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Background Japanese encephalitis (JE) virus is an important cause of neurological disease in Asia. JE vaccine is recommended for travelers with higher JE risk itineraries. Inactivated Vero cell culture-derived JE vaccine (JE-VC) is the only JE vaccine currently available in the United States. An inactivated mouse brain-derived JE vaccine (JE-MB) previously was available but production was discontinued. One JE-VC dose administered to adults previously vaccinated with ≥3 doses of JE-MB provides good short-term protection for at least one month, but data on longer-term protection are limited. We evaluated non-inferiority of the JE virus neutralizing antibody response at 12–23 months in JE-MB-vaccinated adults administered one JE-VC dose compared with JE vaccine-naive adults administered a JE-VC two-dose primary series. Methods We obtained archived sera from U.S. military personnel and performed a 50% plaque reduction neutralization test for anti-JE virus neutralizing antibodies. We compared the geometric mean titer (GMT) and seroprotection rate at 12–23 months after one JE-VC dose in previously JE-MB-vaccinated personnel and after the second JE-VC dose in previously JE vaccine-naive personnel. Non-inferiority was concluded if the lower bound of the two-sided 95% confidence interval (CI) of the GMT ratio in previously vaccinated to vaccine-naive personnel was >1/1.5. Results The GMT in previously JE-MB-vaccinated persons was 75 (95% CI 63–90) and in previously JE vaccine-naive persons was 12 (95% CI 11–14), and seroprotection rates were 94% (235/250) and 54% (135/250), respectively. The ratio of GMTs was 6.3 (95% CI: 5.0–7.7), satisfying the criterion for non-inferiority. Conclusions One JE-VC dose in previously JE-MB-vaccinated military personnel provides good protection for at least 1–2 years. The benefits of administration of a single JE-VC dose in previously JE-MB-vaccinated adults include a shorter time to completion of re-vaccination before travel, a decrease in the risk of adverse events, and reduced costs.

Infections of the central nervous system (CNS) are often acute, with significant morbidity and mortality. Routine diagnosis of such infections is limited in developing countries and requires modern equipment in advanced laboratories that may be unavailable to a number of patients in sub-Saharan Africa. We developed a TaqMan array card (TAC) that detects multiple pathogens simultaneously from cerebrospinal fluid. The 21-pathogen CNS multiple-pathogen TAC (CNS-TAC) assay includes two parasites ( Balamuthia mandrillaris and Acanthamoeba ), six bacterial pathogens ( Streptococcus pneumonia e, Haemophilus influenzae , Neisseria meningitidis , Mycoplasma pneumoniae , Mycobacterium tuberculosis , and Bartonella ), and 13 viruses (parechovirus, dengue virus, Nipah virus, varicella-zoster virus, mumps virus, measles virus, lyssavirus, herpes simplex viruses 1 and 2, Epstein-Barr virus, enterovirus, cytomegalovirus, and chikungunya virus). The card also includes human RNase P as a nucleic acid extraction control and an internal manufacturer control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). This CNS-TAC assay can test up to eight samples for all 21 agents within 2.5 h following nucleic acid extraction. The assay was validated for linearity, limit of detection, sensitivity, and specificity by using either live viruses (dengue, mumps, and measles viruses) or nucleic acid material (Nipah and chikungunya viruses). Of 120 samples tested by individual real-time PCR, 35 were positive for eight different targets, whereas the CNS-TAC assay detected 37 positive samples across nine different targets. The CNS-TAC assays showed 85.6% sensitivity and 96.7% specificity. Therefore, the CNS-TAC assay may be useful for outbreak investigation and surveillance of suspected neurological disease.

Summary Laboratory networks provide accurate and timely laboratory confirmation of infections, an essential component of disease surveillance. The World Health Organization coordinates global laboratory surveillance for many vaccine-preventable diseases. This report describes these laboratory networks and the challenges in sustaining and expanding global laboratory surveillance capacity., Laboratory networks were established to provide accurate and timely laboratory confirmation of infections, an essential component of disease surveillance systems. The World Health Organization (WHO) coordinates global laboratory surveillance of vaccine-preventable diseases (VPDs), including polio, measles and rubella, yellow fever, Japanese encephalitis, rotavirus, and invasive bacterial diseases. In addition to providing high-quality laboratory surveillance data to help guide disease control, elimination, and eradication programs, these global networks provide capacity-building and an infrastructure for public health laboratories. There are major challenges with sustaining and expanding the global laboratory surveillance capacity: limited resources and the need for expansion to meet programmatic goals. Here, we describe the WHO-coordinated laboratory networks supporting VPD surveillance and present a plan for the further development of these networks.

Commercial chikungunya virus (CHIKV)–specific IgM detection kits were evaluated at the Centers for Disease Control and Prevention (CDC), the Public Health Agency of Canada National Microbiology Laboratory, and the Caribbean Public Health Agency (CARPHA). The Euroimmun Anti-CHIKV IgM ELISA kit had ≥ 95% concordance with all three reference laboratory results. The limit of detection for low CHIK IgM+ samples, as measured by serial dilution of seven sera up to 1:12,800 ranged from 1:800 to 1:3,200. The Euroimmun IIFT kit evaluated at CDC and CARPHA performed well, but required more retesting of equivocal results. The InBios CHIKjj Detect MAC-ELISA had 100% and 98% concordance with CDC and CARPHA results, respectively, and had equal sensitivity to the CDC MAC-ELISA to 1:12,800 dilution in serially diluted samples. The Abcam Anti-CHIKV IgM ELISA had high performance at CARPHA, but at CDC, performance was inconsistent between lots. After replacement of the biotinylated IgM antibody controls with serum containing CHIKV-specific IgM and additional quality assurance/control measures, the Abcam kit was rereleased and reevaluated at CDC. The reformatted Abcam kit had 97% concordance with CDC results and limit of detection of 1:800 to 1:3,200. Two rapid tests and three other CHIKV MAC-ELISAs evaluated at CDC had low sensitivity, as the CDC CHIKV IgM in-house positive controls were below the level of detection. In conclusion, laboratories have options for CHIKV serological diagnosis using validated commercial kits.

Japanese encephalitis virus (JEV) is the leading cause of pediatric viral neurological disease in Asia. The JEV-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) in cerebrospinal fluid (CSF) and serum is the recommended method of laboratory diagnosis, but specificity of JEV MAC-ELISA can be low due to cross-reactivity. To increase the specificity of the commercially available JE Detect™ MAC-ELISA (JE Detect), a differential testing algorithm was developed in which samples tested by JE Detect with positive results were subsequently tested by the DEN Detect™ MAC-ELISA (DEN Detect) kit, and results of both tests were used to make the final interpretation. The testing algorithm was evaluated with a reference panel of serum and CSF samples submitted for confirmatory testing. In serum, the false Japanese encephalitis (JE) positive rate was reduced, but sequential testing in CSF resulted in reduced JE specificity, as true JEV+ CSF samples had positive results by both JE Detect and DEN Detect and were classified as JE− (dengue virus [DENV]+). Differential diagnosis of JE by sequential testing with JE Detect and DEN Detect increased specificity for JE in serum, but more data with CSF is needed to make a final determination on the usefulness of this testing algorithm for CSF.

The Centers for Disease Control and Prevention, Arbovirus Reference Collection (ARC) contains viral isolates from both environmental and human sources that are maintained in the laboratory through passage in suckling mouse brain and/or vertebrate and invertebrate cell culture. There has been increased concern regarding the effect of mycoplasma contamination on virus growth and its impact on research and phenotypic analysis. Therefore, quality control testing of virus preparations has become a routine part of the ARC quality assurance program. We compared the performance of three kits - the PCR Mycoplasma Detection Kit (ABM), the VenorGem Mycoplasma Detection Kit (Sigma), and the MycoAlert Mycoplasma Detection Kit (Lonza) - against a reference mycoplasma detection assay from the American Tissue Culture Collection (ATCC) using 744 virus preparations in the ARC, representing 721 unique viruses comprising twelve families and unclassified viruses. We found the ABM kit had the highest sensitivity and specificity, followed by the Sigma kit and Lonza kit, when compared to the ATCC kit. An increase in false positives was observed for the Lonza kit for preparations recently passaged in suckling mouse. Our data supports previously reported observations; that once introduced a specific species of mycoplasma is maintained within a lab.

Yellow fever virus (YFV) is endemic in tropical and sub-tropical regions of the world, with around 180,000 human infections a year occurring in Africa. Serologic testing is the chief laboratory diagnostic means of identifying an outbreak and to inform the decision to commence a vaccination campaign. The World Health Organization disseminates the reagents for YFV testing to African reference laboratories, and the US Centers for Disease Control and Prevention (CDC) is charged with producing and providing these reagents. The CDC M-antibody capture ELISA is a 2-day test, requiring titration of reagents when new lots are received, which leads to inconsistency in testing and wastage of material. Here we describe the development of a kit-based assay (YF MAC-HD) based upon the CDC method, that is completed in approximately 3.5 h, with equivocal samples being reflexed to an overnight protocol. The kit exhibits >90% accuracy when compared to the 2-day test. The kits were designed for use with a minimum of equipment and are stored at 4 °C, removing the need for freezing capacity. This kit is capable of tolerating temporary sub-optimal storage conditions which will ease shipping or power outage concerns, and a shelf life of >6 months was demonstrated with no deterioration in accuracy. All reagents necessary to run the YF MAC-HD are included in the kit and are single-use, with 8 or 24 sample options per kit. Field trials are envisioned for the near future, which will enable refinement of the method. The use of the YF MAC-HD is anticipated to reduce materials wastage, and improve the quality and consistency of YFV serologic testing in endemic areas.

Arboviruses are medically important pathogens that cause human disease ranging from a mild fever to encephalitis. Laboratory diagnosis is essential to differentiate arbovirus infections from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the virus-specific immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Antigen production in cell culture has largely replaced the use of suckling mice; however, the methods are not directly transferable. The development of a cell culture antigen production algorithm for nine arboviruses from the three main arbovirus families, Flaviviridae, Togaviridae, and Bunyaviridae, is described here. Virus cell culture growth and harvest conditions were optimized, inactivation methods were evaluated, and concentration procedures were compared for each virus. Antigen performance was evaluated by the MAC-ELISA at each step of the procedure. The antigen production algorithm is a framework for standardization of methodology and quality control; however, a single antigen production protocol was not applicable to all arboviruses and needed to be optimized for each virus.

Detection of chikungunya virus (CHIKV) or viral RNA is the primary laboratory test used to diagnose infection in serum collected 5 days of illness. Commercially available CHIKV IgM–detection assays include lateral flow rapid tests, IgM antibody capture enzyme-linked immunosorbent assays (MAC-ELISAs), and indirect immunofluorescence tests. Nine commercial CHIKV IgM detection assays were evaluated at 3 reference laboratories to provide guidance to public health diagnostic laboratories on their performance parameters. Sensitivity of the rapid tests and 3MAC-ELISAs was

The U.S. Culture Collection Network was formed in 2012 by a group of culture collection scientists and stakeholders in order to continue the progress established previously through efforts of an ad hoc group. The network is supported by a Research Coordination Network grant from the U.S. National Science Foundation (NSF) and has the goals of promoting interaction among collections, encouraging the adoption of best practices, and protecting endangered or orphaned collections. After prior meetings to discuss best practices, shared data, and synergy with genome programs, the network held a meeting at the U.S. Department of Agriculture (USDA)-Agricultural Research Service (ARS) National Center for Genetic Resources Preservation (NCGRP) in Fort Collins, Colorado in October 2015 specifically to discuss collections that are vulnerable because of changes in funding programs, or are at risk of loss because of retirement or lack of funding. The meeting allowed collection curators who had already backed up their resources at the USDA NCGRP to visit the site, and brought collection owners, managers, and stakeholders together. Eight formal collections have established off-site backups with the USDA-ARS, ensuring that key material will be preserved for future research. All of the collections with backup at the NCGRP are public distributing collections including U.S. NSF-supported genetic stock centers, USDA-ARS collections, and university-supported collections. Facing the retirement of several pioneering researchers, the community discussed the value of preserving personal research collections and agreed that a mechanism to preserve these valuable collections was essential to any future national culture collection system. Additional input from curators of plant and animal collections emphasized that collections of every kind face similar challenges in developing long-range plans for sustainability.

Chikungunya fever a re-emerging infection with expanding geographical boundaries, can mimic symptoms of other infections like dengue, malaria which makes the definitive diagnosis of the infection important. The present study compares the utility of four laboratory diagnostic methods viz. IgM capture ELISA, an in house reverse transcription PCR for the diagnosis of Chikungunya fever, TaqMan real-time PCR, and a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP). Out of the 70 serum samples tested, 29 (41%) were positive for Chikungunya IgM antibody by ELISA and 50 (71%) samples were positive by one of the three molecular assays. CHIKV specific nucleic acid was detected in 33/70 (47%) by reverse transcription PCR, 46/70 (66%) by TaqMan real-time PCR, and 43/70 (62%) by RT-LAMP assay. A majority of the samples (62/70; 89%) were positive by at least one of the four assays used in the study. The molecular assays were more sensitive for diagnosis in the early stages of illness (2-5 days post onset) when antibodies were not detectable. In the later stages of illness, the IgM ELISA is a more sensitive diagnostic test. In conclusion we recommend that the IgM ELISA be used as an initial screening test followed one of the molecular assays in samples that are collected in the early phase of illness and negative for CHIKV IgM antibodies. Such as approach would enable rapid confirmation of the diagnosis and implementation of public health measures especially during outbreaks.

Eastern equine encephalitis virus (EEEV) is a highly virulent, mosquito-borne alphavirus that causes severe and often fatal neurological disease in humans and horses in eastern North American, the Caribbean, and Mexico and throughout Central and South America. EEEV infection is diagnosed serologically by anti-EEEV-specific IgM detection, with confirmation by the plaque reduction neutralization test (PRNT), which is highly specific for alphaviruses. Live virus is used in the PRNT procedure, which currently requires biosafety level 3 containment facilities and select agent security in the case of EEEV. These requirements restrict the ability of public health laboratories to conduct PRNTs. Sindbis virus (SINV)/EEEV recombinant constructs have been engineered to express the immunogenic structural proteins from 2 wild-type EEEV strains in an attenuated form. These SINV/EEEVs, which are not classified as select agents, were evaluated as alternative diagnostic reagents in a PRNT using human, equine, and murine sera. The results indicate that the chimeric viruses exhibit specificity comparable to that of wild-type EEEV, with only a slight reduction in sensitivity. Considering their benefits in increased safety and reduced regulatory requirements, these chimeric viruses should be highly useful in diagnostic laboratories throughout the Americas.

The incidence rate of Japanese encephalitis (JE) in the People's Republic of China has decreased substantially with the wide use of JE vaccine, but the accuracy of JE reporting is uncertain. We established active surveillance for acute meningitis and encephalitis syndrome (AMES) in four prefectures in China during 2006–2008 and performed JE laboratory testing on AMES cases identified from six sentinel hospitals in each prefecture. We estimated JE incidence for each prefecture by applying age-adjusted and season-adjusted JE positivity rates from sentinel hospitals to the total AMES resident cases. We identified 4,513 AMES cases, including 3,561 (79%) among residents of four prefectures. Among 2,294 AMES cases from sentinel hospitals, we identified 213 (9.2%) laboratory-confirmed JE cases. Adjusted estimates of JE incidence per 100,000 persons ranged from 0.08 in Shijiazhuang to 1.58 in Guigang. Active surveillance and laboratory confirmation provides a better estimate of the actual JE disease burden and should be used to further refine JE prevention strategies.

Acute febrile illnesses comprise the majority of the human disease burden in sub-Saharan Africa. We hypothesized that arboviruses comprised a considerable proportion of undiagnosed febrile illnesses in Guinea and sought to determine the frequency of arboviral disease in two hospitals there. Using a standard case definition, 47 suspected cases were detected in approximately 4 months. Immunoglobulin M antibody capture enzyme-linked immunosorbent assays and plaque-reduction neutralization assays revealed that 63% (30/47) of patients were infected with arboviruses, including 11 West Nile, 2 yellow fever, 1 dengue, 8 chikungunya, and 5 Tahyna infections. Except for yellow fever, these are the first reported cases of human disease from these viruses in Guinea and the first reported cases of symptomatic Tahyna infection in Africa. These results strongly suggest that arboviruses circulate and are common causes of disease in Guinea. Improving surveillance and laboratory capacity for arbovirus diagnoses will be integral to understanding the burden posed by these agents in the region.

The plaque reduction neutralization test (PRNT) is a specific serological test used to identify and confirm arbovirus infection in diagnostic laboratories and monitor immunological protection in vaccine recipients. Wild-type (wt) viruses used in the PRNT may be difficult to grow and plaque titrate, such as the dengue viruses (DENV), and/or may require biosafety level 3 (BSL3) containment, such as West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). These requirements preclude their use in diagnostic laboratories with only BSL2 capacity. In addition, wt JEV falls under the jurisdiction of the select-agent program and can be used only in approved laboratories. The chimeric vaccine viruses ChimeriVax-WNV and -SLEV have previously been shown to elicit antibody reactivity comparable to that of parental wt WNV and SLEV. ChimeriVax viruses provide advantages for PRNT, as follows: they grow more rapidly than most wt flaviviruses, produce large plaques, require BSL2 conditions, and are not under select-agent restrictions. We evaluated the ChimeriVax-DENV serotype 1 (DENV1), -DENV2, -DENV3, -DENV4, and -JEV for use in PRNT on sera from DENV- and JEV-infected patients and from JEV vaccine recipients. Serostatus agreement was 100% between the ChimeriVax-DENV serotypes and wt prototype DENV and 97% overall with ChimeriVax-JEV compared to prototype Nakayama JEV, 92% in a subgroup of JEV vaccine recipients, and 100% in serum from encephalitis patients naturally infected with JEV. ChimeriVax-DENV and -JEV plaque phenotype and BSL2 requirements, combined with sensitive and specific reactivity, make them good substitutes for wt DENV and JEV in PRNT in public health diagnostic laboratories.

Since the introduction of West Nile virus into the United States in 1999, there has been a greater awareness of arboviruses, consequently, diagnostic testing for West Nile virus and other arboviruses has increased both in U.S. and international public health laboratories. The Centers for Disease Control and Prevention/Division of Vector-Borne Infectious Diseases/Arbovirus Diagnostic and Reference Laboratory produces and provides the serodiagnostic reagents which are not available commercially. Reagents needed to conduct the enzyme-linked immunoassay (ELISA) include a virus-specific non-infectious antigen. Antigens for Japanese encephalitis and the four dengue virus serotypes have been developed from COS-1 transformed cells that secrete non-infectious, virus-like particles into the cell culture supernatant. Four methods for concentrating the supernatant are discussed here. The methods are ultracentrifugation, polyethylene glycol precipitation, and two ultrafiltration methods: the Stirred Cell (Millipore Corporation, Billerica, MA) and the Pellicon 2 (Millipore Corporation, Billerica, MA). Ultracentrifugation and the Pellicon 2 ultrafiltration system produced antigen at a sufficient concentration for use in the ELISA. Large volumes were concentrated in a shorter time in the Pellicon 2 ultrafiltration system. An additional filtration step was necessary to produce antigen of sufficient concentration for use in the microsphere-based immunoassay, which requires antigen concentrated an additional 10 times.

The emergence of chikungunya virus in the Americas means the affected population is at risk of developing severe, chronic, rheumatologic disease, even months after acute infection. Accurate diagnostic methods for past infections are essential for differential diagnosis and consequence management. This study evaluated three commercially-available chikungunya Immunoglobulin G immunoassays by comparing them to an in-house Enzyme-Linked ImmunoSorbent Assay conducted by the Centers for Disease Control and Prevention (Atlanta, Georgia, United States). Results showed sensitivity and specificity values ranging from 92.8% – 100% and 81.8% – 90.9%, respectively, with a significant number of false-positives ranging from 12.5% – 22%. These findings demonstrate the importance of evaluating commercial kits, especially regarding emerging infectious diseases whose medium and long-term impact on the population is unclear.

BACKGROUND: Japanese encephalitis (JE) virus (JEV) is a mosquito-borne flavivirus found across Asia that is closely related to West Nile virus. There is no known antiviral treatment for any flavivirus. Results from in vitro studies and animal models suggest intravenous immunoglobulin (IVIG) containing virus-specific neutralizing antibody may be effective in improving outcome in viral encephalitis. IVIG's anti-inflammatory properties may also be beneficial. METHODOLOGY/PRINCIPAL FINDINGS: We performed a pilot feasibility randomized double-blind placebo-controlled trial of IVIG containing anti-JEV neutralizing antibody (ImmunoRel, 400mg/kg/day for 5 days) in children with suspected JE at two sites in Nepal; we also examined the effect on serum neutralizing antibody titre and cytokine profiles. 22 children were recruited, 13 of whom had confirmed JE; 11 received IVIG and 11 placebo, with no protocol violations. One child (IVIG group) died during treatment and two (placebo) subsequently following hospital discharge. Overall, there was no difference in outcome between treatment groups at discharge or follow up. Passive transfer of anti-JEV antibody was seen in JEV negative children. JEV positive children treated with IVIG had JEV-specific neutralizing antibody titres approximately 16 times higher than those treated with placebo (p=0.2), which was more than could be explained by passive transfer alone. IL-4 and IL-6 were higher in the IVIG group. CONCLUSIONS/SIGNIFICANCE: A trial of IVIG for JE in Nepal is feasible. IVIG may augment the development of neutralizing antibodies in JEV positive patients. IVIG appears an appealing option for JE treatment that warrants further study. TRIAL REGISTRATION: ClinicalTrials.gov NCT01856205.

Eastern Equine Encephalitis virus (EEEV) is a medically important pathogen that can cause severe encephalitis in humans, with mortality rates ranging from 30 to 80%. Unfortunately there are no antivirals or licensed vaccines available for human use, and laboratory diagnosis is essential to differentiate EEEV infection from other pathogens with similar clinical manifestations. The Arboviral Diseases Branch (ADB) reference laboratory at the CDC Division of Vector-Borne Diseases (DVBD) produces reference antigens used in serological assays such as the EEEV immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). However, EEEV is classified as a HHS select agent and requires biosafety level (BSL) three containment, limiting EEEV antigen production in non-select agent and BSL-2 laboratories. A recombinant Sindbis virus (SINV)/EEEV has been constructed for use under BSL-2 conditions and is not regulated as a select agent. Cell culture production of inactivated EEEV antigen from SINV/EEEV for use in the EEEV MAC-ELISA is reported here. Cell culture conditions and inactivation procedures were analyzed for SINV/EEEV using a recently developed antigen production algorithm, with the MAC-ELISA as the performance indicator.

Surveillance for acute flaccid paralysis with laboratory confirmation has been a key strategy in the global polio eradication initiative, and the laboratory platform established for polio testing has been expanded in many countries to include surveillance for cases of febrile rash illness to identify measles and rubella cases. Vaccine-preventable disease surveillance is essential to detect outbreaks, define disease burden, guide vaccination strategies and assess immunization impact. Vaccines now exist to prevent Japanese encephalitis (JE) and some etiologies of bacterial meningitis.We evaluated the feasibility of expanding polio-measles surveillance and laboratory networks to detect bacterial meningitis and JE, using surveillance for acute meningitis-encephalitis syndrome in Bangladesh and China and acute encephalitis syndrome in India. We developed nine syndromic surveillance performance indicators based on international surveillance guidelines and calculated scores using supervisory visit reports, annual reports, and case-based surveillance data.Scores, variable by country and targeted disease, were highest for the presence of national guidelines, sustainability, training, availability of JE laboratory resources, and effectiveness of using polio-measles networks for JE surveillance. Scores for effectiveness of building on polio-measles networks for bacterial meningitis surveillance and specimen referral were the lowest, because of differences in specimens and techniques.Polio-measles surveillance and laboratory networks provided useful infrastructure for establishing syndromic surveillance and building capacity for JE diagnosis, but were less applicable for bacterial meningitis. Laboratory-supported surveillance for vaccine-preventable bacterial diseases will require substantial technical and financial support to enhance local diagnostic capacity.

The dengue (DEN) viruses are positive-strand RNA viruses in the genus Flavivirus . Dengue fever and dengue hemorrhagic fever/dengue shock syndrome are important human arboviral diseases caused by infection with one of four closely related but serologically distinct DEN viruses, designated DEN-1, DEN-2, DEN-3, and DEN-4 viruses. All four DEN serotypes are currently cocirculating throughout the subtropics and tropics, and genotypic variation occurs among isolates within a serotype. A real-time quantitative nucleic acid amplification assay has been developed to detect viral RNA of a single DEN virus serotype. Each primer-probe set is DEN serotype specific, yet detects all genotypes in a panel of 7 to 10 representative isolates of a serotype. In single reactions and in fourplex reactions (containing four primer-probe sets in a single reaction mixture), standard dilutions of virus equivalent to 0.002 PFU of DEN-2, DEN-3, and DEN-4 viruses were detected; the limit of detection of DEN-1 virus was 0.5 equivalent PFU. Singleplex and fourplex reactions were evaluated in a panel of 40 viremic serum specimens with 10 specimens per serotype, containing 0.002 to 6,000 equivalent PFU/reaction (0.4 to 1.2 × 10 6 PFU/ml). Viral RNA was detected in all viremic serum specimens in singleplex and fourplex reactions. Thus, this serotype-specific, fourplex real-time reverse transcriptase PCR nucleic acid detection assay can be used as a method for differential diagnosis of a specific DEN serotype in viremic dengue patients and as a tool for rapid identification and serotyping of DEN virus isolates.

St. Louis encephalitis (SLE) and West Nile (WN) flaviviruses are genetically closely related and cocirculate in the United States. Virus neutralization tests provide the most specific means for serodiagnosis of infections with these viruses. However, use of wild-type SLE and WN viral strains for laboratory testing is constrained by the biocontainment requirements. We constructed two highly attenuated yellow fever (YF) virus chimeras that contain the premembrane-envelope (prM-E) protein genes from the virulent MSI-7 (isolated in the United States) or the naturally attenuated CorAn9124 (Argentina) SLE strains. The YF/SLE (CorAn version) virus and the previously constructed YF/WN chimera were shown to specifically distinguish between confirmed human SLE and WN cases in a virus neutralization test using patient sera. These chimeras have the potential for use as diagnostic reagents and vaccines against SLE and WN.

A chimeric yellow fever-dengue 1 (ChimeriVax-DEN1) virus was produced by the transfection of Vero cells with chimeric in vitro RNA transcripts. The cell culture supernatant was subjected to plaque purification for the identification of a vaccine candidate without mutations. Of 10 plaque-purified clones, 1 containing no mutation (clone J) was selected for production of the vaccine virus. During subsequent cell culture passaging of this clone for vaccine production, a single amino acid substitution (K to R) occurred in the envelope (E) protein at residue 204 (E204) (F. Guirakhoo, K. Pugachev, Z. Zhang, G. Myers, I. Levenbook, K. Draper, J. Lang, S. Ocran, F. Mitchell, M. Parsons, N. Brown, S. Brandler, C. Fournier, B. Barrere, F. Rizvi, A. Travassos, R. Nichols, D. Trent, and T. Monath, J. Virol. 78: 4761-4775, 2004). The same mutation was observed in another clone (clone E). This mutation attenuated the virus in 4-day-old suckling mice inoculated by the intracerebral (i.c.) route and led to reduced viremia in monkeys inoculated by the subcutaneous or i.c. route. The histopathology scores of lesions in the brain tissue of monkeys inoculated with either the E204K or E204R virus were reduced compared to those for monkeys inoculated with the reference virus, a commercial yellow fever 17D vaccine (YF-VAX). Both viruses grew to significantly lower titers than YF-VAX in HepG2, a human hepatoma cell line. After intrathoracic inoculation into mosquitoes, both viruses grew to a similar level as YF-VAX, which was significantly lower than that of their wild-type DEN1 parent virus. A comparison of the E-protein structures of nonmutant and mutant viruses suggested the appearance of new intramolecular bonds between residues 204R, 261H, and 257E in the mutant virus. These changes may be responsible for virus attenuation through a change in the pH threshold for virus envelope fusion with the host cell membrane.

The vector competence of mosquitoes for chimeric viruses being developed as vaccines to protect against dengue (DEN) virus infection were evaluated in a cooperative agreement with Acambis, Inc. Chimeric viruses have been constructed that contain the premembrane (prM) and envelope (E) genes of each of the wild-type (wt) DEN virus serotypes, DEN-1, DEN-2, DEN-3, and DEN-4, in the yellow fever (YF) vaccine virus (strain 17D) YF-VAX backbone. It was previously shown that the replication profile of ChimeriVax-DEN2 virus in Aedes albopictus C6/36 cells and in vivo in Ae. aegypti mosquitoes corresponded to that of YF-VAX virus; replication was restricted in C6/36 cells, and Ae. aegypti were poorly infected via an artificial infectious blood meal. Thus, there is very little risk of transmission by mosquitoes of ChimeriVax-DEN2 vaccine virus through the bite of a mosquito. However, because ChimeriVax-DEN 1, 2, 3, 4 viruses will be administered to humans simultaneously, growth of a mixture of ChimeriVax-DEN 1, 2, 3, 4 viruses was assessed in both C6/36 cells in culture and in the Ae. aegypti mosquito, which is the primary vector of both YF and DEN viruses. Mosquitoes were intrathoracically (IT) inoculated with virus or fed a virus-laden blood meal, and the replication kinetics of ChimeriVax-DEN 1, 2, 3, 4 were compared with the wt DEN and YF-VAX viruses. A quantitative real-time reverse transcriptase-polymerase chain reaction assay was developed as a method to detect and differentiate replication of each of the four ChimeriVax-DEN serotypes in the ChimeriVax-DEN 1, 2, 3, 4 tetravalent mixture. Growth of the chimeric viruses in C6/36 cells and in IT-inoculated Ae. aegypti was lower than that of YF-VAX virus; in previous studies Ae. aegypti was shown to be refractory to infection by YF-VAX virus. The growth rate of each chimeric virus was similar whether it was a single serotype infection, or part of the tetravalent mixture, and no interference by one chimeric virus over another chimeric serotype was observed. ChimeriVax-DEN viruses infected mosquitoes poorly via an infectious blood meal compared with wt DEN viruses. Therefore, it is unlikely that a mosquito feeding on a viremic vaccinee, would become infected with the chimeric viruses. Thus, there is very little potential for transmission by mosquitoes of the ChimeriVax-DEN vaccine viruses.

To the Editor: Ilheus virus (ILHV) (genus Flavivirus in the Ntaya antigenic complex) is most closely related to Rocio virus. However, antibodies produced during ILHV infection cross-react in serologic assays to other flavivirus antigens, and ILHV was originally classified in the Japanese encephalitis antigenic complex (1–3). ILHV is transmitted in an enzootic cycle between birds and mosquitoes. Since the first isolation of ILHV from a pool of Aedes spp. and Psorophora spp. mosquitoes collected in 1944 at Ilheus City, on the eastern coast of Brazil (4), isolates have been obtained in Central and South America and Trinidad, primarily from Psorophora ferox mosquitoes (5,6). ILHV is not associated with epidemic disease and has been only sporadically isolated from humans (5,7–9). The clinical spectrum of human infections documented by virus isolation ranges from asymptomatic to signs of central nervous system involvement suggestive of encephalitis. Most commonly, patients exhibit a mild febrile illness accompanied by headache, myalgia, arthralgia, and photophobia, symptoms that may result in clinical diagnosis of dengue, Saint Louis encephalitis, yellow fever, or influenza (7). Laboratory diagnosis of ILHV infection may be difficult, unless a virus isolate can be obtained, because of the cross-reactivity in serologic assays to other flaviviruses that circulate in the same area, such as Rocio, dengue, yellow fever, and Saint Louis encephalitis viruses. On March 1, 2004, after 4 days of symptoms, a 20-year-old male soldier stationed in Lorocachi, Ecuador, was admitted to the Hospital de la IV Division del Ejercito “Amazonas” in Puyo, Ecuador. Lorocachi is in the Amazonian province of Pastaza, of which Puyo is the capital. The patient had fever, rash, epistaxis, headache, myalgia, retroocular pain, nausea, vomiting, jaundice, sore throat, and abdominal pain. A blood specimen (containing isolate FSE800) was collected, and an acute-phase serum sample was processed for virus isolation in C6/36 (Aedes albopictus) and Vero cells. After 3 days, cytopathic effects were observed, and cells were screened by an immunofluorescence assay for reactivity against alphaviruses and flaviviruses by using polyclonal antibodies. The cells gave positive results with yellow fever, Saint Louis encephalitis, Rocio, and Ilheus antisera. Viral RNA was then extracted from the patient’s acute-phase serum and cell supernatants and processed for virus sequencing. Amplification of an ≈250-bp product by SyBRgreen real-time reverse transcriptase PCR and subsequent sequencing of the amplicon were conducted with the flavivirus consensus primers FU1 and cFD2 (2). Viruses were identified by BLAST search (www.ncbi.nlm.nih.gov/blast) and alignment to GenBank sequences. FSE800 had 96% identity (182 of 188 bp) with the nonstructural (NS) 5 region of the original ILHV strain {"type":"entrez-nucleotide","attrs":{"text":"AY632539","term_id":"226374365","term_text":"AY632539"}}AY632539 (1). An ILHV original 1944 isolate used as positive control had 100% sequence identity with {"type":"entrez-nucleotide","attrs":{"text":"AY632539","term_id":"226374365","term_text":"AY632539"}}AY632539. Limited sequence information is available in GenBank for ILHV outside of the conserved NS5 region. Therefore, further sequencing of the NS5 region using flavivirus consensus primers FU1 and cFD3 (2) and the complete envelope (E) gene region, based on the complete open reading frame ILHV nucleotide sequence {"type":"entrez-nucleotide","attrs":{"text":"AY632539","term_id":"226374365","term_text":"AY632539"}}AY632539 (1), was conducted on FSE800, as well as a low-passage original 1944 isolate (R. Tesh, University of Texas Medical Branch [UTMB]), 2 mosquito isolates from Iquitos, Peru (R. Tesh, UTMB), and 2 human isolates from Brazil (CDC) (8) (Figure). There was 100% sequence identity among the 2 Brazilian and 2 Peruvian isolates at both gene regions. In the NS5 region, there was 100% identity between Brazilian and original isolates; 95% identity between FSE800 and Brazilian-original isolates; 98% identity between FSE800 and Peruvian isolates; and 96% identity between Peruvian and original-Brazilian isolates. At the protein level, 3 amino acid (aa) differences were found between FSE800 and original-Brazilian isolates, 2 between FSE800-Peruvian and original-Brazilian isolates (aa 3086, Lys-Gln; aa 3138, Ala-Val) and 1 between FSE800 and original-Brazilian-Peruvian isolates (aa 3309, Asp-Asn). Figure Phylogenetic analysis of the NS5 (A) and E gene (B) regions of 6 Ilheus virus (ILHV) isolates. The sequences were aligned in MegAlign (DNASTAR, Inc., Madison, WI, USA); the alignments were then analyzed by using the maximum parsimony method with 500 bootstrap ... In the E gene region, there was 99.9% identity between original and Brazilian isolates; 95.1% and 95% identity between FSE800 and original and Brazilian isolates, respectively; 98.5% identity between FSE800 and Peruvian isolates; and 95.3% identity between Peruvian and original isolates. At the protein level, 2 aa differences were found between original-Brazilian and Peruvian-FSE800 isolates (aa 432, Ile-Thr; aa 652, Lys-Asn), 1 aa change only in Brazilian isolates (aa 570, His-Tyr), and 1 only in Peruvian isolates (aa 675, Asn-Ser). With the exception of the Ala-Val change, all were nonconservative changes. The effect of the amino acid changes has yet to be determined. However, nucleotide sequence comparison between the 6 ILHV isolates has shown that these gene regions are highly conserved geographically and temporally. Ilheus virus is not usually associated with human disease, and human ILHV infections may not be correctly identified without a virus isolate because of the similar clinical symptoms and cross-reactivity in serologic assays to other flaviviruses. As laboratory surveillance is enhanced to monitor the emergence of West Nile virus in Central and South America, detection of ILHV and other flaviviruses may increase.

West Nile virus ecology has yet to be rigorously investigated in the Caribbean Basin. We identified a transmission focus in Puerto Barrios, Guatemala, and established systematic monitoring of avian abundance and infection, seroconversions in domestic poultry, and viral infections in mosquitoes. West Nile virus transmission was detected annually between May and October from 2005 to 2008. High temperature and low rainfall enhanced the probability of chicken seroconversions, which occurred in both urban and rural sites. West Nile virus was isolated from Culex quinquefasciatus and to a lesser extent, from Culex mollis/Culex inflictus, but not from the most abundant Culex mosquito, Culex nigripalpus. A calculation that combined avian abundance, seroprevalence, and vertebrate reservoir competence suggested that great-tailed grackle (Quiscalus mexicanus) is the major amplifying host in this ecosystem. West Nile virus transmission reached moderate levels in sentinel chickens during 2007, but less than that observed during outbreaks of human disease attributed to West Nile virus in the United States.

To differentiate exposure to the newly introduced chikungunya virus from exposure to endemic dengue virus and other pathogens in Haiti.We used a multiplex bead assay to detect immunoglobulin G (IgG) responses to a recombinant chikungunya virus antigen, two dengue virus-like particles and three recombinantOf the samples from the longitudinal cohort, none of the 153 collected between 2011 and 2013 but 78.7% (48/61) of those collected in 2014 were positive for IgG responses to the chikungunya virus antigen. In the cross-sectional sample, such responses were detected in 96 (75.6%) of the children and occurred at similar prevalence across all age groups. In the same sample, responses to malarial antigen were only detected in eight children (6.3%) but the prevalence of IgG responses to dengue virus antigens was 60.6% (77/127) overall and increased steadily with age. Spatial analysis indicated that the prevalence of IgG responses to the chikungunya virus and one of the dengue virus-like particles decreased as the sampling site moved away from the city of Léogâne and towards the ocean.Serological evidence indicates that there had been a rapid and intense dissemination of chikungunya virus in Haiti. The multiplex bead assay appears to be an appropriate serological platform to monitor the seroprevalence of multiple pathogens simultaneously.Différentier l'exposition au virus chikungunya, récemment introduit, de l'exposition au virus de la dengue et à d'autres pathogènes endémiques en Haïti.Nous avons procédé à une analyse multiplex à l'aide de billes pour détecter les réponses de l'immunoglobuline G (IgG) à un antigène recombinant du virus chikungunya, à deux particules pseudo-virales de la dengue et à trois antigènes recombinants deDans la cohorte longitudinale, aucun des 153 échantillons prélevés entre 2011 et 2013 n'affichait de réponse positive de l'IgG à l'antigène du virus chikungunya, mais 78,7% (48/61) de ceux prélevés en 2014 étaient en revanche positifs. Dans l'échantillon transversal, des réponses positives ont été relevées chez 96 enfants (75,6%), avec une prévalence similaire dans tous les groupes d'âge. Sur ce même échantillon, des réponses à l'antigène du paludisme n'ont été détectées que chez huit enfants (6,3%) mais la prévalence globale des réponses de l'IgG aux antigènes du virus de la dengue était de 60,6% (77/127) et augmentait progressivement avec l'âge. L'analyse géographique a révélé que la prévalence des réponses de l'IgG au virus du chikungunya et à l'une des particules pseudo-virales de la dengue diminuait à mesure que le site d'échantillonnage s'éloignait de la ville de Léogâne en direction de l'océan.Les preuves sérologiques indiquent que la propagation du virus du chikungunya en Haïti a été rapide et intense. L'analyse multiplex à l'aide de billes s'est avérée être une plate-forme sérologique appropriée pour contrôler simultanément la séroprévalence de plusieurs pathogènes.Diferenciar la exposición al nuevo virus chikungunya de la exposición al virus endémico del dengue y a otros patógenos en Haití.Se utilizó un ensayo multiplex de microesferas para detectar las respuestas de la inmunoglobulina G (IgG) ante un antígeno recombinante del virus chikungunya, dos partículas similares al virus del dengue y tres antígenos recombinantes deDe las muestras de la cohorte longitudinal, ninguna de las 153 muestras recogidas entre 2011 y 2013 dio un resultado positivo de respuestas de la IgG al antígeno del virus chikungunya, pero sí que lo dieron el 78,7% (48/61) de las muestras recogidas en 2014. En la muestra transversal, se detectaron dichas respuestas en 96 (75,6%) de los niños y se mostró una prevalencia similar en todos los grupos de edades. En la misma muestra, únicamente se detectaron respuestas al antígeno de la malaria en ocho niños (6,3%), aunque la prevalencia de las respuestas de la IgG a los antígenos del virus del dengue fue del 60,6% (77/127) en general y aumentaba de forma estable con la edad. Los análisis territoriales indicaron que la prevalencia de las respuestas de la IgG al virus chikungunya y a una de las partículas similares al virus del dengue se redujo conforme el lugar de la muestra se alejaba de la ciudad de Léogâne y se acercaba al océano.La prueba serológica indica que se ha producido una diseminación rápida e intensa del virus chikungunya en Haití. Parece que el ensayo multiplex de microesferas es una plataforma serológica adecuada para supervisar la seroprevalencia de varios patógenos al mismo tiempo.التمييز بين معدلات التعرض لفيروس شِيكُونْغُونْيا الذي تم الكشف عنه مؤخرًا ومعدلات التعرض لفيروس الضنك المتوطّن والعوامل الأخرى المسببة للأمراض في دولة هايتي.استخدمنا المقايسة المتعددة للحبيبات لاكتشاف أية استجابات من جانب الغلوبولين المناعي G (IgG) إلى مستضد مؤتلف لفيروس شِيكُونْغُونْيا، وجسيمين مشابهين لفيروس الضنك، وثلاثة مستضدات مؤتلفة لطُفيل المتصورة المنجلية. لقد تم جمع معظم عينات الدم (البالغ عددها 217) التي خضعت للاستقصاء من كل 61 طفلًا على مدى فترة طويلة تمتد بين عامي 2011 و2014، ولكن في عام 2014، تم جمع 127 عينة أخرى لقطاعات متعددة من الأطفال.لم تُظهر أي من العينات المُجمّعة فيما بين عامي 2011 و2013 والبالغ عددها 153 نتائج إيجابية عن وجود استجابات من جانب IgG إلى المستضد المؤتلف لفيروس شِيكُونْغُونْيا باستثناء ما يعادل 78.7‏% (‏48/61) من العينات التي تم جمعها في عام 2014 وذلك من أصل العينات المستخرجة من مجموعة الأطفال على مدى فترة طويلة. في العينة المستخرجة من عدة قطاعات، تم اكتشاف تلك الاستجابات في 96 طفلاً (فيما يعادل 75.6%) وكان معدل انتشارها متشابهًا في جميع الفئات العمرية. وفي نفس تلك العينة، لم يتم اكتشاف أية استجابات لمستضد الملاريا سوى في ثمانية أطفال (فيما يعادل 6.3‏%) بينما بلغ إجمالي معدل انتشار الاستجابات من جانب IgG إلى مستضدات فيروس الضنك 60.6‏% (‏77/127) واستمر في الازدياد بثبات مع ازدياد العمر. وأوضح التحليل المكاني أن معدل انتشار الاستجابات من جانب lgG إلى فيروس شِيكُونْغُونْيا وأحد الجسيمين المشابهين لفيروس الضنك قد ازداد بتغيير موقع أخذ العينات من مدينة يوغان انتقالاً باتجاه المحيط.تشير الأدلة المصلية إلى انتشار فيروس شِيكُونْغُونْيا بسرعة وبشدّة في دولة هايتي. كما تُظهر المقايسة المتعددة للحبيبات أن بإمكانها أن تصبح نظامًا مصليًا مناسبًا للإشراف على معدل الانتشار المصلي لعدة عوامل مسببة للأمراض في نفس الوقت.旨在将海地地区接触新流入的基孔肯雅病毒与接触地方性登革病毒以及其他病原体进行区分。.我们采用多重微球试验以检测免疫球蛋白 G (IgG) 对重组基孔肯雅病毒抗原、两种类似登革病毒的微粒和三种重组在纵向群组样本中，2011 年到 2013 年间采集的 153 例样本中没有一例在 lgG 对基孔肯雅病毒抗原有反应中显示阳性，但在 2014 年采集的样本中 78.7% (48/61) 显示阳性。 在横断面样本中，我们发现其中 96 位儿童 (75.6%) 存在此类反应，并且在所有年龄段都有相似概率。 在相同样本中，仅在八位儿童 (6.3%) 中检测到对疟疾抗原有反应。但整体而言，IgG 对登革病毒抗原有反应的概率是 60.6% (77/127)，并且随着年龄增长而稳步增长。 空间分析显示，IgG 对基孔肯雅病毒和其中一种类似登革病毒的微粒有反应的概率随着取样地点从莱奥甘转移至海边而有所降低。.血清学证据表明海地地区的基孔肯亚病毒已经快速密集传播。 多重微球试验似乎是适当的血清学平台，可同时监控多种病原体血清阳性率。.Провести различия между подверженностью воздействию недавно интродуцированного вируса чикунгуньи и воздействию эндемического вируса лихорадки денге и других болезнетворных микроорганизмов в Гаити.С помощью мультиплексного анализа с использованием гранул авторы статьи выявили реакцию иммуноглобулина G (IgG) на антиген рекомбинантного вируса чикунгуньи, две вирусоподобные частицы денге и три антигена рекомбинантного паразита Plasmodium falciparum. Забор большинства (217) исследованных образцов крови совершался в течение нескольких лет у 61 ребенка между 2011 и 2014 годами, а 127 образцов были взяты у детей в рамках профильной пробы в 2014 году.Ни для одного из 153 образцов, отобранных между 2011 и 2013 годами у детей из когорты долгосрочного исследования для анализа на реакцию IgG на антиген вируса чикунгуньи, не был получен положительный результат; однако 78,7% (48/61) образцов, полученных у детей из этой же группы в 2014 году, дали положительный результат. В профильной выборке реакция была обнаружена у 96 (75,6%) детей и наблюдалась со схожей частотностью во всех возрастных группах. В той же выборке реакция на малярийный антиген была обнаружена только у восьми детей (6,3%), однако общая распространенность реакции IgG на антигены вируса денге составила 60,6% (77/127) и с возрастом стабильно увеличивалась. Анализ на распределение по местности показал, что распространенность реакции IgG на вирус чикунгуньи и вирусоподобные частицы денге уменьшалась по мере перемещения места отбора проб от города Леоган в сторону океана.Выявленные серологические реакции свидетельствуют о произошедшем скоротечном и усиленном распространении вируса чикунгуньи в Гаити. Мультиплексный анализ с использованием гранул служит подходящей базой для серодиагностики для одновременного отслеживания распространенности положительных серологических реакций на несколько болезнетворных микроорганизмов.

Background There are no data on the use of inactivated Vero cell culture-derived Japanese encephalitis (JE) vaccine (JE-VC) as a booster among individuals who previously received inactivated mouse brain-derived JE vaccine (JE-MB). Methods Military personnel who received ≥3 doses of JE-MB or were JE vaccine-naive were vaccinated with 2 doses of JE-VC on days 0 and 28. Serum neutralizing antibodies were measured pre-vaccination and 28 days after each dose. Non-inferiority was evaluated for seroprotection rate and geometric mean titer (GMT) between previously vaccinated participants post-dose 1 and vaccine-naive participants post-dose 2. Results Fifty-three previously vaccinated and 70 JE vaccine-naive participants were enrolled. Previously vaccinated participants had significantly higher GMTs pre-vaccination, post-dose 1, and post-dose 2. Seroprotection rates among previously vaccinated participants post-dose 1 (44/44, 100%) were noninferior to those achieved in previously naive participants post-dose 2 (53/57, 93%). The GMT was significantly higher in previously vaccinated participants post-dose 1 (GMT 315; 95% CI 191–520) compared to previously naive participants post-dose 2 (GMT 79; 95% CI 54–114). Conclusions Among military personnel previously vaccinated with ≥3 doses of JE-MB, a single dose of JE-VC adequately boosts neutralizing antibody levels and provides at least short-term protection. Additional studies are needed to confirm these findings in other populations and determine the duration of protection following a single dose of JE-VC in prior recipients of JE-MB.

We evaluated performance of three commercial Japanese encephalitis virus (JEV) IgM antibody capture enzyme-linked immunosorbent assay (MAC ELISA) kits with a panel of serological specimens collected during a surveillance project of acute encephalitis syndrome in India and acute meningitis and encephalitis syndrome in Bangladesh. The serum and cerebral spinal fluid specimens had been referred to the Centers for Disease Control and Prevention (CDC) for confirmatory testing. The CDC results and specimen classifications were considered the reference standard. All three commercial kits had high specificity (95-99.5%), but low sensitivities, ranging from 17-57%, with both serum and cerebrospinal fluid samples. Specific factors contributing to low sensitivity compared with the CDC ELISA could not be determined through further analysis of the limits and dilution end points of IgM detection.

West Nile virus (WNV) is an emerging mosquito-borne flavivirus, which has rapidly spread and is currently widely distributed. Therefore, efforts for WNV early detection and ecological surveillance of this disease agent have been increased around the world. Although virus isolation is known to be the standard method for detection and identification of viruses, the use of RT-PCR assays as routine laboratory tests provides a rapid alterative suitable for the detection of viral RNA on field-collected samples. A method for WNV RNA genome detection in field-collected mosquitoes is presented in this chapter. This method has been designed for virus surveillance in tropical regions endemic for other flaviviruses. Reverse Transcriptase-PCR (RT-PCR) assays, both standard and real time, to detect WNV and other flaviviruses are described. A first screening for flavivirus RNA detection is performed using a conventional RT-PCR with two different sets of flavivirus consensus primers. Mosquito samples are then tested for WNV RNA by a real-time (TaqMan) RT-PCR assay. Sample preparation and RNA extraction procedures are also described.

West Nile virus (WNV) is an emerging mosquito-borne flavivirus, which has rapidly spread and is currently widely distributed. Therefore, efforts for WNV early detection and ecological surveillance of this disease agent have been increased around the world. Although virus isolation is known to be the standard method for detection and identification of viruses, the use of RT-PCR assays as routine laboratory tests provides a rapid alterative suitable for the detection of viral RNA on field-collected samples. A method for WNV RNA genome detection in field-collected mosquitoes is presented in this chapter. This method has been designed for virus surveillance in tropical regions endemic for other flaviviruses. Reverse Transcriptase-PCR (RT-PCR) assays, both standard and real time, to detect WNV and other flaviviruses are described. A first screening for flavivirus RNA detection is performed using a conventional RT-PCR with two different sets of flavivirus consensus primers. Mosquito samples are then tested for WNV RNA by a real-time (TaqMan) RT-PCR assay. Sample preparation and RNA extraction procedures are also described.

Infection with Japanese encephalitis virus (JEV) is a major public health problem in Asia. Detection of JEV-specific IgM in serum and cerebrospinal fluid (CSF) by the IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) is currently the most widely used diagnostic method to detect JEV infection. Because of the possible presence of IgM cross-reactivity with other flaviviruses in serum and the high ratio of inapparent-to-apparent JEV infections, a positive result in serum only suggests a recent infection and not necessarily an encephalitic illness caused by JEV. Consequently, detection of JEV-specific IgM in CSF assumes great diagnostic relevance. We evaluated two commercial JEV MAC-ELISA kits using 60 CSF samples obtained from patients with acute encephalitis syndrome. The Panbio and XCyton kits had sensitivities of 65-80% and 95% and specificities of 90% and 97.5%, respectively. Performance information on these commercial JEV MAC-ELISA kits for CSF should assist in laboratory-based JE surveillance programs.

Two stably transfected COS-1 cell lines that secrete recombinant Japanese encephalitis and dengue virus serotype 4 virus-like particles (VLPs) have been adapted to grow on Cytodex™ 3 microcarriers in an orbital shaker flask platform. The VLPs are used as antigens in diagnostic enzyme-linked immunosorbent assays to detect anti-arboviral IgM and IgG antibodies in human serum samples. Converting from a stationary flask batch system to a microcarrier fed-batch system has led to increases in antigen concentration while decreasing costs by reducing the amount of cell culture medium, disposables, and labor. The cell culture longevity was increased by 48 days in this optimized system, which may be due to a continual supply of nutrients resulting in prolonged survival of the cells on the microcarrier surface. An initial trial using a serum-free medium with this cell line was promising and may lead to reductions in cost, while reducing the variability between batches introduced by fetal bovine serum.

It is hypothesized that previous heterologous flaviviral exposure may modulate clinical illness among persons infected with West Nile virus (WNV). Little is known about the serological response in such persons. In summer 2003, a WNV outbreak occurred in Colorado, the location of the Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases (DVBID). DVBID employees, most previously vaccinated with yellow fever virus (YFV) or Japanese encephalitis virus (JEV) vaccines, were studied to determine whether previous vaccination affected symptom development among those subsequently infected with WNV during the outbreak, as well as their serological response. Serum samples collected in December 2003 and previously banked samples were tested using the plaque reduction neutralization test (PRNT) against WNV, Saint Louis encephalitis virus, dengue- 4 virus, JEV, and YFV. Specimens shown to have WNV antibody by PRNT were tested by IgM and IgG enzymelinked immunosorbent assays (ELISAs). Ten (9%) of 113 serosurvey participants had WNV neutralizing antibody titers in December 2003. PRNT titers from previous specimens showed that one of the ten had seroconverted to WNV before 2003. Of the remaining nine participants, seven reported illness in the summer of 2003, two of which were unvaccinated and five previously vaccinated. In the December 2003 specimens, five persons previously unvaccinated or vaccinated only against YFV had a fourfold or greater neutralizing titer with WNV than with other flaviviruses, whereas no persons previously vaccinated against JEV or JEV and YFV showed a similar difference in neutralizing titers. Eight of nine persons infected in 2003 had negative or indeterminate WNV MAC-ELISA results in the December 2003 sample; the ninth person was vaccinated against YFV one month previously, and was also YFV positive by MAC-ELISA. We conclude that previous flaviviral vaccination does not markedly affect the development of WNV fever and that the IgM antibody response in patients without neuroinvasive WNV disease is transient.

St. Louis encephalitis (SLE) and West Nile (WN) flaviviruses are genetically closely related and cocirculate in the United States. Virus neutralization tests provide the most specific means for serodiagnosis of infections with these viruses. However, use of wild-type SLE and WN viral strains for laboratory testing is constrained by the biocontainment requirements. We constructed two highly attenuated yellow fever (YF) virus chimeras that contain the premembrane-envelope (prM-E) protein genes from the virulent MSI-7 (isolated in the United States) or the naturally attenuated CorAn9124 (Argentina) SLE strains. The YF/SLE (CorAn version) virus and the previously constructed YF/WN chimera were shown to specifically distinguish between confirmed human SLE and WN cases in a virus neutralization test using patient sera. These chimeras have the potential for use as diagnostic reagents and vaccines against SLE and WN.

The vector competence of mosquitoes for chimeric viruses being developed as vaccines to protect against dengue (DEN) virus infection were evaluated in a cooperative agreement with Acambis, Inc. Chimeric viruses have been constructed that contain the premembrane (prM) and envelope (E) genes of each of the wild-type (wt) DEN virus serotypes, DEN-1, DEN-2, DEN-3, and DEN-4, in the yellow fever (YF) vaccine virus (strain 17D) YF-VAX backbone. It was previously shown that the replication profile of ChimeriVax-DEN2 virus in Aedes albopictus C6/36 cells and in vivo in Ae. aegypti mosquitoes corresponded to that of YF-VAX virus; replication was restricted in C6/36 cells, and Ae. aegypti were poorly infected via an artificial infectious blood meal. Thus, there is very little risk of transmission by mosquitoes of ChimeriVax-DEN2 vaccine virus through the bite of a mosquito. However, because ChimeriVax-DEN 1, 2, 3, 4 viruses will be administered to humans simultaneously, growth of a mixture of ChimeriVax-DEN 1, 2, 3, 4 viruses was assessed in both C6/36 cells in culture and in the Ae. aegypti mosquito, which is the primary vector of both YF and DEN viruses. Mosquitoes were intrathoracically (IT) inoculated with virus or fed a virus-laden blood meal, and the replication kinetics of ChimeriVax-DEN 1, 2, 3, 4 were compared with the wt DEN and YF-VAX viruses. A quantitative real-time reverse transcriptase-polymerase chain reaction assay was developed as a method to detect and differentiate replication of each of the four ChimeriVax-DEN serotypes in the ChimeriVax-DEN 1, 2, 3, 4 tetravalent mixture. Growth of the chimeric viruses in C6/36 cells and in IT-inoculated Ae. aegypti was lower than that of YF-VAX virus; in previous studies Ae. aegypti was shown to be refractory to infection by YF-VAX virus. The growth rate of each chimeric virus was similar whether it was a single serotype infection, or part of the tetravalent mixture, and no interference by one chimeric virus over another chimeric serotype was observed. ChimeriVax-DEN viruses infected mosquitoes poorly via an infectious blood meal compared with wt DEN viruses. Therefore, it is unlikely that a mosquito feeding on a viremic vaccinee, would become infected with the chimeric viruses. Thus, there is very little potential for transmission by mosquitoes of the ChimeriVax-DEN vaccine viruses.

In 1999 West Nile (WN) virus was introduced to North America where this flavivirus has spread rapidly among wildlife (especially birds) transmitted by various species of mosquitoes (Diptera: Culicidae). Increasing numbers of cases and deaths among humans, horses and other domestic animals require development of effective vaccines. 'ChimeriVax-West Nile(vet)' is being developed for use as a veterinary vaccine to protect against WN infection. This chimeric virus contains the pre-membrane (prM) and envelope (E) genes from the wild-type WN NY99 virus (isolated from a flamingo in New York zoo during the 1999 WN epidemic) in the backbone of yellow fever (YF) 17D vaccine virus. Replication kinetics of ChimeriVax-WN(vet) virus were evaluated in mosquito cell culture (Aedes albopictus C6/36), in WN vector mosquitoes [Culex tritaeniorhynchus Giles, Cx. nigripalpus Theobald and Cx. quinquefasciatus Say (Diptera: Culicidae)] and in YF vectors [Aedes aegypti (L) and Ae. albopictus (Skuse)], to determine whether these mosquitoes become infected through feeding on a viraemic vaccine, and their potential infectivity to transmit the virus. Growth of ChimeriVax-WN(vet) virus was found to be restricted in mosquitoes, compared to WN virus in Ae. albopictus C6/36 cells. When inoculated intrathoracically, ChimeriVax-WN(vet) and YF 17D viruses did not replicate in Cx. tritaeniorhynchus or Cx. nigripalpus; replication was very restricted compared to the wild-type WN virus in Cx. quinquefasciatus, Ae. aegypti and Ae. albopictus. When fed on hanging drops with ChimeriVax-WN(vet) virus (7.7 log10 PFU/mL), none of the Culex mosquitoes became infected; one Ae. albopictus and 10% of the Ae. aegypti became infected, but the titre was very low and virus did not disseminate to head tissue. ChimeriVax-WN(vet) virus had a replication profile similar to that of the attenuated vaccine virus YF 17D, which is not transmitted by mosquitoes. These results suggest that the natural mosquito vectors of WN and YF viruses, which may incidentally take a bloodmeal from a vaccinated host, will not become infected with ChimeriVax-WN(vet) virus.

Because the potential urban yellow fever (YF) mosquito vectors Aedes aegypti and Ae. albopictus are at historical highs in Brazil, both in terms of density and geographical range, we assessed the risk of an urban YF epidemic in Brazil. We evaluated and confirmed in a laboratory setting the vector competence of Brazilian Ae. aegypti for a currently circulating strain of YF virus, and investigated the potential for Brazilian Ae. albopictus to transmit YF.

The chimeric yellow fever (YF) 17D-dengue type 2 (ChimeriVax-DEN2) vaccine virus developed by Acambis, Inc. (Cambridge, MA) contains the prM and E genes of wild-type (wt) dengue 2 (DEN-2) (strain PUO-218) virus in the YF vaccine virus (strain 17D) backbone. The potential of ChimeriVax-DEN2 virus to infect and be transmitted by Aedes aegypti, the principal DEN and YF virus mosquito vector, and Aedes albopictus, a species that occurs in areas of active transmission of YF and DEN viruses, was evaluated. Mosquitoes were intrathoracically (IT) inoculated with virus or were fed a virus-laden blood meal, and the replication kinetics of ChimeriVax-DEN2 were compared with the wt DEN-2 and YF 17D vaccine viruses. Replication of YF 17D virus is attenuated in cultured Ae. albopictus C6/36 mosquito cells and in Ae. aegypti and Ae. albopictus mosquitoes. Growth of ChimeriVax-DEN2 virus similarly was restricted in C6/36 cells and in mosquitoes. ChimeriVax-DEN2 replicated in 56% of IT inoculated Ae. aegypti, and virus disseminated to head tissue in 36%, with a mean viral titer of 1.8 log10 PFU/mosquito. Of mosquitoes, 16% of Ae. aegypti and 24% of Ae. albopictus were infected 14 days after a blood meal containing ChimeriVax-DEN2, but virus did not disseminate to head tissue. In contrast, DEN-2 replicated in all IT inoculated and orally infected Ae. aegypti (mean titer 5.5 log10 PFU/mosquito), and virus disseminated to head tissue in 95%. Of Ae. albopictus, 84% were infected after a blood meal containing DEN-2 virus; dissemination occurred in 36%. Replication of ChimeriVax-DEN2 virus in mosquitoes corresponded to that of YF 17D vaccine virus, which is restricted in its ability to infect and replicate in mosquitoes. Therefore, transmission of ChimeriVax-DEN2 virus by vector mosquitoes is unlikely.

A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus , Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3′2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5′ end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo .

The first step in poliovirus replication is binding of virus to a cellular receptor. Mouse L cells, which are resistant to poliovirus infection because they do not bear a poliovirus receptor, were transformed with HeLa cell (human) DNA to poliovirus sensitivity at a frequency of approximately 1 in 50,000 transformants. Monoclonal antibody directed against the HeLa cell poliovirus receptor site was used in rosette assays to identify poliovirus-sensitive L-cell transformants in a background of L-cell tk+ transformants. A cloned cell line, CM-1, was isolated that displayed a surface component recognized by the anti-poliovirus receptor antibody. CM-1 cells were susceptible to infection with all three poliovirus serotypes, and infection could be blocked by the antireceptor antibody. Poliovirus formed plaques in CM-1 and HeLa cells with equal efficiency. CM-1 and HeLa cells produced infectious poliovirus at a similar rate, although yield of virus in CM-1 cells was about 33% less than the yield in HeLa cells. These results suggest that DNA encoding the HeLa cell poliovirus receptor has been introduced into mouse cells, resulting in the expression of the receptor and susceptibility to poliovirus infection.