Joseph G. Maffei

High-Throughput Detection of West Nile Virus RNA

ABSTRACT The recent outbreaks of West Nile virus (WNV) in the northeastern United States and other regions of the world have made it essential to develop an efficient protocol for surveillance of WNV. In the present report, we describe a high-throughput procedure that combines automated RNA extraction, amplification, and detection of WNV RNA. The procedure analyzed 96 samples in approximately 4.5 h. A robotic system, the ABI Prism 6700 Automated Nucleic Acid workstation, extracted RNA and set up reactions for real-time reverse transcription (RT)-PCR in a 96-well format. The robot extracted RNA with a recovery as efficient as that of a commercial RNA extraction kit. A real-time RT-PCR assay was used to detect and quantitate WNV RNA. Using in vitro transcribed RNA, we estimated the detection limit of the real-time RT-PCR to be approximately 40 copies of RNA. A standard RT-PCR assay was optimized to a sensitivity similar to that of the real-time RT-PCR. The standard assay can be reliably used to test a small number of samples or to confirm previous test results. Using internal primers in a nested RT-PCR, we increased the sensitivity by approximately 10-fold compared to that of the standard RT-PCR. The results of the study demonstrated for the first time that the use of an automated system for the purpose of large-scale viral RNA surveillance dramatically increased the speed and efficiency of sample throughput for diagnosis.

Detection by Enzyme-Linked Immunosorbent Assay of Antibodies to West Nile virus in Birds

We adapted an indirect immunoglobulin G enzyme-linked immunosorbent assay to facilitate studies of West Nile virus (WNV) and evaluated its application to taxonomically diverse avian species. Anti-WNV antibodies were detected in 23 bird species, including many exotic species, demonstrating its value in studies of WNV epizootiology.

West Nile Virus, Venezuela

To the Editor: West Nile virus (WNV; genus Flavivirus; family Flaviviridae) has been perpetuating in North America since 1999 (1). However, its status as a self-perpetuating pathogen in South America remains uncertain. Infected horses and birds have been reported in various Caribbean Islands, Mexico, and northern Central America (2,3). In South America, isolated reports of infected dead-end hosts (horses) have come from northern Colombia and Argentina but they lack evidence for infection in avian amplifying hosts (4,5). We report serologic evidence of establishment of WNV in South America. Serum samples from birds and horses from 33 locations in Venezuela (Appendix Table) were screened for immunoglobulin G (IgG) antibodies against WNV antigen by ELISA (6) and confirmed by plaque reduction neutralization test (PRNT) as previously described (7). The flavivirus generating the IgG response was identified by using the following criteria: 90% inhibition of virus in serum diluted at least 1:40 and 4-fold greater neutralizing antibody titer compared with closely related flaviviruses. IgG antibody against flavivirus was detected by ELISA in 14 of 576 resident birds, including 5 Turdus leucomelas, 3 Gallus gallus (captive), 2 Campylorhamphus trochilirostris, and 1 each of Elaenia flavogaster, Coereba flaveola, Thraupis palmarum, and Anisognathus flavinucha. WNV was confirmed as the etiologic agent of infection in 5 adult birds (3 T. leucomelas [pale-breasted thrush], 1 C. flaveola [bananaquit], and 1 G. gallus [domestic chicken] with the earliest collection date in February 2006); virus neutralization titers ranged from 80 to 320. One serum sample cross-reacted with other flaviviruses tested, with equivalent titers to WNV, Saint Louis encephalitis virus (SLEV), and Ilheus virus (ILHV) and was thus considered infected with an undetermined flavivirus. Seven serum samples were negative (antibody titers <20), and 1 sample was not tested because of insufficient sample volume. Antibody against flavivirus was detected by ELISA in 141 of 791 horses, and 34 (4.3%) were confirmed positive for WNV infection by PRNT; viral titers ≥640 occurred in half of these horses. The earliest collection date for a WNV-positive horse was February 2004 and the most recent was May 2006. Specific WNV-reactive equine serum samples were distributed in valley regions (prevalence 1.3%), savannah grasslands (2.4%), the western region of Zulia (0.4%) and the Central Lake Basin (0.3%). A total of 46 (5.8%) equine serum samples were positive for neutralizing antibody to SLEV, and 8 (1.0%) samples were positive for neutralizing antibodies to ILHV. Forty-nine samples neutralized at least 2 of the 3 viruses and were classified as undetermined flaviviruses. Serum samples from 2 horses were negative in neutralization assays; 2 others were not tested because of insufficient sample volume. WNV-infected resident birds, rather than an importation event, are the basis of establishment of WNV in South America. We hypothesize that ornithophilic mosquitoes (such as some Culex spp.), which are present in the area in consistently high numbers, acquired the virus through hematophagous feeding on recently infected, migrating birds. Once introduced to local mosquitoes, virus is amplified among susceptible resident birds fed upon by ornithophilic mosquitoes. This pattern allows perpetuation and subsequent establishment of virus in a continuous transmission cycle, as opposed to infection of dead-end hosts, e.g., horses. This is the first report of WNV infection in South American birds and definitive establishment of the virus in South America. We observed varying WNV seroprevalence rates in birds and horses across regions in Venezuela (Figure). These differences reflect the focal and stochastic nature of arbovirus transmission, which depends upon many ecologic factors. One possible explanation for the greater seroprevalence in the central and eastern llanos (savannahs) and valley regions, compared with the coastal western region of Zulia State (p<0.0001, by Pearson’s χ2 test) would be virus introduction by migrating birds by an eastern migration route. Figure Collection sites for West Nile virus (WNV) in Venezuela. Symbols represent results of tests for specific antibodies to WNV in serum samples of birds and horses (viral titers in a 90% plaque reduction neutralization test >40 and a 4-fold differential ... Existence of several closely related flaviviruses in the American tropics (8–10) may convey cross-protection in animals (e.g., ILHV and SLEV) or humans (dengue viruses, yellow fever virus), thereby potentially diminishing disease caused by a newly introduced flavivirus such as WNV. Although ILHV infection has not been detected in Venezuela, this flavivirus is prevalent in Brazil, Peru, French Guyana, Trinidad, and Colombia. Our study demonstrated widespread distribution of ILHV in Venezuela. Other South American flaviviruses, such as Bussuquara, Cacipacore, and Iguape, and as yet undiscovered viruses may also circulate in Venezuela. We encourage those involved in the public and animal health systems in Venezuela to consider zoonotic flaviviruses in the differential diagnoses of human and equine cases of encephalitis and to consider ecologic surveillance for zoonotic flaviviruses in mosquito and vertebrate host populations. We recommend monitoring blood and organ donations for flavivirus infections. Our study sheds light on flavivirus distribution in Venezuela. However, nothing else is known about the ecology of zoonotic flaviviruses in this country. Such knowledge will be essential for designing effective surveillance and control should these viruses be shown to cause human illnesses.

Land Use and West Nile Virus Seroprevalence in Wild Mammals

We examined West Nile virus (WNV) seroprevalence in wild mammals along a forest-to-urban gradient in the US mid-Atlantic region. WNV antibody prevalence increased with age, urbanization, and date of capture for juveniles and varied significantly between species. These findings suggest several requirements for using mammals as indicators of transmission.

Molecular Epidemiology of Eastern Equine Encephalitis Virus, New York

Southern strains are undergoing amplification, perpetuation, and overwintering in New York.

Phylogenetic and evolutionary analyses of St. Louis encephalitis virus genomes

St. Louis encephalitis virus belongs to the Japanese encephalitis virus serocomplex of the genus Flavivirus, family Flaviviridae. Since the first known epidemic in 1933, the virus has been isolated from a variety of geographical, temporal, and host origins. We have sequenced 10,236 nucleotides of the open reading frame (93.6% of the full-length genome) of 23 of these strains, and have used the sequences to conduct phylogenetic analyses, in order to investigate the forces shaping the evolution of St. Louis encephalitis virus. Contrary to previous reports, we found little evidence for recombination in these isolates. Most of the amino acid sites in the SLEV polyprotein appeared to be under negative selection, with some sites evolving neutrally, and a small number under positive selection. The strongest signal for positive selection was evident in the N-linked glycosylation site of the envelope protein. Intra-strain sequence variability within strains was observed at this site, and analyses suggested that it is under selection in vitro. Furthermore, using heterochronous sequence data, we estimated the most recent expansion of St. Louis encephalitis virus in North America to have happened towards the end of the 19th century.

Isolation of Bunyamwera serogroup viruses (Bunyaviridae, Orthobunyavirus) in New York state.

Abstract During routine arbovirus surveillance from 2000 to 2004 in New York state (NYS), 14,788 mosquito pools making up 36 species and nine genera were inoculated onto Vero cell cultures to test for a broad spectrum of viruses. Forty-six percent of viruses isolated in cell culture from species, excluding Culex pipiens L. and Culex restuans Theobald, were identified as Bunyamwera serogroup viruses. Here, we report the distribution and level of Bunyamwera activity in NYS detected during this period. We developed specific primers for Cache Valley virus (family Bunyaviridae, genus Orthobunyavirus, CVV) and Potosi virus (family Bunyaviridae, genus Orthobunyavirus, POTV), to facilitate rapid molecular identification of these viruses. Viral RNA was detected in 12 mosquito species by reverse transcription-polymerase chain reaction, with the majority isolated from Aedes trivittatus (Coquillet). We report the first POTV isolation in NYS and describe the development of specific primers to identify both POTV and CVV.

Unreliable Inactivation of Viruses by Commonly Used Lysis Buffers

There is a common assumption that viral lysis buffers are sufficient to render viruses noninfectious. This assumption has a significant impact on the way biological samples are processed, labeled, and handled for shipment. Several lysis buffers, including TRIzol, AVL, RLT, MagMAX, and easyMAG, were examined for their capacity to inactivate representative viruses from multiple genera, including alphavirus, bunyavirus, flavivirus, adenovirus, enterovirus, influenza B, and simplexvirus. Viruses were noninfectious following treatment with TRIzol, MagMAX, and easyMAG buffers, while some viruses were still viable in cell cultures following treatment with AVL and RLT buffers. These results indicate the need to further evaluate the expectation that lysis buffers render live viruses inactive, allowing handling and processing of these samples under low-level containment conditions.