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Haller, Logan C.
Developement of a micropshere-based immunoassay for the detection of IgM antibodies to West Nile virus and St. Louis Encephalitis virus in sentinel chicken sera
h [electronic resource] /
by Logan C. Haller.
[Tampa, Fla] :
b University of South Florida,
ABSTRACT: West Nile virus (WNV) and St. Louis Encephalitis (SLEV) are arthropod-borne viruses belonging to the genus Flavivirus and are classified as significant human pathogens of global epidemiological importance. Since its introduction into the United States in 1999, WNV has spread throughout most of the country and has caused major epidemics of neuroinvasive disease (Hayes and Gubler, 2005). SLEV is endemic to the United States and is maintained in an enzootic transmission cycle in Florida.The Florida Sentinel Chicken Arboviral Surveillance Network was established in 1978 following a widespread rural epidemic of SLEV in central Florida to monitor the activity of arboviruses (Day and Stark, 1996). This program ultimately impacts vector control strategies and may warrant medical alerts to warn the population.Current serological detection methods for sentinel chickens include hemagglutination inhibition antibody test (HAI), IgM antibody capture enzyme-linkedimmunosorbent assay ( MAC-ELISA), and Plaque Reduction Neutralization Test (PRNT).These serological assays may take over three weeks to generate a final result. A more rapid and equally sensitive test to replace these current serological methods would be of benefit. Microsphere-based immunoassays (MIAs) are a more rapid serological option for laboratory diagnosis of many diseases (Kellar et al, 2001). The objective of this study was to develop and validate a protocol for a MIA to detect antibodies to WNV and SLEV in sentinel chicken sera. A total of 385 sentinel chicken sera from 2005 were assayed using the MIA for WNV and 424 sera from multiple years were assayed for SLEV. The capability of the MIA to multiplex allowed for simultaneous detection of antibodies to WNV and SLEV in sentinel chicken sera.The MIA was found to be more sensitive and specific than both the HAI and MAC-ELISA for the detection of antibodies to WNV, and just as sensitive and specific as the MAC-ELISA for the detection of antibodie s to SLEV in sentinel chicken sera. These results indicate that there is a potential of the MIA to decrease turn-around time and allow for earlier detection and improvement to the current surveillance system.
Thesis (M.A.)--University of South Florida, 2006.
Includes bibliographical references.
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Adviser: Lillian M. Stark, Ph.D.
x Public Health
t USF Electronic Theses and Dissertations.
Development of a Microsphere-based Immunoa ssay for the Detection of IgM Antibodies to West Nile Virus and St. Louis Encephalitis Virus in Sentinel Chicken Sera by Logan C. Haller A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Public Health Department of Global Health College of Public Health University of South Florida Major Professor: Lillian M. Stark, Ph.D. Azliyati Azizan, Ph.D. Andrew Cannons, Ph.D. Date of Approval: April 5, 2006 Keywords: flaviviruses, surveillance, hai, elisa, prnt, luminex Copyright 2006, Logan C. Haller
Dedication To my husband Chad who constantly encourages and motivates me to pursue my dream.
Acknowledgments This has truly been an amazing experience and I have been extremely fortunate to conduct my research with some of the fine st faculty, students, and colleagues in the public health field. I am especially grateful to Lillian Stark, Ph.D., MPH for all of her encouragement, expertise, and the opportunity in assisting me to make a difference in the public health field. I would also like to thank Azliyati Azizan, Ph.D. and Andrew Cannons, Ph.D. for their guidance and valuable advice. I extend my thanks to Mark Sweat, Ph.D. for his guidance and knowledge Deno Kazanis, Ph.D. for ordering all the supplies and the CDC Fort Collins for suppl ying me with the antigen for this study. Without the hard work of the Arboserology De partment at the Florida Department of Health, this study would not be possible. I w ould especially like to thank Rita Judge and Maribel Castaneda for their excellent work and dedication to the Sentinel Chicken Surveillance Program, and Ann Mutilinksy for her assistance with the IgM capture enzyme-linked immunosorbent assay. I grea tly appreciate Christy Ottendorfer for her support and assistance with the Plaque Reduc tion Neutralization Test and helping me with my experimental design and completion. I would also like to thank Angela Butler for all of her help and dedication with the statistical analysis. I extend my thanks to everyone in my thesis support group: Jazmin e Mateus, Jennifer Gemmer, & Dana Longo. Special thanks are also extended to my families, the Wolpins and the Hallers for all of their support, and for always believing in me.
i Table of Contents List of Tables iii List of Figures v List of Symbols and Abbreviations vii Abstract viii Introduction 1 Arboviruses 1 Flaviviruses 2 West Nile Virus 4 Discovery 4 Epidemiology 4 Transmission 5 Clinical Features 5 Immune Response 6 St. Louis Encephalitis Virus 6 Treatment and Prevention 8 Surveillance 8 Florida Sentinel Chicken Program 9 Serological Detection Methods 10 Hemagglutination Inhibition Test 11 IgM Antibody Capture Enzyme-Linked Immunosorbent Assay 12 Plaque Reduction Neutralization Test 13 Microsphere-based Immunoassays 14 Objectives 18 Materials and Methods 20 Sentinel Chicken Sera Submission and Processing 20 Sample Selection and Serum Specimens 20 Serum Analysis 25 HAI, MAC-ELISA and PRNT Assays 25 Microsphere-based Immunoassay 28
ii Standardization of Microsphere-based Immunoassay 28 Flavivirus Monoclonal An tibody Coupled Microspheres 29 Addition of Antigen to Bead Sets 29 Microsphere-based I mmunoassay Protocol (Sentinel Chicken Sera) 32 Detection of Antibodies to WNV and SLEV 38 Classification of the Micros phere-based Immunoassay Results 40 Results 43 IgG Depletion of Sentinel Chicken Sera 44 Classification of the Microsphe rebased Immunoassay Result 44 Detection of Antibodies to West N ile Virus and St. Louis Encephalitis Virus 48 Hemagglutination Inhibition Test 48 Flavivirus 48 West Nile Virus 48 St. Louis Encephalitis Virus 51 IgM Antibody Capture Enzyme-Linked Immunosorbent Assay 51 West Nile Virus 51 St. Louis Encephalitis Virus 53 Microsphere-based Immunoassay 56 West Nile Virus 56 St. Louis Encephalitis Virus 56 Correlation of Assay Sensitivity, Specificity, Positive Predictive Value, and Negative Predictive Value with Prevalence by Month for West Nile Virus 59 Discussion 65 References 79 Bibliography 86
iii List of Tables Table 1 Illustration of Sentinel Chicken Sera from Multiple Years Tested for the Presence of IgM Antibodies to St. Louis Encephalitis Virus 24 Table 2 Standardization of Micros phere-based Immunoassay Reagents for the Detection of IgM Antibodies to WNV and SLEV in Sentinel Chicken Sera 30 Table 3 Standardization of Primary and Secondary Antibodies in Combination to Optimize the Detection of Viral Specific IgM Antibodies in Sentinel Chicken Sera 31 Table 4 WNV Antigen Coupled Bead Set Sample Mix 33 Table 5 SLEV Antigen Coupled Bead Set Sample Mix 34 Table 6 Detection of Antibodies to Flavivirus Group Using the HAI 49 Table 7 Detection of Antibodies to Flavivirus Group in the HAI Compared to the WNV True Result 50 Table 8 Detection of Antibodies to Flavivirus Group in the HAI Compared to the SLEV True Result 52 Table 9 Detection of Antibodies to WNV in the MAC-ELISA vs. the True Result 53 Table 10 Detection of Antibodies to SLEV in the MAC-ELISA vs. the True Result 55 Table 11 Detection of Antibodies to WNV in the MIA vs. the True Result 57 Table 12 Detection of Antibodies to SLEV in the MIA vs. the True Result 58
iv Table 13 Summary of Statistical Analyses for the Hemagglutination Inhibition Assay (HAI), MAC-ELISA (ELISA), and Microsphere-based Immunoassay (MIA). 60 Table 14 Cost Analysis of th e MIA for WNV and SLEV Based on 40 Samples 76
v List of Figures Figure 1 Flowchart Algorithm for Se ntinel Chicken Sera Testing 21 Figure 2 Flowchart Algorithm for Sentinel Chicken Sera Testing for Antibodies to WNV 23 Figure 3 Flowchart Algorithm for Se ntinel Chicken Sera Testing for Antibodies to SLEV 26 Figure 4 Plate Format of the Microsphere-based Immunoassay for Sentinel Chicken Sera, Adapted from Johnson et al, 2005 36 Figure 5 96-Well Filter Plate Showi ng the Combination of WNV and SLEV Bead Sets 37 Figure 6 MIA Flowchart for Sentinel Chicken Sera 39 Figure 7 Illustration of the Lumine x Systems Dual Laser Detection of IgM Antibodies to WNV 41 Figure 8 WNV ROC Curve Illustrati ng the Determination of the WNV Cut-off Value 46 Figure 9 SLEV ROC Curve Illustrating the Determination of the SLEV Cut-off Value 47 Figure 10 Correlation of Prevalence with Sensitivity, Specificity, PPV, and NPV for the Hemagglutina tion Inhibition Antibody Test 62 Figure 11 Correlation of Prevalence with Sensitivity, Specificity, PPV, and NPV for the MAC-ELISA 63 Figure 12 Correlation of Prevalence with Sensitivity, Specificity, PPV, and NPV for the Microsphere-based Immunoassay 64
vi Figure 13 Comparison of the Sens itivity and Specificity for the Hemagglutination Inhibition Test, MAC-ELISA, and Microsphere-based Immunoassay for the Detection of Antibodies to WNV 70 Figure 14 Comparison of Positive Predictive Value and Negative Predictive Value for the Hema gglutination Inhibition Test, MAC-ELISA, and Microsphere-b ased Immunoassay for the Detection of Antibodies to WNV 71 Figure 15 Comparison of Sensitivity and Specificity for the Hemagglutination Inhibition Test, MAC-ELISA, and Microsphere-based Immunoassay for the Detection of Antibodies to SLEV 72 Figure 16 Comparison of Positive Predictive Value and Negative Predictive Value for the Hema gglutination Inhibition Test, MAC-ELISA, and Microsphere-b ased Immunoassay for the Detection of Antibodies to SLEV 73 Figure 17 Combination of MIA and HAI Results for West Nile Virus in Five Chicken Sera 74
vii List of Symbols and Abbreviations Symbol and Abbreviations Description % Percent C Degrees Centigrade Ab Antibody Ag Antigen CDC Centers for Disease Control and Prevention CSF Cerebrospinal Fluid EEEV Eastern Equine Encephalomyelitis Virus FBE Florida Bureau of Epidemiology FDOH Florida Department of Health g gravity HAI Hemagglutination Inhibition Assay HJV Highlands J Virus IgG Immunoglobulin G IgM Immunoglobulin M JE Japanese Encephalitis Virus MIA Microsphere-based Immunoassay MAC-ELISA IgM Antibody Capture Enzyme-Linked mRNA Messenger Ribonucleic Acid g microgram L microliter mL milliliter min minute NPV Negative Predicted Value PPV Positive Predicted Value PRNT Serum Neutrali zation Plaque Reduction Test SLEV St. Louis Encephalitis Virus
viii Development of a Microsphere-based Immunoa ssay for the Detection of IgM Antibodies to West Nile Virus and St. Louis Encephalitis Virus in Sentinel Chicken Sera Logan C. Haller ABSTRACT West Nile virus (WNV) and St. Louis Encephalitis (SLEV) are arthropod-borne viruses belonging to the genus Flavivirus and are classified as significant human pathogens of global epidemiological importan ce. Since its introduction into the United States in 1999, WNV has spread throughout mo st of the country and has caused major epidemics of neuroinvasive disease (Hayes a nd Gubler, 2005). SLEV is endemic to the United States and is maintained in an enz ootic transmission cycle in Florida. The Florida Sentinel Chicken Arboviral Surveillance Network was established in 1978 following a widespread rural epidemic of SLEV in centr al Florida to monitor the activity of arboviruses (Day and Stark, 1996). This program ultimately impacts vector control strategies and may warrant medi cal alerts to warn the population. Current serological detection methods for sentinel chickens include hemagglutination inhibition an tibody test (HAI), IgM anti body capture enzyme-linked immunosorbent assay (MAC-ELI SA), and Plaque Reduction Ne utralization Test (PRNT). These serological assays may take over three weeks to generate a final result. A more rapid and equally sensitive test to replace th ese current serological methods would be of benefit.
ix Microsphere-based immunoassays (MIAs) ar e a more rapid serological option for laboratory diagnosis of many di seases (Kellar et al, 2001). The objective of this study was to develop and validate a protocol for a MIA to detect antibodi es to WNV and SLEV in sentinel chicken sera. A total of 385 sentinel chicke n sera from 2005 were assayed using the MIA for WNV and 424 sera from mu ltiple years were assayed for SLEV. The capability of the MIA to multiplex allowed fo r simultaneous detection of antibodies to WNV and SLEV in sent inel chicken sera. The MIA was found to be more sensitive and specific than both the HAI and MAC-ELISA for the detection of antibodies to WNV, and just as sens itive and specific as the MAC-ELISA for the detection of antibodies to SLEV in sentinel chicken sera. These results indicate that there is a potential of the MIA to decrease turn-around time and allow for earlier detection a nd improvement to the current surveillance system.
1 Introduction Arboviruses Arboviruses ( ar thropodbo rne viruses) are viruses ma intained in nature through cycles involving hematophagous (blood sucking) arthropod vectors and vertebrate hosts (Beaty, et al, 1989). There are more than 534 viruses registered in the International Catalogue of Arboviruses; 134 (25%) have been documented as causing illness in humans (Karabatsos, 1985). The medically important arboviruses belong to three virus families: the Bunyaviridae, Flaviviridae, and Togaviridae (Gubler & Roehrig, 1998). Arboviral activity can be classified as enzootic and involves sporadic, intermittent, or epidemic cases resulting in significant outbreaks in humans, horses, and birds. In the United States, birds and rodents may serve as amplifying hosts and mosquitoes play an important role in transm ission. Infected mosqu itoes can spread the virus from the enzootic amplifying host to hor ses, other animals and people. For most arboviruses, humans and domes tic animals are considered dead-end or incidental hosts because they do not develop high enough levels of viremia to infect the arthropod vector and continue the transmission cycle. De ngue virus is one exception and has adapted completely to humans and is maintained in a mosquito-human-mosquito transmission cycle in urban centers of the tropics and sub-tropics (Gubler, 2002).
2 Arboviruses are distributed worldwide, but are commonly found in tropical areas where the climate can support year-round transmission by cold-blooded arthropods (Gubler, 2002). The presence of these arboviruses in a particular area depends on the availability of specific species of mosqu itoes and changing epidemiological trends. Population growth, uncontrolle d urbanization, new irrigation systems, and deforestation in tropical developing countri es have especially contri buted to the emergence and resurgence of arboviral diseases in several regions of the worl d (Gubler, 2001). This is a major public health concern, as th ese viruses can cause outbreaks of disease in areas where virus transmission has not occurred, or was previously controlle d. For example, in 1999, West Nile Virus (WNV) was introduced in to North America. Unlike WNV strains found in the eastern hemisphere, the North American strain is highly pathogenic, virulent, and has caused severe neurological disease epidemic s and epizootics in humans, birds, and horses (Gubler, 2002). This highly pa thogenic WNV strain evolved from a less pathogenic strain found in the Middle East, Israel, India, France, and South America (Lanciotti et al, 1999). Flaviviruses Many flaviviruses (a genus of the family Flaviviridae) are significant human pathogens, including the four serotypes of dengue virus, yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis vi rus, St. Louis encephal itis virus, and West Nile virus (Burke & Monath, 2001). These viruses are transmitted to humans from the bite of an arthropod vector. WNV and S LEV belong to the Japanese Encephalitis Antigenic Serocomplex.
3 Flaviviruses are spherical, lipid-enveloped RNA viruses approximately 50 nm in diameter with a 30 nm core. The genome c onsists of single-stranded positive sense RNA of approximately 11,000 nucleotides. This R NA encodes structural capsid (C) protein, envelope (E) protein, membrane/matrix (M) protein and seven non structural (NS) proteins (Mackenzie, Gubler, and Petersen, 2004). E protein, NS1, and NS3 are the most immunogenic proteins during a flavivirus infe ction (Hill et al., 1993). Envelope proteins are the major surface proteins and considered to be an important factor in receptor binding and membrane fusion with host cells The E protein stim ulates neutralizing antibody response and changes in this protei n affect WNV virulence (Hayes & Gubler, 2005). Membrane/matrix proteins are respon sible for the maturation of immature viral particles into infectious forms and C prot eins help build nucleocapsids (Knipe and Hawley, 2001). The binding of E protein to a host cell r eceptor allows the viron to enter the cell via receptor mediated endocytosis. Once inside the cell, vira l nucleopcapsids are dissembled. Transcription of the virons into messenger RNA (mRNA) is followed by translation into various protei ns (Chambers et al, 1990). A large polyprotein results from the translation of mRNA and is later divided by proteases into ten or more separate products. The RNA genome is replicated in th e cytoplasm, viral particles assemble and mature inside the lumen of endoplasmic reticul um and the virion is re leased from the host cell (Knipe and Hawley, 2001).
4 West Nile Virus (WNV) Discovery West Nile virus was first isolated from the blood of a febrile patient in the West Nile region of Uganda in 1937 (Smithburn et al, 1940). Thirteen y ears later, additional isolates were obtained from the blood of thr ee apparently healthy children (Melnick et al, 1951). Researchers were able to show that WNV was antigenically related to two other arboviruses known to cause encephatilitis, St. Louis encephalitis virus (SLEV) and Japanese encephalitis (JE) virus (Smithburn, 1942). Epidemiology In Africa and Asia, WNV is endemic w ith outbreaks occurring every few years during the late summer and fall months. Significant epidemics were reported in Israel in the 1950s, the Rhone delta region of France in 1962, South Africa in 1974, and Romania in 1996 (Hayes 1989). West Nile virus was first detected in No rth America in 1999 during an outbreak of encephalitis in New York City (Hayes and G ubler, 2005). At first mistaken for St. Louis encephalitis virus (SLEV), researchers identi fied WNV by its high mortality in birds. The virus appeared to be a highly virulent st rain of WNV introduced possibly from Israel (Lanciotti et al, 1999). Since its 1999 app earance in New York City, WNV has spread across North America and into Canada, Latin America, and the Caribbean. The largest epidemics of neuroinvasive WNV disease ever reported occurred in the United States in 2002 and 2003 (Hayes and Gubler, 2005). West Nile virus has emerged as a major public
5 health threat and rapidly impacted human s, horses, and birds throughout the western hemisphere. Transmission The rapid spread of WNV may be due in part to its transmission cycle. WNV is maintained in an enzootic transmission cycle between birds and Culex mosquitoes, with humans and horses as incidental hosts (Blackmore et al, 2003 ). Infectious mosquitoes carry virus particles in thei r salivary glands and infect susceptible birds during blood meal-feeding. In the temperate zone, transmi ssion occurs during the warmer months with peak activity from July-October (OLeary et al, 2004). The main competent bird reservoirs are corvids (crows, jays and magpies), house sparrows, house finches, and grackles (Komar, 2003). Field studies c onducted in different geographical areas corroborated the importance of birds in the transmission cycle of WNV based on the presence of high antibody rates in birds (Hayes 2001). It is thought that the spread of WNV throughout the United States is due to the migration patterns of these bird reservoirs (Rappole and Hubalek, 2003). Allig ators may also serve as a reservoir for WNV in the southeastern United States (Klenk et al, 2004). Since 2002, it has been noted that human-to-human transmissi on of WNV can also occur through blood transfusion, organ transplantations, trans-placentally, and pos sibly through breastfeeding (Pearler et al, 2003). Clinical Features WNV infection is characterized by an acute onset of fever, headache, malaise, fatigue, weakness, muscle pain, and difficulty concentrating. However, approximately
6 80% of WNV infections are asymptomatic, 20% result in self limiting West Nile virus fever (WNF), and <1% results in neuroinvasive disease (Hayes, 1989). Symptomatic illness develops 2-14 days after virus transmission in humans (Mackenzie, Gubler, and Petersen, 2004). Immune Response WNV replicates in dendritic cells at the site of infection, spreads to regional lymph nodes, and then progresses to the blood stream. In one study, Wang et al. (2004) showed evidence that binding of viral RNA to toll-like receptor-3 induces permeability of the blood-brain barrier, which allows viral penetration of the central nervous system in mice. This indicates that WNV can directly invade neurons, deep nuclei of the brain, and interior horn cells in the spinal cord. It has also been noted that CD8 T cells are involved in both the immunopathology of and recovery fr om WNV infection (Wang, et al., 2003). These studies have aided in the identificati on of the clinical symptoms that may be present with a WNV infection. An array of antibody types and subc lasses is produced by the normal host humoral response to viral in fection. IgM antibody is pr oduced early in the immune response and may indicate an acute recen t infection (Martin et al, 2000). St. Louis Encephalitis Virus (SLEV) Prior to the outbreak of WNV in New York City, St. Louis encephalitis virus (SLEV) was the most important agent of epid emic viral encephalitis in North America and the only mosquito-borne human pathogen in the family Flaviviridae found in this continent (Hunt et al, 2002; Tsai & Monath, 1987). SLEV was first identified as the
7 cause of human disease in North America af ter a large urban epidemic in St. Louis, Missouri, during the summer of 1933 (Day 2001). Over the past 70 years, SLEV has been responsible for important epidemics th roughout the United States (Monath & Heinz, 1996). The last major outbreak of SLEV in the United States occurred in 1974 to 1977, when more than 2500 human cases were repor ted (Johnson et al, 2005). The severity of SLEV disease in humans is dependent on age. During epidemics, incidence of disease in people older than 60 is generally 5-40 times gr eater than in those le ss than 10 years old. Frequency of encephalitis (the most severe symptom associated with SLEV) is also agedependent, increasing from 56%, for those age 20 or younger, to 87% for those over 60. In addition, mortality is 7-24% among those over 50, and less than 5% for those under 50 (Shroyer, 1990). The first indication that S LEV was a threat to the human population of Florida came in 1952 when the virus was is olated from a 30-year-old Miami man (Sanders et al, 1953). Major SLEV epidemics occurred in Florida in 1959, 1961, 1962, 1977 and 1990 (Shroyer, 1990). However, enzootic SLEV transmission is silent in nature with no reports of avian mortality, unlike the high rates of avian mortality associated with WNV in the western hemisphere (Lanciotti and Kerst et al, 2001). SLEV is maintained in an enzootic transmission cycle between birds and Culex mosquitoes. In tropical America, SLEV has also been isolated from many nonCulex mosquito species (Charrel et al, 1999). The case fatality ratio for human disease is variable depending upon different geographical locations and conditions (Monath and Heinz, 1996). WNV and SLEV infections ofte n present with simila r clinical profiles, sudden onset of fever, headache, and myalgi a, and have similar prevention measures
8 (Johnson et al, 2005). These closely related viruses sh are many antigenic, genetic, and ecologic characteristics (Chambers et al, 1990). Treatment and Prevention Currently, there are no approved human v accines for WNV or SLEV and clinical options for treatment of infection are limite d. Arroyo et al. (2004) investigated the immunogenicity, safety, and e fficacy of a ChimeriVax-West Nile Virus live attenuated vaccine. Pre-clinical studies are in progress on this vaccine and further investigation is required before its approval for widespread vaccination of humans (Arroyo et al, 2004). Although there are no current WNV vaccines approved for use in humans, there are two commercially available recombinant WNV DNA vaccines for horses (Davis et al., 2001). The main mode of prevention is to contro l the vector population (mosquitoes) through integrated vector control management programs and persona l protection behaviors. A surveillance network of federa l, state, and local health departments monitors arboviral activity in wildlife hosts, vector s, and humans (Gubler, 2002). Surveillance Surveillance systems serve as an early wa rning for the transmission of disease and aim to limit or prevent human cases. Ar bovirus surveillance programs have used antibody development in sentinel birds to monitor transmission cycles for decades (CDC, 1993). A few states use sentin el chicken flocks scattered throughout regions at greatest risk for WNV, SLEV, and other arboviruse s (where Florida is a primary monitoring region). WNV and SLEV are maintained in na ture by birds and therefore infections in avian hosts should occur more frequently (and earlier) than disease in people or horses.
9 Transmission of WNV and SLEV to sentinel chickens has be en seen year round (Day and Stark, 2000). The ideal sentinel bird is a species that is uniformly susceptible to infection, resistant to disease, rapidly develops a de tectable immune response, easily maintained, presents negligible health ri sks to handlers, does not contribute to transmission cycles, and seroconverts to the ta rget pathogen prior to the onset of disease outbreaks in the community (Komar, 2001). Ch ickens are often chosen as sentinels because they exhibit most of these characte ristics (Langevin et al, 2001). Domestic chickens are one of the most widely used sentinel animals for the detection of arboviruses. However, one ideal captive avia n sentinel for all arboviruses may not truly exist (CDC, 1993). The use of sentinel chickens allows sentinel data to be collected in real time and assesses the relative risk of mosquito-borne arbovirus transmission (Day, 2001). Florida Sentinel Chicken Program The Florida Sentinel Chicken Arboviral Su rveillance Network was established in 1978 following a widespread rural epidemic of SLEV in centr al Florida (Day and Stark, 1996). This surveillance program has formed statewide partnerships between the Florida Department of Health, local mosquito ma nagement districts, the Department of Agriculture and Consumer Services and other governmental agencies that monitor arboviral activity Florida guidelines recommend that se ntinels be placed in cages near potential mosquito breeding grounds and that all chickens in a flock are sampled weekly (FBE & FDOH, 2000). Seroconversion (devel opment of measurab le antibodies after
10 exposure to an infectious agent) rates in sentinel chickens serve as indicators and predictors of arbovirus transmission and id entify high risk areas (Komar, 2001). Floridas sentinel chicken surveillance program serves as an excellent early warning system not only for endemic diseas es like SLEV, but also allows for the detection of new or re-emerging diseases by presence of viral antibodies (Blackmore et al, 2003). It is very important to use surv eillance data to differentiate between the presence of SLEV and WNV since SLEV is ende mic to Florida. An increase in arboviral activity above historical baseline levels imme diately impacts vector control strategies and may warrant medical alerts to warn the popul ation. In 2005, there were 3,081 individual sentinel birds assayed (4 7, 542 serum samples). 414 chic kens developed antibody to WNV (10.9%), 5 to SLEV (0.3%), 414 (9.0%) to Eastern Equine Encephalomyelitis virus (EEEV) (9.0%), and 108 to Highlands Jay virus (HJV) (2.84%) (Stark, 2005). Serological Detection Methods Serological detection methods are comple x due to close antigenic relationships between the flaviviruses. Specialized diagnostic tests are required to differentiate between viruses, especially cross-reactions between WNV and SLEV, such that acute and convalescent paired sera samp les from patients are often ne cessary to analyze antibody response (Petersen and Roehrig, 2001). Curre nt serological detec tion methods include Hemagglutination Inhibition Antibody Test (HAI), IgM Antibody Capture EnzymeLinked Immunosorbent Assa y (MAC-ELISA), IgG ELISA Indirect Fluorescent Antibody Test (IFA) and Plaque Reduc tion Neutralization Test (PRNT).
11 Hemagglutination Inhibition Test (HAI) Hemagglutination is a characteristic harbored by most arboviruses, and hemagglutinins have specific requirements for type of erythrocyte and pH (Sabin & Buescher, 1950; Sabin, 1951; Chanock & Sabin, 1953, 1954a, 1954b; Sweet & Sabin, 1954). Arboviruses agglutinate goo se erythrocytes and antivir al antibodies in sera can specifically inhibit the hemagglu tination reaction such that this test can be utilized for diagnostic purposes (Clarke & Casals, 1958). Suckling mice are inoculated with virus, and the brain tissue, which contains high titers of virus, is harvested. The brains are used as antigens in the HAI assay after processing with sucrose-acetone. Acetone extraction and treatment of sera with protamine sulfate remove non-specific lipoprot ein inhibitors and broadens the pH range for hemagglutination activity. The test is pe rformed with treated sera that are serially diluted and incubated overnight with a st andardized amount of antigen allowing for antigen-antibody binding. The presence of antibody bound to the antigen inhibits the agglutination of the goose red blood cells as indicated by a button of red cells showing the inhibition of agglutinati on (Clarke & Casals, 1958). The endpoint of the HAI activity is the reciprocal of the highest serum dilution showing complete inhibition of hemaglutination (Lennette et al, 1995). The HAI test is inexpensive and easy to perform, used for a variety of etiologic agents, and can test large numbers of specimens at one time. Another benefit of this test is that it is not restricted to species type. Currently, the HAI test is used as a screening tool to detect the presence of antibodies to flaviviruses and alphaviruses in sentinel
12 chicken sera by the Florida Department of Heal th, Bureau of Laboratories. The HAI test is cost-effective, reproducible, has a high se nsitivity, and is usef ul for analyzing the 50,000 chicken sera received each year for testing. However, the HAI test cannot be performed rapidly, especially when large numbers of sera are tested. Results may not be reported for a week and may effect local agencies response time to initiate or intens ify control measures (Olsen et al, 1991). Cross-reactivity within a virus group (SLEV & WNV) is common and can complicate interpretation of HAI test re sults. The HAI test does not distinguish between antibodies to SLEV and to WNV, and for this reason this test is only used as a screening tool. Positive sera are then confirmed using other serological methods. IgM Antibody Capture Enzymed-Linked Immunoasorbent Assay (MAC-ELISA) The MAC-ELISA test can distinguish between viruses by indicating the infecting strain rather than just virus group. Th e immunoglobulin M antibody capture enzymelinked immunosorbent assay (MAC -ELISA) is a confirmatory test used to detect IgM antibodies to WNV and SLEV (Martin et al, 2000). Elevated levels of IgM usually indicate recent exposure to an tigen or a recent infection (Ben jamini et al, 2000). This is very important in determining current and increased arboviral activity. The MAC-ELISA has proven to be an excellent technique for measuring IgM antibodies in response to vira l infection (Duermeyer et al 1979; Hofmann et al, 1979; Schmitz et al, 1980; Roggendorf et al, 1981; Burke & Nisalak, 1982; Jamnback et al, 1982; Monath et al, 1984; Olson et al, 1991; Martin et al, 2000; Johnson et al, 2003). This standardized diagnostic method allo ws for a consistent rapid approach for
13 monitoring arboviral disease but is restricted by species type (Martin et al, 2000). The MAC-ELISA is very advantageous as it reduc es the need for a convalescent-phase serum which may be hard to obtain (Schmidt & Emmons, 1989). The MAC-ELISA test is performed as follows: anti-species IgM capture antibody (i.e. chicken) is coated on 96 well microplat es, and the wells are blocked with milk to decrease nonspecific background absorbance. Serum from the animal species is added (sentinel chicken sera) followed by non-infectio us viral antigen. The presence of antigen is detected using an enzyme-conjugated an ti-species antibody that interacts with a chromogenic substrate to generate a colorimetric result (Martin et al, 2000). The MAC-ELISA is very sensitive and speci fic. One shortcoming is that the MAC-ELISA is a two day test that requi res about 4 hours of hands-on time for a 40-sample test (Johnson et al, 2005). Since the ELISA is a confirmation test the number of sera tested is greatly reduced from the amount assayed in the HAI. Plaque Reduction Neutralization Test (PRNT) The Plaque Reduction Neutralization Test (PRNT) is by far the most sensitive and specific assay. It can be used with sera from any animal species and is considered to be the gold standard of reliability. Sera that are negative or equivocal in the MAC-ELISA are tested in the PRNT, which serves as the final confirmatory test. The standard PRNT is performed in Vero (African green monke y kidney) cell cultures and utilizes live infectious virus. If neutralizing antibody, re sulting from viral inf ection or immunization, is present in a serum sample it will prevent the virus from infecting Vero cells. Plaques form as colorless round areas where the cells have been killed by the virus. A reduction
14 in number of plaques in the pr esence of a serum sample, when compared to the controls, is indicative of neutralizati on (Schmidt & Emmons, 1989). A drawback of PRNT is that it uses live infectious virus, expensive, and takes at least two weeks to obtain confirmed results. The PRNT requires precision in pipetting and must be performed under stringent bi osafety requirements (Beaty et al, 1989). This described series of serological assays (HAI, ELISA, PRNT) may take over three weeks to generate a final result. A more rapid and equally sensitive test to replace these current serological met hods would be of benefit in decreasing turnaround time to initiate prevention strategies and early warning systems in a timely manner. Microsphere-based Immunoassays (MIAs) Microsphere-based immunoassays (MIAs) are increasingly popular as a serological option for laboratory diagnosis of many diseases (Kellar et al, 2001). The MIA technology merges the concepts of enzyme-linked immunosorbent assay (ELISA) and flow cytometry. While the full potential of this approach has only been realized recently, microspheres and flow cytometry have had a long history (Vignali, 2000). In 1981, fluorescent labeled latex particles were used in the measurement of phagocytosis by neutrophils and macrophages (Dunn and Tyrer, 1981). A year later, fluorescent beads were used as the standard for counting cells in the blood (Stewart and Steinkamp, 1982). Monoclonal antibodies coupled to beads gave rise to the idea that microsphere-based flow cytometric assays could be a viable alternative to the microtitre plate-based ELISA (Vignali, 2000).
15 An early attempt to use the microsphere -based flow cytometric technique to diagnose disease was utilized in an assay designed to quantify human IgG in sera. However, this study was not very successful as it was performed without any washing steps (Lisi et al, 1982). Since then, a number of MIA assays have been used to successfully measure antibodies against Helicobacter pylori (Best et al, 1992), hepatitis C virus (McHugh et al, 1997), immunoglobulin and immune comple xes (McHugh et al, 1986; Syrjala et al., 1991; Labus and Peters en, 1992), and phospholipid s (Stewart et al, 1993). In some instances these assays were mo re sensitive than th e conventional ELISA and could resolve indeterminate clinical samples (Vignali, 2000; McHugh et al, 1997). A multiplex cytokine assay has also been de veloped to detect 15 different cytokines simultaneously (Jager et al, 2002). All of these assays utilized the Bio-Plex instrument (Bio-Rad Laboratories, Hercules, CA). The Bio-Plex system combines the principle of immunoassay with Luminex fluorescent-bead-based technology (Jager et al, 2002). The Luminex system merges ELISA and flow cytometry and involves the de tection and analysis of a reaction attached to microspheres or beads (Johnson et al, 2005 ). The carboxylated surface of the bead allows for binding to many different biologi cal agents. Proteins, oligonucleotides, polysaccharides, lipids, antibodies, and small peptides have been adsorbed or chemically coupled to the surface of microspheres to capture analytes that are subsequently measured by a fluorochrome-conjugated detection molecule (Kellar & Iannone, 2002). A possibility of 100 different bead sets (pr oduced by the Luminex Corporation, Austin, Texas) allow for a single small volume of sa mple to be tested for several different
16 parameters at one time. Each bead set cont ains a mixture of red and infrared dyes and possesses its own unique spectral properties. This feature allows multiple bead sets to be simultaneously used in one tes ting well (Waterboer et al, 2005). The Luminex software has the ability to id entify each individual bead set by the combination of two lasers. The red laser exci tes the red and infrared dyes within the bead set which allows it to classify each bead set. The green laser measures the florescence associated with the binding between the bead and the fluorochrome-conjugated detection molecule. A main advantage of this met hodology is the ability to multiplex by testing many different biological agents simultaneously and the speed at which it can be accomplished (Waterboer et al, 2005). A single small volume of sample can generate a large amount of information from one single assay. MIAs have the potential to be especially applicable in arbovirus serology because viruses of the same genus can share sim ilar formats (Johnson et al, 2005). One study conducted by Wong et al. (2003a) shows the ab ility of MIAs to detect antibodies to flavivirus E proteins in human sera. This me thod involves a recombinant WNV envelope (E) protein antigen covalently coupled to mi crospheres (beads). The beads are incubated with human serum or cerebral spinal flui d (CSF) and antibodies bound to the E-protein antigen are detected with a fluorescently labeled anti-human i mmunoglobulin antibody. Flaviruses (especially WNV and SLEV) are antigeni cally similar and related in structure and contain the E protein. For this reason, th is assay can only detect antibodies to the group flavivirus, and cannot differentiate between WNV and SLEV. High crossreactivity of the E protein among flaviviruses limits the specificity of the assay and a
17 confirmation test is needed to identify the infecting virus (Wong et al, 2003). A later study by Wong et al. (2003b) used similar me thodology to target nonstructural protein 5 to differentiate WNV infection from Dengue and SLEV infection, and from flavivirus vaccination. Wong identified that nonstructural protein 5 is less cross reactive than the envelope protein, and has the ability to di scriminate a WNV infection from SLEV and Dengue (Wong et al, 2003b). A recent study by Johnson et al has shown the ability of the MIA to detect IgM antibodies to WNV and SLEV in human se ra (2005). This duplex method uses a flavivirus group-reactive monoclonal antibody coupled to two different bead sets. The use of this monoclonal antibody al lows for different types of antigen to be attached to the different bead sets. Human serum is added and detection of virus specific IgM antibody is done with a single primary antibody (Antihuman IgM) that is conjugated to the detection molecule phycoerythrin. These duplex MIA results compared favorably to those of the PRNT and MAC-ELISA. An advant age of this method is that it only takes 3.5 hours to complete a 40 sample plate test a nd several plates can be tested within one day. The duplex MIA represents a significan t improvement over the way in which WNV and SLEV serology is currently being perfor med, especially with respect to decreased turn-around time and the generation of a single result (Johnson et al, 2005).
18 Objectives Rapid and accurate West Nile virus serologi cal testing is a public health priority prompted by the dramatic increase in WNV in fections in the United States, and by the evidence that the virus can be transmitted by blood and organ donations (Wong et. al, 2003a). The use of sentinel chickens for su rveillance of arbovirus transmission is well established. The Florida sen tinel chicken program serves as a powerful tool for the detection of arbovirus transmission and identifi es prevention and cont rol strategi es that should be implemented (Blackmore et al, 2001). Currently, sentinel chicken sera are te sted using three different serological methods. The HAI assay is used as a screen ing tool, MAC-ELISA as a confirmatory test, and the PRNT as the gold standard final confirmatory test. The HAI is very sensitive although it uses group antigens so that closely re lated viruses often cross-react. MACELISA is a much more sensitive and specific as say that can identify the infecting virus, but it is limited by species specific reagents. The PRNT is the most sensitive and specific assay, but it is very expensive, a use live infectious virus, and is labor intensive. The combination of these three serological methods takes 2 to 3 weeks before participating counties can receive confirmed results. The MIA test has been shown to be just as sensitive and specific as the MAC-ELISA a nd PRNT when testing human sera, but has several advantages over these trad itional serological assays.
19 The hypothesis of this study is that a MI A can be developed (based on the method used by Johnson et al for human sera, 2005) th at is as sensitive and more specific than current serological screening methods to de tect IgM antibodies to WNV and SLEV in sentinel chicken sera. The development of an MIA to detect IgM an tibodies in sentinel chicken sera will decrease turn-around time for accurate results and allow prevention and control measures to be implemented in a more timely manner. The MIA has the potential and capability to handle a la rge number of sera for a sent inel surveillance program. This study has three specific aims: 1) To design and validate a protocol for a microsphere-based immunoassay to detect IgM antibodies to WNV and S LEV in sentinel chicken sera. 2) To determine the sensitivity, specificity, positive predictive value, and negative predictive value of the assa y compared to the MAC-ELISA and HAI tests. 3) To determine a cost and time analysis comparing the current series of serological detection methods to the adapted IgM MIA. The overall goal of this study is to e nhance public health by improving current serological detection methods that are used for the surveillance of WNV and SLEV in sentinel chickens.
20 Materials and Methods Sentinel Chicken Sera Subm ission and Processing Sentinel chicken sera are received on a weekly basis at the Florida Department of Health, Tampa Branch Laboratory as part of Florida s sentinel chicken surveillance program. A total of 3,081 adult sentinel chicke ns were maintained at sites throughout the state of Florida and 47,542 serum samples were submitted between 1/01/2005 to 12/31/05. Upon submission, samples were sc reened for the pres ence of antibody to flavivirus (SLE) and alphavirus (EEE) group antigens with the hemagglutination inhibition antibody test (HAI). Sentinels that were flavivir us group positive for the first time were assayed using the IgM antibody cap ture enzyme-linked immunosorbent assay (MAC-ELISA) and stored at 4 0 C for future MIA testing. Additional HAI negative sera (titer < 10) were also stored for future analysis (Figure 1). Sample Selection and Serum Specimens A total sample size of 368 sentinel chicke n sera was selected for the microspherebased immunoassay to detect IgM antibodies to WNV. This was determined using the sample size calculator designed by Camer on and Baldock (1998) based upon a population size of 3,081 individual sentinel chickens, 80% expected sensitivity and specificity, 12.1% expected prevalence (based on annual hi storical data), leve l of significance ( = 0.05), and power of 95% (http://www.ausvet.com.au/content.php? page=res_software#freecalc, 1998).
Figure 1 Flowchart Algorithm for Sentinel Ch icken Sera Testing. Sentinel chicken sera were first tested in the HAI antibody test with flavivirus group antigen. Sera were confirmed using the MAC-ELISA with WNV and SLEV antigen. MAC-ELISA negative or equivocal sera were tested in the PRNT. All sera samples were tested in the MIA to de tect IgM antibodies to WNV and SLEV. 21 Sentinel Chicken Sera HAI Positive HAI Negative MAC-ELISA MAC-ELISA Positive Negative or Equivocal Positive Negative or Equivocal PRNT Microsphereb ased Immunoassay PRNT Microsphereb ased Immunoassay Microsphere-based Immunoassay Microsphere-based Immunoassay Sentinel Chicken Sera Testing Algorithm
22 A total of 385 sentinel ch icken sera were assayed in the MIA for WNV infection and SLEV infection. 273 flavivirus group positive sera samples from the HAI assay (titer > 1:10 and reactive) were confirmed WNV pos itive in the MAC-ELI SA (P/N > 2.0) and tested in the MIA assay. An additional 36 samples were also HAI flavivirus positive but tested negative or equivocal in the MAC-ELI SA (P/N < 2.0) and were assayed in the PRNT and the MIA. 76 HAI negative samples (titer <10) were tested in the MACELISA, PRNT and MIA (Figure 2). The prevalence of SLEV is very low lead ing to a large predic ted sample size of 950 sentinel chickens. This was determined using the same sample size calculator as above, based upon an average population size of 2,820 individual sentinel chickens from five years (1999, 2000, 2003, 2004, and 2005), 80% e xpected sensitivity and specificity, 7.3% expected prevalence, level of significance ( = 0.05), and power of 95% (http://www.ausvet.com.au/content.php?pa ge=res_software#freecalc, 1998). The expected prevalence was determined based upon annual historical pr evalence data from the Florida Department of Health, Tampa Bran ch Laboratories. A total of 424 sentinel chicken sera from multiple years were a ssayed in the microsphere-based immunoassay for SLEV (Table 1). In 1999, 6 sera were HAI flavivirus group positive (titer > 1:10) and confirmed in the PRNT for SLEV. 28 sentinel chicken sera were HAI flavivirus group positive and confirmed SLEV positive in the MAC-ELISA in 2000. A total of 5 sera from 2003 were HAI flavivirus group positive and confirmed MAC-ELISA positive for SLEV. One sample from 2004 was HAI flavivirus group positive and SLEV confirmed
Figure 2 Flowchart Algorithm for Sentinel Chic ken Sera Testing for Antibodies to WNV. Sentinel chicken sera were firs t tested in the HAI antibody test with flavivirus group antigen. Sera were confirmed using the MAC-ELI SA with WNV antigen. MAC-ELISA negative or equivocal sera (Neg or Equiv) were tested in the PRNT. All sera samples were tested in the MIA to detect IgM antibodies to WNV. 23 Sentinel Chicken Sera (385) HAI Positive (309) HAI Negative (76) MAC-ELISA (309) MAC-ELISA (76) Positive (273) Neg or Equiv (36) Positive (6) Neg or Equiv (70) PRNT (36) Microsphere-based Immunoassay (6) PRNT (70) Microsphere-based Immunoassay (273) Microsphere-based Immunoassay (36) Microsphere-based Immunoassay (70) Sentinel Chicken Sera Testing Algorithm for West Nile Virus
24 Table 1 Illustration of Sentinel Chicken Sera from Multiple Years Tested for the Presence of IgM Antibodies to St. Louis Encephalitis Virus. Chicken sera were classified into four different groups: flavivirus ( Flav. ) hemagglutination inhibition antibody test positive (HAI +) and MAC-ELISA positive (ELISA +), HAI + and ELISA negative (-), HAI and ELISA -, and HAI + and PRNT +. St. Louis Encephalitis Chicken Sera Year Flav. HAI +, ELISA + Flav. HAI +, ELISA Flav. HAI -, ELISA -, Flav. HAI +, PRNT + 1999 0 0 0 6 2000 28 0 0 0 2003 5 0 0 0 2004 0 0 0 1 2005 3 305 76 0
25 positive by the PRNT. 308 sera were HAI flavivirus group positive: 3 sera were confirmed in the MAC-ELISA for SLEV and 305 were MACELISA negative in 2005. 76 HAI negative sera were tested and confir med negative for SLEV in the MACELISA. All negative or equivocal sera in the MAC-ELISA were test ed in the PRNT (Figure 3). All sentinel chicken sera in this stud y were tested for the presence of IgM antibodies to WNV and SLEV simultaneous ly in the microsphere-based immunoassay. Serum Analysis HAI, MAC-ELISA, and PRNT Assays Sentinel chicken sera were received each week and centrifuged at 2000 xg for 10 min, 4 0 C (Beckman Coulter, Allegra 6R) for tes ting in the hemagglutination inhibition antibody test (HAI). Samples were treated with pr otamine sulfate (Holden et al., 1966), acetone extracted and assayed by the method of Clarke and Casals (1958) in microtiter plates. Antigens (TBH 28 SM11 11/5/04) fo r the HAI antibody test were prepared from suckling mouse brains by the sucroseacetone-extraction (Schmidt, 1979) and betapropriolactone-inactivation (Sever et al, 1964) method. Sentinel chicken sera that were flavivirus group positive for the first time in the HAI assay were tested for virusspecific IgM antibodies to WNV or SLEV in the MAC-ELISA, using the protocol developed by Martin et al., 2000).
Figure 3 Flowchart Algorithm for Sentinel Chicken Sera Testing for Antibodies to SLEV. Sentinel chicken sera were first tested in th e HAI antibody test with flavivirus group antigen. Sera were confirmed using the MAC-ELISA with SLEV antigen. MAC-ELISA negative or equivocal sera (Neg or Equiv) were tested in the PRNT. All sera samples were tested in the MIA to detect IgM antibodies to SLEV. 26 Sentinel Chicken Sera (424) HAI Positive (348) HAI Negative (76) MAC-ELISA (348) MAC-ELISA (76) Positive (40) Neg or Equiv (308) Positive (0) Neg or Equiv (76) Microsphere-based Immunoassay (0) PRNT (76) Microsphere-based Immunoassay (40) Microsphere-based Immunoassay (308) Microsphere-based Immunoassay (76) Sentinel Chicken Sera Testing Algorithm for St. Louis Encephalitis Virus
27 Antigens used in the MAC-ELISA included SLEV viral mouse brain antigen (TBH-28, cat# M29797) and normal antigen (for WNV and SLEV cat# M29714) that were obtained from the Centers for Disease Co ntrol and Prevention (CDC), Fort Collins, Colorado. West Nile viral antigen was prepar ed by sucrose-acetone extraction of suckling mouse brain at the Florida Department of Health, Tampa Branch Laboratory (WN EG101 SMB SA-antigen, lot# TBL 1, 2/6/03). Fl avivirus group peroxidase conjugated monoclonal antibodies (6B6C-1) (Cat# Vs2372, lot# 99-0074L) were obtained from the CDC, Atlanta, Georgia. Briefly, the MAC-ELISA was performed as follows: capture antibody, goat antichicken IgM, lyophilized (MP Biomedicals, cat# 64395, lot# 8155H), was diluted 1:1000 in carbonate-bicarbonate buffer (0.015M sodi um carbonate, 0.035 M s odium bicarbonate, pH 9.6). Viral antigens were diluted in wash buffer (0.05% Tween 20 PBS solution), WNV + antigen (1:160) and SLEV + antigen (1 :800). Chicken sera and test controls were also diluted in wash buffer (1:400). Se ra were assayed in duplicate, known positive and negative control chicken sera were included on each test plate. There were separate assay plates for each antigen tested (WNV and SLEV). MAC-ELISA results were determined by calculating positive to negative ratios (P/N). The P/N value for test serum was co mputed by dividing the mean Optical Density (OD) of the test serum with viral antigen by the mean OD of the negative control serum with viral antigen. A specimen was considered positive for IgM antibodies if the P/N > 2. A specimen was considered to have an equivocal result if the P/N value was in the range of 1.6 to 1.999. The validity of each sample was determined by dividing the OD of
28 the test sera with viral antigen by the OD of the test sera with negative antigen. Sera were considered valid if this value was > 2. Sera samples positive for IgM antibodies to WNV or SLEV were reported positive for inf ection. IgM-negative or equivocal chickens were tested in the PRNT to detect neutralizing antibody titers, using a previously described method (Schmidt, 1979; Beaty et al, 1989). Both positive and negative sera were then tested in the micr osphere-based immunoassay. Microsphere-based Immunoassay (MIA) Standardization of Microsphere-based Immunoassay The Microsphere-based immunoassay devel oped for this study was based on the method used by Johnson et al (2005) for the detection of IgM an tibodies to WNV and SLEV in human sera, and adapted to test sentin el chicken sera in this study. The clinical MIA used to assay human sera required a single anti-human IgM phycoerythrin conjugated primary antibody. This type of antibody was not available for chicken sera; therefore, a two step combination of a primary antibody followed by a secondary antibody was used to enhance detection. The secondary antibody was attached to the detector molecule and measured the detec tion of IgM antibodies to WNV and SLEV in sentinel chicken sera. A total of 20 MA C-ELISA WNV positive sera, 8 SLEV positive sera, and 20 negative sera were initially tested in the MI A to determine the optimal concentrations for primary and secondary an tibody, antigen/bead set mixtures, and test serum samples. Several dilution series were perfor med for each parameter individually (Table 2). Varying dilutions of the primary and seconda ry antibody were tested in combination with sentinel chicken sera, which was diluted 1:400 in MIA running buffer
29 (Table 3). Optimal concentrations were 2 g/ml for the primary antibody, 1 g/ml for the secondary antibody, 1:10 dilution for the antigen/bead set mixtures and a 1:400 dilution for the sentinel chicken sera samples. These concentrations were then used to test all specimens. The clinical MIA developed by Johnson et al, (2005) depleted the human sera of IgG prior to testing the human sera for the pr esence of IgM antibodies. A total of 20 MAC-ELISA WNV positive sera, 10 SLEV pos itive sera, and 20 negative sera were depleted of IgG using protein G sepharos e following the manufacturers instructions (Amersham Biosciences, cat# 17-0618-01) prior to tested in the MIA. These sera were also tested without depletion of IgG. Flavivirus Monoclonal Antibody Coupled Microspheres (Bead Sets) Two different bead sets were chemically coupled to a flavivirus group reactive monoclonal antibody (6B6C-1) and were purchased from Radix, Biosolutions, Georgetown, Texas. One bead set was used for WNV (bead set 32-6B6C-1. lot# C050330rG-11) and another bead set used for SLEV (bead set 57-6B6C-1, lot# C050330RG-12). Either control or positive vi ral antigens were then added to these monoclonal antibody coupled bead sets in order to perform the microsphere-based immunoassay. Negative control antigens were necessary to ensure that nonspecific factors or inhibitors were not pr esent in the test sera. Addition of Antige n to Bead Sets In two separate 4ml brown bottles (Nalgene, HDPE lot #2004-915), West Nile viral protein antigen, E-prM protein expresse d in COS-1 cells (obt ained from CDC Fort
Table 2 Standardization of Microsphere-ba sed Immunoassay Reagents for the Detection of IgM Antibodies to WNV and SLEV in Sentinel Chicken Sera. Each reagent was tested in duplicate at the specified dilution series for sera and antigen/bead set mixtures to optimize the performan ce of the MIA. For example, sera were diluted at a 1:160 and assayed with each dilution of the antigen/bead set mixtures (1:5, 1:10, 1:20). The concen tration of the primary and secondary antibody was held constant at 2 g/ml and 1 g/ml respectively. Microsphere-based Immunoassay Reagents 30 Sera Antigen Beadset 1:160 1:5, 1:10, 1:20 1:320 1:5, 1:10, 1:20 1:400 1:5, 1:10, 1:20 1:640 1:5, 1:10, 1:20 Dilution Series 1:1280 1:5, 1:10, 1:20
Table 3 Standardization of Primary and Secondary Antibodies in Combination to Optimize the Detection of Viral Specific Ig M Antibodies in Sentinel Chicken Sera. Different concentrations of each antibody were combined and tested against sentinel chicken sera (1:400 dilution in MIA running buffer) to minimize nonspecific reactions that influence fluorescence intensit y. The concentration of the antigen/bead set mixture was also held constant at a 1:10 dilution. 31 Antibody Concentrations ( g/ml) Primary Antibody Secondary Antibody (Goat Anti-Chicken IgM) (PorcineAnti-Goat IgG-PE) Combination 1 4 2 Combination 2 2 2 Combination 3 2 1
32 Collins-Focus technologies, lot# 02-VPA-RD 10274) and negative COS-1 antigen (obtained from CDC Fort Collins, 2005 lot) were sepa rately coupled to bead set 32 containing the 6B6C-1 monoclonal antibody (6 B6C-1/beadset 32) in MIA running buffer (PBS 1% BSA, Sigma cat # P3688) [see Ta ble 4]. The mixture was incubated on a rotating labquake tube rotator (VWR cat # 56264-302) at room temperature for one hour and then stored for up to one month at 4 0 C (Johnson et al, 2005). St. Louis Encephalitis virus suckling mouse brain antigen (obtaine d from CDC, Fort Collins FC-M29 715-04-0, cat # 0057) and normal control suckling mouse brain antigen (negativ e antigen, obtained from CDC, Fort Collins, cat# 0006) were coupled separately to bead set 57 containing the 6B6C-1 monoclonal antibody (6B6C-1/beadset 57) using the same procedure [see Table 5]. The final concentration for each antigen/bead set mixture was 500 beads/ l (Johnson et al, 2005). Microsphere-based Immunoassay Pr otocol (Sentinel Chicken Sera) The microsphere-based immunoassay differs from the traditional ELISA format primarily in the type of plate used. The 96 well filter plates are specifically developed for compatibility with bead-based assays (Millipore cat # MABVN1250, lot# F5HN65106). Individual wells are used as reaction vessels and allow fo r repeated incubations and filtrations using the vacuum manifold system (VWR cat# 16003-836). The beads are trapped on the filter and are not removed during filtration. Serum samples, primary and secondary antibody, and antigen/bead set mixtures were diluted to their working concentrations (Tables 4, 5). Sera, including positive and
33 Table 4 WNV Antigen Coupled Bead Set Sample Mix. Reagents and volumes listed are sufficient for 60 reactions. The protein concentration of the WNV positive recombinant antigen required the usage of a higher volume due to the nature of the antigen. Reagent Volumes for Coupling Positive and Negative Viral Antigens to Bead Set 32 Reagents WNV (+) Ag/6B6C-1* WNV (-) Ag/6B6C-1* WNV (+) Recombinant E-prM Antigen 17.5 _________ WNV(-) Recombinant Antigen ________ 15.5 Bead set 32/6B6C-1 50 50 Running Buffer 432.7 434.5 Volumes for all reagents are in l.
Table 5 SLEV Antigen Coupled Bead Set Sample Mix. Reagents and volumes listed are sufficient for 60 reactions Reagent Volumes for Coupling Positive and Negative Viral Antigens to Bead Set 57 Reagents SLE (-) Ag/6B6C-1 SLE (+) Ag/6B6C-1 SLEV (+) SMB Antigen 5 _________ SLEV Normal Mouse Brain Antigen ________ 5 Bead set 57/6B6C-1 50 50 Running Buffer 445 Volumes for all reagents are in l. 34 445
35 negative controls, were diluted in MIA running buffer (1:400). Primary antibody, goat anti-chicken IgM serum lyophilized (MP Bi omedicals, cat# 64395, lot# 8155H), was diluted to 2 g/ml in MIA running buffer Secondary antibody, porcine anti-goat IgGphycoerythrin (PE) (R&D Systems, cat# F0106, lot# LNG06), was diluted to 1 g/ml in MIA running buffer. The antigen/bead set mixtures (6B6C-1/bead set 32/WNV+Ag, 6B6C-1/bead set 32/WNV-Ag, 6B6C-1/bead set 57/SLEV+Ag, 6B6C-1/bead set 57/SLEV-Ag) were also dilute d in MIA running buffer (1:10) Working dilutions were prepared the same day as the assay and kept on ice until use. The bead/antigen mixtures and secondary antibody are light sensitive and were covered with aluminum foil. The left side of the plate was designate d for positive viral antigen and the right side was for negative viral or normal antige n. Each side included control sera in duplicate: WNV positive control serum, SLEV positive control serum, and negative control serum (Figure 4). This allowed for 40 sentinel chicken sera to be tested on one plate at ambient temperature. MIA running buffer (100 l) was added to each of the 96 wells and incubated for 5 minutes. Vacuum was applied to a manifo ld suctioning buffer from the wells. The positive SLEV and WNV bead/antigen mixtures were combined and added to each well in the left side of the plate (viral antigen side). Negative SLEV and WNV bead/antigen mixtures were combined and added to each well in the right side of the plate (Figure 5). The buffer was immediately removed by vacuum The beads were then washed twice with 100 l of MIA running buffer. Positive, negative, or test sera were added to each
Figure 4 Plate Format of the Microsphere-ba sed Immunoassay for Sentinel Chicken Sera, Adapted from Johnson et al, 2005. The pl ate was divided in half and the left side contained WNV and SLEV viral anti gens and the right side contained the negative or normal WNV and SLEV antigens. Positive Viral Antigen Negative Viral Antigen WNV positive sentinel chicken control sera SLEV positive sentinel chicken control sera SLEV/WNV negative control sera Sentinel Chicken test sera 36
Figure 5 96-well Filter Plate Showing the Co mbination of WNV and SLEV Bead Sets. WNV/6B6C-1/bead set 32 a nd SLEV/6B6C-1/bead set 57 were combined and added to each well of the filter plate. The left side of the plate contained WNV and SLEV positive antigen bead set/mixt ures and each well of the right side contained WNV and SLEV negati ve antigen bead set/mixtures. Positive Antigen Viral Antigen Negative Viral Antigen 37 WNV bead set32 6B6C-1/(+)WNV Ag SLE bead set57 6B6C-1/(+)SMB Ag WNV bead set32 6B6C-1/(-)WNV Ag SLE bead set57 6B6C-1/(-)SMB Ag
38 well in 50 l aliquots of the working dilution and th e plate was sealed with tape (Marsh Biomedicals, cat#1044-39-4) and covered with aluminum foil to protect the light sensitive beads. The plate was placed on a plate shaker platform and rotated at 1100 rpm for 30 sec to re-suspend and mix the beads/sera in each well. The plate shaker was then slowed to 300 rpm for 1 hour. After the one hour incubation, the we lls were washed twice with 100 l of MIA running buffer using the v acuum manifold, and 50 l of goat anti-chi cken IgM antibody was added to each well. The plate was placed on the shaker platform for 30 min. After the 30 min incubation, the wells were washed twice with 100 l of MIA running buffer, after which, 50 l of secondary antibody was added to each well. The plate was placed on the shaker for 30 minutes. After this 30 minute incubation, the wells were vacuumed and washed twice with 100 l of MIA running buffer. After the second wash, 100 l of MIA running buffer was added to each well of the plate. The plate was placed on the plate shaker at 1100 rpm for 30 sec to resuspend the beads (Figure 6). Detection of IgM Anti bodies to WNV and SLEV The Luminex instrument (BioRad (Bi oplex) cat # 171-203060) was turned on at least 30 minutes prior to plate reading to wa rm up the lasers. Start up and calibration steps on the instrument were performed fo llowing the recommended guidelines from the manufacturer (Bioplex Luminex). After the machine was calibrated, sample information including plate format, and pr otocol set up were added into the Luminex system. This protocol identified the two bead sets to be targeted (6B6C-1/32 and 6B6C-1/57) and the
Figure 6 MIA Flowchart for Sentinel Chicken Sera Bead sets coupled with Monoclonal Antibody and +/-Ag Addition of Sentinel chicken sera Addition of 10Ab Goat Anti-Chicken IgM Incubate 30 min Addition of 2oAb Anti-Goat-PE Incubate 30 min Read Plate using Luminex System Incubate for 1 hr Wash 2X (vacuum manifold) Wash 2X (vacuum manifold) Wash 2X (vacuum manifold)Resuspension 39
40 total sample volume. The plate was then placed into the machine and read under these conditions. The combination of two lasers within the Luminex machine simultaneously identified the individual bead sets and a ssociated antigen-antibody binding reactions. The red laser detected the bead set (32 or 57) while the green laser detected and measured the median fluorescent intensity associated with the bond between th e phycoerythrin (PE) molecule (secondary antibody) and the sample to antigen/bead set mi xture (Figure 7). A median fluorescent intensity (MFI) was given for each sample. These MFI numbers were exported to a Microsoft Excel file for further analysis. Classification of the Microsphere-based Immunoassay Results Classification was defined as the resu lt of the WNV/ SLEV MIA for a sample based upon transformed data. The median fl uorescent intensity (MFI) for each sample was transformed into an adjusted value by dividing the MFI of the sample reacted on viral antigen by the MFI of the negative contro l on viral antigen for each plate. Receiver Operator Characteristic (ROC) curves were used to define the optimal cut-off for the adjusted MIA values and further characteri zed the adjusted MFI as WNV positive, SLEV positive or negative. These curves were generated using the Analyse-it Software, LTD (a Microsoft Excel add-in) to vi sually assess a diagnostic test's ability to discriminate between normal and abnormal subjects (i.e. chicken sera) At each possible decision threshold, a curve plotted the percentage of abnormal subjects correctly diagnosed (true positives) against the percentage of normal subjects incorrectly diagnosed as abnormal (false positives). The ROC curve analysis determined the cutoffs for adjusted WNV and
Figure 7 Illustration of the Luminex Systems Dual Laser Detection of IgM Antibodies to WNV. If antibody to WNV was present in the sera it bound to the bead set coupled to the WNV viral antigen. Binding was detected with the combination of the green and red lasers of the Lu minex machine. This laser technology will also detect SLEV antigen/bead sets th at are also present in the same well. Detection of Antibodies in Chicken Sera PE 2oAb (anti-goat IgG-PE) WNV bead set32 6B6C-1/(+)WNV Ag Ab in chicken sera1oAb (goat anti-chicken IgM) LuminexGreen Laser Red Laser 41
42 SLEV MFI values based on sensitivity and sp ecificity. At each cut-off point, the ROC curve determines the amount of true positiv es, true negatives, false positives and false negatives expected. The comparisons of true values were defined by the current testing algorithm. For example, sentinel chicken sera that were HAI positive and MAC-ELISA positive were considered to have a positive true value. Sera that were HAI positive, MAC-ELISA negative and PRNT positive had a true value of positive and sera that were HAI negative MAC-ELISA negative and PRNT negative were classified as true negatives.
43 Results As previously determined by the Cameron and Baldock sample calculator, a total of 368 sentinel chicken sera were calculated for the WNV sample size and 950 sera for the SLEV sample size for this study. A total of 385 sentinel chicken sera were assayed as part of the WNV sample size. However, due to the low prevalence of SLEV, only 424 sera were available for testing and thus did not meet the expe cted sample size of 950. All sera were tested for viral antibodies to WNV and SLEV simultaneously using the microsphere-based immunoassay (MIA) developed for this study, and the ability of the assay to distinguish between the two viruses was evaluated. Using two-by-two tables, the sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NPV) were determined for each serological assay used to dete ct the presence of antibodies to WNV and SLEV (HAI, MAC-ELI SA, MIA). Results of the serological assays were compared to the true result as determined by the current testing algorithm. Univariate comparison of dichotomou s outcomes in the HAI, MAC-ELISA, and MIA was performed using McNemars Test (A nalyse It LTD, for Microsoft Excel). McNemars test is a statistical equivalency test in which the null and alternative hypotheses are reversed, thus looking for disc ordance. A p value less than 0.05 indicates that the two testing methods being compared are not equivalent. Kappa measure of agreement, Pearsons Correlation, and Youden s J, Discordance, and Concordance are statistical tests which look at equivalency and measure the association between variables
44 with dichotomous outcomes (Simple Inter active Statistical An alysis, Diagnostic effectiveness, http://home.clara.net/sisa/diaghlp.htm). The Kappa measure of agreement takes on a value of zero if there is no agreement between tests, and a va lue of 1 if there is perfect agreement indicating th e test always correctly predicts the outcome. The Kappa statistic has the following characteristics: less than 0.4 poor agreement, 0.4 to 0.75 fair to good, and greater than 0.75 excellent agreemen t. A correlation between the sensitivity, specificity, PPV, and NPV of the three serological assays with the prevalence of WNV per season and month were generated using Microsoft Excel. Statistical analyses were performed with the assistan ce of Angela E. Butler, MSPH. IgG Depletion of Sentinel Chicken Sera A total of 20 MAC-ELISA WNV positive sera, ten SLEV positive sera and 20 negative sera were depleted of IgG (describ ed above) and tested in the MIA. These results were compared to the non-depletion resu lts using a paired sample t-test (Microsoft Excel). It was determined that there was no significant difference between depleted and non-depleted chicken sera (p-value for all the paired > 0.05). As a result, all of the sera further analyzed in the MIA were not depl eted of IgG before testing. Classification of the Microsphere-based Immunoassay Results The raw median fluorescent intensity (MFI) values for the sentinel chicken sera reacted with viral antigen reached a maximum of 10,000 for antibodies to WNV and a maximum of 5,984.5 for antibodies to SLEV. Negative controls r eacted with viral antigen typically had MFI values less th an 120 and negative controls reacted with negative antigen resulted in MFI values less than 100. MFI values for positive controls
45 were approximately 4,000 for WNV and 2,000 for SLEV reacting with viral antigens and below 200 for negative antigens. The raw MF I values were adjusted by dividing the MFI of the sample reacting with vi ral antigen by the MFI of the negative control reacting with viral antigen (MIA Adjusted Value) for each plate. This adjusted value was then compared to the true value (determined by the current testing al gorithm), using Receiver Operator Characteristics (ROC) curves to de termine the optimal cut-off for the sentinel chicken sera. The point on the curve correspond ing to the best sensi tivity and specificity that yielded the highest numb er of true positives and true negatives, and the lowest number of false positives and false negativ es was selected. Based on 92.8% sensitivity and 80% specificity the cut-off for a sa mple with antibodies to WNV was 1.74545 and greater (Figure 8). This value represents the point on the curve (c ut-off point) where the sensitivity, specificity, PPV, and NPV for the pr ediction of the true value was the highest. The cut-off for a SLEV positive sample was 3.73214 and greater, based upon 93.2% sensitivity and 92.2% specificity (Figure 9). Samples with adjusted values below the cutoff were classified as negative. Since cross-reactivity was present among WNV and SLEV the sample was considered positive fo r the virus which had the higher adjusted MFI value or two or more times greater the ad justed MFI value. For example, if a serum had a WNV adjusted MFI of 3.5 and a SLEV adjusted MFI value of 3.8, the sera would be considered positive for WNV since 3.5 is two times greater than 1.74545 and 3.8 is just slightly above the cut-off for SLEV (3.73214).
Figure 8 WNV ROC Curve Illustrating the Determination of the WNV Cut-off Value (1.74545). The sensitivity, por trayed on the y axis, was plotted against 1specificity, x axis, to determine cutoff values corresponding to different sensitivities and specificities. The optim al cutoff for the det ection of IgM antibodies to WNV corresponding to high e xpected value of true positives and true negatives and a low value of fa lse positives and false negatives was established with a sensitivity of 92.8% and specificity of 80%. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 00.20.40.60.81 1 Specificity (false positives)Sensitivity (true positives) No discrimination WN ADJ VALUE 46
Figure 9 SLEV ROC Curve for the Determ ination of the SLEV Cut-off Value (3.73214). The sensitivity, portrayed on th e y axis, was plotted against 1specificity, x axis, to de termine cut-off values corresponding to different sensitivities and specificities. The optimal cutoff for the detection of IgM antibodies to SELV corres ponding to high expected value of true positives and true negatives and a low value of false positives and false negatives was established with a sensitivity of 93.2% and specificity of 92.2%. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 00.20.40.60.81 1 Specificity (false positives)Sensitivity (true positives) No discrimination SLE ADJ VALUE 47
48 Detection of Antibodies to West Nile Virus and St. Louis Encephalitis Virus Hemagglutination Inhibition Test (HAI) Flavivirus A total of 424 sentinel chic ken sera were assayed in the HAI for the presence of antibodies to the flavivirus group (WNV & SLEV) and 351 sera were flavivirus group positive. The HAI correctly classified 338 sera as flavivirus group positive and misclassified 13 sera as flavivirus group negative. A total of 73 negative sentinel chicken sera were assayed using the HAI a nd 23 were correctly classified as flavivirus group positive. Comparison of these two results in a 2 X 2 table yielded a 93 .6 % sensitivity, 79% specificity, 96% PPV, and 68% NPV for the de tection of antibodies to the flavivirus group (Table 6). West Nile Virus From a sample size of 385 sentinel chic kens, the HAI classified 312 sentinel chicken sera as having antibodies to the flavivirus group. Compared to the true results, 295 sentinel chicken sera were positive for W NV, 17 sera were flavivirus group positive, but negative for WNV: 3 of these sera were positive for antibodies to SLEV and 14 were negative for antibodies to both WNV and SLEV. The HAI test identified 73 sera that were flavivirus group negative, but when fu rther tested using the other serological methods (MAC-ELISA, PRNT) 22 sera were cl assified as having antibodies to WNV. In a 2x2 comparison of these results, sens itivity of the HAI assay for the detection of antibodies to WNV is 93%, specificity 75% PPV 94%, and NPV 70% (Table 7). The
Table 6 Detection of Antibodies to the Flavivirus Group Using the Hemagglutination Inhibition Antibody Test. This 2x2 table demonstrates the ability of the MIA to classify chicken sera as flavivirus group positive or negative. ( Flav + or Flav. ). Sensitivity, specificity, positive predictive value and negative predictive value for the HAI for the detection of antibodies to WNV were calculated based upon this 2x2 table. 49 ALGORITHM TRUE RESULT HAI Flav. + Flav. Totals Flavivirus + 338 13 351 Flavivirus 23 50 73 Sensitivity 94% Specificity 79% Totals 361 63 424 PPV 94% NPV 68%
Table 7 Detection of Antibodies to Flavivirus Group in the HAI Compared to the WNV True Result. The true results we re determined by the current testing algorithm. Sensitivity, specificity, positive predictive value and negative predictive value for the HAI for the detection of antibodies to WNV were calculated based upon this 2x2 table. 50 ALGORITHM TRUE RESULT HAI WNV + WNV Totals Flavivirus + 295 17 312 Flavivirus 22 51 73 Sensitivity 93% Specificity 75% Totals 317 68 385 PPV 94% NPV 70%
51 detection of antibodies to WNV in sentinel ch icken sera by the HAI co mpared to the true result was shown to be statistically equiva lent by McNemars test (p value = 0.8238). The Kappa measure of agreement was 0.661 indicating a fair to good agreement. St. Louis Encephalitis Virus From a sample size of 424 sentinel chic kens, the HAI classified 351 sentinel chicken sera as having antibodies to the flavivirus group. Compared to the true results, 43 sentinel chicken sera were positive for SLEV. 308 sera were negative for SLEV: 264 were positive for antibodies to WNV and 44 were negative for antibodies to both WNV and SLEV. The HAI test identified 73 sera th at were flavivirus group negative, but when further tested using the PRNT, one serum was cl assified as having antibodies to SLEV. HAI values were compared to the tr ue result in a 2x2 table showing 98% sensitivity, 19% specificity, 12% PPV and 99% NPV for the detection of antibodies to SLEV (Table 8). The detection of antibodies to SLEV in sentinel chicken sera by the HAI compared to the true resu lt was shown to be non-equivalent by McNemars test (p < 0.0001). The Kappa measure of agreement was 0.041 indicating a poor agreement. IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA) West Nile Virus A total of 302 sentinel ch icken sera were classified as MAC-ELISA positive or equivocal (P/N > 1.6). Compared to the true result, 290 sera were correctly classified as having antibodies to WNV (true positives) and 12 sera were cl assified as WNV
Table 8 Detection of Antibodies to Flavivirus Group in the HAI Compared to the SLEV True Result. Sensitivity, specificity, positive predictive value and negative predictive value for the HAI for the detection of antibodies to SLEV were calculated based upon this 2x2 table. 52 ALGORITHM TRUE RESULT HAI SLEV + SLEV Totals Flavivirus + 43 308 351 Flavivirus 1 72 73 Sensitivity 98% Specificity 19% Totals 44 380 424 PPV 12% NPV 99%
53 negative. 83 sera were classified as MAC-ELISA negative: 27 of these sera were identified positive for WNV and 56 sera were classified as negative for WNV in the PRNT (true negatives). A 2x2 comparison of the MAC-ELISA resu lts with the true results yielded a sensitivity of 91%, 82% specificity, 96% PPV and 67% NPV (Table 9). The detection of antibodies to WNV by the MAC-ELISA as compared to the true result showed that they were statistically no t equivalent by McNemars test (p = 0.0237). However, the Kappa measure of agreement was 0.679 indicating a fair to good agreement. St. Louis Encephalitis Virus 43 sera were classified as MAC-ELISA positive or equivocal for SLEV (P/N > 1.6) from a sample size of 424 sera. Compared to the true results, 42 of these sera were correctly classified as SLEV positive and one serum was classified as negative. 381 sera were identified as MAC-ELISA negative: two of these sera were identified positive for SLEV and 379 sera were confirmed negative fo r SLEV in the PRNT (true negatives). A 2x2 table comparison of the SLEV MAC-ELISA result with th e true results showed the sensitivity to be 95%, 99.7% specificit y, 98% PPV, and 99% NPV (Table 10). The detection of antibodies to SLEV in sentin el chicken sera by the MAC-ELISA compared to the true results were shown to be statistically equivalent by McNemars test (p value = 1.0000) and an excellent measure of agreement (K = 0.962).
Table 9 Detection of Antibodies to WNV in the MAC-ELISA vs. the True Result. Sensitivity, specificity, and predictiv e values were calculated based upon this 2x2 comparison for the detection of antibodies to WNV. ALGORITHM TRUE RESULT MAC-ELISA WNV + WNV Totals WNV+/ equiv 290 12 302 WNV 27 56 83 Sensitivity 91% Specificity 82% PPV 96% 385 Totals 317 68 54 NPV 67%
Table 10 Detection of Antibodies to SLEV in the MAC-ELISA vs. the True Result. Sensitivity, specificity, and predic tive values were calculated based upon this 2x2 comparison for the detection of antibodies to SLEV. ALGORITHM TRUE RESULT MAC-ELISA SLEV + SLEV Totals SLEV+/ equiv 42 1 43 SLEV 2 379 381 55 Sensitivity 95% Specificity 99.7% PPV 98% Totals 44 380 424 NPV 99%
56 Microsphere-based Immunoassay (MIA) West Nile Virus Based on a sample of 385 sera, a total of 315 sentinel chicken sera were classified as WNV positive by Microsphere-based Immunoassa y. Compared to the true results, 306 sera were identified as true positives for WNV (MIA positive and true result positive) and nine sera were determined to have a true negative result (MIA positive and true result negative). 70 sera were cl assified WNV negative by MIA. After comparison to the true result, 11 were WNV positive (false negative) and 59 were correctly classified as a true negative (MIA negativ e and true result negative). The 2x2 comparison of the MIA results with the true results yielded a 96% sensitivity, 87% specificity, PPV 97%, and 84% NPV (Table 11). The detection of antibodi es to WNV in sentinel chicken sera by the MIA as compared with the true results wa s statistically equivalent by McNemars test (p = 0.8238). The Kappa measure of agreement was 0.937 indicating an excellent agreement. St. Louis Encephalitis Virus The MIA detected antibodies to SLEV in 45 of the 424 sentinel chicken sera assayed. 42 sera were correctly classified as SLEV positive (MIA positive and true result positive) and three were misclassified (MIA positive and true result negative). A total of 379 sera were negative for antibodies to S LEV in the MIA: 377 sera were correctly classified as negative (MIA negative and true result negativ e) and two sera positive MIA result and a negative true result. The 2 x 2 comparison of the MIA results with the true results yielded 95% sensitivity, 99% specific ity, 93% PPV, and 99% NPV (Table 12).
Table 11 Detection of Antibodies to WNV in the MIA vs. the True Result. Sensitivity, Specificity, and predictive values were calculated based upon this 2x2 comparison for the detecti on of IgM antibodies to WNV. ALGORITHM TRUE RESULT MIA WNV + WNV Totals WNV+ 306 9 315 WNV 11 59 70 Sensitivity 96% Specificity 87% 385 Totals 317 68 57 PPV 97% NPV 84%
Table 12 Detection of Antibodies to SLEV in the MIA vs. the True Result. Sensitivity, Specificity, and predictive values were calculated based upon this 2x2 comparison for the detecti on of IgM antibodies to SLEV. ALGORITHM TRUE RESULT MIA SLEV + SLEV Totals SLEV+ 42 3 58 45 SLEV 2 377 379 Sensitivity 95% Specificity 99% Totals 44 380 424 PPV 93% NPV 99%
59 Detection of antibodies to SLEV in sentinel chicken sera by the MIA compared with the true result was statistically equivalent by McNemars test (p = 1.0000) and showed an excellent measure of agreement (K = 0.937). See Table 13 for a summary of statistical analyses. Correlation of Assay Sensitivity, Specificity, Positive Predictive Value, and Negative Predictive Value with Prevalence by Month for West Nile Virus Sentinel chicken sera tested in this study, were grouped by month and sensitivity, specificity, PPV, NPV, and prevalence were ca lculated for each month. The prevalence of each month was based upon historical se ntinel chicken surveillance data. A correlation between the prevalence and sensit ivity, specificity, PPV and NPV for each assay (HAI, MAC-ELISA and the MIA) were conducted. As prevalence of WNV infection increas ed, the sensitivity and NPV of the HAI remained consistent. However, as the prevalence increased, the specificity and PPV increased. Inversely, as the prevalence of WNV infection decreased in the sentinel chickens, the specificity and PPV of the HAI test decreased (Figure 10). Correlation for the MAC-ELISA showed that as prevalence increased, the sensitivity and PPV remained consistent As prevalence decreased the NPV and specificity decreased however, the data contained extreme values causing the trend line not to have a good fit with the data points (F igure 11). In comparison, as the prevalence increased the sensitivity and PPV remained consistent and the specificity slightly increased for the MIA. The NPV slightly decreased as prevalence increased (Figure 12).
Table 13 Summary of Statistical Analyses for the Hemagglutinati on Inhibition Assay (HAI), MAC-ELISA (ELISA), and Mi crospehere-based Immunoassay (MIA). McNemars Test, Pearsons Correlation, Youdens J, Kappa measurement of agreement, Discordance, and Concordance were used to show the equivalence, agreement and measure the associa tion between variables with dichotomous outcomes. The HAI, ELISA, and MIA were compared to the True Results for each statistical test. WNV Statistical Analyses Assay McNemar Pearsons Youdens J Kappa Discordance Concordance x 2 =0.013 0.684 p=0.000 0.681 0.661 10.1% HAI 89.9% p=0.522 x 2 =0.039 0.662 p=0.000 0.738 0.679 10.1% ELISA 89.9% p=0.024 x 2 =0.005 0.937 MIA 0.947 0.937 5.2% 94.8% p=0.824 p=0.000 SLEV Statistical Analyses x 2 =0.724 p<0.000 0.135 p=0.005 0.167 0.041 72.9% HAI 27.1% x 2 =0.002 0.961 p=0.000 0.952 0.962 0.7% ELISA 99.2% p=1.000 x 2 =0.002 0.937 MIA 0.947 0.937 1.2% p=1.000 p=0.000 60 98.8%
61 A correlation of WNV prevalence with sensitivity, specificity, PPV and NPV by season was calculated and found to have extreme values and too few data points and was considered to be statistica lly invalid. The month by m onth correlation was a better indicator to determine the influence of prev alence on the sensitivity, specificity, PPV and NPV for each serological assay. The prevalen ce of SLEV was too low to calculate the correlation by month or season and more data was needed to determine a statistically valid result.
Figure 10 Correlation of Prevalence with Se nsitivity, Specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) for the Hemagglutination Inhibition Antibody Test (HAI). R2 = 0.0378 0 20 40 60 80 100 024681 0 PrevalenceSensitivity (%) R2 = 0.5501 0 20 40 60 80 100 024681 PrevalenceSpecificity (%) 0 R2 = 0.1745 0 20 40 60 80 100 024681 0 PrevalencePPV (%) R2 = 0.016 0 20 40 60 80 100 024681 PrevalenceNPV (%) 0 62
Figure 11 Correlation of Prevalence with Sens itivity, Specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) for the MAC-ELISA. R2 = 0.029 0 20 40 60 80 100 024681 63 0 PrevalenceSensitivit y ( % ) R2 = 0.0013 0 20 40 60 80 100 024681 0 Prevalence S p ec ific it y ( % ) R2 = 0.0008 0 20 40 60 80 100 02468 1 0 Prevalence PPV ( % ) R2 = 0.0648 0 20 40 60 80 100 024681 0 PrevalenceNPV ( % )
Figure 12 Correlation of Prevalence with Sens itivity, Specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) for the Microsphere-based Immunoassay. 64 R2 = 0.003 0 20 40 60 80 100 02468 1 PrevalenceSensitiv 0 it y ( % ) R2 = 0.0135 0 20 40 60 80 100 024681 0 Prevalenceificity (%) Spec R2 = 0.0265 0 20 40 60 80 100 02468 1 PrevalencePPV 0 ( % ) R2 = 0.0348 0 20 40 60 80 100 024681 0 Prevalence NPV ( % )
65 Discussion This study describes the development of a Microsphere-based Immunoassay for the detection of IgM anti bodies to WNV and SLEV in sentinel chicken sera. Seroconversion rates in sentinel chickens ar e useful predictors of arboviral transmission activity in an area and the success of a sen tinel chicken surveillance program has been well-established in Florida (Blackmore, 2003). However, current serological detection methods may take up to 2-3 weeks to obtain a confirmed result. Unlike these assays, the MIA has the capability of testing a single small volume of sample for many parameters at the same time, dramatically decreasing turn -around-time by the labor atory (Johnson et al, 2005). Consequently, the development and impl ementation of a MIA for the detection of antibodies to WNV and SLEV would be especially beneficial to a sentinel chicken surveillance program as a tool to rapidl y identify arboviral transmission activity and implement vector control strategies. The MIA previously described by Johnson et al (2005) was shown to be capable of detecting IgM antibodies to WNV and SLEV in human sera and cerebral spinal fluid (CSF). Further adaptation of this method is necessary for other species and several combinations of test reagents were assayed for this projec t. In the clinical method, human sera were first depleted of IgG prior to testing. Af ter comparison of depleted and non-depleted chicken sera, it was shown that depletion of IgG was not necessary for testing of sentinel chicken sera. This may have been due to the different types of IgG
66 found in human and chicken sera. The IgG pres ent in chicken sera (also referred to as IgY) has a different molecular weight and structural properties than the human IgG (Johnson et al, 2005). In addition, the adapted MIA for chicken sera required a combination of primary and secondary antibody to enhance detection, which differed from the single antibody requirement for the clinical MIA. It was s hown (in this study), that a combination of antibodies provided the least background (as measured by the luminex instrument) & detection was not possible with a single antibody. However, the combination of two antibodies may have influenced the raw MFI values for each sample. Although raw MFI values for positive controls reacting with vira l antigen were similar to values shown in the clinical method, negative cont rol raw MFI values were sli ghtly higher in the MIA for sentinel chicken sera. Consequently, a separate testi ng algorithm and protocol was developed for the chicken MIA, as the raw MFI results of the negative chicken sera were different from the human sera and significantly impacted analysis. The statistical analysis program desc ribed by Johnson et al (2005) used MIA historical human data as a comparison group, and was developed for human sera only. Since the results indicated that chicken sera MFI values were different from the human sera MFI values, this statistical method wa s not used. In addition, historical data on chicken sera assayed by the MIA was not available for use as the comparison group. Unlike the statistical program used for clinic al sera analysis, raw MFI values were not used to determine test validity. Instead, ra w MFI values and ROC curves were used in this study to generate the cut-off values. Sera were then classified as WNV positive,
67 SLEV positive, or negative based on these adjusted MFI values and cut-off values. These adjusted MFI values for chicken sera may pr ovide historical or baseline data for a statistical analysis program to be developed in the future. The HAI antibody test is used as a scr eening tool and detects antibodies to the flavivirus group. The sensitivity, specificity, PPV, and NPV for the detection of antibodies to WNV was greater when using the MIA compared to the HAI (Figures 13 and 14). Sensitivity of the HAI (98%) for th e detection of antibodies to SLEV when compared to the true value was higher than the MIA (95%). However, the specificity of the HAI was extremely low (19%) indicating that the HAI test alone does not distinguish between SLEV and WNV (Figure 15). The low specificity is due to th e fact that 70% of the flavivirus group positive sera were WNV positive, and not SLEV positive The MIA, on the other hand, demonstrated a much highe r specificity of 87%. The PPV (99%) and NPV (93%) of the MIA for the detection of antibodies to SLEV were higher than the HAI, 99% & 12%, respectively (Figure 16). This illustrates the ability of the MIA to identify a true positive and a true ne gative sample for both WNV and SLEV. The HAI assay detects total antibody (Ig M and IgG) and requires a confirmation test to identify a recent infection (IgM), and to distinguish between WNV and SLEV. The detection of IgM antibodies may not be e qually as effective as the detection of IgG antibodies in the HAI. One advantage of the MIA is that it detects IgM antibodies and is able to distinguish between WNV and SLEV. For example, 11 negative HAI sera tested WNV positive in the MAC-ELISA, MIA, and PRNT. The convalescent phase of these sera later seroconverted in the HAI ( flavivirus positive) and confirmed in the MAC-
68 ELISA, MIA and PRNT. The MIA was able to detect antibodies to WNV in these samples seven to thirty-six days before the HAI (Figure 17) Two out of these 11 negative sera never seroconverted in th e HAI, despite detection in the MIA (and confirmation in the PRNT). This example demonstrates that the MIA was capable of detecting low levels of IgM antibodies in these sera, which could not be detected by the HAI. These results may be due to a limitati on of the HAI that requires a highly skilled technician to read and interpret the test resu lts, whereas the MIA is very simple to use, and interpretation of results are automated. Therefore, the MIA would serve as an excellent screening test because it is very se nsitive, specific, detects IgM antibodies, and identifies early antibody response, much superi or to the current HAI screening technique. MAC-ELISA is used as a confirmation test and distinguis hes between WNV and SLEV. The MAC-ELISA and MIA share sim ilar serological concepts and both can detect IgM antibodies. The MIA was more sensitive (97%) and specific (84%) than the MAC-ELISA (91% & 82%, respectively) for the detection of IgM antibodies to WNV (Figure 13). The specificity of the MIA establ ishes the ability of this assay to identify a true negative result and reduces the amount of false negatives by the high sensitivity. McNemars Test (p = 0.8238) and Kappa measure of agreement (K= 0.937) statistically showed that the MIA is an excellent predic tor of the true value. The PPV (97%) and NPV (84%) were also higher in the MIA co mpared to the MAC-ELISA (96% & 67%, respectively) (Figure 14). These results indicate that the MIA reduces the amount of false positives and false negatives. For example, 16 sentinel chicken sera that were negative in the MAC-ELISA
69 were WNV positive in the MIA, and confirmed by PRNT. Two sera were MAC-ELISA and HAI negative and were classified as SLEV positive in the MIA. Only one of the sera was confirmed SLEV positive with PRNT and demonstrated the sensitivity and specificity of the MIA to correctly classi fy a serum sample. The MAC-ELISA and MIA had very similar sensitivity, specificit y, PPV, and NPV for the detection of IgM antibodies to SLEV in sentinel chicken sera (Figure 15 and 16). Due to the low prevalence of SLEV since the introduction of WNV in Florida, the recommended sample size of 950 sera was not reached. Consequent ly, this smaller sample size may have influenced the results of the MIA for the detection of antibodies to SLEV. The sensitivity, specificity, PPV and NPV were determined by comparing the results of each assay, HAI, ELISA, MIA, to a tr ue value. The true value in this study was determined by the combination of the MAC-ELISA and PRNT result, following the current testing algorithm. HAI positive sera were assayed in the MAC-ELISA, and if negative in MAC-ELISA, these sera were then tested in the PRNT The PRNT, like the HAI, detects total antibody (primarily IgG ne utralizing antibody) in the sera. If low levels of IgM were present in the sera, the PRNT may not be able to detect the presence of these antibodies. Nine sera were WNV positive in the MIA, but negative in the MACELISA and PRNT negative. Consequently, these nine sera could not be confirmed positive and were classified as false positiv es (true value negative). The convalescent phase of these sera were not available for testing.
Figure 13 Comparison of Sensitivity and Specific ity for Hemagglutination Inhibition Test (HAI), MAC-ELISA (ELISA), a nd Microsphere-based Immunoassay (MIA) for the detection of antibodies to WNV. 91 97 75 87 93 82 0 20 40 60 80 100 HAI ELISA MIA Serolo g ical Assa y Percent (%) Sensitivity Specificity 70
Figure 14 Comparison of Positive Predictive Value and Negative Predictive Value for the Hemagglutination Inhibition Te st (HAI), MAC-ELISA (ELISA), and Microsphere-based Immunoassay for the detection of antibodies to WNV. 96 97 70 84 95 67 0 20 40 60 80 100 HAI ELISA MIA Serological Assay Percent (%) PPV NPV 71
Figure 15 Comparison of Sensitivity and Specificity Comparison of Positive Predictive for the Hemagglutination Inhibiti on Test (HAI), MAC-ELISA (ELISA), and Microsphere-based I mmunoassay for the detection of antibodies to SLEV. 95 95 19 99 98 100 0 20 40 60 80 100 HAI ELISA MIA Serolo g ical Assa y Percent (%) Sensitivity Specificity 72
Figure 16 Comparison of Positive Predictive Value and Negative Predictive Value for the Hemagglutination Inhibition Te st (HAI), MAC-ELISA (ELISA), and Microsphere-based Immunoassay for the detection of antibodies to SLEV. 98 93 99 99 12 99 0 20 40 60 80 100 HAI ELISA MIA Serolo g ical Assa y Percent (%) PPV NPV 73
Figure 17 Combination of MIA a nd HAI Results for West Nile Virus in Five Chicken Sera. Each chicken was tested twi ce in the MIA and these adjusted MFI results are depicted as bars. HAI antibody titers are also shown for each chicken and represent first time seroconversions (line of the same color for MIA numbers). Sera samples were not tested at ev ery time point. Once the HAI assay detected antibodies in sera for the first tim e, additional convalescent sera were not evaluated further. This data illust rates the ability of the MIA to detect low levels of virus specific antibody earlier than the HAI. 0 20 40 60 80 100 1 7 17 21 28 36 Time (Days)MIA Adjusted MFI Value0 1 2 3 4 5Natural Log (ln) of HAI Antibody Titer (Total Ab) MIA #1 MIA #2 MIA #3 MIA #4 MIA #5 HAI #1 HAI #2 HAI #3 HAI #4 HAI #5 74 A Combination of MIA and HAI Results for WNV in Five Sentinel Chickens
75 A recommendation would be to conduct parallel testing with the current testing algorithm to identify the true values of the sera and al low for a truly random sample to be tested. Parallel testing would also help to determine the appropriate confirmation algorithm needed for the MIA. The correlation of prevalence with sensitivity, specificity, PPV, and NPV by month for WNV showed similar results in MIA. The sensitivity and PPV of the MIA and MAC-ELISA are more robust to changes in pr evalence and the specificity and PPV of the HAI is negatively influenced by prevalence. The data for the NPV and specificity for the MAC-ELISA showed extreme values, which may be due to the samples that were HAI and PRNT positive, but MAC-ELISA negative (11 sera). These results indicated that the MIA test produced accurate results as the prevalence changed from month to month. The results indicate that the MIA is an excellent alterati ve to the current serological methods. A total of 18 sentinel ch icken sera can be assayed on a 96 well plate for the detection of antibodies to WNV using the MAC-ELISA. A separate plate is needed for the SLEV antigen. The HAI can assay 24 sera on a 96 well plate for SLEV antigen only. The MIA also uses a 96 we ll format but can test 40 samples for the presence of IgM antibodies to WNV and S LEV simultaneously. A cost analysis was evaluated to compare the HAI, MAC-ELISA, and MIA. The current cost per sample for the HAI is $6.00 for the flavivirus group, $12.00 for MAC-ELISA for WNV and SLEV, and $50.00/antigen pair (WNV and SLEV) for the PRNT (Personal communication with Dr. Lillian Stark, 2005). It was determined th at the cost per sample for the MIA is $9.30 for the WNV and SLEV antigen pair (Table 14) This price is very comparable to the
Table 14 Cost Analysis of the MIA for WNV and SLEV Based on 40 Samples. Reagents/Supplies Cost in dollars for 40 samples Coupled Bead Sets 32 and 57 216 Goat anti-chicken IgM 2 Porcine Anti-goat IgG-Phycoerythrin 45 Multiscreen BV filter plates 11 Plate sealer 2 PBS w/ 1% BSA 1 Multi-channel pipette tips 16 Nalgene brown bottles/ Reagent reservoirs 3 Hands on time (Labor 25.00$/hr) 75 TOTAL 371 MEAN COST PER SAMPLE 76 9.30
77 other serological methods, especially if this MIA could replace one or two of the current serological assays. An important consideration in implementi ng a new test in a diagnostic setting is the length of time needed to complete the a ssay and report results. Current serological assays are complex and require more than a single day to complete. The estimated completion times for the current serological methods are as follows: HAI, 3-5 days, MAC-ELISA, 2-3 days, and PRNT, 2-3 weeks. The MIA can be finished within one day and requires approximately 3 hours of hands on time. The most time consuming part of this assay was the reading of the plates by the Luminex instrument, especially when more than one plate was used. A full 96 well plat e takes about 30 minutes to read in the instrument. Multiple bead sets will also si gnificantly increase read time and should be taken into consideration when developing future multiplex assays. However, several plates can be finished within one day; a maxi mum of four plates per day were analyzed in this study. One plate can be read at a tim e while the other plates are covered with aluminum foil and placed in a drawer at room temperature until the instrument is available. Further research should be conducte d to determine the tota l number of plates, with multiple bead sets, the instrument can read in one day and the length of time the plates can be placed a room temperature before reading. This study focused on the MIA for the detection of flaviviruses in sentinel chicken sera. The current surveillance system includes screening for alphaviruses It is therefore recommended that a future study be conducted to develop a MIA to detect antibodies to alphaviruses in sentinel chicken sera. Ability of the MIA to multiplex may allow for the
78 development of an MIA that detects antibodies to flaviviruses and alphaviruses simultaneously and independently. The sentinel chicken surveillance program is a vital component for the control and prevention of arboviruses in the state of Florida. This surveillance system is particularly necessary in the state of Fl orida where the transmission of arboviruses is influenced by environmental factors such as climate, heavy rainfall, and hurricanes. Accurate and rapid testing is critical for an immediate public he alth response in order to identify high risk areas, issue medical alerts/advisories, and implement vector control measures. The implementation of the MIA into the already we ll established sentinel chicken surveillance program will decrease turn-around time and pr ovide a rapid and accurate detection tool necessary for a surveillance system. In conclusion, a sentinel chicken surveillance program is important for the development and implementation of prevention and control measures to protect the population. The MIA has the potential to rapidly and accurately detect recent WNV and SLEV infections (IgM antibodies), where it ma y eliminate the necessity of multiple assays, and would be especially advantageous to diagnostic laborat ories. The MIA was found to be more sensitive and specific th an the HAI and MAC-ELISA for the detection of antibodies to WNV and just as sensitiv e and specific as the MAC-ELISA for the detection of antibodies to SLEV in sentinel chicken sera. Early detection is very important to a surveillance system and allo ws prevention and control measures to be implemented in a timely manner. This study concludes that the MIA may be capable of providing earlier detection to protect the population.
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