Antibodies to Puumala virus in humans determined by neutralization test.

An assay for detection of neutralizing antibodies to Puumala virus using 96-well microtiter plates (NT-ELISA) was developed and evaluated. The test proved to have similar sensitivity and specificity as an IgG ELISA and indirect immunofluorescence test, when screening 187 sera (with an antibody prevalence rate of 19%) from normal populations in an endemic area of Nephropathia epidemica (NE) in Sweden. NE-patients monitored for 2 years had neutralizing antibodies in early sera collected 1-4 days after the onset of disease with a continuous increase in neutralizing antibodies with time. Furthermore, high titers of neutralizing antibodies were detected 10-20 years post-infection. This neutralization assay was also evaluated as a screening method in the production of monoclonal antibodies. The format of the NT-ELISA makes it feasible to screen a large number of specimens with results similar to the standard plaque or focus-reduction neutralization tests.     

A simplified and standardized neutralization enzyme immunoassay for the quantification of measles neutralizing antibody

A simplified and standardized neutralization enzyme immunoassay (Nt-EIA) was developed to detect measles virus growth in Vero cells and to quantify measles neutralizing antibody. Heat-inactivated sera were diluted serially 4-fold and tested in duplicate. The 50% reduction point (50%RP) of virus growth was calculated using the Reed-Muench formula and the neutralizing antibody titre of test sera was converted into mIU/ml by comparing their 50%RP with that of the international standard serum. The optimal virus input and incubation time were found to be 50-100 plaque forming unit (PFU)/well and 64-72 h, respectively. The simplified Nt-EIA had a good reproducibility with only 3.7-4.2% of duplicate tests having a ratio > 4 in an evaluation of intra assay variation and the coefficients of variance were 2-9% in an evaluation of inter assay variation. In addition, the simplified Nt-EIA had a high sensitivity(98.6%), specificity (100%) and agreement (98.8%) in qualitative comparison with plaque reduction neutralization test (PRNT). In quantitative comparison, the correlation coefficient between Nt-EIA and PRNT was 0.83 without log transformation or 0.77 after log transformation and 90% of 61 positive sera had a ratio < 4 between antibody titre tested by the two methods. The simplified Nt-EIA is thus a suitable alternative to the PRNT for the quantification of measles neutralizing antibody.     

Development of a tissue-culture-based enzyme-immunoassay method for the quantitation of anti-vaccinia-neutralizing antibodies in human sera

Vaccination with vaccinia virus is carried out in order to induce protection against variola virus, the causative agent of smallpox. Serum titer of vaccinia virus-neutralizing antibodies is considered to be well-correlated with in vivo protection. Plaque reduction neutralization test (PRNT) is the gold standard for detecting and quantifying vaccinia virus-neutralizing antibodies in sera of vaccinees. However, PRNT is time and labor consuming, which does not allow large-scale screening needed for a population survey. A simplified, sensitive, standardized, reproducible and rapid method, neutralization tissue-culture enzyme immunoassay (NTC–EIA) was developed for quantitation of neutralizing antibodies against vaccinia virus. The assay consists of the following steps: neutralization of the virus with serially diluted sera, infection of cells in culture and measurement of residual virus replication using an enzyme immunoassay. The assay can be used for animal (rabbit) or human sera. Titer averages obtained using NTC–EIA were highly correlated (R² = 0.9994) to those obtained using PRNT. The assay is carried out in 96-well plates and takes only 2 days to complete. With the appropriate setup, it can be automated fully to allow screening of a large number of sera.     

Development and evaluation of an automatable focus reduction neutralisation test for the detection of measles virus antibodies using imaging analysis

A plaque reduction neutralisation test (PRNT) is still regarded as the gold standard for the investigation of anti-measles immunity. In this study, an alternative simplified automatable focus reduction neutralisation test (AFRNT) based on the classical PRNT was developed. The AFRNT uses the conventional Edmonston strain of measles, immunoperoxidase staining with monoclonal antibodies, and automated plaque counts performed with AID ViruSpot software. The assay is performed in 96-well plates, requires 2 days, and is fully automatable. The AFRNT was evaluated in comparison with PRNT and Enzygnost anti-measles enzyme immunoassay (EIA). A total of 130 samples, which included two available WHO international anti-measles standards, sera from 90 patients, and 38 different lots of immunoglobulin products, were tested. Overall, good agreement was observed between EIA and both neutralisation tests; however, the EIA values for the immunoglobulin products and international standards were slightly but significantly higher than those of the neutralisation tests. The Bland-Altman analysis showed excellent agreement between AFRNT and PRNT. AFRNT is a fully automatable high-throughput neutralisation assay, which can be performed with measles and other types of viruses, including wild-type strains. It is perfectly suited for epidemiological and vaccine studies.     

Detection of West Nile virus in the tissues of specific pathogen free chickens and serological response to laboratory infection: a comparative study.

Using an isolate of West Nile virus (WNV) from lineage 1 (Goose/Israel 1998), groups of specific pathogen free chickens were experimentally infected via the subcutaneous or intravenous routes. To evaluate the relative efficiency of detecting the virus in the infected chickens, samples from a range of tissues and organs were examined by virus isolation tests in tissue culture, including Vero, primary chicken embryo liver and fibroblast cells, and polymerase chain reaction (PCR) analyses. Additionally, in order to investigate the serological response of the chickens and produce WNV monospecific antibodies, serum samples were collected from the birds during the trial and analysed for antibodies by virus neutralization (VN) and the plaque-reduction neutralization test (PRNT). No clinical signs or gross pathological changes were seen in any of the inoculated chickens throughout the study. The nested PCR used in the study appeared to be significantly more sensitive at detecting the presence of the virus in both the tissues and the inoculated Vero cell cultures compared with the detection of gross cytopathic changes as observed in infected Vero cell culture. No cytopathic changes were seen in the inoculated avian cell cultures. Following primary inoculation of the chickens there was a weak antibody response 15 days post-inoculation. However, following re-inoculation with inactivated WNV and adjuvant there was a substantial increase in the neutralizing antibody titres when tested 2 weeks later. The results obtained suggested that the PRNT was more sensitive than the conventional VN test. Based on detection of virus and serology there was no evidence of viral transmission to the close contact controls. It can be concluded that the PCR used in this study was more sensitive than virus isolation for the detection of WNV while the PRNT also appeared more sensitive than the conventional VN test.     

Real-time monitoring of flavivirus induced cytopathogenesis using cell electric impedance technology

A real-time cell analysis (RTCA) system based on cell-substrate electric impedance technology was used to monitor cytopathic effects (CPE) in Vero cell cultures infected with West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) at infectious doses ranging from 10¹ to 10⁶ plaque forming units (PFU) of virus. A kinetic parameter characterizing virus-induced CPE, CIT₅₀ or the time to 50% decrease in cell impedance, was inversely proportional to virus infectious dose. In WNV-infected cells, the onset and rate of CPE was earlier and faster than in SLEV-infected cells, which was consistent with viral cytolytic activity. A mathematical model simulating impedance-based CPE kinetic curves indicated that the replication rate of WNV was about 3 times faster than SLEV. The RTCA system also was used for quantifying the level of cell protection by specific neutralizing antibodies against WNV and SLEV. The onset of WNV or SLEV-induced CPE was delayed in the presence of specific anti-sera, and this delay in the CIT₅₀ was well correlated with the titer of the neutralizing antibody as measured independently by plaque reduction neutralization tests (PRNT). The RTCA system provided a high throughput and quantitative method for real-time monitoring viral growth in cell culture and its inhibition by neutralizing antibodies.     

Evaluation of a diagnostic algorithm using immunoglobulin M enzyme-linked immunosorbent assay to differentiate human West Nile Virus and St. Louis Encephalitis virus infections during the 2002 West Nile Virus epidemic in the United States

A diagnostic algorithm was developed to differentiate between human infections of West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) using positive-to-negative (P/N) ratios derived from the immunoglobulin M capture enzyme-linked immunosorbent assay (MAC-ELISA). To validate this algorithm, we tested 1,418 serum and cerebrospinal fluid (CSF) samples from confirmed WNV and SLEV infections collected during the WNV epidemic of 2002 in the United States. WNV P/N-to-SLEV P/N ratios (W/S ratios) were calculated and used to identify the infecting virus. These results were compared to results from the plaque reduction neutralization test (PRNT), which is currently the standard assay used to discriminate between closely related flavivirus infections. If the W/S ratio was > or =1, the predictive value positive (PNP) for WNV was 97.8%, where 95% of flavivirus cases were due to WNV infection and only 3.7% of specimens would require PRNT to differentiate WNV from SLEV infection. Use of the W/S ratio as part of the testing algorithm to interpret MAC-ELISA results generates reportable probable cases quickly, alleviating the need for PRNT in most instances.     

Humoral immunity and correlation between ELISA, hemagglutination inhibition, and neutralization tests after vaccination against tick-borne encephalitis virus in children

Vaccination against tick-borne encephalitis (TBE) virus is the measure of choice for disease control in endemic areas, as no treatment is available. In Italy, the province of Belluno is one of the most active TBE virus infection foci. In this study sera were examined from vaccinated children living in areas around Belluno in order to monitor the immune response after anti-TBE vaccination. For the assessment of neutralizing antibodies, a plaque reduction neutralization test (PRNT) was optimized and the correlation between enzyme-linked immunosorbent assay (ELISA), hemaglutination inhibition (HI), and neutralizing antibodies titers was evaluated. All children had high serum levels of TBE IgG in ELISA test after the vaccination, in agreement with previous studies. HI and PRNT titers ranged between very low and high levels. A good correlation between HI and PRNT titers, and with IgG ELISA titers, was observed. PRNT is an useful assay for monitoring protective immunity after the completion of anti-TBE vaccination. This type-specific assay is an important tool for differential diagnosis in cases where the presence of cross-reactive antibodies due to other flavivirus infections or vaccinations cannot be excluded.     

Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization.

A newly modified semimicro plaque reduction neutralization test (PRNT) in BHK cells was compared with a standard PRNT in bottles with LLC-MK2 monolayers and with an LLC-MK2 PRNT adapted to semimicro methods. The BHK semimicro PRNT compared favorably in terms of sensitivity in detecting dengue antibody (96%), specificity at a screening dilution (95%), and ability to detect seroconversion to dengue viruses of three serotypes (93%). Disagreements between the BHK test and the LLC-MK2 tests were attributed to greater sensitivity of the BHK test in detecting dengue type 2 (DEN-2) antibody in acute-phase sera and to apparent low-level DEN-1/DEN-3 cross-reactions in some sera in all three tests. The BHK PRNT was easier, faster, and more economical than either of the LLC-MK2 tests. Many of the benefits of the BHK PRNT derive from the fact that cells are infected while still in suspension, at the time of cell splitting, hence the term "BHK suspension test."     

Serological tests for detecting Rift Valley fever viral antibodies in sheep from the Nile Delta.

To determine the accuracy of serological methods in detecting Rift Valley fever (RVF) viral antibodies, we examined serum samples obtained from 418 sheep in the Nile Delta by using five tests. The plaque reduction neutralization test (PRNT) was considered the standard serological method against which the four other tests were compared. Twenty-four serum samples had RVF viral antibodies detected by PRNT. Hemagglutination inhibition and enzyme-linked immunosorbent assay antibodies to RVF virus were also present in the same 24 serum samples. Indirect immunofluorescence was less sensitive in comparison with PRNT, and complement fixation was the least sensitive. These results extend observations made with laboratory animals to a large field-collected group of Egyptian sheep.     

Differential dengue cross-reactive and neutralizing antibody responses in BALB/c and Swiss albino mice induced by immunization with flaviviral vaccines and by infection with homotypic dengue-2 virus strains

We investigated whether cross-reactive and/or cross-protective antibodies against dengue virus could be generated in 6-week-old BALB/c mice by immunization with currently approved flaviviral vaccines, i.e., Japanese encephalitis (JE) BIKEN and yellow fever (YF) 17D. Cross-reactivity with dengue antigens was apparent in at least one-third each of JE-vaccinated mouse sera and of JE/YF-vaccinated mouse sera by dengue enzyme immunoassay, but was not detected in sera of mice immunized with YF vaccine alone. All the immunized BALB/c mice failed to generate neutralizing antibodies against the New Guinea C laboratory (NGC-lab) strain of dengue virus type 2. In addition, we determined the specificity of neutralizing antibodies elicited in 3-week-old Swiss albino mice against two homotypic dengue-2 strains, i.e., NGC-lab and Singapore 1999 (SING/99). Although sera from virus-inoculated mice displayed better neutralization against the corresponding strain, antibodies elicited by NGC-lab exhibited a significantly poorer neutralizing response against the SING/99 strain compared to antibodies elicited by SING/99 against NGC-lab. The differences may be related to sequence variations of approximately 3% between the envelope proteins of both strains. Amino acid disparities at positions 71 (Glu --> Ala), 112 (Ser --> Gly) and 124 (Ile --> Asn), which are found in dengue-2 neutralization escape mutants, were also found in the SING/99 strain. The envelope sequence differences may explain diminished binding of NGC-lab-induced neutralizing antibodies to neutralizing epitopes within the envelope of the SING/99 strain, resulting in a lower titer of neutralizing antibodies against another strain of the same serotype.     

Presence of neutralizing antibodies to heterologous human immunodeficiency virus type 1 isolates in sera of infected individuals is not predictive of rate of disease progression.

These studies were undertaken to examine whether the presence of human immunodeficiency virus type 1 (HIV-1)-neutralizing antibodies in sera of infected individuals would alter the rate of disease progression. HIV-1-infected individuals (n = 87) were initially examined for neutralizing activity in vitro against both laboratory and tissue culture-adapted clinical heterologous HIV-1 isolates. The neutralizing activities of sera were determined by a 90% or greater reduction in HIV-1 p24 levels in vitro. In a cross-sectional analysis of all infected individuals, we observed that sera from asymptomatic individuals neutralized a significantly greater number of heterologous HIV-1 isolates than sera from symptomatic patients. Patients who could be followed up longitudinally (n = 24) were then studied to determine the impact of neutralizing antibodies on the rate of disease progression. We observed no significant difference between the numbers of HIV-1 isolates neutralized in vitro by sera from patients who remained clinically stable and by those from patients who progressed rapidly. Our data indicated that the presence or absence of neutralizing antibodies to heterologous HIV-1 isolates was not associated with the rate of disease progression.     

Comparison of Plaque- and Enzyme-Linked Immunospot-Based Assays To Measure the Neutralizing Activities of Monoclonal Antibodies Specific to Domain III of Dengue Virus Envelope Protein

The plaque reduction neutralization test (PRNT) is used widely to measure the neutralization activity of anti-dengue virus (DENV) antibodies, but it is time-consuming and labor-intensive and has low sample throughput. For fast and convenient measurement of neutralizing antibodies, especially in evaluating the efficiency of the DENV vaccines on a large scale, a new method is needed to replace PRNT. In recent decades, several microneutralization assays have been developed to overcome the limitations of PRNT. In the present study, we evaluated one of these, the enzyme-linked immunospot microneutralization test (ELISPOT-MNT), in comparison with PRNT. ELISPOT-MNT is performed in 96-well format, and the plaques are developed after 2 to 4 days using an ELISA to transform them into spots, which are detected automatically with an ELISPOT instrument. The assay is faster than PRNT, has a high throughput, and is more objective. We used 10 monoclonal antibodies (MAbs) against domain III of the DENV envelope protein (EDIII) to evaluate the two assays; all of these MAbs cross-react with all four serotypes of DENV as measured by immunofluorescence assay. The two neutralization assays were performed simultaneously to measure the 50% inhibitory concentration (IC₅₀) of these MAbs. Using PRNT as the reference and treating IC₅₀ values higher than 50 μg/ml of MAbs as negative, ELISPOT-MNT showed a sensitivity of 95.6% and specificity of 88.24% when 10 MAbs were tested against four DENV serotype strains. A good correlation (R² = 0.672; P = 0.000) was observed between the two assays, making ELISPOT-MNT a potentially valuable method for measure of neutralizing antibodies against DENV.     

An immunocytometric assay based on dengue infection via DC-SIGN permits rapid measurement of anti-dengue neutralizing antibodies

Dengue remains a global public health threat and development of a safe and effective vaccine is a principal public health goal. The primary correlate of immunity is thought to be neutralizing antibodies. Currently, the plaque reduction neutralization test (PRNT) is the gold standard measure of dengue neutralizing antibody responses, but this test is limited by time-consuming performance. In addition, some feel that use of viral strains adapted to grow in Vero or BHK cells may not accurately reflect protective responses. A human cell line transfected to express a putative natural dengue receptor, DC-SIGN (CD209), was used to measure antibody-mediated dengue neutralization. Using neutralizing monoclonal antibodies, immune sera, and laboratory adapted dengue viruses, serotype-specific neutralizing activity was demonstrated similar to that seen in the Vero PRNT. Importantly, serotype-specific neutralizing activity against recently isolated dengue strains with less heterotypic cross-neutralization than laboratory adapted viruses was also demonstrated.     

Performance of immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays using a West Nile virus recombinant antigen (preM/E) for detection of West Nile virus- and other flavivirus-specific antibodies

Focus Technologies developed an indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a mu-capture IgM ELISA for the detection of West Nile virus (WNV)-specific antibodies based on a WNV preM/E protein recombinant antigen. Normal and disease state serum panels were used to assess the performance characteristics of the two WNV ELISA kits. Totals of 807 and 1,423 sera were used to assess the IgG ELISA and IgM ELISA kits, respectively. The Focus Technologies IgG ELISA had a sensitivity of 97.6% and a specificity of 92.1% (excluding non-WNV flavivirus sera). The comparative method for WNV IgG may lack sensitivity in detecting IgG in early WNV infection, so the specificity of the Focus IgG ELISA may be higher than 92.1%. When sera from patients either infected with or vaccinated against other flaviviruses were tested on the WNV IgG assay, 35% of the sera reacted as positive for WNV IgG. Yellow fever and Japanese encephalitis vaccinees were less reactive in the IgG ELISA than St. Louis and dengue fever patients. The Focus Technologies IgM ELISA had a sensitivity and a specificity of 99.3% (excluding the non-WNV flavivirus sera). The overall cross-reactivity for the IgM ELISA to flavivirus sera was 12%, with 31% of St. Louis encephalitis patients found to be WNV IgM positive and no yellow fever vaccinees found to be WNV IgM positive. In a selected population of 706 sera, 15 false-positive WNV IgM sera were identified. The use of a background subtraction method for the IgM ELISA eliminated all 15 false-positive results, giving a specificity of 100% for the Focus IgM ELISA.     

Use of immunoglobulin G avidity assays for differentiation of primary from previous infections with West Nile virus

Since its introduction in 1999, West Nile virus (WNV) infections have spread rapidly across the North American continent. Diagnosis of acute WNV infection by detection of WNV-specific immunoglobulin M (IgM) is complicated by the persistence of detectable IgM for more than 1 year in some patients. IgG antibody avidity testing was assessed as a supplemental assay in the diagnosis of current infections. Three groups of serum samples were assayed in parallel by two different IgG avidity test systems (indirect immunofluorescence test [IIFT] and prototype enzyme-linked immunosorbent assay [ELISA]; EUROIMMUN, Luebeck, Germany). Group I (40 sera taken between 2 and 9 days after the onset of influenza-like symptoms) and group II (40 sera taken between 10 and 43 days after onset) were acute and convalescent specimens from patients with a positive anti-WNV IgM test (ELISA; Focus Diagnostics, Cypress, CA). Group III consisted of 43 patient sera collected between 6 and 12 months after infection. IgG antibodies specific for WNV were detected in 38% (ELISA) and 50% (IIFT) of group I sera, in 90% (ELISA and IIFT) of group II sera, and in 100% (ELISA and IIFT) of group III sera. Low-avidity IgG antibodies were demonstrated in 86% (ELISA) and 95% (IIFT) of IgG-positive patient samples taken between 2 and 43 days after the onset of symptoms (groups I and II). High-avidity IgG antibodies were detected in 100% of group III sera obtained 6 months or more after the onset of symptoms (ELISA and IIFT). IgG avidity tests for WNV infections are rapid and simple to perform. The determination of IgG avidity provides additional diagnostic certainty in differentiating between recently acquired and previous infections with WNV.     

Neutralizing antibodies for sandfly fever Naples virus in human sera on the island of Mljet

Sera from apparently healthy residents of various age from different localities of the Adriatic island of Mljet were tested by the plaque reduction neutralization test (PRNT) for antibodies to sandfly fever Naples virus (SFN). An average of 51.4% of the sera examined was found to have neutralizing antibodies. Reactors to SFN virus were found in all the age groups examined and the antibody prevalence appeared to increase with age. The presence of SFN neutralizing antibodies in all age groups indicates that the virus must have been present endemically and that it is still active on the island of Mljet.     

Evaluation of an enzyme-linked immunosorbent assay for quantitation of antibodies to Junin virus in human sera

An enzyme-linked immunosorbent assay (ELISA) was evaluated for the quantitation of anti-Junin virus (JV) antibodies, in 83 selected cases of Argentine haemorrhagic fever (AHF). Serum samples were studied in two groups to facilitate comparative analysis; the first group was ELISA with indirect immunofluorescence (IF) test, in the second ELISA with plaque reduction neutralization test (PRINT). From the results obtained by using ELISA and IF on the same serum samples, a clear tendency of ELISA to demonstrate seroconversion for JV earlier and at higher frequency than IF test was noted. Simultaneous titration of specific antibodies by ELISA and PRNT tests rendered significantly correlated titers (r = 0.81), both methods being equivalently specific (100%). The demonstration of specific antibodies by ELISA in two cases that were undetected by the PRNT test resulted in a higher sensitivity index for ELISA than for PRNT (100% vs 97%). It is concluded that ELISA could efficiently replace IF and PRNT tests for the diagnosis of AHF.     

Kinetics of the Neutralizing Antibody Response to Respiratory Syncytial Virus Infections in a Birth Cohort

The kinetics of respiratory syncytial virus (RSV) neutralizing antibodies following birth, primary and secondary infections are poorly defined. The aims of the study were to measure and compare neutralizing antibody responses at different time points in a birth cohort followed‐up over three RSV epidemics. Rural Kenyan children, recruited at birth between 2002 and 2003, were monitored for RSV infection over three epidemic seasons. Cord and 3‐monthly sera, and acute and convalescent sera following RSV infection, were assayed in 28 children by plaque reduction neutralization test (PRNT). Relative to the neutralizing antibody titers of pre‐exposure control sera (1.8 log₁₀ PRNT), antibody titers following primary infection were (i) no different in sera collected between 0 and 0.4 months post‐infection (1.9 log₁₀ PRNT_, P = 0.146), (ii) higher in sera collected between 0.5 and 0.9 (2.8 log₁₀ PRNT_, P < 0.0001), 1.0–1.9 (2.5 log₁₀ PRNT, P < 0.0001), and 2.0–2.9 (2.3 log₁₀ PRNT, P < 0.001) months post‐infection, and (iii) no different in sera collected at between 3.0 and 3.9 months post‐infection (2.0 log₁₀ PRNT, P = 0.052). The early serum neutralizing response to secondary infection (3.02 log₁₀ PRNT) was significantly greater than the early primary response (1.9 log₁₀ PRNT, P < 0.0001). Variation in population‐level virus transmission corresponded with changes in the mean cohort‐level neutralizing titers. It is concluded that following primary RSV infection the neutralizing antibody response declines to pre‐infection levels rapidly (～3 months) which may facilitate repeat infection. The kinetics of the aggregate levels of acquired antibody reflect seasonal RSV occurrence, age, and infection history. J Med. Virol. 85:2020–2025, 2013. © 2013 The Authors. Journal of Medical Virology published by Wiley Periodicals, Inc.