Culex flavivirus isolates from mosquitoes in Guatemala.

A new strain of Culex flavivirus (family Flaviviridae, genus Flavivirus, CxFV), an insect virus first described in Japan, was isolated from adult Culex quinquefasciatus Say (Diptera: Culicidae) collected in 2006 from Izabal Department on the Caribbean coast of Guatemala. Mosquito pools were assayed for flavivirus RNA by using flavivirus group-specific primers that amplified a 720-bp region of the nonstructural (NS) 5 gene by standard reverse transcriptase-polymerase chain reaction. From 210 pools (1,699 mosquitoes), eight tested positive, and six of these mosquito pools produced virus isolates in Aedes albopictus Skuse C6/36 cells. Nucleotide sequence comparison of the eight flavivirus RNA-positive pools showed that there was 100% identity among them, and phylogenetic analysis of the NS5 and envelope gene regions indicated that they represent a strain of the recently described CxFV from Japan. This is the first report of an insect flavivirus from Central America.

West Nile virus in livestock and wildlife.

WN virus is one of the most ubiquitous arboviruses occurring over a broad geographical range and in a wide diversity of vertebrate host and vector species. The virus appears to be maintained in endemic foci on the African continent and is transported annually to temperate climates to the north in Europe and to the south in South Africa. Reports of clinical disease due to natural WN virus infection in wild or domestic animals were much less common than reports of infection (virus isolation or antibody detection). Until recently, records of morbidity and mortality in wild birds were confined to a small number of cases and infections causing encephalitis, sometimes fatal, in horses were reported infrequently. In the period 1996-2001, there was an increase in outbreaks of illness due to WN virus in animals as well as humans. Within the traditional range of WN virus, encephalitis was reported in horses in Italy in 1998 and in France in 2000. The first report of disease and deaths caused by WN virus infection in domestic birds was reported in Israel in 1997-1999, involving hundreds of young geese. In 1999 WN virus reached North America and caused an outbreak of encephalitis in humans in the New York area at the same time as a number of cases of equine encephalitis and deaths in American crows and a variety of other bird species, both North American natives and exotics. Multi-state surveillance for WN virus has been in place since April 2000 and has resulted in the detection of WN virus in thousands of dead birds from an increasing number of species in North America, and also in several species of mammals. The surveillance system that has developed in North America because of the utility of testing dead birds for the rapid detection of WN virus presence has been a unique integration of public health and wildlife health agencies. It has been suggested that the recent upsurge in clinical WN virus infection in wild and domestic animals as well as in humans may be related to the emergence of one or more new strains of WN virus. Virus isolated in New York in 1999 was found to be identical to that from Israel. It was alarming for WN virus to so easily invade the United States and surprising that it became established so quickly in the temperature climate of New York. Its persistence and rapid expansion in the United States leave a number of unanswered questions. New disease characteristics and patterns have occurred and more are evolving as WN virus further invades the western hemisphere. Additional animal research is needed to answer these questions. Some of the research needs include bird migration as a mechanism of virus dispersal, vector and vertebrate host relationships, virus persistence mechanisms, laboratory diagnosis, viral pathogenesis, risk factor studies, vaccine development, and WN virus impact on wildlife (CDC 2001a). Determination of the primary reservoir host species that are involved in the epidemiology of WN virus and the suitable sentinel species for active surveillance are also important research areas.

Serologic evidence for West Nile virus infection in birds in the New York City vicinity during an outbreak in 1999.

As part of an investigation of an encephalitis outbreak in New York City, we sampled 430 birds, representing 18 species in four orders, during September 13-23, 1999, in Queens and surrounding counties. Overall, 33% were positive for West Nile (WN) virus-neutralizing antibodies, and 0.5% were positive for St. Louis encephalitis virus-neutralizing antibodies. By county, Queens had the most seropositive birds for WN virus (50%); species with the greatest seropositivity for WN virus (sample sizes were at least six) were Domestic Goose, Domestic Chicken, House Sparrow, Canada Goose, and Rock Dove. One sampled bird, a captive adult Domestic Goose, showed signs of illness; WN virus infection was confirmed. Our results support the concept that chickens and House Sparrows are good arbovirus sentinels. This study also implicates the House Sparrow as an important vertebrate reservoir host.

Crow deaths as a sentinel surveillance system for West Nile virus in the northeastern United States, 1999.

In addition to human encephalitis and meningitis cases, the West Nile (WN) virus outbreak in the summer and fall of 1999 in New York State resulted in bird deaths in New York, New Jersey, and Connecticut. From August to December 1999, 295 dead birds were laboratory-confirmed with WN virus infection; 262 (89%) were American Crows (Corvus brachyrhynchos). The New York State Department of Health received reports of 17,339 dead birds, including 5,697 (33%) crows; in Connecticut 1,040 dead crows were reported. Bird deaths were critical in identifying WN virus as the cause of the human outbreak and defining its geographic and temporal limits. If established before a WN virus outbreak, a surveillance system based on bird deaths may provide a sensitive method of detecting WN virus.

Experimental infection of chickens as candidate sentinels for West Nile virus.

We evaluated the susceptibility, duration and intensity of viremia, and serologic responses of chickens to West Nile (WN) virus (WNV-NY99) infection by needle, mosquito, or oral inoculation. None of 21 infected chickens developed clinical disease, and all these developed neutralizing antibodies. Although viremias were detectable in all but one chicken, the magnitude (mean peak viremia <10,000 PFU/mL) was deemed insufficient to infect vector mosquitoes. WNV-NY99 was detected in cloacal and/or throat swabs from 13 of these chickens, and direct transmission of WNV-NY99 between chickens occurred once (in 16 trials), from a needle-inoculated bird. Nine chickens that ingested WNV-NY99 failed to become infected. The domestic chickens in this study were susceptible to WN virus infection, developed detectable antibodies, survived infection, and with one exception failed to infect cage mates. These are all considered positive attributes of a sentinel species for WN virus surveillance programs.

West Nile virus outbreak among horses in New York State, 1999 and 2000.

West Nile (WN) virus was identified in the Western Hemisphere in 1999. Along with human encephalitis cases, 20 equine cases of WN virus were detected in 1999 and 23 equine cases in 2000 in New York. During both years, the equine cases occurred after human cases in New York had been identified.

Exposure of domestic mammals to West Nile virus during an outbreak of human encephalitis, New York City, 1999.

We evaluated West Nile (WN) virus seroprevalence in healthy horses, dogs, and cats in New York City after an outbreak of human WN virus encephalitis in 1999. Two (3%) of 73 horses, 10 (5%) of 189 dogs, and none of 12 cats tested positive for WN virus-neutralizing antibodies. Domestic mammals should be evaluated as sentinels for local WN virus activity and predictors of the infection in humans.

Comparative West Nile virus detection in organs of naturally infected American Crows (Corvus brachyrhynchos).

Widespread deaths of American Crows (Corvus brachyrhynchos)were associated with the 1999 outbreak of West Nile (WN) virus in the New York City region. We compared six organs from 20 crow carcasses as targets for WN virus detection. Half the carcasses had at least one positive test result for WN virus infection. The brain was the most sensitive test organ; it was the only positive organ for three of the positive crows. The sensitivity of crow organs as targets for WN virus detection makes crow death useful for WN virus surveillance.

Arbovirus infection increases with group size.

Buggy Creek (BCR) virus is an arthropod-borne alphavirus that is naturally transmitted to its vertebrate host the cliff swallow (Petrochelidon pyrrhonota) by an invertebrate vector, namely the cimicid swallow bug (Oeciacus vicarius). We examined how the prevalence of the virus varied with the group size of both its vector and host. The study was conducted in southwestern Nebraska where cliff swallows breed in colonies ranging from one to 3700 nests and the bug populations at a site vary directly with the cliff swallow colony size. The percentage of cliff swallow nests containing bugs infected with BCR virus increased significantly with colony size at a site in the current year and at the site in the previous year. This result could not be explained by differences in the bug sampling methods, date of sampling, sample size of the bugs, age structure of the bugs or the presence of an alternate host, the house sparrow (Passer domesticus). Colony sites that were reused by cliff swallows showed a positive autocorrelation in the percentage of nests with infected bugs between year t and year t+1, but the spatial autocorrelation broke down for year t+2. The increased prevalence of BCR virus at larger cliff swallow colonies probably reflects the larger bug populations there, which are less likely to decline in size and lead to virus extinction. To the authors' knowledge this is the first demonstration of arbovirus infection increasing with group size and one of the few known predictive ecological relationships between an arbovirus and its vectors/hosts. The results have implications for both understanding the fitness consequences of coloniality for cliff swallows and understanding the temporal and spatial variation in arboviral epidemics.

Response to and efficacy of vaccination against eastern equine encephalomyelitis virus in emus.

OBJECTIVE: To evaluate humoral immune responses of emus vaccinated with commercially available equine polyvalent or experimental monovalent eastern equine encephalomyelitis (EEE) virus and western equine encephalomyelitis (WEE) virus vaccines and to determine whether vaccinated emus were protected against challenge with EEE virus. DESIGN: Cohort study. ANIMALS: 25 emus. PROCEDURE: Birds were randomly assigned to groups (n = 5/group) and vaccinated with 1 of 2 commercially available polyvalent equine vaccines, a monovalent EEE virus vaccine, or a monovalent WEE virus vaccine or were not vaccinated. Neutralizing antibody responses against EEE and WEE viruses were examined at regular intervals for up to 9 months. All emus vaccinated with the equine vaccines and 2 unvaccinated control birds were challenged with EEE virus. An additional unvaccinated bird was housed with the control birds to assess the possibility of contact transmission. RESULTS: All 4 vaccines induced detectable neutralizing antibody titers, and all birds vaccinated with the equine vaccines were fully protected against an otherwise lethal dose of EEE virus. Unvaccinated challenged birds developed viremia (> 10(9) plaque-forming units/ml of blood) and shed virus in feces, oral secretions, and regurgitated material. The unvaccinated pen-mate became infected in the absence of mosquito vectors, presumably as a result of direct virus transmission between birds. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that emus infected with EEE virus develop a high-titer viremia and suggest that they may serve as important virus reservoirs. Infected emus shed EEE virus in secretions and excretions, making them a direct hazard to pen-mates and attending humans. Commercially available polyvalent equine vaccines protect emus against EEE virus infection.

Interrupted blood-feeding by Culiseta melanura (Diptera: Culicidae) on European starlings.

To determine whether Culiseta melanura (Coquillett) mosquitoes tend to take multiple blood meals when birds of certain species serve as hosts, we compared the frequencies with which such mosquitoes fed upon caged starlings and robins and determined whether similar volumes of blood were imbibed from each. The blood of robins (Turdus migratorius) and European starlings (Sturnus vulgaris) was marked contrastingly by injecting birds with rubidium or cesium salts. Caged birds were placed together in a natural wetland setting overnight. Mosquitoes captured nearby on the following morning were analyzed for each of the elemental markers. Where marked robins and starlings were equally abundant, 43% of freshly engorged Cs. melanura fed on more than or equal to two hosts. More Cs. melanura fed on robins than on starlings. Individual mosquitoes tended to contain far more robin- than starling-associated marker, indicating that mosquitoes "feasted" on robins but only "nibbled" on starlings. Mosquitoes marked with both elements apparently fed meagerly on the starlings then abundantly on the robins. Our estimates of bloodmeal volume indicate that 85% of mosquitoes that fed on marked starlings obtained < 0.5 microliter of blood from them. We suggest that defensive behavior by starlings interrupts mosquito blood-feeding and that, in a communal roost of starlings, each mosquito will tend to feed on more than one bird, thereby promoting rapid transmission of such ornithonotic arboviruses as eastern equine encephalomyelitis virus and West Nile virus.

Serologic evidence for West Nile virus infection in birds in Staten Island, New York, after an outbreak in 2000.

After an outbreak of West Nile virus (WNV) infections in people, horses, and wildlife in Staten Island, NY, during the summer of 2000, we surveyed the bird population of the island for evidence of infection. Neutralizing antibodies were detected in 59 of 257 (23.0%) resident birds and none of 96 transient (migrating) birds sampled in early October. Species with the greatest seroprevalence were northern cardinal (Cardinalis cardinalis) (69.2%) and rock dove (Columba livia) (54.5%). House sparrows (Passer domesticus) and chickens (Gallus gallus) had lower than expected seroprevalences, 8.6% and 5.5%, respectively. The geographic distribution of seropositivity suggested focal transmission at several locations on the island. The concentration of seropositive birds among resident bird populations on Staten Island supports the concept that many birds survive WNV infection and that some of these play an important role in the WNV-bird-mosquito transmission cycle.

Bunyavirus infections in North Carolina white-tailed deer (Odocoileus virginianus).

We analyzed serum from white-tailed deer (Odocoileus virginianus) collected in southeastern North Carolina in 1991 for neutralizing antibodies to six mosquito-borne bunyaviruses (Lacrosse, Jamestown Canyon, Keystone,Cache Valley, Potosi, and Tensaw), including several of public health importance. Evidence was found for all six to be locally transmitted, although greatest seroprevalence was found for Potosi, Jamestown Canyon, and Cache Valley viruses.

West Nile virus surveillance using sentinel birds.

Captive and free-ranging birds have been used for decades as living sentinels in arbovirus surveillance programs. This review summarizes information relevant to selecting sentinel bird species for use in surveillance of West Nile (WN) virus. Although experience using avian sentinels for WN virus surveillance is limited, sentinels should be useful for both detecting and monitoring WN virus transmission; however, sentinel bird surveillance systems have yet to be adequately tested for use with the North American strain of WN virus. Captive chickens are typically used for arbovirus surveillance, but other captive species may be used as well. Serosurvey and experimental infection data suggest that both chickens and pigeons show promise as useful captive sentinels; both species were naturally exposed during the epizootics in New York City, 1999-2000, and both species develop antibodies after infection without becoming highly infectious to Culex pipiens vectors. Wild bird species that should be targeted for use as free-ranging sentinels include house sparrows and pigeons. The ideal wild bird should be determined locally on the basis of seroprevalence studies. Interpreting serological data generated from studies using free-ranging sentinel birds is complex, however. Sentinel bird monitoring sites should be selected in enzootic transmission foci. Several years of observation may be required for selection of effective sentinel monitoring sites.

Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay.

The authors report on the development and application of a rapid TaqMan assay for the detection of West Nile (WN) virus in a variety of human clinical specimens and field-collected specimens. Oligonucleotide primers and FAM- and TAMRA-labeled WN virus-specific probes were designed by using the nucleotide sequence of the New York 1999 WN virus isolate. The TaqMan assay was compared to a traditional reverse transcriptase (RT)-PCR assay and to virus isolation in Vero cells with a large number ( approximately 500) of specimens obtained from humans (serum, cerebrospinal fluid, and brain tissue), field-collected mosquitoes, and avian tissue samples. The TaqMan assay was specific for WN virus and demonstrated a greater sensitivity than the traditional RT-PCR method and correctly identified WN virus in 100% of the culture-positive mosquito pools and 98% of the culture-positive avian tissue samples. The assay should be of utility in the diagnostic laboratory to complement existing human diagnostic testing and as a tool to conduct WN virus surveillance in the United States.

Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York.

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.

Competence of American robins as reservoir hosts for Lyme disease spirochetes.

To explore the competence of American robins as a reservoir for Lyme disease spirochetes, we determined the susceptibility of these birds to tickborne spirochetes and their subsequent infectivity for larval vector ticks. Robins acquired infection and became infectious to almost all xenodiagnostic ticks soon after exposure to infected nymphal ticks. Although infectivity waned after 2 months, the robins remained susceptible to reinfection, became infectious again, and permitted repeated feeding by vector ticks. In addition, spirochetes passaged through birds retained infectivity for mammalian hosts. American robins become as infectious for vector ticks as do reservoir mice, but infectivity in robins wanes more rapidly.

West Nile virus in the United States: guidelines for detection, prevention, and control.

The epidemic/epizootic of West Nile (WN) encephalitis in the northeastern United States in the summer and fall of 1999 was an unprecedented event, underscoring the ease with which emerging infectious pathogens can be introduced into new geographic areas in today's era of rapid transportation and increased movement of people, animals, and commodities. This epidemic/epizootic and the increased frequency of other exotic pathogens being imported into the United States raises the issue of whether local, state, and national public health agencies are prepared to deal with epidemics/epizootics of vector-borne infectious diseases. The overwintering of WN virus and the epizootic transmission in the summer of 2000 reinforces the need to rebuild the public health infrastructure to deal with vector-borne diseases in this country. This article summarizes guidelines for surveillance, prevention, and control of WN virus that were drafted in December 1999 to help prepare state and local health departments for monitoring WN virus activity in the spring and summer of 2000 and also summarizes the data collected from those surveillance systems through September 2000.

West Nile viral encephalitis.

West Nile virus (WNV) has emerged in recent years in temperate regions of Europe and North America, presenting a threat to both public and animal health. The most serious manifestation of infection is fatal encephalitis in humans and horses, as well as mortality in certain domestic and wild birds. A recent development in the epizootiology of this mosquito-borne flavivirus was the occurrence of a severe outbreak in New York City and surrounding areas. During this outbreak, mortality was observed in humans, horses, a cat and numerous species of wild birds, particularly members of the family Corvidae (crows). The author reviews basic information and summarises recent developments in the epidemiology and epizootiology of WNV.