Expansion of Surveillance for Vaccine-preventable Diseases: Building on the Global Polio Laboratory Network and the Global Measles and Rubella Laboratory Network Platforms.

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.

Evaluation of a TaqMan Array Card for Detection of Central Nervous System Infections.

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 pneumoniae, 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.

Evaluation of three commercially-available chikungunya virus immunoglobulin G immunoassays.

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.

Laboratory Diagnosis of Chikungunya Virus Infections and Commercial Sources for Diagnostic Assays.

Detection of chikungunya virus (CHIKV) or viral RNA is the primary laboratory test used to diagnose infection in serum collected <6 days after onset of illness. Two real-time reverse transcription-polymerase chain reaction (RT-PCR) kits are available commercially, but validity data are limited. There are 2 commercial sources of inactivated positive-control CHIKV RNA to be used with purchased primers. The Centers for Disease Control and Prevention provides viral RNA-positive controls and primer and probe nucleotide sequences for real-time RT-PCR testing. Detection of CHIKV-specific immunoglobulin M (IgM) antibody becomes a sensitive test for samples collected approximately >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 3 MAC-ELISAs was <50%, and thus these assays are not recommended. Three of the MAC-ELISA kits and 1 indirect immunofluorescence kit had comparable performance to the reference assays. In summary, commercial assays with performance comparable to reference assays are available for molecular and serological diagnosis of CHIKV infections.

Measuring Haitian children's exposure to chikungunya, dengue and malaria.

OBJECTIVE: To differentiate exposure to the newly introduced chikungunya virus from exposure to endemic dengue virus and other pathogens in Haiti. METHODS: 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 recombinant Plasmodium falciparum antigens. Most (217) of the blood samples investigated were collected longitudinally, from each of 61 children, between 2011 and 2014 but another 127 were collected from a cross-sectional sample of children in 2014. FINDINGS: Of 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. CONCLUSION: 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.

The U.S. Culture Collection Network Lays the Foundation for Progress in Preservation of Valuable Microbial Resources.

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.

Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of Chikungunya fever.

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.

Laboratory evaluation of RealStar Yellow Fever Virus RT-PCR kit 1.0 for potential use in the global yellow fever laboratory network.

BACKGROUND: Early detection of human yellow fever (YF) infection in YF-endemic regions is critical to timely outbreak mitigation. African National Laboratories chiefly rely on serological assays that require confirmation at Regional Reference Laboratories, thus delaying results, which themselves are not always definitive often due to antibody cross-reactivity. A positive molecular test result is confirmatory for YF; therefore, a standardized YF molecular assay would facilitate immediate confirmation at National Laboratories. The WHO-coordinated global Eliminate Yellow Fever Epidemics Laboratory Technical Working Group sought to independently evaluate the quality and performance of commercial YF molecular assays relevant to use in countries with endemic YF, in the absence of stringent premarket assessments. This report details a limited laboratory WHO-coordinated evaluation of the altona Diagnostics RealStar Yellow Fever Virus RT-PCR kit 1.0. METHODOLOGY AND PRINCIPAL FINDINGS: Specific objectives were to assess the assay's ability to detect YF virus strains in human serum from YF-endemic regions, determine the potential for interference and cross-reactions, verify the performance claims as stated by the manufacturer, and assess usability. RNA extracted from normal human serum spiked with YF virus showed the assay to be precise with minimal lot-to-lot variation. The 95% limit of detection calculated was approximately 1,245 RNA copies/ml [95% confidence interval 497 to 1,640 copies/ml]. Positive results were obtained with spatially and temporally diverse YF strains. The assay was specific for YF virus, was not subject to endogenous or exogenous interferents, and was clinically sensitive and specific. A review of operational characteristics revealed that a positivity cutoff was not defined in the instructions for use, but otherwise the assay was user-friendly. CONCLUSIONS AND SIGNIFICANCE: The RealStar Yellow Fever Virus RT-PCR kit 1.0 has performance characteristics consistent with the manufacturer's claims and is suitable for use in YF-endemic regions. Its use is expected to decrease YF outbreak detection times and be instrumental in saving lives.

Mycoplasma detection in a historical arbovirus repository: Commercial kit comparison and implications for improved repository management.

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.

Immune response at 12-23 months following a single dose of Vero cell culture-derived Japanese encephalitis (JE) vaccine in adults previously vaccinated with mouse brain-derived JE vaccine.

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-naïve 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-naïve 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-naïve 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-naïve 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.

Microcarrier culture of COS-1 cells producing Japanese encephalitis and dengue virus serotype 4 recombinant virus-like particles.

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.

A comparison of concentration methods applied to non-infectious flavivirus recombinant antigens for use in diagnostic serological assays.

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.

Differential Diagnosis of Japanese Encephalitis Virus Infections with the Inbios JE Detect™ and DEN Detect™ MAC-ELISA 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 JEDetect™ MAC-ELISA (JEDetect), a differential testing algorithm was developed in which samples tested by JEDetect with positive results were subsequently tested by the DENDetect™ MAC-ELISA (DENDetect) 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 JEDetect and DENDetect and were classified as JE- (dengue virus [DENV]+). Differential diagnosis of JE by sequential testing with JEDetect and DENDetect 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.

Evaluation of Commercially Available Chikungunya Virus Immunoglobulin M Detection Assays.

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.

Development and validation of an ELISA kit (YF MAC-HD) to detect IgM to yellow fever virus.

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 3.5h, 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.

Expansion of syndromic vaccine preventable disease surveillance to include bacterial meningitis and Japanese encephalitis: evaluation of adapting polio and measles laboratory networks in Bangladesh, China and India, 2007-2008.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Spot the difference-development of a syndrome based protein microarray for specific serological detection of multiple flavivirus infections in travelers.

BACKGROUND: The family Flaviviridae, genus Flavivirus, holds many of the world's most prevalent arboviral diseases that are also considered the most important travel related arboviral infections. In most cases, flavivirus diagnosis in travelers is primarily based on serology as viremia is often low and typically has already been reduced to undetectable levels when symptoms set in and patients seek medical attention. Serological differentiation between flaviviruses and the false-positive results caused by vaccination and cross-reactivity among the different species, are problematic for surveillance and diagnostics of flaviviruses. Their partially overlapping geographic distribution and symptoms, combined with increase in travel, and preexisting antibodies due to flavivirus vaccinations, expand the need for rapid and reliable multiplex diagnostic tests to supplement currently used methods. GOAL: We describe the development of a multiplex serological protein microarray using recombinant NS1 proteins for detection of medically important viruses within the genus Flavivirus. Sera from clinical flavivirus patients were used for primary development of the protein microarray. RESULTS: Results show a high IgG and IgM sensitivity and specificity for individual NS1 antigens, and limited cross reactivity, even within serocomplexes. In addition, the serology based on this array allows for discrimination between infection and vaccination response for JEV vaccine, and no cross-reactivity with TBEV and YFV vaccine induced antibodies when testing for antibodies to other flaviviruses. CONCLUSION: Based on these data, multiplex NS1-based protein microarray is a promising tool for surveillance and diagnosis of flaviviruses.

Analysis of the replication kinetics of the ChimeriVax-DEN 1, 2, 3, 4 tetravalent virus mixture in Aedes aegypti by real-time reverse transcriptase-polymerase chain reaction.

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.

Growth characteristics of ChimeriVax-DEN2 vaccine virus in Aedes aegypti and Aedes albopictus mosquitoes.

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.

Construction of yellow fever/St. Louis encephalitis chimeric virus and the use of chimeras as a diagnostic tool.

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.