Incidence of West Nile virus infection in the Dallas-Fort Worth metropolitan area during the 2012 epidemic.

The 2012 West Nile virus (WNV) epidemic was the largest since 2003 and the North Texas region was the most heavily impacted. We conducted a serosurvey of blood donors from four counties in the Dallas-Fort Worth area to characterize the epidemic. Blood donor specimens collected in November 2012 were tested for WNV-specific antibodies. Donors positive for WNV-specific IgG, IgM, and neutralizing antibodies were considered to have been infected in 2012. This number was adjusted using a multi-step process that accounted for timing of IgM seroreversion determined from previous longitudinal studies of WNV-infected donors. Of 4971 donations screened, 139 (2·8%) were confirmed WNV IgG positive, and 69 (1·4%) had IgM indicating infection in 2012. After adjusting for timing of sampling and potential seroreversion, we estimated that 1·8% [95% confidence interval (CI) 1·5-2·2] of the adult population in the Dallas-Fort Worth area were infected during 2012. The resulting overall estimate for the ratio of infections to reported WNV neuroinvasive disease (WNND) cases was 238:1 (95% CI 192-290), with significantly increased risk of WNND in older age groups. These findings were very similar to previous estimates of infections per WNND case, indicating no change in virulence as WNV evolved into an endemic infection in the United States.

Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern United States.

In late summer 1999, an outbreak of human encephalitis occurred in the northeastern United States that was concurrent with extensive mortality in crows (Corvus species) as well as the deaths of several exotic birds at a zoological park in the same area. Complete genome sequencing of a flavivirus isolated from the brain of a dead Chilean flamingo (Phoenicopterus chilensis), together with partial sequence analysis of envelope glycoprotein (E-glycoprotein) genes amplified from several other species including mosquitoes and two fatal human cases, revealed that West Nile (WN) virus circulated in natural transmission cycles and was responsible for the human disease. Antigenic mapping with E-glycoprotein-specific monoclonal antibodies and E-glycoprotein phylogenetic analysis confirmed these viruses as WN. This North American WN virus was most closely related to a WN virus isolated from a dead goose in Israel in 1998.

The emergence of West Nile virus in North America: ecology, epidemiology, and surveillance.

In late summer 1999, the first domestically acquired human cases of WN encephalitis were documented in the USA. Aggressive vector-control and public education efforts by state and local public health officials limited the extent of human involvement. The discovery of virus-infected, overwintering mosquitoes during the winter of 1999-2000, predicted renewed virus activity for the following spring, and prompted early season vector-control activities and disease surveillance efforts in NYC and the surrounding areas. These surveillance efforts were focused on identifying WN virus infections in birds and mosquitoes as predictors of the potential risk of transmission to humans. By the end of the 2000 mosquito-borne disease transmission season, WN virus activity had been documented as far north as the states of Vermont and New Hampshire, and as far south as the state of North Carolina. The ongoing impacts that WN virus will have on wildlife, domestic animal and human populations of the western hemisphere are not yet known. Plans are in place for public health officials and scientists to monitor the further expansion of WN virus with the establishment or enhancement of vector-borne disease surveillance and control programs throughout the eastern seaboard. The valuable lessons learned from the detection and response to the introduction of WN virus into NYC should prove useful if and when subsequent intrusions of new disease agents occur.

First field evidence for natural vertical transmission of West Nile virus in Culex univittatus complex mosquitoes from Rift Valley province, Kenya.

West Nile virus is a mosquito borne flavivirus endemic over a large geographic area including Africa, Asia, and the Middle East. Although the virus generally causes a mild, self-limiting febrile illness in humans, it has sporadically caused central nervous system infections during epidemics. An isolate of West Nile virus was obtained from a pool of four male Culex univittatus complex mosquitoes while we were conducting an investigation of Rift Valley fever along the Kenya-Uganda border in February-March 1998. This represents the first field isolation of West Nile virus from male mosquitoes and strongly suggests that vertical transmission of the virus occurs in the primary maintenance mosquito vector in Kenya. A phylogenetic analysis of the complete amino acid sequence of the viral envelope glycoprotein demonstrated a sister relationship with a Culex pipiens mosquito isolate from Romania made in 1996. This unexpected finding probably reflects the role of migratory birds in disseminating West Nile virus between Africa and Europe.

Direct sequencing of large flavivirus PCR products for analysis of genome variation and molecular epidemiological investigations.

The polymerase chain reaction (PCR) was used to amplify viral cDNAs from selected regions of dengue genomic RNA by using appropriate 'consensus' primers. DNA amplicons containing the structural genes from all 4 dengue serotypes were prepared and directly sequenced using dengue-virus-specific primers. This method can characterize reliably flavivirus field isolates at the molecular level without extensive virus propagation and molecular cloning, and will be a valuable tool for molecular epidemiological studies.

Detection of West Nile virus RNA and antibody in frozen plasma components from a voluntary market withdrawal during the 2002 peak epidemic.

BACKGROUND: The US West Nile virus (WNV) epidemic in the summer and fall of 2002 included the first documented cases of transfusion-transmitted WNV infection. In December 2002, the FDA supported a voluntary market withdrawal by the blood banking community of frozen blood components collected in WNV high-activity areas. At the time, the prevalence of viremia and serologic markers for WNV in the blood supply was undefined. STUDY DESIGN AND METHODS: In collaboration with America's Blood Centers, 1468 frozen plasma components (of approx. 60,000 frozen units voluntarily withdrawn from the market) were selectively retrieved from the peak epidemic regions and season (June 23, 2002-September 28, 2002). These units were unlinked, subaliquoted, and tested by WNV enzyme immunoassays (EIAs; Focus Technologies and Abbott Laboratories) and nucleic acid amplification tests (NATs; Gen-Probe Inc. and Roche Molecular Systems). RESULTS: Of the 1468 EIA results from Abbott and Focus, 7 were anti-immunoglobulin M (IgM)- and anti-immunoglobulin G (IgG)-reactive by both assays, 8 and 1 were IgM-only-reactive, and 8 and 23 were IgG-only-reactive, respectively. NAT by Gen-Probe and Roche Molecular Systems yielded one RNA-positive, antibody-negative unit containing approximately 440 RNA copies per mL. An additional 10-fold replicate NAT testing by Gen-Probe on 14 of 15 IgM-reactive specimens yielded 2 additional IgM- and IgG-reactive units with low-level viremia (i.e., 7/10 and 2/10 replicates tested reactive). CONCLUSION: The prevalence of acute (RNA-positive) and recent (IgM-seroreactive) WNV infections indicates that transfusion risk in high-risk areas could have been considerable and that voluntary market withdrawal of frozen components likely averted some WNV transfusion transmissions. The existence of very-low-level viremic units raises concerns, because WNV minipool NAT screening will miss such units and individual NAT may not completely correct this situation.

Nucleic acid sequence-based amplification assays for rapid detection of West Nile and St. Louis encephalitis viruses.

The development and application of nucleic acid sequence-based amplification (NASBA) assays for the detection of West Nile (WN) and St. Louis encephalitis (SLE) viruses are reported. Two unique detection formats were developed for the NASBA assays: a postamplification detection step with a virus-specific internal capture probe and electrochemiluminescence (NASBA-ECL assay) and a real-time assay with 6-carboxyfluorescein-labeled virus-specific molecular beacon probes (NASBA-beacon assay). The sensitivities and specificities of these NASBA assays were compared to those of a newly described standard reverse transcription (RT)-PCR and TaqMan assays for SLE virus and to a previously published TaqMan assay for WN virus. The NASBA assays demonstrated exceptional sensitivities and specificities compared to those of virus isolation, the TaqMan assays, and standard RT-PCR, with the NASBA-beacon assay yielding results in less than 1 h. These assays should be of utility in the diagnostic laboratory to complement existing diagnostic testing methodologies and as a tool in conducting flavivirus surveillance in the United States.

Molecular evolution and phylogeny of dengue-4 viruses.

Nucleotide sequences of the envelope protein genes of 19 geographically and temporally distinct dengue (DEN)-4 viruses were determined. Nucleic acid sequence comparison revealed that the identity among the DEN-4 viruses was greater than 92%. Similarity among deduced amino acids was between 96 and 100%; in most cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis generated phylogenetic trees, which indicated that geographically independent evolution of DEN-4 viruses had occurred. DEN-4 viruses were separated into two genetically distinct subtypes (genotypes). Genotype-1 contains viruses from the Philippines, Thailand and Sri Lanka; genotype-2 consists of viruses from Indonesia, Tahiti, the Caribbean Islands (Puerto Rico, Dominica) and Central and South America.

Detection of Colorado tick fever virus by using reverse transcriptase PCR and application of the technique in laboratory diagnosis.

Colorado tick fever (CTF) virus elicits an acute illness in humans, producing nonspecific flu-like symptoms and a biphasic fever in approximately 50% of patients. The disease is transmitted by the adult Rocky Mountain wood tick (Dermacentor andersoni), and therefore incidence is limited by the habitat and life cycle of that vector. The early symptoms of infection are difficult to distinguish from those of several other agents, especially Rickettsia rickettsii. Serologic testing is usually unable to provide evidence of CTF viral infection during the acute phase because of the late appearance of the various antibodies. Here we report the development and clinical application of a test to diagnose this disease during the acute stages. Oligonucleotide primers to the S2 segment of CTF (Florio) virus were made, and these were used in the amplification of a 528-bp fragment of DNA, transcribed from the double-stranded CTF virus RNA template by reverse transcriptase PCR. RNAs processed from 16 CTF virus isolates yielded similar results when analyzed on agarose gels. These were distinguishable from their antigenic relatives Eyach, S6-14-03, and T5-2092 and from other coltiviruses and an orbivirus but not from the antigenically distinct CTF virus-related isolate 720896. A mouse model demonstrated the utility of this method with whole-blood specimens, and CTF virus was successfully detected in human sera from the initial day of the onset of symptoms to 8 days later. The reverse transcriptase PCR method is a promising tool for the early diagnosis of CTF viral infection, or for ruling out CTF virus as the etiologic agent, in order to facilitate appropriate medical support.

Molecular evolution and epidemiology of dengue-3 viruses.

The nucleic acid sequences of the pre-membrane/membrane and envelope protein genes of 23 geographically and temporally distinct dengue (DEN)-3 viruses were determined. This was accomplished by reverse transcriptase-PCR amplification of the structural genes followed by automated DNA sequence analysis. Comparison of nucleic acid sequences revealed that similarity among the viruses was greater than 90%. The similarity among deduced amino acids was between 95% and 100%, and in many cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis allowed the generation of phylogenetic trees, demonstrating that geographically independent evolution of DEN-3 viruses had occurred. The DEN-3 viruses were separated into four genetically distinct subtypes. Subtype I consists of viruses from Indonesia, Malaysia, the Philippines and the South Pacific islands; subtype II consists of viruses from Thailand; subtype III consists of viruses from Sri Lanka, India, Africa and Samoa; subtype IV consists of viruses from Puerto Rico and the 1965 Tahiti virus. Phylogenetic analysis has also contributed to our understanding of the molecular epidemiology and worldwide distribution of DEN-3 viruses.

Inactivation of human immunodeficiency virus type 1 in blood samples stored as high-salt lysates.

Blood samples to be tested for the presence of parasite DNA by using specific DNA probes are routinely stored in our laboratory as high-salt lysates (HSL). To safeguard against the risk of accidental infection with etiological agents such as the human immunodeficiency virus type 1 (HIV-1) while manipulating large numbers of blood samples in preparation for DNA probing, we determined the residual infectivity of HIV-1 after exposure to HSL components. Both high-titer virus stocks or provirus-carrying cells, suspended either in tissue culture medium or freshly drawn blood, were completely inactivated upon contact with the HSL components. This was verified by the absence of any detectable HIV-1-specific antigen in the supernatants of long-term cultures and the absence of virus-specific DNA fragments after amplification by polymerase chain reaction with DNA from such cultures as target DNA. These results support the conclusion that the virus is in fact completely inactivated by contact with the HSL components, rendering blood specimens stored as HSL noninfectious in regard to HIV-1.

Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction.

We report on the development and application of a rapid assay for detecting and typing dengue viruses. Oligonucleotide consensus primers were designed to anneal to any of the four dengue virus types and amplify a 511-bp product in a reverse transcriptase-polymerase chain reaction (PCR). First, we produced a cDNA copy of a portion of the viral genome in a reverse transcriptase reaction in the presence of primer D2 and then carried out a standard PCR (35 cycles of heat denaturation, annealing, and primer extension) with the addition of primer D1. The resulting double-stranded DNA product of the RT-PCR was typed by two methods: dot blot hybridization of the 511-bp amplified product to dengue virus type-specific probes or a second round of PCR amplification (nested PCR) with type-specific primers, yielding DNA products the unique sizes of which were diagnostic for each dengue virus serotype. The accumulated data demonstrated that dengue viruses can be accurately detected and typed from viremic human serum samples.

Comparative West Nile virus detection in organs of naturally infected American Crows (Corvus brachyrhynchos).

Widespread deaths of American Crows (Corvus brachyrhynchos)were associated with the 1999 outbreak of West Nile (WN) virus in the New York City region. We compared six organs from 20 crow carcasses as targets for WN virus detection. Half the carcasses had at least one positive test result for WN virus infection. The brain was the most sensitive test organ; it was the only positive organ for three of the positive crows. The sensitivity of crow organs as targets for WN virus detection makes crow death useful for WN virus surveillance.

Phylogenetic relationships of dengue-2 viruses.

RNA oligonucleotide fingerprinting studies on a large number of virus isolates previously demonstrated considerable genetic variation in isolates of dengue (DEN)-2 serotype. We report the entire envelope (E) glycoprotein gene and deduced amino acid sequences of 16 DEN-2 viruses and the phylogenetic relationships of these, plus 17 additional published DEN E gene sequences. Comparison of DEN-2 E glycoprotein gene sequences revealed base substitutions scattered throughout the entire gene with as much as 22% sequence divergence. Aligned E glycoprotein amino acid sequences revealed the viruses differed by as much as 10%. There appeared to be constraints on the overall structure of the E protein to maintain biological function. Clusters of amino acid substitutions were present in the hydrophobic membrane anchor region at the carboxyl terminal end of the protein. Maximum parsimony analysis of the E gene sequences allowed construction of a phylogram indicating evolutionary relationships of the virus isolates within the DEN-2 serotype. Five genetic subtypes were identified. Phylogenetic relationships of the DEN-2 serotype and other flaviviruses based on E protein sequences reflected traditional antigenic and serologic classifications.

West Nile encephalitis in Israel, 1999: the New York connection.

We describe two cases of West Nile (WN) encephalitis in a married couple in Tel Aviv, Israel, in 1999. Reverse transcription-polymerase chain reaction performed on a brain specimen from the husband detected a WN viral strain nearly identical to avian strains recovered in Israel in 1998 (99.9% genomic sequence homology) and in New York in 1999 (99.8%). This result supports the hypothesis that the 1999 WN virus epidemic in the United States originated from the introduction of a strain that had been circulating in Israel.

West Nile virus outbreak among horses in New York State, 1999 and 2000.

West Nile (WN) virus was identified in the Western Hemisphere in 1999. Along with human encephalitis cases, 20 equine cases of WN virus were detected in 1999 and 23 equine cases in 2000 in New York. During both years, the equine cases occurred after human cases in New York had been identified.

Emergence of epidemic O'nyong-nyong fever in Uganda after a 35-year absence: genetic characterization of the virus.

O'nyong-nyong (ONN) virus is an alphavirus (family Togaviridae, genus Alphavirus) classified in the Semliki Forest virus (SFV) antigenic complex. ONN was initially isolated in northern Uganda in 1959 during the early stages of an explosive arbovirus epidemic in which > 2 million cases were reported. No additional epidemics or human isolations of ONN were reported until 1996, when it was isolated from an epidemic in southern Uganda. We report the complete nucleotide and deduced amino acid sequence of one of these 1996-1997 ONN isolates (SG650) and that of the related alphavirus Igbo Ora virus. The data indicate that the recent ONN virus isolate is closely related to the previously published ONN strain isolated in 1959. In addition, phylogenetic analysis of the sequence data reveals that Igbo Ora virus, previously thought to be a separate virus closely related to ONN and Chikungunya (CHIK), clearly is a strain of ONN. The sequence data also reveal that unlike the published ONN (1959) sequence, all ONN strains from the 1996-1997 epidemic possess a stop codon at the nsp3-nsp4 junction.

Rapid detection of west nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase-PCR assay.

The authors report on the development and application of a rapid TaqMan assay for the detection of West Nile (WN) virus in a variety of human clinical specimens and field-collected specimens. Oligonucleotide primers and FAM- and TAMRA-labeled WN virus-specific probes were designed by using the nucleotide sequence of the New York 1999 WN virus isolate. The TaqMan assay was compared to a traditional reverse transcriptase (RT)-PCR assay and to virus isolation in Vero cells with a large number ( approximately 500) of specimens obtained from humans (serum, cerebrospinal fluid, and brain tissue), field-collected mosquitoes, and avian tissue samples. The TaqMan assay was specific for WN virus and demonstrated a greater sensitivity than the traditional RT-PCR method and correctly identified WN virus in 100% of the culture-positive mosquito pools and 98% of the culture-positive avian tissue samples. The assay should be of utility in the diagnostic laboratory to complement existing human diagnostic testing and as a tool to conduct WN virus surveillance in the United States.

Outbreak of West Nile virus infection, Volgograd Region, Russia, 1999.

From July 25 to October 1, 1999, 826 patients were admitted to Volgograd Region, Russia, hospitals with acute aseptic meningoencephalitis, meningitis, or fever consistent with arboviral infection. Of 84 cases of meningoencephalitis, 40 were fatal. Fourteen brain specimens were positive in reverse transcriptase-polymerase chain reaction assays, confirming the presence of West Nile/Kunjin virus.

West Nile virus in overwintering Culex mosquitoes, New York City, 2000.

After the 1999 West Nile (WN) encephalitis outbreak in New York, 2,300 overwintering adult mosquitoes were tested for WN virus by cell culture and reverse transcriptase-polymerase chain reaction. WN viral RNA and live virus were found in pools of Culex mosquitoes. Persistence in overwintering Cx. pipiens may be important in the maintenance of WN virus in the northeastern United States.