Kiet A. Ngo

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.

Identification of Mosquito Bloodmeals Using Polymerase Chain Reaction (PCR) With Order-Specific Primers

Abstract A polymerase chain reaction (PCR) protocol was developed to identify host bloodmeals from mosquitoes. Primers for the cytochrome b gene were designed to distinguish between mammalian and avian bloodmeals and further differentiate among four avian orders: passeriformes, falconiformes, columbiformes, and galliformes. The assay was validated by testing tissues from 18 species of passeriformes, three species of falconiformes, three species of columbiformes, and two species of galliformes. American crows were distinguished from other passeriformes by restriction enzyme digestion. Host bloodmeals from engorged mosquitoes collected in New York State were identified to avian order level. PCR was able to detect the mosquito bloodmeal for up to 3 d after feeding on a quail. Significantly, these studies use order-specific primers in a single PCR test to identify mosquito bloodmeals.

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.

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.

Consequences of in vitro host shift for St. Louis encephalitis virus.

Understanding the potential for host range shifts and expansions of RNA viruses is critical to predicting the evolutionary and epidemiological paths of these pathogens. As arthropod-borne viruses (arboviruses) experience frequent spillover from their amplification cycles and are generalists by nature, they are likely to experience a relatively high frequency of success in a range of host environments. Despite this, the potential for host expansion, the genetic correlates of adaptation to novel environments and the costs of such adaptations in originally competent hosts are still not characterized fully for arboviruses. In the studies presented here, we utilized experimental evolution of St. Louis encephalitis virus (SLEV; family Flaviviridae, genus Flavivirus) in vitro in the Dermacentor andersoni line of tick cells to model adaptation to a novel invertebrate host. Our results demonstrated that levels of adaptation and costs in alternate hosts are highly variable among lineages, but also that significant fitness increases in tick cells are achievable with only modest change in consensus genetic sequence. In addition, although accumulation of diversity may at times buffer against phenotypic costs within the SLEV swarm, an increased proportion of variants with an impaired capacity to infect and spread on vertebrate cell culture accumulated with tick cell passage. Isolation and characterization of a subset of these variants implicates the NS3 gene as an important host range determinant for SLEV.

High levels of local inter- and intra-host genetic variation of West Nile virus and evidence of fine-scale evolutionary pressures

West Nile virus (WNV; Flaviviridae, Flavivirus) has been endemic in New York State (NYS) since its 1999 introduction, yet prevalence in Culex mosquitoes varies substantially over small spatial and temporal scales. It is unclear if viral genetics plays a role in this variability, as genetic and phenotypic characterization on local scales has generally been lacking. In addition, intrahost diversity of circulating strains have not been fully characterized despite the documented role of minority variants in viral fitness and virulence. In an effort to characterize WNV variability within epidemiologically relevant scales, we performed phylogenetic analyses on NYS isolates from 1999 to 2012. In addition, we performed full-genome, deep-sequencing and genetic analyses on 15 WNV strains isolated in 2012 from Cx. pipiens in an endemic focus of Suffolk County, NY. Our results indicate continued evolution and seasonal maintenance in NYS, yet also widespread mixing and high levels of genetic diversity within geographic foci and individual seasons. Well supported local clusters with shared amino acid differences were identified and suggest local evolutionary pressures and the potential for phenotypic variability. Intrahost diversity of focal isolates was also high, with polymorphism at levels >1.0% identified in approximately 10% of the WNV genome. Although most minority mutations were unique, mutational hotspots shared among local isolates were identified, particularly in C, NS1 and NS2A genes. The most polymorphic region, positions 3198-3388 of the NS1 gene, was comprised predominately of non-synonymous mutations, suggesting a selective advantage for amino acid diversity in this region.

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.