Landscape-Genetic Analysis of Population Structure in the Texas Gray Fox Oral Rabies Vaccination Zone

In west-central Texas, USA, abatement efforts for the gray fox (Urocyon cinereoargenteus) rabies epizootic illustrate the difficulties inherent in large-scale management of wildlife disease. The rabies epizootic has been managed through a cooperative oral rabies vaccination program (ORV) since 1996. Millions of edible baits containing a rabies vaccine have been distributed annually in a 16-km to 24-km zone around the perimeter of the epizootic, which encompasses a geographic area >4 x 105 km2. The ORV program successfully halted expansion of the epizootic into metropolitan areas but has not achieved the ultimate goal of eradication. Rabies activity in gray fox continues to occur periodically outside the ORV zone, preventing ORV zone contraction and dissipation of the epizootic. We employed a landscape-genetic approach to assess gray fox population structure and dispersal in the affected area, with the aim of assisting rabies management efforts. No unique genetic clusters or population boundaries were detected. Instead, foxes were weakly structured over the entire region in an isolation by distance pattern. Local subpopulations appeared to be genetically non-independent over distances >30 km, implying that long-distance movements or dispersal may have been common in the region. We concluded that gray foxes in west-central Texas have a high potential for long-distance rabies virus trafficking. Thus, a 16-km to 24-km ORV zone may be too narrow to contain the fox rabies epizootic. Continued expansion of the ORV zone, although costly, may be critical to the long-term goal of eliminating the Texas fox rabies virus variant from the United States.

Landscape Genetics of Raccoons (Procyon lotor) Associated with Ridges and Valleys of Pennsylvania: Implications for Oral Rabies Vaccination Programs

Raccoons are the reservoir for the raccoon rabies virus variant in the United States. To combat this threat, oral rabies vaccination (ORV) programs are conducted in many eastern states. To aid in these efforts, the genetic structure of raccoons (Procyon lotor) was assessed in southwestern Pennsylvania to determine if select geographic features (i.e., ridges and valleys) serve as corridors or hindrances to raccoon gene flow (e.g., movement) and, therefore, rabies virus trafficking in this physiographic region. Raccoon DNA samples (n = 185) were collected from one ridge site and two adjacent valleys in southwestern Pennsylvania (Westmoreland, Cambria, Fayette, and Somerset counties). Raccoon genetic structure within and among these study sites was characterized at nine microsatellite loci. Results indicated that there was little population subdivision among any sites sampled. Furthermore, analyses using a model-based clustering approach indicated one essentially panmictic population was present among all the raccoons sampled over a reasonably broad geographic area (e.g., sites up to 36 km apart). However, a signature of isolation by distance was detected, suggesting that widths of ORV zones are critical for success. Combined, these data indicate that geographic features within this landscape influence raccoon gene flow only to a limited extent, suggesting that ridges of this physiographic system will not provide substantial long-term natural barriers to rabies virus trafficking. These results may be of value for future ORV efforts in Pennsylvania and other eastern states with similar landscapes.

Rabies in Experimentally Infected Bears, Ursus spp., with Epizootiologic Notes

Ten black bears, Ursus americanus Pallas, and three brown bears, U. arctos Linnaeus, were inoculated with rabies virus from naturally infected foxes in Alaska. The bears were more resistant than canine species, requiring at least 1,000 MLD50 of virus for infection. Low titres or negative results were obtained in salivary glands titrated in mice. Clinical course of the disease, post mortem findings, and microscopic lesions are described. Microscopic lesions were more· severe in brown bears, in which the inflammatory response was distinguished by the presence of numerous eosinophils in the perivascular infiltrate and among cells diffusely infiltrating the parenchyma. In both species, inclusion bodies were found only in the Purkinje cells of the cerebellum. Rabies is discounted as a factor in unprovoked attacks by bears on man at high latitudes. The epizootiology of rabies in a region where bears are numerous is discussed, with the conclusion that rabid foxes usually do not excrete sufficient quantities of virus in the saliva to infect bears. German title: Tollwut bei experimentell infizierten Bären, Ursus spp., mit epizootiologischen Anmerkungen German abtract: Zehn Schwarzbären, Ursus americanus Pallas, und drei Braunbären, U. arctos Linnaeus, wurden mit einem von Füchsen in Alaska isolierten Feldstamm des Tollwutvirus infiziert. Die Untersuchungsergebnisse lassen erkennen, daß Bären eine größere Resistenz gegenüber Tollwutinfektion aufweisen als hundeartige Karnivoren, und zwar konnten sie nicht mit weniger als 1000 MLD50 des Tollwutvirus infiziert werden. Das Virus war selten nachweisbar in den Speicheldrüsen der tollwuterkrankten Bären. Klinik und Pathologie der Tollwut bei Bären wurden kurz beschrieben. Die im Gehirn vorkommenden entzündlichen Veränderungen waren bei Braunbären besonders schwer und unterschieden sich durch die Häufigkeit der eosinophilen Leukozyten in den perivasculären und Gewebs-Infiltraten. Bei beiden Arten wurden Einschlußkörperchen nur in den Purkinje-Zellen beobachtet. Die Epizootiologie der Tollwut auf der Alaska-Halbinsel, wo Bären häufig vorkommen, wurde besprochen. Die Ergebnisse deuten an, daß Füchse wenig Virus mit dem Speichel ausscheiden, und selten soviel, daß es für die Infektion von Bären ausreicht. French title: La rage expérimentale chez les ours, Ursus spp., avec observations épizootiologiques French abstract: Dix ours noirs, Ursus americanus Pallas, et trois ours bruns, U. arctos Linnaeus, ont été inoculés avec de virus rabique provenant des renards infectés naturellement dans l'Alaska. Les ours Ont été plus résistants au virus que des espèces canines, et pour produire l'infection chez les ours, au moins 1000 MLD50 ont été requis. La titration des glandes salivaires chez des souris a données des titres peu éléves ou des résultats négatifs. La course clinique de la maladie, les observations des autopsies, et les lésions microscopiques sont decrites. Les lésions microscopiques les plus sévères ont été observées chez les ours bruns, dans lesquels la réponse inflammatoire a été distinguée par la présence de nombreux éosinophiles dans l'infiltration périvasculaire et parmi les cellules infiltrées diffusément dans Ie parenchyme. Chez les deux espèces des ours, des corps d'inclusion ont été trouvés seulement dans les cellules de Purkinje du cervelet. On a discuté l'épizootiologie de la rage dans une région où des ours sont nombreux, avec la conclusion qu'il y a dans la salive des renards rabiques une quantité de virus insuffisante pour infecter les ours. é ó í á ú Spanish title: Rabia en osos, Ursus spp., infectados experimentalmente, con anotaciones epizootológicas Spanish abstract: Diez osos negros, Ursus americanus Pallas, y tres osos pardos, U. arctos Linea, se infectaron con una estirpe campal de virus rábico aislada de zorros en Alasca. Los resultados de la experiencia permiten reconocer que los osos presentan una resistencia mayor frente a la infección rábica que los carnívoros cánidos, pues no se pudieron infectar con menos de 1.000 DML50 de virus rábico. El virus era muy raras veces identificable en las glándulas salivales de los osos enfermos de rabia. Se describen sucintamente la clínica y patología de la rabia en los osos. Las modificaciones inflamatorias en el cerebro eran muy graves en el oso pardo y se distinguían por la frecuencia de los leucocitos eosinófilos en los infiltrados perivasculares e hísticos. En ambas especies solo se hallaron corpúsculos de inclusión en las células de Purkinje. Se discute la epizootología de la rabia en la península de Alasca, donde es frecuente Ia presencia de osos. Los resultados señalan que los zorros eliminan poco virus con la saliva y casi nunca en cantidad tal que fuese suficiente para infectar los osos.

Observations on Some Natural-Focal Zoonoses in Alaska

In Alaska, as in arctic and subarctic Eurasia, important natural-focal zoonoses are rabies, brucellosis, tularemia, trichinosis, alveolar hydatid disease, cystic hydatid disease, and diphyllobothriasis. Most frequently affected are aboriginal peoples in villages within biocenoses that include the natural parasite-host assemblages. Pathogens are transmitted to man from wild animals and from dogs, which are important as synanthropic hosts. The prevalence and rate of transmission of certain pathogens in natural foci are related to the numerical density of small mammals, especially rodents, which may themselves be involved as hosts, and on which the numbers of their predators ultimately depend, such as is evident in the natural cycles of Echinococcus multilocularis and of rabies virus. Some pathogens in northern regions exhibit biological Characteristics that separate them from morphologically indistinguishable strains at lower latitudes (e.g., Trichinella spiralis and E. granulosus). Host-parasite relationships may also differ, as in the Arctic where rabies virus is maintained in populations of foxes, without significant involvement of mammals of other groups. Faunal interchanges during and after the Pleistocene period have influenced the distribution of parasite-host assemblages in Alaska.

Characterization of a monothiol glutaredoxin encoded by Chlorella virus PBCV-1

Annotation of the 330-kb Chlorella virus PBCV-1 genome identified a 237 nucleotide gene (a438l) that codes for a protein with ~35% amino acid identity to glutaredoxins (Grx) found in other organisms. The PBCV-1 protein resembles classical Grxs in both size (9 kDa) and location of the active site (N-terminus). However, the PBCV-1 Grx is unusual because it contains a monothiol active site (CPYS) rather than the typical dithiol active site (CPYC). To examine this unique active site, four sitespecific mutants (CPYC, CPYA, SPYC, and SPYS) were constructed to determine if the N-terminal cysteine is necessary for enzyme activity. Wild type and both mutants containing N-terminal cysteines catalyzed the reduction of disulfides in a coupled system with GSH, NADPH, and glutathione reductase. However, both mutants with an altered N-terminal cysteine were inactive. The grx gene is common in the Chlorella viruses. Molecular phylogenetic analyses of the PBCV-1 enzyme support its relatedness to those from other Chlorella viruses and yet demonstrate the divergence of the Grx molecule.

QUANTITATIVE TRAIT LOCI FOR AGRONOMIC AND END-USE QUALITY PERFORMANCE AND THE EFFECT OF SOILBORNE WHEAT MOSAIC VIRUS IN A HARD WINTER WHEAT POPULATION IN NEBRASKA

To better understand agronomic and end-use quality in wheat (Triticum aestivum L.) we developed a population containing 154 F6:8 recombinant inbred lines (RILs) from the cross TAM107-R7/Arlin. The parental lines and RILs were phenotyped at six environments in Nebraska and differed for resistance to Wheat soilborne mosaic virus (WSBMV), morphological, agronomic, and end-use quality traits. Additionally, a 2300 cM genome-wide linkage map was created for quantitative trait loci (QTL) analysis. Based on our results across multiple environments, the best RILs could be used for cultivar improvement. The population and marker data are publicly available for interested researchers for future research. The population was used to determine the effect of WSBMV on agronomic and end-use quality and for the mapping of a resistance locus. Results from two infected environments showed that all but two agronomic traits were significantly affected by the disease. Specifically, the disease reduced grain yield by 30% of susceptible RILs and they flowered 5 d later and were 11 cm shorter. End-use quality traits were not negatively affected but flour protein content was increased in susceptible RILs. The resistance locus SbmTmr1 mapped to 27.1 cM near marker wPt-5870 on chromosome 5DL using ELISA data. Finally, we investigated how WSBMV affected QTL detection in the population. QTLs were mapped at two WSBMV infected environments, four uninfected environments, and in the resistant and susceptible RIL subpopulations in the infected environments. Fifty-two significant (LOD≥3) QTLs were mapped in RILs at uninfected environments. Many of the QTLs were pleiotropic or closely linked at 6 chromosomal regions. Forty-seven QTLs were mapped in RILs at WSBMV infected environments. Comparisons between uninfected and infected environments identified 20 common QTLs and 21 environmentally specific QTLs. Finally, 24 QTLs were determined to be affected by WSBMV by comparing the subpopulations in QTL analyses within the same environment. The comparisons were statistically validated using marker by disease interactions. These results showed that QTLs can be affected by WSBMV and careful interpretation of QTL results is needed where biotic stresses are present. Finally, beneficial QTLs not affected by WSBMV or the environment are candidates for marker-assisted selection.

Monitoring Raccoon Rabies in Alabama: The Potential Effects of Habitat and Demographics

Density, morphometrics, and disease prevalence of raccoon populations were determined in 4 habitats (agriculture, riverine, managed, and forested) in central Alabama. In addition we monitored 71 collared raccoons to determine survival. Density estimates were similar in the agriculture (ag) and riverine habitats in central Alabama with 8 raccoons/km2, and lower in the forested habitat at 5 raccoons/ km2. Retention of juveniles did not appear to contribute to observed higher populations in the riverine and ag habitat. Although the riverine and ag, possibly due to supplemental resources, likely provide better habitat for raccoons, we found only body size in female raccoons to be different across habitats (P = 0.001). Human-caused mortality (either hunting or missing and presumed killed) was the main cause of mortality in several raccoon populations during fall; however, fall survival did not differ between the habitats (χ = 1.47, d.f. = 3, P = 0.69). Although rabies and distemper virus were prevalent in all habitats, they did not appear to contribute to mortality even with a high proportion of the population exhibiting positive CDV titers (ag – 44%, managed- 50%) and rabies titers (managed- 57% and riverine habitat-60%).

INFLUENCE OF TYPE 2 BOVINE VIRAL DIARRHEA VIRUS NPRO ON ENHANCEMENT OF BOVINE RESPIRATORY SYNCYTIAL VIRUS REPLICATION MEDIATED BY ANTAGONISM OF HOST CELL INTERFERON TYPE I RESPONSES

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, Family Flaviviridae. The virus can infect many species of animals of the order Artiodactyla. The BVDV genome encodes an auto protease, Npro, that degrades interferon regulatory factor-3 (IRF-3) reducing type I interferon (IFN-I) production from host cells. Bovine respiratory syncytial virus (BRSV) is a member of the genus Pneumovirus, Family Paramyxoviridae. Concurrent infection with BVDV and BRSV causes more severe respiratory and enteric disease than infection with either virus alone. Our hypothesis was that Npro modulates the innate immune responses to BVDV infection and enhances replication of BVDV or BRSV co-infection. The noncytopathic BVDV2 viruses NY93/c N- Npro 18 EGFP (a mutant with modified Npro fused with enhanced green fluorescent protein), NY93 infectious clone (NY93/c), wild-type NY93-BVDV2 (NY93-wt), and BRSV were evaluated in this study. The objectives of this study were: (1) to characterize the replication kinetics and IFN-I induction in Madin-Darby bovine kidney (MDBK) cells following infection with each of the BVDV isolates, and (2) to characterize the influence of BVDV-mediated IFN-I antagonism on enhancement of BRSV replication in bovine turbinate (BT) cells. NY93/c N- Npro 18 EGFP replicated 0.4 – 1.6 TCID50 logs lower than NY93-wt in MDBK cells. NY93/c N- Npro 18 EGFP-infected MDBK cells synthesized IFN-I significantly higher than NY93/c- and NY93-wt-infected MDBK cells. BT cells co-infected with NY93/c N- Npro 18 EGFP/BRSV or NY93-wt/BRSV were evaluated to determine the effects of co-infection on BRSV replication and IFN-I induction in BT cells. BRSV RNA levels in NY93-wt/BRSV co-infected BT cells were 2.49, 2.79, and 2.89 copy number logs significantly greater than in NY93/c N- Npro 18 EGFP/BRSV co-infected BT cells on days 5, 7, and 9 post-infection, respectively. BVDV RNA levels in NY93/c N- Npro 18 EGFP-infected BT cells were 1.64 – 4.38 copy number logs lower than in NY93-wt-infected BT cells. NY93/c N- Npro 18 EGFP single and co-infected BT cells synthesized IFN-I significantly higher than NY93-wt single and co-infected BT cells. In summary, these findings suggest: (1) NY93/c N- Npro 18 EGFP BVDV2 induced higher levels of IFN-I than BVDV2-wt and may be useful as a safer, replicating BVDV vaccine, and (2) Enhancement of BRSV infection by BVDV co-infection is mediated by antagonism of IFN-I.