Abstract. West Nile fever caused fatal disease in humans, horses, and birds in the northeastern United States during 1999. We studied birds from two wildlife facilities in New York City, New York, that died or were euthanatized and were suspected to have West Nile virus infections. Using standard histologic and ultrastructural methods, virus isolation, immunohistochemistry, in situ hybridization and reverse-transcriptase polymerase chain reaction, we identified West Nile virus as the cause of clinical disease, severe pathologic changes, and death in 27 birds representing eight orders and 14 species. Virus was detected in 23/26 brains (88%), 24/ 25 hearts (96%), 15/18 spleens (83%), 14/20 livers (70%), 20/20 kidneys (100%), 10/13 adrenals (77%), 13/ 14 intestines (93%), 10/12 pancreata (83%), 5/12 lungs (42%), and 4/8 ovaries (50%) by one or more methods. Cellular targets included neurons and glial cells in the brain, spinal cord, and peripheral ganglia; myocardial fibers; macrophages and blood monocytes; renal tubular epithelium; adrenal cortical cells; pancreatic acinar cells and islet cells; intestinal crypt epithelium; oocytes; and fibroblasts and smooth muscle cells. Purkinje cells were especially targeted, except in crows and magpies. Gross hemorrhage of the brain, splenomegaly, meningoencephalitis, and myocarditis were the most prominent lesions. Immunohistochemistry was an efficient and reliable method for identifying infected cases, but the polyclonal antibody cross-reacted with St. Louis encephalitis virus and other flaviviruses. In contrast, the in situ hybridization probe pWNV-E (WN-USAMRIID99) reacted only with West Nile virus. These methods should aid diagnosticians faced with the emergence of West Nile virus in the United States.

Diagnosis of alphaviral arthritides is complicated by nonspecific symptoms and the lack of commercial serodiagnostic kits, except for Ross River and Barmah Forest virus infections in Australia. Differential diagnoses should be actively pursued, especially if symptoms persist. Treatment with nonsteroidal anti-inflammatory drugs appears largely effective, with no evidence of long-term sequelae or relapse.

Ross River virus (RRV) is the aetiological agent of epidemic polyarthritis (EPA) a predominantly rheumatic disease afflicting up to 5000 Australians annually. We show here for the first time that macrophages can be productively infected by RRV. Subneutralizing titres of anti-RRV IgG (but not IgM) also showed classical antibody-dependent enhancement (ADE) of RRV infection in macrophage and monocyte cell lines. No correlation between development of EPA and the preexistence of ADE titres was apparent, nor could sera raised against a related arbovirus, Barmah Forest, enhance RRV infection. Tumour necrosis factor-a, implicated in the immunopathogenesis of rheumatoid arthritis, was not secreted by RRV-infected monocytes or macrophages. Macrophage cell lines infected with RRV were, however, capable of producing virus for over 50 days. RRV-induced arthritis may therefore be due to the persistent productive infection of macrophages, perhaps established by a brief period of ADE early in infection.Ross River virus (RRV) is a mosquito-borne alphavirus endemic to Australia and New Guinea and is the aetiological agent of epidemic polyarthritis (EPA). In Australia, where EPA is a notifiable disease, up to 5000 cases are reported annually. In 1979/1980 an explosive epidemic also swept through several islands of the South Pacific resulting in tens of thousands of cases (Aaskov & Doherty, 1994). The principal symptom of EPA is arthritis/arthralgia, which is often severe and usually lasts for 30 to 40 weeks, with about 25 % of EPA patients experiencing symptoms for a year or more (Fraser, 1986). Approximately half of the EPA patients also experience a rash, fever, myalgia and/or lethargy. The current treatment with non-steroidal anti-inflammatory agents can provide relief, but it is often inadequate. EPA is rare in children and overall reports for the ratio of asymptomatic to symptomatic infections (resulting in EPA) range from 50:1 to approximately 2:1 (Aaskov & Doherty, 1994;Fraser, 1986).The rheumatic synovial exudates from EPA patients are largely devoid of neutrophils and contains a mononuclear cell infiltrate, predominantly composed of monocytes, vacuolated and phagocytic macrophages, T cells and B cells. RRV can be isolated from the peripheral * Author for correspondence. Fax +61 7 33620106. e-mail andreasS@qimr.edu.au blood and viral antigens can be detected in synovial monocyte/macrophages during the first week after the onset of symptoms (Fraser, 1986). Elevated interferon-7 (IFN-7) levels were also found in exudates taken during this time (J. R. E. Fraser & A. L. Cunningham, personal communication).RRV has a large host range and can infect many cell types (Aaskov & Doherty, 1994) including murine muscle cells, and human epidermal and synovial ceils (Seay et al., 1981 ;Fraser, 1986). Using the RRV prototype strain T48, we found that the following human cell types could be productively infected with RRV resulting in overt cytopathic effect (CPE): human primary dermal and synovial fibroblasts, primary endothelial cells,...

The serine proteinase inhibitor (serpin) plasminogen activator inhibitor type 2 (PAI-2) is well characterized as an inhibitor of extracellular urokinase-type plasminogen activator. Here we show that intracellular, but not extracellular, PAI-2 protected cells from the rapid cytopathic effects of alphavirus infection. This protection did not appear to be related to an effect on apoptosis but was associated with a PAI-2–mediated induction of constitutive low-level interferon (IFN)-α/β production and IFN-stimulated gene factor 3 (ISGF3) activation, which primed the cells for rapid induction of antiviral genes. This primed phenotype was associated with a rapid development of resistance to infection by the PAI-2 transfected cells and the establishment of a persistent productive infection. PAI-2 was also induced in macrophages in response to viral RNA suggesting that PAI-2 is a virus response gene. These observations, together with the recently demonstrated PAI-2–mediated inhibition of tumor necrosis factor-α induced apoptosis, (a) illustrate that PAI-2 has an additional and distinct function as an intracellular regulator of signal transduction pathway(s) and (b) demonstrate a novel activity for a eukaryotic serpin.

A novel alphavirus was isolated from the louse Lepidophthirus macrorhini, collected from southern elephant seals, Mirounga leonina, on Macquarie Island, Australia. The virus displayed classic alphavirus ultrastructure and appeared to be serologically different from known Australasian alphaviruses. Nearly all Macquarie Island elephant seals tested had neutralizing antibodies against the virus, but no virus-associated pathology has been identified. Antarctic Division personnel who have worked extensively with elephant seals showed no serological evidence of exposure to the virus. Sequence analysis illustrated that the southern elephant seal (SES) virus segregates with the Semliki Forest group of Australasian alphaviruses. Phylogenetic analysis of known alphaviruses suggests that alphaviruses might be grouped according to their enzootic vertebrate host class. The SES virus represents the first arbovirus of marine mammals and illustrates that alphaviruses can inhabit Antarctica and that alphaviruses can be transmitted by lice.The genus Alphavirus in the family Togoviridae represents a group of enveloped, plus-strand viruses comprising over 40 known members. Alphaviruses are classified as arboviruses since they are maintained in nature by a biological transmission cycle between susceptible vertebrate hosts and hematophagous arthropods, usually ticks or mosquitoes. Alphaviruses have been grouped by geographic distribution into Old and New World viruses (28). The New World alphaviruses, which include Venezuelan equine encephalitis (VEE), eastern equine encephalitis (EEE), and western equine encephalitis (WEE) viruses are pathogenic for humans, horses, and certain bird species (26,28). The Old World alphaviruses are associated with rheumatic disease in humans and include the Australian Ross River (RR) and Barmah Forest viruses, the Asian/African chikungunya virus, the African o'nyong-nyong virus, and the European Ockelbo virus, which is a subtype of Sindbis virus (28). Weaver et al. (39) hypothesized that alphaviruses may have originated a few thousand years ago in the New World, possibly through recombination with plant viruses (9), and spread to the Old World via bird migration. Recently, two fish alphaviruses have been described, salmon pancreas disease virus (40) and rainbow trout sleeping disease virus (37), suggesting either aquatic origins for alphaviruses or an invasion of the marine environment by terrestrial alphaviruses.Macquarie Island is located some 2,000 km south (54°30'S, 159°E) off the Australian mainland. The island has been a rich source of tick-borne arboviruses. A flavivirus of penguins (22) has been identified on Macquarie Island, as have flaviviruses, bunyaviruses, and orbiviruses, whose enzootic hosts are also likely to be birds (3, 23, 33). The island is also home to about 12% of the world's population of southern elephant seals (Mirounga leonina), roughly 78,000 animals (16), which are known to be infested with the blood-sucking louse Lepidophthirus macrorhini (25). The Macquarie Island elepha...

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Abstract. Chelonian intranuclear coccidiosis has been reported once, in two radiated tortoises (Geochelone radiata), and is apparently rare. We describe intranuclear coccidiosis diagnosed histologically in two radiated tortoises, three Travancore tortoises (Indotestudo forstenii), two leopard tortoises (Geochelone pardalis), one bowsprit tortoise (Chersina angulata), and one impressed tortoise (Manouria impressa). Infection was systemic and involved alimentary, urogenital, respiratory, lymphoid, endocrine, and integumentary systems. Trophozoites, meronts, merozoites, macrogametocytes, microgametocytes, and nonsporulated oocysts were seen histologically or by electron microscopy. Intracytoplasmic and extracellular stages of parasite development also were identified histologically. Sequencing of a coccidial 18S rRNA consensus polymerase chain reaction (PCR) product revealed a novel sequence that provided phylogenetic information and may be useful for further diagnostic test design. Intranuclear coccidiosis was associated with variable degrees of inflammation in all cases, was considered the cause of death in six tortoises, and was a substantial contributing factor to the cause of death in two tortoises.

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