Virology 2006 May 16; (Read article online)

Mason PW, Shustov AV, Frolov I

To develop new vaccine candidates for flavivirus infections, we have engineered two flaviviruses, yellow fever virus (YFV) and West Nile virus (WNV), that are deficient in replication. These defective pseudoinfectious viruses (PIVs) lack a functional copy of the capsid (C) gene in their genomes and are incapable of causing spreading infection upon infection of cells both in vivo and in vitro. However, they produce extracellular E protein in form of secreted subviral particles (SVPs) that are known to be an effective immunogen. PIVs can be efficiently propagated in trans-complementing cell lines making high levels of C or all three viral structural proteins. PIVs derived from YFV and WNV, demonstrated very high safety and immunization produced high levels of neutralizing antibodies and protective immune response. Such defective flaviviruses can be produced in large scale under low biocontainment conditions and should be useful for diagnostic or vaccine applications.

Microbiol Immunol 2006; 50(5): 379-87 (Read article online)

Toriniwa H, Komiya T

We established a rapid, quantitative real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the envelope gene of Japanese encephalitis virus. The RTLAMP enabled us to detect the target product within 1 hr by only reacting reverse transcriptase and Bst DNA polymerase in a single tube at an isothermal temperature. The detection sensitivity of the RT-LAMP for Japanese encephalitis virus was 1 PFU, similar to that of conventional reverse transcription-polymerase chain reaction (RT-PCR). Flaviviruses of the Japanese encephalitis virus group, such as Dengue virus and West Nile virus, could not be detected. This confirmed the specificity of the RT-LAMP assay for Japanese encephalitis virus. A standard curve was constructed by plotting viral titer against the time for virus detection by the RT-LAMP, validating the quantitative accuracy of the assay. In addition, the amount of virus estimated by RT-LAMP was strongly correlated (r = 0.902) with that determined by plaque assay, a conventional method for virus quantification. These results indicate that the RT-LAMP assay established in this study is specific for Japanese encephalitis virus, and allows more rapid detection and quantification of the virus.

Int J Health Geogr 2006 May 16; 5(1): 21 (Read article online)

Tachiiri K, Klinkenberg B, Mak S, Kazmi J

ABSTRACT: BACKGROUND: West Nile virus (WNv) has recently emerged as a health threat to the North American population. After the initial disease outbreak in New York City in 1999, WNv has spread widely and quickly across North America to every contiguous American state and Canadian province, with the exceptions of British Columbia (BC), Prince Edward Island and Newfoundland. In this study we develop models of mosquito population dynamics for Culex tarsalis and C. pipiens, and create a spatial risk assessment of WNv prior to its arrival in BC by creating a raster-based mosquito abundance model using basic geographic and temperature data. Among the parameters included in the model are spatial factors determined from the locations of BC Centre for Disease Control mosquito traps (e.g., distance of the trap from the closest wetland or lake), while other parameters were obtained from the literature. Factors not considered in the current assessment but which could influence the results are also discussed. RESULTS: Since the model performs much better for C. tarsalis than for C. pipiens, the risk assessment is carried out using the output of C. tarsalis model. The result of the spatially-explicit mosquito abundance model indicates that the Okanagan Valley, the Thompson Region, Greater Vancouver, the Fraser Valley and southeastern Vancouver Island have the highest potential abundance of the mosquitoes. After including human population data, Greater Vancouver, due to its high population density, increases in significance relative to the other areas. CONCLUSIONS: Creating a raster-based mosquito abundance map enabled us to quantitatively evaluate WNv risk throughout BC and to identify the areas of greatest potential risk, prior to WNv introduction. In producing the map important gaps in our knowledge related to mosquito ecology in BC were identified, as well, it became evident that increased efforts in bird and mosquito surveillance are required if more accurate models and maps are to be produced. Access to real time climatic data is the key for developing a real time early warning system for forecasting vector borne disease outbreaks, while including social factors is important when producing a detailed assessment in urban areas.

Ecol Lett 2006 Jun; 9(6): 706-25 (Read article online)

Wonham MJ, Lewis MA, Rencławowicz J, van den Driessche P

This review synthesizes the conflicting outbreak predictions generated by different biological assumptions in host-vector disease models. It is motivated by the North American outbreak of West Nile virus, an emerging infectious disease that has prompted at least five dynamical modelling studies. Mathematical models have long proven successful in investigating the dynamics and control of infectious disease systems. The underlying assumptions in these epidemiological models determine their mathematical structure, and therefore influence their predictions. A crucial assumption is the host-vector interaction encapsulated in the disease-transmission term, and a key prediction is the basic reproduction number, R(0). We connect these two model elements by demonstrating how the choice of transmission term qualitatively and quantitatively alters R(0) and therefore alters predicted disease dynamics and control implications. Whereas some transmission terms predict that reducing the host population will reduce disease outbreaks, others predict that this will exacerbate infection risk. These conflicting predictions are reconciled by understanding that different transmission terms apply biologically only at certain population densities, outside which they can generate erroneous predictions. For West Nile virus, R(0) estimates for six common North American bird species indicate that all would be effective outbreak hosts.

Emerg Infect Dis 2006 Mar; 12(3): 514-6 (Read article online)

Sejvar JJ, Bode AV, Marfin AA, Campbell GL, Pape J, Biggerstaff BJ, Petersen LR

We report 1-year follow-up data from a longitudinal prospective cohort study of patients with West Nile virus-associated paralysis. As in the 4-month follow-up, a variety of recovery patterns were observed, but persistent weakness was frequent. Respiratory involvement was associated with considerable illness and death.

Emerg Infect Dis 2006 Apr; 12(4): 549-55 (Read article online)

Gould EA

Arboviruses have evolved a number of strategies to survive environmental challenges. This review examines the factors that may determine arbovirus emergence, provides examples of arboviruses that have emerged into new habitats, reviews the arbovirus situation in western Europe in detail, discusses potential arthropod vectors, and attempts to predict the risk for arbovirus emergence in the United Kingdom. We conclude that climate change is probably the most important requirement for the emergence of arthropodborne diseases such as dengue fever, yellow fever, Rift Valley fever, Japanese encephalitis, Crimean-Congo hemorrhagic fever, bluetongue, and African horse sickness in the United Kingdom. While other arboviruses, such as West Nile virus, Sindbis virus, Tahyna virus, and Louping ill virus, apparently circulate in the United Kingdom, they do not appear to present an imminent threat to humans or animals.

Emerg Infect Dis 2006 Mar; 12(3): 395-402 (Read article online)

Busch MP, Wright DJ, Custer B, Tobler LH, Stramer SL, Kleinman SH, Prince HE, Bianco C, Foster G, Petersen LR, Nemo G, Glynn SA

National blood donor screening for West Nile virus (WNV) RNA using minipool nucleic acid amplification testing (MP-NAT) was implemented in the United States in July 2003. We compiled national NAT yield data and performed WNV immunoglobulin M (IgM) testing in 1 WNV-epidemic region (North Dakota). State-specific MP-NAT yield, antibody seroprevalence, and the average time RNA is detectable by MP-NAT were used to estimate incident infections in 2003. WNV donor screening yielded 944 confirmed viremic donors. MP-NAT yield peaked in August with >0.5% of donations positive for WNV RNA in 4 states. Peak IgM seroprevalence for North Dakota was 5.2% in late September. The average time viremia is detectable by MP-NAT was 6.9 days (95% confidence interval [CI] 3.0-10.7). An estimated 735,000 (95% CI 322,000-1,147,000) infections occurred in 2003, with 256 (95% CI 112-401) infections per neuroinvasive case. In addition to preventing transfusion-transmitted WNV infection, donor screening can serve as a tool to monitor seasonal incidence in the general population.

Emerg Infect Dis 2006 Apr; 12(4): 556-61 (Read article online)

Kahn LH

Many of the emerging infectious diseases, including those caused by bioterrorist agents, are zoonoses. Since zoonoses can infect both animals and humans, the medical and veterinary communities should work closely together in clinical, public health, and research settings. In the clinical setting, input from both professions would improve assessments of the risk-benefit ratios of pet ownership, particularly for pet owners who are immunocompromised. In public health, human and animal disease surveillance systems are important in tracking and controlling zoonoses such as avian influenza virus, West Nile virus, and foodborne pathogens. Comparative medicine is the study of disease processes across species, including humans. Physician and veterinarian comparative medicine research teams should be promoted and encouraged to study zoonotic agent-host interactions. These efforts would increase our understanding of how zoonoses expand their host range and would, ultimately, improve prevention and control strategies.

Emerg Infect Dis 2006 Mar; 12(3): 375-80 (Read article online)

Zohrabian A, Hayes EB, Petersen LR

West Nile virus (WNV) was first detected in the Western Hemisphere in 1999 in New York City. From 1999 through 2004, >16,600 cases of WNV-related illnesses were reported in the United States, of which >7,000 were neuroinvasive disease and >600 were fatal. Several approaches are under way to develop a human vaccine. Through simulations and sensitivity analysis that incorporated uncertainties regarding future transmission patterns of WNV and costs of health outcomes, we estimated that the range of values for the cost per case of WNV illness prevented by vaccination was US 20,000 dollars-59,000 dollars(mean 36,000 dollars). Cost-effectiveness was most sensitive to changes in the risk for infection, probability of symptomatic illness, and vaccination cost. Analysis indicated that universal vaccination against WNV disease would be unlikely to result in societal monetary savings unless disease incidence increases substantially over what has been seen in the past 6 years.

Emerg Infect Dis 2006 Apr; 12(4): 618-23 (Read article online)

Bakonyi T

Two different West Nile virus (WNV) strains caused lethal encephalitis in a flock of geese and a goshawk in southeastern Hungary in 2003 and 2004, respectively. During the outbreak in geese, 14 confirmed human cases of WNV encephalitis and meningitis were reported in the same area. Sequencing of complete genomes of both WNV strains and phylogenetic analyses showed that the goose-derived strain exhibits closest genetic relationship to strains isolated in 1998 in Israel and to the strain that emerged in 1999 in the United States. WNV derived from the goshawk showed the highest identity to WNV strains of lineage 2 isolated in central Africa. The same strain reemerged in 2005 in the same location, which suggests that the virus may have overwintered in Europe. The emergence of an exotic WNV strain in Hungary emphasizes the role of migrating birds in introducing new viruses to Europe.

Emerg Infect Dis 2006 Mar; 12(3): 468-74 (Read article online)

Molaei G, Andreadis TG, Armstrong PM, Anderson JF, Vossbrinck CR

To evaluate the role of Culex mosquitoes as enzootic and epidemic vectors for WNV, we identified the source of vertebrate blood by polymerase chain reaction amplification and sequencing portions of the cytochrome b gene of mitochondrial DNA. All Cx. restuans and 93% of Cx. pipiens acquired blood from avian hosts; Cx. salinarius fed frequently on both mammals (53%) and birds (36%). Mixed-blood meals were detected in 11% and 4% of Cx. salinarius and Cx. pipiens, respectively. American robin was the most common source of vertebrate blood for Cx. pipiens (38%) and Cx. restuans (37%). American crow represented <1% of the blood meals in Cx. pipiens and none in Cx. restuans. Human-derived blood meals were identified from 2 Cx. salinarius and 1 Cx. pipiens. Results suggest that Cx. salinarius is an important bridge vector to humans, while Cx. pipiens and Cx. restuans are more efficient enzootic vectors in the northeastern United States.

J Wildl Dis 2006 Jan; 42(1): 92-106 (Read article online)

Ward MR, Stallknecht DE, Willis J, Conroy MJ, Davidson WR

Surveillance targeting dead wild birds, in particular American crows (Corvus brachyrhynchos), plays a critical role in West Nile virus (WNV) surveillance in the United States. Using crow decoy surrogates, detection and reporting of crow carcasses within urban and rural environments of DeKalb County, Georgia were assessed for potential biases that might occur in the county's WNV surveillance program. In each of two replicated trials, during July and September 2003, 400 decoys were labeled with reporting instructions and distributed along randomly chosen routes throughout designated urban and rural areas within DeKalb County. Information-theoretic methods were used to compare alternative models incorporating the effects of area and trial on probabilities of detection and reporting. The model with the best empirical support included the effects of area on both detection and reporting of decoys. The proportion of decoys detected in the urban area (0.605, SE=0.024) was approximately twice that of the rural area (0.293, SE=0.023), and the proportion of decoys reported in the urban area (0.273, SE=0.023) was approximately three times that of the rural area (0.103, SE=0.028). These results suggest that human density and associated factors can substantially influence dead crow detection and reporting and, thus, the perceived distribution of WNV. In a second and separate study, the persistence and fate of American crow and house sparrow (Passer domesticus) carcasses were assessed in urban and rural environments in Athens-Clarke, Madison, and Oconee counties, Georgia. Two replicated trials using 96 carcasses of each species were conducted during July and September 2004. For a portion of the carcasses, motion sensitive cameras were used to monitor scavenging species visits. Most carcasses (82%) disappeared or were decayed by the end of the 6-day study. Carcass persistence averaged 1.6 days in rural areas and 2.1 days in urban areas. We analyzed carcass persistence rates using a known-fate model framework in program MARK. Model selection based on Akaike's Information Criteria (AIC) indicated that the best model explaining carcass persistence rates included species and number of days of exposure; however, the model including area and number of days of exposure received approximately equal support. Model-averaged carcass persistence rates were higher for urban areas and for crow carcasses. Six mammalian and one avian species were documented scavenging upon carcasses. Dead wild birds could represent potential sources of oral WNV exposure to these scavenging species. Species composition of the scavenger assemblage was similar in urban and rural areas but "scavenging pressure" was greater in rural areas.

Eur J Epidemiol 2006; 21(4): 323-4 (Read article online)

Hubálek Z, Lukácová L, Halouzka J, Sirůcek P, Januska J, Precechtelová J, Procházka P

We report West Nile virus infection of the central nervous system in a 69-year-old man, residing in North Moravia (Czech Republic), who visited the USA from 6 July to 31 August 2002. He developed fever with fatigue at the end of his US stay, and was hospitalized in Ostrava after his return on 3 September with fever (up to 39.5 degrees C), fatigue, anorexia, moderate laryngotracheitis, dizziness, insomnia, blurred speech, and a marked bradypsychism. EEG demonstrated a slow bifrontal theta-delta activity, and CT of the brain a slight hydrocephalus. A significant increase of antibodies neutralizing West Nile virus was detected between the first (1:16) and second (1:256) blood serum sample. The patient recovered gradually and was released from hospital on 16 September. This is the first recorded human case of West Nile fever (WNF) imported to the Czech Republic. Nine similar cases of WNF import from the USA have already been reported in other European countries - France, Denmark, the Netherlands, and Germany.

J Wildl Dis 2006 Jan; 42(1): 133-41 (Read article online)

Travis EK, Vargas FH, Merkel J, Gottdenker N, Miller RE, Parker PG

The flightless cormorant (Phalacrocorax harrisi) is an endemic species of the Galápagos Islands, Ecuador. Health studies of the species have not previously been conducted. In August 2003, baseline samples were collected from flightless cormorant colonies on the islands of Isabela and Fernandina. Seventy-six birds, from nestlings to adults, were evaluated. Genetic sexing of 70 cormorants revealed 37 females and 33 males. Hematology assessment consisted of packed cell volume (n=19), leukograms (n=69), and blood smear evaluation (n=69). Microscopic evaluation of blood smears revealed microfilaria in 33% (23/69) of the cormorants. Plasma chemistries were performed on 46 cormorants. There was no significant difference in chemistry values or complete blood counts between male and female cormorants or between age groups. Based on a serologic survey to assess exposure to avian pathogens, birds (n=69) were seronegative for West Nile virus, avian paramyxovirus type 1 (Newcastle disease virus), avian paramyxovirus types 2 and 3, avian influenza, infectious bursal disease, infectious bronchitis, Marek's disease (herpes), reovirus, avian encephalomyelitis, and avian adenovirus type 2. Antibodies to avian adenovirus type 1 and Chlamydophila psittaci were found in 31% (21/68) and 11% (7/65) of flightless cormorants respectively. Chlamydophila psittaci was detected via polymerase chain reaction in 6% (2/33) of the cormorants. The overall negative serologic findings of this research suggest that the flightless cormorant is an immunologically naïve species, which may have a reduced capacity to cope with the introduction of novel pathogens.

J Wildl Dis 2006 Jan; 42(1): 107-14 (Read article online)

Allender MC, Mitchell MA, Phillips CA, Gruszynski K, Beasley VR

During the 2004 field season, blood was collected from Eastern massasauga rattlesnakes (Sistrurus catenatus catenatus) in the Carlyle Lake (Carlyle, Illinois, USA) and Allerton Park (Monticello, Illinois, USA) populations to derive baseline complete blood count and plasma biochemistry data and to assess the prevalence of antibodies to West Nile virus (WNV) and ophidian paramyxovirus (OPMV). Massasaugas were located for sampling through visual encounter surveys. Body weight, snout-vent length, total protein, globulins, sodium, and potassium were normally distributed among the survey population. Aspartate aminotransferase, creatine kinase, albumin, calcium, uric acid, white blood cell count, heterophils, lymphocytes, monocytes, eosinophils, and basophils were non-normally distributed within these animals. Female snakes had significantly shorter tail lengths; lower blood glucose, packed cell volumes, and absolute azurophil counts; and higher plasma calcium and phosphorus concentrations than did males. None of the snakes tested (n=21) were seropositive for WNV, whereas all (n=20) were seropositive for OPMV.

J Virol 2006 Jun; 80(11): 5338-48 (Read article online)

Shrestha B, Wang T, Samuel MA, Whitby K, Craft J, Fikrig E, Diamond MS

West Nile virus (WNV) causes a severe central nervous system (CNS) infection in humans, primarily in the elderly and immunocompromised. Prior studies have established an essential protective role of several innate immune response elements, including alpha/beta interferon (IFN-alpha/beta), immunoglobulin M, gammadelta T cells, and complement against WNV infection. In this study, we demonstrate that a lack of IFN-gamma production or signaling results in increased vulnerability to lethal WNV infection by a subcutaneous route in mice, with a rise in mortality from 30% (wild-type mice) to 90% (IFN-gamma(-/-) or IFN-gammaR(-/-) mice) and a decrease in the average survival time. This survival pattern in IFN-gamma(-/-) and IFN-gammaR(-/-) mice correlated with higher viremia and greater viral replication in lymphoid tissues. The increase in peripheral infection led to early CNS seeding since infectious WNV was detected several days earlier in the brains and spinal cords of IFN-gamma(-/-) or IFN-gammaR(-/-) mice. Bone marrow reconstitution experiments showed that gammadelta T cells require IFN-gamma to limit dissemination by WNV. Moreover, treatment of primary dendritic cells with IFN-gamma reduced WNV production by 130-fold. Collectively, our experiments suggest that the dominant protective role of IFN-gamma against WNV is antiviral in nature, occurs in peripheral lymphoid tissues, and prevents viral dissemination to the CNS.

J Wildl Dis 2006 Jan; 42(1): 14-22 (Read article online)

Clark L, Hall J, McLean R, Dunbar M, Klenk K, Bowen R, Smeraski CA

Populations of greater sage-grouse (Centrocercus urophasianus) have declined 45-80% in North America since 1950. Although much of this decline has been attributed to habitat loss, recent field studies have indicated that West Nile virus (WNV) has had a significant negative impact on local populations of grouse. We confirm the susceptibility of greater sage-grouse to WNV infection in laboratory experimental studies. Grouse were challenged by subcutaneous injection of WNV (10(3.2) plaque-forming units [PFUs]). All grouse died within 6 days of infection. The Kaplan-Meier estimate for 50% survival was 4.5 days. Mean peak viremia for nonvaccinated birds was 10(6.4) PFUs/ml (+/-10(0.2) PFUs/ml, standard error of the mean [SEM]). Virus was shed cloacally and orally. Four of the five vaccinated grouse died, but survival time was increased (50% survival=9.5 days), with 1 grouse surviving to the end-point of the experiment (14 days) with no signs of illness. Mean peak viremia for the vaccinated birds was 10(2.3) PFUs/ml (+/-10(0.6) PFUs/ml, SEM). Two birds cleared the virus from their blood before death or euthanasia. These data emphasize the high susceptibility of greater sage-grouse to infection with WNV.

Am J Trop Med Hyg 2006 May; 74(5): 908-914 (Read article online)

Farfán-Ale JA, Blitvich BJ, Marlenee NL, Loroño-Pino MA, Puerto-Manzano F, García-Rejón JE, Rosado-Paredes EP, Flores-Flores LF, Ortega-Salazar A, Chávez-Medina J, Cremieux-Grimaldi JC, Correa-Morales F, Hernández-Gaona G, Méndez-Galván JF, Beaty BJ

Surveillance for evidence of West Nile virus (WNV) infection in taxonomically diverse vertebrates was conducted in the Yucatan Peninsula of Mexico in 2003 and 2004. Sera from 144 horses on Cozumel Island, Quintana Roo State, 415 vertebrates (257 birds, 52 mammals, and 106 reptiles) belonging to 61 species from the Merida Zoo, Yucatan State, and 7 farmed crocodiles in Ciudad del Carmen, Campeche State were assayed for antibodies to flaviviruses. Ninety (62%) horses on Cozumel Island had epitope-blocking enzyme-linked immunosorbent assay (ELISA) antibodies to flaviviruses, of which 75 (52%) were seropositive for WNV by plaque reduction neutralization test (PRNT). Blocking ELISA antibodies to flaviviruses also were detected in 13 (3%) animals in the Merida Zoo, including 7 birds and 2 mammals (a jaguar and coyote) seropositive for WNV by PRNT. Six (86%) crocodiles in Campeche State had PRNT-confirmed WNV infections. All animals were healthy at the time of serum collections and none had a history of WNV-like illness.

J Virol 2006 Jun; 80(11): 5451-64 (Read article online)

Uchil PD, Kumar AV, Satchidanandam V

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.

J Wildl Dis 2006 Jan; 42(1): 1-13 (Read article online)

Nemeth N, Gould D, Bowen R, Komar N

We studied the effects of natural and/or experimental infections of West Nile virus (WNV) in five raptor species from July 2002 to March 2004, including American kestrels (Falco sparverius), golden eagles (Aquila chrysaetos), red-tailed hawks (Buteo jamaicensis), barn owls (Tyto alba), and great horned owls (Bubo virginianus). Birds were infected per mosquito bite, per os, or percutaneously by needle. Many experimentally infected birds developed mosquito-infectious levels of viremia (>10(5) WNV plaque forming units per ml serum) within 5 days postinoculation (DPI), and/ or shed virus per os or per cloaca. Infection of organs 15-27 days postinoculation was infrequently detected by virus isolation from spleen, kidney, skin, heart, brain, and eye in convalescent birds. Histopathologic findings varied among species and by method of infection. The most common histopathologic lesions were subacute myocarditis and encephalitis. Several birds had a more acute, severe disease condition represented by arteritis and associated with tissue degeneration and necrosis. This study demonstrates that raptor species vary in their response to WNV infection and that several modes of exposure (e.g., oral) may result in infection. Wildlife managers should recognize that, although many WNV infections are sublethal to raptors, subacute lesions could potentially reduce viability of populations. We recommend that raptor handlers consider raptors as a potential source of WNV contamination due to oral and cloacal shedding.