Molecular characterization of Haemaphysalis longicornis-borne rickettsiae, Republic of Korea and China.

Haemaphysalis longicornis, the cattle tick or bush tick, has an extended distribution throughout Asia and the Pacific region, including China, Russia, the Republic of Korea (ROK), Japan, Australia, New Zealand, and the South Pacific islands. It is an obligate ectoparasite found commonly on medium to large sized wild and domestic animals, with humans as an accidental host. Haemaphysalis longicornis transmits a number of pathogens, including severe fever with thrombocytopenia syndrome and tick-borne encephalitis viruses, bacteria, helminths, and protozoans, that impact on veterinary (wild and domestic animals) and human health. Surveys of rickettsial pathogens associated with H. longicornis from China, the ROK, and Japan have resulted in the discovery of more than 35 incompletely characterized molecular isolates of Rickettsia. In response to the increased global threat of tick-borne rickettsial diseases, H. longicornis collected in the ROK and China were assessed in our laboratory and two additional Rickettsia spp. isolates (ROK-HL727 and XinXian HL9) were identified. These agents were fully characterized by multilocus sequence typing using partial gene fragment sequences of rrs, gltA, ompA, ompB, and sca4. Phylogenetic comparisons of these Rickettsia isolates with known Rickettsia species and other molecular isolates identified from H. longicornis were performed to better understand their interrelationships. Phylogenetic analysis of the sequences from these 5 gene fragments showed that ROK-HL727 was closely related to rickettsial isolates of H. longicornis previously reported from China, the ROK and Japan, but distinct from any currently recognized Rickettsia species. It therefore qualifies genetically as a new species, introduced herein as Candidatus Rickettsia longicornii. The XinXian-HL9 isolate detected from China was determined to be genetically similar to the human pathogen Rickettsia heilongjiangensis. People living and working in areas where H. longicornis is endemic should be aware of the potential for rickettsial diseases.

Development of conventional and real-time reverse transcription polymerase chain reaction assays to detect Tembusu virus in Culex tarsalis mosquitoes.

Tembusu virus (TMUV) is an important emerging arthropod-borne virus that may cause encephalitis in humans and has been isolated in regions of southeast Asia, including Malaysia, Thailand, and China. Currently, detection and identification of TMUV are limited to research laboratories, because quantitative rapid diagnostic assays for the virus do not exist. We describe the development of sensitive and specific conventional and real-time quantitative reverse transcription polymerase chain reaction assays for detecting TMUV RNA in infected cell culture supernatant and Culex tarsalis mosquitoes. We used this assay to document the replication of TMUV in Cx. tarsalis, where titers increased 1,000-fold 5 days after inoculation. These assays resulted in the detection of virus-specific RNA in the presence of copurified mosquito nucleic acids. The use of these rapid diagnostic assays may have future applications for field pathogen surveillance and may assist in early detection, diagnosis, and control of the associated arthropod-borne pathogens.

Field detection of Tembusu virus in western Thailand by rt-PCR and vector competence determination of select culex mosquitoes for transmission of the virus.

Tembusu virus (TMUV; Ntaya serocomplex) was detected in two pools of mosquitoes captured near Sangkhlaburi, Thailand, as well as from sera from sentinel ducks from the same area. Although TMUV has been isolated from several mosquito species in Asia, no studies have ever shown competent vectors for this virus. Therefore, we allowed mosquitoes captured near Sangkhlaburi to feed on young chickens that had been infected with TMUV. These mosquitoes were tested approximately 2 weeks later to determine infection, dissemination, and transmission rates. Culex vishnui developed high viral titers after feeding on TMUV-infected chicks and readily transmitted virus to naïve chickens. In contrast, Cx. fuscocephala seemed less susceptible to infection, and more importantly, zero of five fuscocephala with a disseminated infection transmitted virus by bite, indicating a salivary gland barrier. These results provide evidence for the involvement of Culex mosquitoes in the transmission of TMUV in the environment.

Isolation and genomic characterization of Chaoyang virus strain ROK144 from Aedes vexans nipponii from the Republic of Korea.

During June 2003, mosquito surveillance was conducted at a US Army installation and a US Military training site 2 km south of the demilitarized zone, Republic of Korea. Mosquitoes were collected using Mosquito Magnets™, sorted to species, and assayed for the presence of arboviruses. From the 3,149 mosquitoes that were sorted into 126 pools, one Aedes vexans nipponii pool (out of 73 pools) tested positive for flavivirus RNA by reverse transcription-PCR. After isolation from C6/36 cell culture supernatant, the viral genome was sequenced and found to be 98.9% related to Chaoyang virus, a potential arthropod-specific flavivirus. This report details the first identification of Chaoyang virus in the Republic of Korea and highlights its relationship to other flaviviruses.

Evaluation of a field-portable DNA microarray platform and nucleic acid amplification strategies for the detection of arboviruses, arthropods, and bloodmeals.

Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors.

Development of three quantitative real-time PCR assays for the detection of Rickettsia raoultii, Rickettsia slovaca, and Rickettsia aeschlimannii and their validation with ticks from the country of Georgia and the Republic of Azerbaijan.

A previous surveillance study of human pathogens within ticks collected in the country of Georgia showed a relatively high infection rate for Rickettsia raoultii, R. slovaca, and R. aeschlimannii. These 3 spotted fever group rickettsiae are human pathogens: R. raoultii and R. slovaca cause tick-borne lymphadenopathy (TIBOLA), and R. aeschlimannii causes an infection characterized by fever and maculopapular rash. Three quantitative real-time polymerase chain reaction (qPCR) assays, Rraoul, Rslov, and Raesch were developed and optimized to detect R. raoultii, R. slovaca, and R. aeschlimannii, respectively, by targeting fragments of the outer membrane protein B gene (ompB) using species-specific molecular beacon or TaqMan probes. The 3 qPCR assays showed 100% specificity when tested against a rickettsiae DNA panel (n=20) and a bacteria DNA panel (n=12). The limit of detection was found to be at least 3 copies per reaction for all assays. Validation of the assays using previously investigated tick nucleic acid preparations, which included Rickettsia-free tick samples, tick samples that contain R. raoultii, R. slovaca, R. aeschlimannii, and other Rickettsia spp., gave 100% sensitivity for all 3 qPCR assays. In addition, a total of 65 tick nucleic acid preparations (representing 259 individual ticks) collected from the country of Georgia and the Republic of Azerbaijan in 2009 was tested using the 3 qPCR assays. R. raoultii, R. slovaca, and R. aeschlimannii were not detected in any ticks (n=31) from the Republic of Azerbaijan, but in the ticks from the country of Georgia (n=228) the minimal infection rate for R. raoultii and R. slovaca in Dermacentor marginatus was 10% and 4%, respectively, and for R. aeschlimannii in Haemaphysalis sulcata and Hyalomma spp. it was 1.9% and 20%, respectively.

Hantaan virus surveillance in small mammals at firing points 10 and 60, Yeoncheon, Gyeonggi Province, Republic of Korea.

We used epidemiological data and indirect fluorescent antibody tests to determine the Hantaan virus (HTNV) antibody-positive (Ab+) prevalence in small mammals captured at firing point 10 (FP-10) and firing point 60 (FP-60), Gyeonggi Province, near the demilitarized zone, Republic of Korea (ROK), from 2001 to 2005. We used these data, combined with the partial M segment amplified from HTNV recovered from lung tissues of Apodemus agrarius, to clarify the genetic diversity and phylogenetic relationships among HTNV strains in the ROK. Of the eight species of rodents and one insectivore species captured, A. agrarius accounted for 93.4% and 88.5% at FP-10 and FP-60, respectively. Only two species of rodents, A. agrarius and Micromys minutus, were HTNV Ab+. The overall HTNV Ab+ prevalence for A. agrarius captured at FP-10 and FP-60 was 23.3% (121/520) and 14.5% (94/647), respectively. The hantaviral reverse transcription-polymerase chain reaction-positive rate of Ab+ A. agrarius was 74.2% (167/215), and the phylogenetic trees, based on the 269-nucleotide G2-encoding M segment, demonstrated that HTNV strains from FP-10 and FP-60 were distantly segregated from HTNV of other geographic regions in Korea and China. These data are useful in the development of risk reduction strategies for the prevention of hantavirus infections among military personnel, especially during training or the event of hostilities, and civilian populations.

Hantaan virus surveillance targeting small mammals at Dagmar North Training Area, Gyeonggi Province, Republic of Korea, 2001-2005.

In response to a hemorrhagic fever with renal syndrome case in November 2000, a seasonal rodent-borne disease surveillance program was initiated at Dagmar North Training Area (DNTA), Gyeonggi Province, Republic of Korea. From April 2001-December 2005, 1,848 small mammals were captured. Apodemus agrarius accounted for 92.5%, followed by Mus musculus (3.6%), Crocidura lasiura (2.1%), and Microtus fortis (1.1%). Three species of rodents were found to be antibody-positive (Ab+) for Hantaan virus (HTNV): A. agrarius (22.3%), M. musculus (9.1%), and M. fortis (5.0%). Ab+ rates for A. agrarius increased with increasing weight (age), except for those weighing <10 g. The peak HTNV transmission period in Korea coincided with the peak reproductive potential of A. agrarius during the fall (August/September) surveys. HTNV strains from DNTA were distinct from HTNV strains from the People's Republic of China. From these studies, more accurate risk assessments can be developed to better protect personnel from rodent-borne diseases.

Ecological surveillance of small mammals at Dagmar North Training Area, Gyeonggi Province, Republic of Korea, 2001-2005.

A seasonal rodent-borne disease surveillance program was established at Dagmar North Training Area located near the demilitarized zone, Republic of Korea, from 2001 through 2005. Selected habitats surveyed included earthen banks separating rice paddies, fighting positions along a 5 m rock-faced earthen berm, and extensive tall grasses with various degrees of herbaceous and scrub vegetation associated with dirt roads, rice paddies, ditches, ponds, or the Imjin River. Of the nine species of small mammals captured, the striped field mouse (Apodemus agrarius), the primary reservoir for Hantaan virus, was the most frequently collected, representing 92.5% of the 1,848 small mammals captured. Males were captured similarly to females during the spring and summer seasons but were captured less frequently during the fall and winter seasons. Gravid rates were highest in the fall (25.5-57.3%) with the lowest rates during the summer (0.0-2.2%). Capture rates were the lowest along earthen banks separating rice paddies (5.5%) and highest in unmanaged tall grasses and crawling vegetation (15.3-43.5%). An increased knowledge of ecological factors that impact the abundance and distribution of small mammals and the associated ectoparasites and pathogens they harbor is critical for developing accurate disease risk assessments and mitigation strategies for preventing vector- and rodent-borne diseases among soldiers training in field environments.

Sequential Rift Valley fever outbreaks in eastern Africa caused by multiple lineages of the virus.

BACKGROUND: During the Rift Valley fever (RVF) epidemic of 2006-2007 in eastern Africa, spatial mapping of the outbreaks across Kenya, Somalia, and Tanzania was performed and the RVF viruses were isolated and genetically characterized. METHODS: Following confirmation of the RVF epidemic in Kenya on 19 December 2006 and in Tanzania on 2 February 2007, teams were sent to the field for case finding. Human, livestock, and mosquito specimens were collected and viruses isolated. The World Health Organization response team in Kenya worked with the WHO's polio surveillance team inside Somalia to collect information and specimens from Somalia. RESULTS: Seven geographical foci that reported hundreds of livestock and >25 cases in humans between December 2006 and June 2007 were identified. The onset of RVF cases in each epidemic focus was preceded by heavy rainfall and flooding for at least 10 days. Full-length genome analysis of 16 RVF virus isolates recovered from humans, livestock, and mosquitoes in 5 of the 7 outbreak foci revealed 3 distinct lineages of the viruses within and across outbreak foci. CONCLUSION: The findings indicate that the sequential RVF epidemics in the region were caused by multiple lineages of the RVF virus, sometimes independently activated or introduced in distinct outbreak foci.

Detection of Crimean-Congo hemorrhagic fever, Hanta, and sandfly fever viruses by real-time RT-PCR.

The development of sensitive and specific nucleic acid diagnostic assays for viral pathogens is essential for proper medical intervention. This chapter describes four fluorescence-based PCR assays to detect the Crimean-Congo Hemorrhagic Fever (CCHFV), Andes (ANDV), Hantaan (HANV), and Sandfly Fever Sicilian (SFSV) Viruses. These assays are based on species-specific hydrolysis probes targeting the nucleocapsid protein gene for CCHFV and SFSV and the glycoprotein gene for ANDV and HANV. All four assays were optimized for LightCycler 2.0 (Roche Diagnostics, Indianapolis, IN) or Ruggedized Advanced Pathogen Identification Device (R.A.P.I.D.; Idaho Technology Inc., Salt Lake City, UT). The assays were evaluated using the protocols described in the Subheading 3. The limits of detection were approximately 5, 2, 2, and 5 plaque-forming units (PFUs) for CCHFV, ANDV, HTNV, and SFSV assays, respectively. The sensitivity and specificity of the assays were evaluated with test panels that consisted of 20-60 known positive and 30-135 known negative samples, representing 7-34 genetically diverse bacterial and viral species. The CCHFV assay detected 59 out of the 60 positive samples and no false positives, resulting in 98.3% sensitivity at LOD of 5 PFU and 100% specificity. The ANDV and HTNV assays correctly identified all the positive samples with no false positive reactions; therefore, the sensitivity and specificity of these assays were determined to be 100% at LOD of 2 PFU. The SFSV assay missed three positive samples and cross-reacted with one of 48 negative samples, resulting in 95% sensitivity at LOD of 5 PFU and 98% specificity.

Rift Valley fever virus epidemic in Kenya, 2006/2007: the entomologic investigations.

In December 2006, Rift Valley fever (RVF) was diagnosed in humans in Garissa Hospital, Kenya and an outbreak reported affecting 11 districts. Entomologic surveillance was performed in four districts to determine the epidemic/epizootic vectors of RVF virus (RVFV). Approximately 297,000 mosquitoes were collected, 164,626 identified to species, 72,058 sorted into 3,003 pools and tested for RVFV by reverse transcription-polymerase chain reaction. Seventy-seven pools representing 10 species tested positive for RVFV, including Aedes mcintoshi/circumluteolus (26 pools), Aedes ochraceus (23 pools), Mansonia uniformis (15 pools); Culex poicilipes, Culex bitaeniorhynchus (3 pools each); Anopheles squamosus, Mansonia africana (2 pools each); Culex quinquefasciatus, Culex univittatus, Aedes pembaensis (1 pool each). Positive Ae. pembaensis, Cx. univittatus, and Cx. bitaeniorhynchus was a first time observation. Species composition, densities, and infection varied among districts supporting hypothesis that different mosquito species serve as epizootic/epidemic vectors of RVFV in diverse ecologies, creating a complex epidemiologic pattern in East Africa.

Serological surveillance of scrub typhus, murine typhus, and leptospirosis in small mammals captured at firing points 10 and 60, Gyeonggi province, Republic of Korea, 2001-2005.

Soldiers from the Republic of Korea and the United States conducting peacetime military operations at various training sites and multiple range complexes located near the demilitarized zone separating North and South Korea are exposed to rodents and their potentially disease-carrying ectoparasites. These diseases include scrub typhus, murine typhus, and leptospirosis. Many of the training sites are rural or semi-rural, surrounded or co-located with various forms of agriculture, and are infested with rodents and insectivores (as well as their ectoparasites), which are commonly found in association with unmanaged tall grasses, scrub, and crawling vegetation habitats. For 5 years, rodents and insectivores were collected seasonally (spring, summer, fall, and winter) at firing points 10 and 60 near the demilitarized zone and serologically tested for the presence of scrub typhus, murine typhus, and leptospirosis antibodies. Of the nine species of small mammals collected, Apodemus agrarius, the common striped field mouse and known reservoir of scrub typhus, was the most frequently collected (90.6%). Only four of the nine species captured, A. agrarius (60.9%), Micromys minutus (100%), Mus musculus (55.6%), and Rattus norvegicus (46.7%), were positive for scrub typhus. Of all the small mammals captured, only A. agrarius was positive for murine typhus (0.3%) and leptospirosis (1.3%). Seasonal and annual prevalence rates based on weight and sex are presented.

Hemorrhagic fever with renal syndrome in 4 US soldiers, South Korea, 2005.

Four US soldiers acquired hemorrhagic fever with renal syndrome while training near the Demilitarized Zone, South Korea, in 2005. Hantaan virus sequences were amplified by reverse transcription-PCR from patient serum samples and from lung tissues of striped field mice (Apodemus agrarius) captured at training sites. Epidemiologic investigations specified the ecology of possible sites of patient infection.

Development of field-based real-time reverse transcription-polymerase chain reaction assays for detection of Chikungunya and O'nyong-nyong viruses in mosquitoes.

Chikungunya (CHIK) and O'nyong-nyong (ONN) are important emerging arthropod-borne diseases. Molecular diagnosis of these two viruses in mosquitoes has not been evaluated, and the effects of extraneous mosquito tissue on assay performance have not been tested. Additionally, no real-time reverse transcription-polymerase chain reaction (RT-PCR) assay exists for detecting ONN virus (ONNV) RNA. We describe the development of sensitive and specific real-time RT-PCR assays for detecting CHIK and ONN viral RNA in mosquitoes, which have application for field use. In addition, we compared three methods for primer/probe design for assay development by evaluating their sensitivity and specificity. This comparison resulted in development of virus-specific assays that could detect less than one plaque-forming unit equivalent of each of the viruses in mosquitoes. The use of these assays will aid in arthropod-borne disease surveillance and in the control of the associated diseases.

Impact of phlebotomine sand flies on U.S. military operations at Tallil Air Base, Iraq: 4. Detection and identification of leishmania parasites in sand flies.

Sand flies collected between April 2003 and November 2004 at Tallil Air Base, Iraq, were evaluated for the presence of Leishmania parasites using a combination of a real-time Leishmania-generic polymerase chain reaction (PCR) assay and sequencing of a 360-bp fragment of the glucose-6-phosphate-isomerase (GPI) gene. A total of 2,505 pools containing 26,574 sand flies were tested using the real-time PCR assay. Leishmania DNA was initially detected in 536 pools; however, after extensive retesting with the real-time PCR assay, a total of 456 pools were considered positive and 80 were considered indeterminate. A total of 532 samples were evaluated for Leishmania GPI by sequencing, to include 439 PCR-positive samples, 80 PCR-indeterminate samples, and 13 PCR-negative samples. Leishmania GPI was detected in 284 samples that were sequenced, to include 281 (64%) of the PCR-positive samples and 3 (4%) of the PCR-indeterminate samples. Of the 284 sequences identified as Leishmania, 261 (91.9%) were L. tarentolae, 18 (6.3%) were L. donovani-complex parasites, 3 (1.1%) were L. tropica, and 2 were similar to both L. major and L. tropica. Minimum field infection rates were 0.09% for L. donovani-complex parasites, 0.02% for L. tropica, and 0.01% for the L. major/tropica-like parasite. Subsequent sequencing of a 600-bp region of the "Hyper" gene of 12 of the L. donovani-complex parasites showed that all 12 parasites were L. infantum. These data suggest that L. infantum was the primary leishmanial threat to U.S. military personnel deployed to Tallil Air Base. The implications of these findings are discussed.

Characterization of Imjin virus, a newly isolated hantavirus from the Ussuri white-toothed shrew (Crocidura lasiura).

Until recently, the single known exception to the rodent-hantavirus association was Thottapalayam virus (TPMV), a long-unclassified virus isolated from the Asian house shrew (Suncus murinus). Robust gene amplification techniques have now uncovered several genetically distinct hantaviruses from shrews in widely separated geographic regions. Here, we report the characterization of a newly identified hantavirus, designated Imjin virus (MJNV), isolated from the lung tissues of Ussuri white-toothed shrews of the species Crocidura lasiura (order Soricomorpha, family Soricidae, subfamily Crocidurinae) captured near the demilitarized zone in the Republic of Korea during 2004 and 2005. Seasonal trapping revealed the highest prevalence of MJNV infection during the autumn, with evidence of infected shrews' clustering in distinct foci. Also, marked male predominance among anti-MJNV immunoglobulin G antibody-positive Ussuri shrews was found, whereas the male-to-female ratio among seronegative Ussuri shrews was near 1. Plaque reduction neutralization tests showed no cross neutralization for MJNV and rodent-borne hantaviruses but one-way cross neutralization for MJNV and TPMV. The nucleotide and deduced amino acid sequences for the different MJNV genomic segments revealed nearly the same calculated distances from hantaviruses harbored by rodents in the subfamilies Murinae, Arvicolinae, Neotominae, and Sigmodontinae. Phylogenetic analyses of full-length S, M, and L segment sequences demonstrated that MJNV shared a common ancestry with TPMV and remained in a distinct out-group, suggesting early evolutionary divergence. Studies are in progress to determine if MJNV is pathogenic for humans.

Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents.

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.

Ecological surveillance of small mammals at Firing Points 10 and 60, Gyeonggi Province, Republic of Korea, 2001-2005.

Throughout Korea, small mammals are hosts to a number of disease-causing agents that pose a health threat to U.S. and Korean military forces while they conduct field-training exercises. A seasonal rodent-borne disease surveillance program was established at two firing points (FP), FP-10, and FP-60, and conducted over five years from 2001 through 2005 in response to hantavirus cases among U.S. soldiers. The ecology of these sites consisted primarily of tall grasses associated with semi-permanent and temporary water sources (drainage ditches and a small stream) and dry-land agriculture farming. Eight species of rodents and one species of insectivore were collected, including Apodemus agrarius, Micromys minutus, Mus musculus, Rattus norvegicus, Tscherskia triton, Microtus fortis, Myodes regulus, and Crocidura lasiura. The striped field mouse, A. agrarius, (primary reservoir for Hantaan virus, the causative agent of Korean hemorrhagic fever), was the most frequently collected, representing 90.6% of the 1,288 small mammals captured at both sites. Reported herein are the ecological parameters, seasonal population densities, and seasonal population characteristics associated with small mammals collected at two military training sites in the Republic of Korea.

Genetic analysis of South American eastern equine encephalomyelitis viruses isolated from mosquitoes collected in the Amazon Basin region of Peru.

Identifying viral isolates from field-collected mosquitoes can be difficult and time-consuming, particularly in regions of the world where numerous closely related viruses are co-circulating (e.g., the Amazon Basin region of Peru). The use of molecular techniques may provide rapid and efficient methods for identifying these viruses in the laboratory. Therefore, we determined the complete nucleotide sequence of two South American eastern equine encephalomyelitis viruses (EEEVs): one member from the Peru-Brazil (Lineage II) clade and one member from the Argentina-Panama (Lineage III) clade. In addition, we determined the nucleotide sequence for the nonstructural P3 protein (nsP3) and envelope 2 (E2) protein genes of 36 additional isolates of EEEV from mosquitoes captured in Peru between 1996 and 2001. The 38 isolates were evenly distributed between lineages II and III virus groupings. However, analysis of the nsP3 gene for lineage III strongly suggested that the 19 isolates from this lineage could be divided into two sub-clades, designated as lineages III and IIIA. Compared with North American EEEV (lineage I, GA97 strain), we found that the length of the nsP3 gene was shorter in the strains isolated from South America. A total of 60 nucleotides was deleted in lineage II, 69 in lineage III, and 72 in lineage IIIA. On the basis of the sequences we determined for South American EEEVs and those for other viruses detected in the same area, we developed a series of primers for characterizing these viruses.