Gregory D. Ebel

Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus

Neutralization of West Nile virus (WNV) in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Using random mutagenesis and yeast surface display, we defined individual contact residues of 14 newly generated monoclonal antibodies against domain III of the WNV E protein. Monoclonal antibodies that strongly neutralized WNV localized to a surface patch on the lateral face of domain III. Convalescent antibodies from individuals who had recovered from WNV infection also detected this epitope. One monoclonal antibody, E16, neutralized 10 different strains in vitro, and showed therapeutic efficacy in mice, even when administered as a single dose 5 d after infection. A humanized version of E16 was generated that retained antigen specificity, avidity and neutralizing activity. In postexposure therapeutic trials in mice, a single dose of humanized E16 protected mice against WNV-induced mortality, and may therefore be a viable treatment option against WNV infection in humans.

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.

Dynamics of Flavivirus Infection in Mosquitoes

Abstract Mosquito-borne flaviviruses are emerging as the cause of some of the most serious and widespread arthropod-borne viral diseases in the world. Flavivirus outbreaks are influenced by intrinsic (e.g., viral strain, vector competence, host susceptibility) and extrinsic (e.g., temperature, rainfall, human land use) factors that affect mosquito biology in complex ways. The concept of vectorial capacity organizes and integrates these factors, enabling a clearer understanding of their complex interrelationships, how they affect transmission of vector-borne disease, and how they impact human health. This review focuses on the components of vectorial capacity, providing an update on our current understanding of how selected aspects of mosquito biology, such as longevity, feeding behavior, oviposition habits, and nutrition of adult and immature stages, impact flavivirus transmission cycles and human disease. The influence of extrinsic factors, such as temperature, rainfall, seasonal and multiyear weather patterns, and human behavior that affects mosquito biology, and therefore flavivirus transmission, is explored. Mechanisms of flaviviral perpetuation over adverse seasons and years are addressed. This review also discusses vector competence, recent advances in mosquito genetics, and vector control as they relate to flavivirus transmission and human health.

Culex restuans (Diptera: Culicidae) relative abundance and vector competence for West Nile Virus.

Abstract The abundance and vector competence of Culex restuans Theobald and Culex pipiens L. were compared to determine the relative importance of these species as West Nile virus (WNV) vectors in the northeastern United States. Abundance was estimated from egg raft collections at 12 sites in Albany, Suffolk, and Richmond counties, New York, during July, August, and September 2002 and 2003. Cx. restuans was more abundant than Cx. pipiens in both urban and rural areas, comprising 86% of 1,623 egg rafts collected. Vector competence for WNV was estimated after feeding on an artificial bloodmeal and in vitro transmission assays. The vector competence of the two species for WNV was similar, but the dynamics of infection seems to be mosquito species dependent. These findings suggest an important role for Cx. restuans in WNV transmission cycles in New York.

Impact of Trap Elevation on Estimates of Abundance, Parity Rates, and Body Size of Culex pipiens and Culex restuans (Diptera: Culicidae)

Abstract Trapping success, abundance, parity rate, and body size indices of Culex pipiens (L.) and Culex restuans Theobald, important vectors of West Nile virus (family Flaviviridae, genus Flavivirus, WNV), were determined for specimens captured from both ground level (≈1-m) and elevated (≈6- to 7-m) CO2-baited CDC miniature light traps. Mosquitoes were collected from six study sites in Albany County, New York, from mid-May to mid-October 2004. There was no significant difference in abundance or mean parity for either Cx. pipiens or Cx. restuans between the elevated and the ground-level traps. Mosquitoes collected in elevated traps tended to be larger than mosquitoes collected at ground level. Elevated traps captured significantly fewer mosquitoes per trap-night than did ground traps, but a greater proportion of those captured were Culex spp. Therefore, elevated traps more efficiently captured Cx. pipiens and Cx. restuans with similar reproductive status and body size and reduced the time necessary to sort and identify collections. These findings may be beneficial to researchers or surveillance programs focusing on Cx. pipiens or Cx. restuans.

Sentinel chickens as a surveillance tool for West Nile virus in New York City, 2000.

West Nile (WN) virus was first identified in the Western Hemisphere during an outbreak of encephalitis in New York City (NYC) in 1999. Prior to 1999, NYC had not had a locally acquired human arboviral infection since the 1800s (yellow fever), although eastern equine and St. Louis encephalitis have occurred in surrounding areas. There was also no existing surveillance system for arthropod-borne viruses and no citywide mosquito control program in NYC prior to 1999. The identification of this new agent prompted the NYC Department of Health to develop surveillance and control programs to prevent future outbreaks of WN and other arboviral diseases. Among the systems implemented for the 2000 mosquito season were sentinel chicken flocks. Experimental evidence has shown that domestic chickens are readily infected with isolates of NY-99 WN virus, shed virus for only a brief period of time, and experience a viremia high enough to infect mosquitoes for only about two days.1,2 The Centers for Disease Control and Prevention supported the use of sentinel chickens as a surveillance tool for the local presence and transmission of WN virus during the 2000 mosquito season.1