Elizabeth B. Kauffman

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.

Inhibition of Flavivirus Infections by Antisense Oligomers Specifically Suppressing Viral Translation and RNA Replication

ABSTRACT RNA elements within flavivirus genomes are potential targets for antiviral therapy. A panel of phosphorodiamidate morpholino oligomers (PMOs), whose sequences are complementary to RNA elements located in the 5′- and 3′-termini of the West Nile (WN) virus genome, were designed to anneal to important cis-acting elements and potentially to inhibit WN infection. A novel Arg-rich peptide was conjugated to each PMO for efficient cellular delivery. These PMOs exhibited various degrees of antiviral activity upon incubation with a WN virus luciferase-replicon-containing cell line. Among them, PMOs targeting the 5′-terminal 20 nucleotides (5′End) or targeting the 3′-terminal element involved in a potential genome cyclizing interaction (3′CSI) exhibited the greatest potency. When cells infected with an epidemic strain of WN virus were treated with the 5′End or 3′CSI PMO, virus titers were reduced by approximately 5 to 6 logs at a 5 μM concentration without apparent cytotoxicity. The 3′CSI PMO also inhibited mosquito-borne flaviviruses other than WN virus, and the antiviral potency correlated with the conservation of the targeted 3′CSI sequences of specific viruses. Mode-of-action analyses showed that the 5′End and 3′CSI PMOs suppressed viral infection through two distinct mechanisms. The 5′End PMO inhibited viral translation, whereas the 3′CSI PMO did not significantly affect viral translation but suppressed RNA replication. The results suggest that antisense PMO-mediated blocking of cis-acting elements of flavivirus genomes can potentially be developed into an anti-flavivirus therapy. In addition, we report that although a full-length WN virus containing a luciferase reporter (engineered at the 3′ untranslated region of the genome) is not stable, an early passage of this reporting virus can be used to screen for inhibitors against any step of the virus life cycle.

Virus Detection Protocols for West Nile Virus in Vertebrate and Mosquito Specimens

ABSTRACT The recent outbreaks of West Nile virus (WNV) infection in the northeastern United States and other regions of the world have made it essential to develop efficient, sensitive, and rapid protocols for virus surveillance. Laboratory testing is the backbone of any surveillance program. Protocols to detect the presence of WNV have been refined since 1999 for sensitivity, speed, efficiency, and specificity. This paper presents the protocols currently used by the New York State Department of Health to handle vertebrate and mosquito specimens that have been submitted for WNV testing to the Arbovirus Laboratories of the Wadsworth Center.

VecTest as Diagnostic and Surveillance Tool for West Nile Virus in Dead Birds

The VecTest WNV assay is adequate for diagnostic and surveillance purposes in American Crows, Blue Jays, and House Sparrows.

Assays to Detect West Nile Virus in Dead Birds

Using oral swab samples to detect West Nile virus in dead birds, we compared the Rapid Analyte Measurement Platform (RAMP) assay with VecTest and real-time reverse-transcriptase–polymerase chain reaction. The sensitivities of RAMP and VecTest for testing corvid species were 91.0% and 82.1%, respectively.

Vector Competence of New Zealand Mosquitoes for Selected Arboviruses

New Zealand (NZ) historically has been free of arboviral activity with the exception of Whataroa virus (Togaviridae: Alphavirus), which is established in bird populations and is transmitted by local mosquitoes. This naive situation is threatened by global warming, invasive mosquitoes, and tourism. To determine the threat of selected medically important arboviruses to NZ, vector competence assays were conducted using field collected endemic and introduced mosquito species. Four alphaviruses (Togaviridae): Barmah Forest virus, Chikungunya virus, Ross River virus, and Sindbis virus, and five flaviviruses (Flaviviridae): Dengue virus 2, Japanese encephalitis virus, Murray Valley encephalitis virus, West Nile virus, and Yellow fever virus were evaluated. Results indicate some NZ mosquito species are highly competent vectors of selected arboviruses, particularly alphaviruses, and may pose a threat were one of these arboviruses introduced at a time when the vector was prevalent and the climatic conditions favorable for virus transmission.

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.

Zika Virus Mosquito Vectors: Competence, Biology, and Vector Control

Zika virus (ZIKV) (Flaviviridae, Flavivirus) has become one of the most medically important mosquito-borne viruses because of its ability to cause microcephaly in utero and Guillain-Barré syndrome in adults. This virus emerged from its sylvatic cycle in Africa to cause an outbreak in Yap, Federated States of Micronesia in 2007, French Polynesia in 2014, and most recently South America in 2015. The rapid expansion of ZIKV in the Americas largely has been due to the biology and behavior of its vector, Aedes aegypti. Other arboviruses transmitted by Ae. aegypti include the 2 flaviviruses dengue virus and yellow fever virus and the alphavirus chikungunya virus, which are also (re)emerging viruses in the Americas. This mosquito vector is highly domesticated, living in close association with humans in urban households. Its eggs are desiccation resistant, and the larvae develop rapidly in subtropical and tropical environments. Climate warming is facilitating range expansion of Ae. aegypti, adding to the threat this mosquito poses to human health, especially in light of the difficulty controlling it. Aedes albopictus, another highly invasive arbovirus vector that has only been implicated in one country (Gabon), is an important vector of ZIKV, but because of its wide geographic distribution may become a more important vector in the future. This article discusses the historical background of ZIKV and the biology and ecology of these 2 vectors.

Rearing of Culex spp. and Aedes spp. Mosquitoes

Mosquito-transmitted pathogens cause major public health problems and contribute substantially to the global burden of disease. Aedes mosquitoes transmit dengue, Zika, yellow fever, and Chikungunya viruses; Culex mosquitoes transmit West Nile, Japanese encephalitis, and Saint Louis encephalitis viruses, among others. Experiments utilizing laboratory-reared colonized mosquitoes can address many issues such as vector biology, vector competence, vector-pathogen interaction, and vector control. The establishment of healthy and standardized mosquito colonies requires generation and implementation of protocols, attention to detail, and an understanding of the factors that affect mosquito fitness, such as temperature and humidity, nutrient quality and availability, population density, blood feeding and mating behavior, and egg-laying requirements. Here, we present a standard protocol for the rearing of Culex spp. and Aedes spp. mosquitoes and maintenance of the mosquito colony.

Detection Protocols for West Nile Virus in Mosquitoes, Birds, and Nonhuman Mammals

West Nile virus is the most widespread mosquito-borne virus in the world, and the most common cause of encephalitis in the USA. Surveillance for this medially important mosquito-borne pathogen is an important part of public health practice. Here we present protocols for testing environmental samples such as mosquitoes, nonvertebrate mammals, and birds for this virus, including RT-PCR, virus isolation in cell culture, and antigenic assays, as well as serologic assays for antibody detection.