Kristen A. Bernard

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.

An adenosine nucleoside inhibitor of dengue virus

Dengue virus (DENV), a mosquito-borne flavivirus, is a major public health threat. The virus poses risk to 2.5 billion people worldwide and causes 50 to 100 million human infections each year. Neither a vaccine nor an antiviral therapy is currently available for prevention and treatment of DENV infection. Here, we report a previously undescribed adenosine analog, NITD008, that potently inhibits DENV both in vitro and in vivo. In addition to the 4 serotypes of DENV, NITD008 inhibits other flaviviruses, including West Nile virus, yellow fever virus, and Powassan virus. The compound also suppresses hepatitis C virus, but it does not inhibit nonflaviviruses, such as Western equine encephalitis virus and vesicular stomatitis virus. A triphosphate form of NITD008 directly inhibits the RNA-dependent RNA polymerase activity of DENV, indicating that the compound functions as a chain terminator during viral RNA synthesis. NITD008 has good in vivo pharmacokinetic properties and is biologically available through oral administration. Treatment of DENV-infected mice with NITD008 suppressed peak viremia, reduced cytokine elevation, and completely prevented the infected mice from death. No observed adverse effect level (NOAEL) was achieved when rats were orally dosed with NITD008 at 50 mg/kg daily for 1 week. However, NOAEL could not be accomplished when rats and dogs were dosed daily for 2 weeks. Nevertheless, our results have proved the concept that a nucleoside inhibitor could be developed for potential treatment of flavivirus infections.

Infectious cDNA Clone of the Epidemic West Nile Virus from New York City

ABSTRACT We report the first full-length infectious clone of the current epidemic, lineage I, strain of West Nile virus (WNV). The full-length cDNA was constructed from reverse transcription-PCR products of viral RNA from an isolate collected during the year 2000 outbreak in New York City. It was cloned into plasmid pBR322 under the control of a T7 promoter and stably amplified in Escherichia coli HB101. RNA transcribed from the full-length cDNA clone was highly infectious upon transfection into BHK-21 cells, resulting in progeny virus with titers of 1 × 109 to 5 × 109 PFU/ml. The cDNA clone was engineered to contain three silent nucleotide changes to create a StyI site (C to A and A to G at nucleotides [nt] 8859 and 8862, respectively) and to knock out an EcoRI site (A to G at nt 8880). These genetic markers were retained in the recovered progeny virus. Deletion of the 3′-terminal 199 nt of the cDNA transcript abolished the infectivity of the RNA. The plaque morphology, in vitro growth characteristics in mammalian and insect cells, and virulence in adult mice were indistinguishable for the parental and recombinant viruses. The stable infectious cDNA clone of the epidemic lineage I strain will provide a valuable experimental system to study the pathogenesis and replication of WNV.

West Nile Virus Infection in Birds and Mammals

Abstract: West Nile virus (WNV) was found throughout New York State in year 2000. The epicenter was located in New York City with a high level of activity in the immediately surrounding counties, including Rockland, Westchester, Nassau, and Suffolk. During 2000, WNV testing was performed by the Wadsworth Center on 3,687 dead birds, representing 153 species, 46 families, and 18 orders. There were 1,203 WNV‐positive birds, representing 63 species, 30 families and 14 orders. The percentage of WNV‐positive birds was 33% for all birds tested throughout the state, with no significant difference in infection rates in migratory versus resident birds, although significantly more resident birds were submitted for testing. The highest apparent mortality for the entire season was observed in American crows in Staten Island, a location that also showed the highest minimal infection rate in Culex pipiens complex mosquitoes. Studies examining tissue tropism of WNV in corvids and noncorvids from the epicenter and from remote locations indicated that the kidney was the most consistently infected tissue in birds, regardless of level of infection. The brain was the next most consistently positive tissue. The differences in infection among the tissues were most apparent when low levels of virus were present. Experimental mouse inoculation demonstrated a classical flavivirus infection pattern.

Induction of Sterilizing Immunity against West Nile Virus (WNV), by Immunization with WNV-Like Particles Produced in Insect Cells

No specific vaccine for West Nile virus (WNV) is currently available for human use. In the present study, we describe the generation of WNV-like particles (WNV-LPs) in insect cells by use of recombinant baculoviruses expressing the WNV structural proteins prME or CprME. BALB/c mice immunized with purified WNV-LPs developed WNV-specific antibodies that had potent neutralizing activities. Mice immunized with prME-like particles (prME-LPs) showed no morbidity or mortality after challenge with WNV. Immunization with prME-LPs can induce sterilizing immunity without producing any evidence of viremia or viral RNA in the spleen or brain. These results suggest that WNV-LPs hold promise as a vaccine candidate for WNV infection.

An Attenuating Mutation in nsP1 of the Sindbis-Group Virus S.A.AR86 Accelerates Nonstructural Protein Processing and Up-Regulates Viral 26S RNA Synthesis

ABSTRACT The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.

West Nile virus—an old virus learning new tricks?

West Nile virus (WNV) has spread across the United States causing annual outbreaks since its emergence in 1999. Although severe disease develops only in about 1% of infections, WNV has claimed a total of 564 lives in the 5 years from 1999 to 2003. Observation of flaccid paralysis due to WNV infection at a higher incidence than previously documented and the devastating mortality recorded in infected American bird species triggered concerns about a potentially enhanced virulence of this virus. Here we summarize recent observations made during the American outbreaks regarding host range and transmission modes of WNV, and discuss epidemiological aspects of the emergence of this pathogen in the new habitat.

Bluetongue virus serotype 17 sequence variation associated with neutralization.

Bluetongue virus (BTV) is an insect-transmitted orbivirus of importance to the cattle and sheep industry. The VP2 protein, encoded by L2, contains neutralizing epitopes. Previously, a panel of neutralizing monoclonal antibodies (MAbs) to the BTV serotype 17 (BTV-17) prototype strain was generated and it was determined that the neutralization domain consists of three overlapping epitopes. Over 30 amino acid changes were found between a neutralized BTV-17 prototype strain and a non-neutralized BTV-17 198 strain. In this study, the L2 genes from eight additional strains, representing both the neutralized and non-neutralized groups of BTV-17, were sequenced to determine the degree of conservation of the previously characterized differences. Comparison of the deduced amino acid sequences showed that 91% (30/33) of the previously noted changes were conserved within each group. The sequence of the M5 gene that encodes VP5 was also examined, since this surface protein has also been shown to affect neutralization. No consistent changes were noted between the neutralized and non-neutralized groups of BTV-17 by analysis of the VP5 protein. Finally, the L2 sequences of five MAb neutralization escape mutants were determined to identify specific amino acids involved in neutralization and perhaps virulence. All five mutants contained 1–3 amino acid changes that were in close proximity to a previously described variable region. These amino acid changes likely define critical sites in the overlapping neutralization domains previously described. This is the first description of two BT virus populations that have distinct neutralization characteristics co-circulating in a defined geographical region.

Production of immunogenic West Nile virus-like particles using a herpes simplex virus 1 recombinant vector

West Nile virus (WNV) is a flavivirus that swept rapidly across North America in 1999, declined in prevalence, and then resurged in 2012. To date, no vaccine is available to prevent infection in the human population. Herpes simplex virus (HSV) replication-defective vaccine vectors induce a durable immunity characterized by strong antibody and CD8(+) T cell responses even in HSV-immune animals. In this study, a WNV protein expression cassette was optimized for virus-like particle (VLP) production in transfection studies, and the cassette was recombined into an HSV-1 d106-WNV virus vector, which produced extracellular VLPs, as confirmed by immunoelectron microscopy. Immunization of mice with the d106-WNV recombinant vector elicited a specific anti-WNV IgG response. This study highlights the flavivirus coding sequences needed for efficient assembly of virus-like particles. This information will facilitate generation of additional vaccine vectors against other flaviviruses including the recently emerged Zika virus.