The Australian paralysis may be the missing link tick in the transmission of Hendra virus from bats to horses to humans

Hendra virus is a new virus of the family Paramyxoviridae. This virus was first detected in Queensland, Australia, in 1994; although, it seems that the virus has infected fruit-eating bats (flying-foxes) for a very long time. At least 2 humans and 15 horses have been killed by this virus since it first emerged as a virus that may infect mammals other than flying-foxes. Hendra virus is thought to have moved from flying-foxes to horses, and then from horses to people. There is a reasonably strong hypothesis for horse-to-human transmission: transmission of virus via nasal discharge, saliva and/or urine. In contrast, there is no strong hypothesis for flying-fox-to-human transmission. I present evidence that the Australian paralysis tick, Ixodes holocyclus, which has apparently only recently become a parasite of flying-foxes, may transmit Hendra virus and perhaps related viruses from flying-foxes to horses and other mammals. (C) 2003 Elsevier Science Ltd. All rights reserved.

Detection of West Nile Virus Infection in birds in the United States by blocking ELISA and immunohistochemistry

A blocking ELISA targeting an immunodominant West Nile epitope on the West Nile Virus NS1 protein was assessed for the detection of West Nile-specific antibodies in blood samples collected from 584 sentinel chickens and 238 wild birds collected in-New Jersey from May-December 2000. Ten mallard ducks (Anas platyrhynchos) experimentally infected with West Nile virus and six uninfected controls were also tested. The ELISA proved specific in detecting WNV antibodies in 9/10 chickens and 4/4 wild birds previously confirmed as positive by Plaque Reduction Neutralization test (PRNT) at the Center for Disease Control, Division of Vector Borne Diseases, Fort Collins, CO, USA (CDC). Nine out of the ten experimentally infected mallard ducks also tested positive for WN antibodies in the blocking ELISA, while 6/6 uninfected controls did not. Additionally, 1705 wild birds, collected in New Jersey from December 2000-November 2001 and Long Island, New York between November 1999 and August 2001 were also tested for WN antibodies by the blocking ELISA. These tests identified 30 positive specimens, 12 of which had formalin-fixed tissues available to allow detection of WN specific viral antigen in various tissues by WNV-specific immunohistochemistry. Our results indicate that rapid and specific detection of antibodies to WN virus in sera from a range of avian species by blocking ELISA is an effective strategy for WN Virus surveillance in avian hosts. In combination with detection of WN-specific antigens in tissues by immunohistochemistry (IHC) the blocking ELISA will also be useful for confirming WN infection in diseased birds.

Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry

Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC50 (50% inhibition) was only 0.5 mug/ml for heparin, 1 mug/ml for dextran sulfate, and 5-10 mug/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate. (C) 2003 Elsevier Science (USA). All rights reserved.

Epizootic activity of Murray Valley encephalitis and Kunjin viruses in an aboriginal community in the Southeast Kimberley region of Western Australia: Results of mosquito fauna and virus isolation studies

We undertook annual surveys of flavivirus virus activity in the community of Billiluna of Western Australia in the southeast Kimberley region between 1989 and 2001. Culex annulirostris was the dominant mosquito species, particularly in years of above average rains and flooding. Murray Valley encephalitis (MVE) virus was isolated in 8 of the 13 years of the study from seven mosquito species, but more than 90% of the isolates were from Cx. annulirostris. The results suggest that MVE virus is epizootic in the region, with activity only apparent in years with average or above average rainfall and increased numbers of Cx. annulirostris. High levels of MVE virus activity and associated human cases were detected only once (in 1993) during the survey period. Activity of MVE virus could only be partially correlated with wet season rainfall and flooding, suggesting that a number of other factors must also be considered to accurately predict MVE virus activity at such communities.

Distribution of Sugarcane mosaic virus in sugarcane plants

Variation in the concentration of virus in different parts of the plant has implications for virus-indexing programs. To allow more reliable detection of Sugarcane mosaic virus (SCMV), the distribution of the virus in sugarcane plants after artificial inoculation was studied using a reverse transcription polymerase chain reaction (RT-PCR) assay. Leaves of susceptible and moderately resistant sugarcane were mechanically inoculated with SCMV 6 weeks after planting. Weekly for 8 weeks after inoculation, plants were examined for mosaic symptoms and samples of leaves, roots and tillers were tested by RT-PCR to detect virus. SCMV moved from the point of inoculation to younger leaves, roots and tillers and eventually to leaves that emerged prior to inoculation. The pattern of SCMV distribution in moderately resistant and susceptible cultivars was not substantially different. However, the virus moved more slowly in the moderately resistant than in the susceptible cultivar. Young leaves proved to be the most suitable tissue for testing.

Structural characterization of respiratory syncytial virus fusion inhibitor escape mutants: homology model of the F protein and a syncytium formation assay

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors. (C) 2003 Elsevier Science (USA). All rights reserved.

Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture

Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of tine of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can he reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the V formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.

Low temperature induced spikelet sterility in rice. I. Nitrogen fertilisation and sensitive reproductive period

Low temperature during panicle development in rice increases spikelet sterility. This effect is exacerbated by high rates of nitrogen (N) application in the field. Spikelet sterility induced by low temperature and N fertilisation was examined in glasshouse experiments to clarify the mechanisms involved. In two glasshouse experiments, 12-h periods of low (18/13degreesC) and high (28/23degreesC) day/night temperatures were imposed over periods of 5-7 days during panicle development, to determine the effects of low temperature and N fertilisation on spikelet sterility. In one experiment, 50% sunlight was imposed together with low temperature to investigate the additive effects of reduced solar radiation and low temperature. The effect of increased tillering due to N fertilisation was examined by a tiller removal treatment in the same experiment. Pollen grain number and spikelet sterility were recorded at heading and harvest, respectively. Although there was no significant effect of low temperature on spikelet sterility in the absence of applied N, low temperature greatly increased spikelet sterility as a result of a reduction in the number of engorged pollen grains per anther in the presence of applied N. Spikelet sterility was strongly correlated with the number of engorged pollen grains per anther. Low temperature during very early ( late stage of spikelet differentiation-pollen mother cell stage) and peak ( second meiotic division stage-early stage of extine formation) microspore development caused a severe reduction in engorged pollen production mainly as a result of reduced total pollen production. Unlike low temperature, the effect of shading was rather small. The increased tillering due to application of high rates of N, increased both spikelet number per plant and spikelet sterility under low temperature conditions. The removal of tillers as they appeared reduced the number of total spikelets per plant and maintained a large number of engorged pollen grains per anther which, in turn, reduced spikelet sterility. The number of engorged pollen grains per anther determined the numbers of intercepted and germinated pollen grains on the stigma. It is concluded that N increased tillering and spikelet number per plant and this, in turn, reduced the number of engorged pollen grains per anther, leading into increased spikelet sterility under low temperature condition.

Low temperature induced spikelet sterility in rice. II. Effects of panicle and root temperatures

Low temperatures impose restrictions on rice (Oryza sativa L.) production at high latitudes. This study is related to low temperature damage that can arise mid-season during the panicle development phase. The objective of this study was to determine whether low temperature experienced by the root, panicle, or foliage is responsible for increased spikelet sterility. In temperature-controlled glasshouse experiments, water depth, and water and air temperatures, were changed independently to investigate the effects of low temperature in the root, panicle, and foliage during microspore development on spikelet sterility. The total number of pollen and number of engorged pollen grains per anther, and the number of intercepted and germinated pollen grains per stigma, were measured. Spikelet sterility was then analysed in relation to the total number of pollen grains per spikelet and the efficiency with which these pollen grains became engorged, were intercepted by the stigma, germinated, and were involved in fertilisation. There was a significant combined effect of average minimum panicle and root temperatures on spikelet sterility that accounted for 86% of the variation in spikelet sterility. Total number of pollen grains per anther was reduced by low panicle temperature, but not by low root temperature. Whereas engorgement efficiency ( the percentage of pollen grains that were engorged) was determined by both root and panicle temperature, germination efficiency (the percentage of germinated pollen grains relative to the number of engorged pollen grains intercepted by the stigma) was determined only by root temperature. Interception efficiency (i.e. percentage of engorged pollen grains intercepted by the stigma), however, was not affected by either root or panicle temperature. Engorgement efficiency was the dominant factor explaining the variation in spikelet sterility. It is concluded that both panicle and root temperature affect spikelet sterility in rice when the plant encounters low temperatures during the microspore development stage.