenous to feral swine, supports the hypothesis that these viruses are primarily transmitted venereally, and not by the respiratory route as is common in domestic swine, in which the trigeminal ganglia are the predominant sites of virus latency.

A Novel Gammaherpesvirus Associated with Genital Lesions in a Blainville's Beaked Whale (Mesoplodon densirostris)

An adult male Blainvilles beaked whale (Mesoplodon densirostris) was found stranded on the Atlantic coast of the USA on 28 January 2004. Necropsy revealed a focal papilloma-like penile lesion, the cells from which revealed single 4–6 μm basophilic intranuclear inclusions. Total DNA extracted from lesion material was tested using a pan-herpesvirus PCR assay that targets the DNA polymerase gene and found to be positive. When the amplified DNA fragment was cloned, sequenced, and compared to GenBank-deposited herpesvirus DNA polymerase sequences, the detected virus was determined to be a distinct member of the Gammaherpesvirinae subfamily of herpesviruses. This new virus, tentatively named Ziphiid herpesvirus type 1, was associated with but not determined to be the cause of genital disease in the Blainvilles beaked whale.

Real-time RT-PCR assays for the rapid and differential detection of dolphin and porpoise morbilliviruses.

Real-time RT-PCR (rtRT-PCR) assays for identifying and differentiating infections caused by dolphin morbillivirus (DMV) and porpoise morbillivirus (PMV) were developed by targeting the hypervariable C-terminal domain of the nucleocapsid (N) gene. Total DMV and PMV RNA extracted from infected Vero cells expressing the canine signaling lymphocyte-activation molecule (SLAM) produced positive cycle threshold (C(T)) values after the 17th and 25th cycles, respectively. The assays were then validated using infected cetacean tissue RNA. The assays were specific for either DMV or PMV and did not cross-react with canine distemper virus (CDV), phocid distemper virus (PDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV) and measles virus (MV). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was targeted as control for RNA quality, and a consensus GAPDH probe that reacted with 11 different marine mammal species, generating positive C(T) values ranging from the 21st to the 37th cycle was used. The rtRT-PCR assays have advantages over conventional assays in that they are rapid, easier to scale up, and are less prone to cross-contamination and have improved the limit of detection and specificity.

PROLIFERATIVE DERMATITIS ASSOCIATED WITH A NOVEL ALPHAHERPESVIRUS IN AN ATLANTIC BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS)

Abstract Herpesviruses and herpes-like viruses have been reported in only a small number of species of cetaceans, and, to date, clinical manifestations have been either as a life-threatening, disseminated infection or as a non-life-threatening dermatitis. A stranded juvenile Atlantic bottlenose dolphin, Tursiops truncatus, was admitted to the Dolphin and Whale Hospital for rehabilitation. On initial physical examination, the rostral skin had multifocal regions of hyperplasia, and the skin of the dorsum contained a large number of small papules. Histologically, epithelial hyperplasia was evident, and clusters of epithelial cells contained 5–15-μm intranuclear inclusion bodies. Transmission electron microscopic investigation revealed numerous 170–190-nm enveloped virions in both the intracellular spaces and the cytoplasm of epithelial cells, with numerous nucleocapsids noted in epithelial cell nuclei. Consensus primer polymerase chain reaction identified the presence of a novel herpesvirus associated with the lesions. Phylogenetic analysis of the deduced amino acid sequences of the herpesvirus DNA polymerase gene fragment showed it to align with alphaherpesvirus sequences from humans and domestic animals. Although clearly distinct, it was most closely related to two previously described alphaherpesviruses of dolphins. This case represents the first documentation of herpesvirus dermatitis in the Atlantic bottlenose dolphin.