Bruce C. Cropp

West Nile Virus Recombinant DNA Vaccine Protects Mouse and Horse from Virus Challenge and Expresses In Vitro a Noninfectious Recombinant Antigen That Can Be Used in Enzyme-Linked Immunosorbent Assays

ABSTRACT Introduction of West Nile (WN) virus into the United States in 1999 created major human and animal health concerns. Currently, no human or veterinary vaccine is available to prevent WN viral infection, and mosquito control is the only practical strategy to combat the spread of disease. Starting with a previously designed eukaryotic expression vector, we constructed a recombinant plasmid (pCBWN) that expressed the WN virus prM and E proteins. A single intramuscular injection of pCBWN DNA induced protective immunity, preventing WN virus infection in mice and horses. Recombinant plasmid-transformed COS-1 cells expressed and secreted high levels of WN virus prM and E proteins into the culture medium. The medium was treated with polyethylene glycol to concentrate proteins. The resultant, containing high-titered recombinant WN virus antigen, proved to be an excellent alternative to the more traditional suckling-mouse brain WN virus antigen used in the immunoglobulin M (IgM) antibody-capture and indirect IgG enzyme-linked immunosorbent assays. This recombinant antigen has great potential to become the antigen of choice and will facilitate the standardization of reagents and implementation of WN virus surveillance in the United States and elsewhere.

Fever and multisystem organ failure associated with 17D-204 yellow fever vaccination: a report of four cases

BACKGROUND In 1998, the US Centers for Disease Control and Prevention was notified of three patients who developed severe illnesses days after yellow fever vaccination. A similar case occurred in 1996. All four patients were more than 63 years old. METHODS Vaccine strains of yellow fever virus, isolated from the plasma of two patients and the cerebrospinal fluid of one, were characterised by genomic sequencing. Clinical samples were subjected to neutralisation assays, and an immunohistochemical analysis was done on one sample of liver obtained at biopsy. FINDINGS The clinical presentations were characterised by fever, myalgia, headache, and confusion, followed by severe multisystemic illnesses. Three patients died. Vaccine-related variants of yellow fever virus were found in plasma and cerebrospinal fluid of one vaccinee. The convalescent serum samples of two vaccinees showed antibody responses of at least 1:10240. Immunohistochemical assay of liver tissue showed yellow fever antigen in the Kuppfer cells of the liver sample. INTERPRETATION The clinical features, their temporal association with vaccination, recovery of vaccine-related virus, antibody responses, and immunohistochemical assay collectively suggest a possible causal relation between the illnesses and yellow fever vaccination. Yellow fever remains an important cause of illness and death in South America and Africa; hence, vaccination should be maintained until the frequency of these events is quantified.

Imported yellow fever in a United States citizen.

The last imported case of yellow fever seen in this country was in 1924. We report a case of yellow fever acquired by an American tourist who visited the jungles of Brazil along the Rio Negro and Amazon Rivers. The patient died 6 days after hospital admission and 10 days after his first symptoms appeared. Yellow fever virus was recovered from clinical specimens, and the isolate was genetically similar to the E genotype IIB of South American yellow fever viruses. This patients illness represents a case of vaccine-preventable death since he failed to be immunized with a recommended preexposure yellow fever vaccine.

Longitudinal Studies of West Nile Virus Infection in Avians, Yucatán State, México

Following the introduction of West Nile virus (WNV) into North America in 1999, surveillance for evidence of infection with this virus in migratory and resident birds was established in Yucatán State, México in March 2000. Overall, 8611 birds representing 182 species and 14 orders were captured and assayed for antibodies to WNV. Of these, 5066 (59%) birds were residents and 3545 (41%) birds were migrants. Twenty-one (0.24%) birds exhibited evidence of flavivirus infection. Of these, 8 birds had antibodies to WNV by epitope-blocking enzyme-linked immunosorbent assay. Five (0.06%) birds (gray catbird, brown-crested flycatcher, rose-breasted grosbeak, blue bunting and indigo bunting) were confirmed to have WNV infections by plaque reduction neutralization test. The WNV-infected birds were sampled in December 2002 and January 2003. The brown-crested flycatcher and blue bunting presumably were resident birds; the other WNV seropositive birds were migrants. These data provide evidence of WNV transmission among birds in the Yucatán Peninsula.

Serologic evidence of west nile virus infection in birds, Tamaulipas State, México

Following the introduction of West Nile virus (WNV) into North America in 1999, surveillance for WNV in migratory and resident birds was established in Tamaulipas State, northern México in December 2001. Overall, 796 birds representing 70 species and 10 orders were captured and assayed for antibodies to WNV. Nine birds had flavivirus-specific antibodies by epitope-blocking enzyme-linked immunosorbent assay; four were confirmed to have antibody to WNV by plaque reduction neutralization test. The WNV-infected birds were a house wren, mourning dove, verdin and Bewicks wren. The house wren is a migratory species; the other WNV-infected birds are presumably residents. The WNV-infected birds were all captured in March 2003. These data provide the first indirect evidence of WNV transmission among birds in northern México.

Nucleic Acid Amplification Assays for Detection of La Crosse Virus RNA

ABSTRACT We report the development of nucleic acid sequence-based amplification (NASBA) and quantitative real-time reverse transcription (RT)-PCR assays for the detection of La Crosse (LAC) virus in field-collected vector mosquito samples and human clinical samples. The sensitivities of these assays were compared to that of a standard plaque assay in Vero cells. The NASBA and quantitative real-time RT-PCR assays demonstrated sensitivities greater than that of the standard plaque assay. The specificities of these assays were determined by testing a battery of reference strain viruses, including representative strains of LAC virus and other arthropod-borne viruses. Additionally, these assays were used to detect LAC viral RNA in mosquito pool samples and human brain tissue samples and yielded results within less than 4 h. The NASBA and quantitative real-time RT-PCR assays detect LAC viral RNA in a sensitive, specific, and rapid manner; these findings support the use of these assays in surveillance and diagnostic laboratory systems.