NS1 Antibodies Research Articles

Ultrasensitive Immunosensor Based on Langmuir-Blodgett Deposited Ordered Graphene Assemblies for Dengue Detection.

In this manuscript partially reduced graphene oxide (RGO) nanosheet-based electrodes have been utilized for quantification of the NS1 protein and subsequently for dengue detection. NS1 is the biomarker found circulating in the body of dengue-infected persons on or after first day of the appearance of disease symptoms. Graphene oxide (GO) has been synthesized using a modified Hummer's method, and its ordered nanostructured films have been electrophoretically deposited on indium tin oxide (ITO)-coated glass substrates using Langmuir-Blodgett (LB) deposition. Deposited LB films of GO have been reduced with hydrazine vapors to obtain RGO-coated ITO electrodes. NS1 antibodies have been grafted onto the ordered thin films using covalent linking, and the bioelectrodes have been utilized for the specific detection of NS1 antigen. The electrochemical performance of the fabricated bioelectrodes for NS1 antigen detection has been explored in standard and spiked sera samples. The limit of detection for the standard samples and spiked serum samples is found to be 0.069 ng mL-1 and 0.081 ng mL-1, respectively, with a sensitivity of 8.41 and 36.75 Ω per ng mL, respectively, in the detection range of 101 to 107 ng mL-1.

Development of Zika NS1 ELISA methodology for seroprevalence detection in a cohort of Mexican patients in an endemic region

BackgroundZika (ZIKV) is a mosquito-borne virus that has caused multiple outbreaks in Americas. The early and accurate diagnosis of ZIKV is the key to minimize the health burden imposed on the infected patients. Many commercial ZIKV diagnosis kit are available based on ZIKV envelope antigen with varying sensitivity and specificity. ObjectiveThe aim of this study was to develop sensitive ELISA methodology based on recombinant ZIKV NS1 and NS1 β-ladder antigens for seroprevalence detection in a cohort of patients in an arbovirus endemic Mexican region in comparison to a commercial kit. Study designWe obtained serum samples from 60 patients presenting with febrile illness and from 10 healthy donors in Michoacán state in 2016–2017. These samples were screened for ZIKV by RT-PCR and used to develop NS1 based ELISA and compared to a commercial kit. ResultsOur results indicate that both ZIKV sNS1 and β-ladder-based ELISA can reliably detect anti-ZIKV NS1 antibodies in infected patients, relevant for the serodiagnosis of ZIKV. Determination of antibody titers showed that it offered higher sensitivity than a commercially available ZIKV ELISA. Over 90% seropositivity against ZIKV for the febrile patients was detected in Lázaro Cárdenas which is an arbovirus endemic region while lower seropositivity was observed in the healthy volunteers in Morelia (non-endemic area). ConclusionWe conclude that our simple and sensitive in-house NS1 based ELISA used in this study has excellent sensitivity, is easy to use and can provide fast results suitable for larger population-based seroprevalence studies in the future.

Avaliação dos testes rápidos para diagnóstico da dengue no Brasil

Introdução: O aumento do número de casos de dengue no mundo estimulou o desenvolvimento e a disponibilização no mercado nacional e internacional de testes de execução rápida e simples para o diagnóstico da doença. Objetivo: Avaliar a sensibilidade e especificidade diagnóstica de Testes Rápidos (TR) imunocromatográficos para detecção de antígeno (Ag) NS1 e de anticorpos (Ac) das classes G (IgG) e M (IgM) e detecção combinada de Ag e Ac (NS1/ IgG/IgM) do vírus da dengue (DENV), encaminhados para análise prévia no INCQS/Fiocruz, no período de maio de 2016 a dezembro de 2018 para obtenção de registro junto à Anvisa do Brasil. Método: A sensibilidade e a especificidade foram avaliadas frente a painéis de amostras verdadeiramente positivas e verdadeiramente negativas para Ag NS1, Ac IgM e IgG do DENV, painéis de desempenho e padrão internacional do National Institute for Biological Standards and Control/Organização Mundial da Saúde (NIBSC/OMS). Os TR que apresentaram valores de sensibilidade e especificidade superiores ou iguais aos declarados pelos fabricantes foram considerados satisfatórios e os com valores inferiores, insatisfatórios. Resultados: Do total de 32 TR avaliados, 23 (71,9%) foram satisfatórios para sensibilidade e especificidade, destes, nove (39,1%), para NS1, 11 (47,8%) para IgG/IgM e três (13,0%), para os testes combinados NS1/IgG/IgM. Dos nove TR insatisfatórios, quatro (44,4%) foram para detecção de NS1; dois (22,2%), para IgG/IgM e três (33,3%), para NS1/IgG/IgM. Os TR considerados insatisfatórios não foram registrados no Brasil. Conclusões: A análise prévia como prevista na legislação brasileira é de grande importância para a manutenção da qualidade dos TR ofertados ao mercado nacional.

Dengue Virus is Hyperendemic in Nigeria from 2009 to 2020: A Contemporary Systematic Review.

BackroundData on Dengue virus (DENV) infection prevalence, geographic distribution and risk factors are necessary to direct appropriate utilization of existing and emerging control strategies. This study aimed to determine the pooled prevalence, risk factors of DENV infection and the circulating serotypes within Nigeria from January 1, 2009 to December 31, 2020.Materials and methodsTwenty-one studies out of 2,215 available articles were eligible and included for this systematic review. Relevant articles were searched, screened and included in this study according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) criteria. The risk of bias in primary studies was assessed by Cochrane's method. Heterogeneity of pooled prevalence was calculated using the chi-square test on Cochrane's Q statistic, which was quantified by I-square values. The random-effects analyses of proportions were used to determine the pooled prevalence of DENV antibodies, antigen and RNA from eligible studies.ResultsOf these, 3 studies reported co-circulation of all the 4 serotypes, while 2 separately reported co-circulation of DENV-1 &2 and DENV-1 to -3. All the antibody-based studies had significantly high heterogeneity (I2 >90%, P <0.05), while the NS1 and PCR-based studies had low heterogeneity (I2 <25%, P >0.05). The pooled prevalence of DENV IgM, IgG, RNA, NS1 and neutralizing antibodies were 16.8%, 34.7%, 7.7%, 7.7% and 0.7%, respectively. South-east Nigeria had the highest pooled DENV-IgG seropositivity, 77.1%. Marital status, gender, educational level and occupation status, the proximity of residence to refuse dumpsite, frequent use of trousers and long sleeve shirts were significantly associated with DENV IgG seropositivity (P <0.05).ConclusionBased on these findings, it can be inferred that Nigeria is hyperendemic for Dengue fever and needs concerted efforts to control its spread within and outside the country.

A Retrospective Study in Dengue Positive Cases - Hematological and Biochemical Parameters (Liver Function Tests, Renal Function Tests) Correlation

Dengue fever is the most common arboviral infection in the world, and it is a disease with a broad clinical spectrum and a wide variety of presentations, ranging from asymptomatic to an undifferentiated fever (viral syndrome) to the more severe forms such as severe dengue (SD) or Dengue hemorrhagic fever (DHF). The diagnosis of dengue fever is carried out based on clinical, epidemiological and laboratory data. Among laboratory tests, both non-specific [blood count, platelet count, tourniquet test, prothrombin time (PT), activated partial thromboplastin time (APTT), liver function tests and serum albumin concentration] and specific tests (viral isolation tests and serology for antibody examination) are used. The principle aim of our study is to assess and compare the biochemical and haematological parameters of patients with dengue. In our study population, the peak frequency of the patients who were dengue positive was on the age group between 21 and 30 years (44%), and it was more common among males. The most common serological indicator of dengue fever was the presence of NS1 antibody. High haematocrit (56%) was found in most of the patients, and the next common finding was thrombocytopenia (51%). Elevated Serum glutamic oxaloacetic transaminase seen in 53% of cases and Elevated Serum glutamic pyruvic transaminase, Alkaline Phosphatase, Serum Bilirubin, Serum creatinine and Serum urea were found in 41%, 11%,7%, 4%, 3%of cases respectively. The knowledge about these parameters helps the clinician in early diagnosis of the cases and initiation of the management measures before the drastic clinical features of the disease sets.

English

BACKGROUND Dengue is an acute febrile illness caused by mosquito-borne dengue viruses (DENV S) consisting of four serotypes (DENV 1 - 4) from flaviviridae family, genus flavivirus. These four are antigenically related serotypes designated as DEN V - 1, DEN V - 2, DEN V - 3 and DEN V – 4. In this context, the present study focuses on the circulating serotypes of dengue in coastal Andhra Pradesh. METHODS Study was done at Andhra Medical College, Visakhapatnam, teaching hospital in Andhra Pradesh. Acute phase dengue serum samples were collected and tested for NS1 antigen and antihuman IgM antibodies by enzyme linked –immunosorbent assay (ELISA). NS1 positive samples were further serotyped by reverse transcriptase real time polymerase chain reaction (R RT - PCR). RESULTS A total of 796 serum samples were included in the study. 300 (37.7 % ) samples were positive for NS1 and IgM antibodies. 192 NS1 antigen positive samples were further processed for serotyping by r RT PCR. Among these samples 72 were negative by r RT PCR. DENV-2 (41 %) was the predominant serotype followed by DENV-4 (37 %), DENV-3 (12 %) and DENV-1 (10 %) in the descending order. CONCLUSIONS All the four dengue serotypes are in co-circulation. Among all the four types, DENV-2 was predominant, followed by DENV-4. By knowing the predominant serotype in circulation, we can forecast dengue outbreaks and take necessary measures like control of vectors. KEY WORDS Andhra Pradesh, Dengue Virus, Dengue Virus - 2, Dengue Virus - 4, Outbreak, Serotypes

Assessment of ns1, igg and igm antibodies in suspected dengue patients

Assessment of ns1, igg and igm antibodies in suspected dengue patients

Evaluation of Bluetongue Virus (BTV) Antibodies for the Immunohistochemical Detection of BTV and Other Orbiviruses.

The detection of bluetongue virus (BTV) antigens in formalin-fixed tissues has been challenging; therefore, only a limited number of studies on suitable immunohistochemical approaches have been reported. This study details the successful application of antibodies for the immunohistochemical detection of BTV in BSR variant baby hamster kidney cells (BHK-BSR) and infected sheep lungs that were formalin-fixed and paraffin-embedded (FFPE). BTV reactive antibodies raised against non-structural (NS) proteins 1, 2, and 3/3a and viral structural protein 7 (VP7) were first evaluated on FFPE BTV-infected cell pellets for their ability to detect BTV serotype 1 (BTV-1). Antibodies that were successful in immunolabelling BTV-1 infected cell pellets were further tested, using similar methods, to determine their broader immunoreactivity against a diverse range of BTV and other orbiviruses. Antibodies specific for NS1, NS2, and NS3/3a were able to detect all BTV isolates tested, and the VP7 antibody cross-reacted with all BTV isolates, except BTV-15. The NS1 antibodies were BTV serogroup-specific, while the NS2, NS3/3a, and VP7 antibodies demonstrated immunologic cross-reactivity to related orbiviruses. These antibodies also detected viral antigens in BTV-3 infected sheep lung. This study demonstrates the utility of FFPE-infected cell pellets for the development and validation of BTV immunohistochemistry.

Clinico-Laboratory Profile of Dengue Patients at Sir T. Hospital, Bhavnagar, India

Dengue fever is caused by any one of four types of dengue viruses (DEN1-DEN4), spread mainly by Aedes aegypti. India had the largest number of dengue cases, with about 33 million apparent and another 100 million asymptomatic infections occurring annually. The patients typically present with the sudden onset of fever, frontal headache, retroorbital pain. The laboratory diagnosis can be made by IgM ELISA or by NS1 antigen-detection ELISA during the acute phase. This research was conducted from January 2018 to December 2018 at Sir T. hospital and Government Medical College in Bhavnagar, Gujarat. The patients having complaints of fever, headache, myalgia, arthralgia or rash, were clinically examined, and laboratory investigated for dengue with NS1 and/or IgM dengue antibody. A total of 536 patients was screened, of which 112 patients were diagnosed as dengue fever at 21% dengue positivity rate, based on detection of NS1 (46/304, 15%), and antidengue IgM (66/232, 28%) in their sera. The majority of the patients were males (77/112, 69%). The majority of patients were in 11–30 years’ age group (66/316, 21%). Fever (100%) was the chief presenting complaint, followed by headache (83, 93%), and myalgia (79, 89%). The highest number (28) of dengue patients was observed in the month of October 2018. According to this study results, the physicians in the dengue-endemic area should be aware of dengue in acute febrile illnesses and use the appropriate laboratory tests such as NS1 antigen and IgM antibodies for early dengue diagnosis. This can help clinicians to prevent morbidity and mortality associated with dengue.

The Immunoprobe Aggregation State is Central to Dipstick Immunoassay Performance.

As new infectious disease outbreaks become more likely, it is important to be able to develop and deploy appropriate testing in time. Paper-based immunoassays are rapid, cheap, and easy to produce at scale and relatively user friendly but often suffer from low selectivity and sensitivity. Understanding the molecular mechanisms of paper immunoassays may help improve and hasten development and therefore production and market availability. Here, we study how the behavior of nanoparticle-antibody immunoprobes in paper dipstick immunoassays is impacted by synthesis strategy and surface chemistry architecture. We conjugate gold nanoparticles to polyclonal anti-immunoglobulin G (IgG) and anti-zika NS1 antibodies by electrostatic adsorption and N-hydroxysuccinimide (NHS) and hydrazide (Hz) chemistries. The immunoprobes were used in paper immunoassays and the effective affinity for the antigen was quantified from the test line intensities, as well as the distribution of the immunoprobes throughout the strips. The results show that nanoparticle colloidal stability, both post synthesis and during antigen binding, is a key factor and affects immunoassay results and performance, often through reduction or loss of signal.

Verification of dengue fever cases imported to the territory of the south Far East.

On biomaterial from 70 people, verification of imported cases of Dengue fever into the south of the Far East from 2012 to 2019 is shown. Serological and virological methods were used, as well as PCR. Using the immunochromatographic rapid test, the Dengue virus (DENV) NS1 antigen and antibodies to DENV (IgM and IgG) were detected in human blood. We examined 12 patients from the infectious diseases department with unknown fever and the blood of 58 people who applied to clinics in Vladivostok after returning from tourist trips. Dengue fever was diagnosed in 23 patients (32.8%), of which antigen was detected in 56%, IgM antibodies in 91.3% and IgG in 52.1%. In 2 cases (8.7%), only antigen was detected in patients. Three strains of the pathogen were isolated by virological methods from 18 blood samples, two of which turned out to be the DENV of the 1st genotype and one - of the DENV of the 2nd genotype. Using RT-PCR, 38 blood samples were tested positive in the immunochromatographic rapid test, of which in 16 cases (42.1%) a DENV marker was detected, in 11 cases it was genotype 1, in three cases genotype 2, and one each - genotypes 3 and 4. 1. The most reliable method of rapid verification (in 100%) the primary infection DENV was the comprehensive determination of antigen and antibodies of the IgM class; 2. With antigenemia, blood should be used to isolate the virus, as well as to diagnose the disease by PCR and to establish the genotype of the DENV; 3. When using only PCR to indicate Dengue virus, a significant proportion of the disease cases will not be diagnosed.

Renal Involvement in Children with Dengue Fever: A Study in Tertiary Care Hospital of Bangladesh.

Background and Objective: Dengue has emerged globally as the most relevant viral infection transmitted by a mosquito bite and represents a major threat to public health. Dengue-related renal manifestations such as proteinuria, hematuria, acute kidney injury (AKI), and rhabdomyolysis are not uncommon, and acute kidney injury (AKI) is a serious complication of dengue fever. There is relatively few data on the renal manifestations of dengue fever in children. Hence, this study was conducted to evaluate the incidence, characteristics, and clinical outcome of dengue fever with renal manifestations. Method. This prospective cross sectional study was conducted in Dr. M R Khan Children Hospital and Institute of Child Health, Dhaka, over a period of 1 year from January 2018 to December 2018. The study was approved by the ethical committee of the institute. A total number of 316 patients were admitted with the diagnosis of dengue fever either NS1 positive or antibody IgM positive or both IgM and IgG positive. Data were collected in a structured questionnaire form and were analyzed by SPSS version 20.0. The disease severity was classified according to the World Health Organization criteria. Renal manifestations were divided into AKI groups using pRIFLE criteria. Proteinuria was defined as urinary protein >1+ (30 mg/dL) by dipstick test. Hematuria was defined as red blood cell (RBC) >5/μL in a fresh uncentrifuged urine specimen. Result. Among 316 dengue patients, thirty-one patients (9.8%) had renal involvement. Most of the patients (54.83%) with renal manifestations were aged between 1 and 5 years. A total of 14 patients were found to have proteinuria (4.4%). Nephrotic-range proteinuria was seen in only one patient (0.3%). AKI was defined by pRIFLE criteria and was seen in 13 patients (4.1%); among AKI 6 (46.15%) had risk, three patients (23.07%) had injury and 4 (30.7%) had failure and needed peritoneal dialysis. Death occurred in 3 patients (9.6%) in dengue with AKI who had failure. The incidence of renal manifestations (proteinuria, hematuria, and AKI) is as high as 9.8% among patients with dengue, and those with AKI had significant morbidity and mortality. Conclusion. Renal involvement in children with dengue is not uncommon. Dengue associated with AKI had significant mortality and morbidity.

Comparative assessment of commercial enzyme-linked immunosorbent assay & rapid diagnostic tests used for dengue diagnosis in India.

Background & objectives:Dengue diagnosis is routinely carried out by detection of dengue virus (DENV) antigen NS1 and/or anti-DENV IgM antibodies using enzyme-linked immunosorbent assays (ELISAs) and rapid diagnostic tests (RDTs). This study was aimed at evaluation of quality of diagnostic assays currently in use in India for the identification of DENV infection.Methods:During 2016 dengue season (July-November) in Pune, India, comparative assessment of a few immunoassays was undertaken using (i) WHO-approved Panbio-Dengue-Early-(NS1)-ELISA and Panbio-Dengue-IgM-Capture-ELISA as reference tests, and (ii) Bayesian latent class analysis (BLCA) which assumes that no test is perfect. The assays included J.Mitra-Dengue-NS1-Ag-MICROLISA (JME-NS1), J.Mitra-Dengue-IgM-MICROLISA (JME-IgM), and two RDTs, namely, J.Mitra-Dengue-Day- 1-Test (JM-RDT) and SD-BIOLINE-Dengue-Duo (SDB-RDT). Serum samples from patients seeking dengue diagnosis (n=809) were tested using the diagnostic kits. The presence of NS1 and/or IgM was taken as evidence for dengue-positive diagnosis.Results:Panbio-NS1/IgM-ELISAs identified 38.6 per cent patients as dengue positive. With Panbio-ELISA as reference, all the tests were less sensitive for IgM detection, while for NS1, JM-RDT was less sensitive. For combined diagnosis (both markers), sensitivity of all the tests was low (55.7-76.6%). According to BLCA, Panbio-ELISA was 84 per cent sensitive for NS1, 86 per cent specific for IgM and 87 per cent specific for combined diagnosis. Accordingly, performance of the other tests was substantially improved with BLCA; however, sensitivity of both the RDTs for IgM detection remained unacceptable. The NS1 ELISAs and RDTs detected all four DENV serotypes, JME being most efficient. All IgM tests exhibited higher sensitivity in secondary infections.Interpretation & conclusions:These results confirmed superiority of ELISAs, and testing for both NS1 and IgM markers for dengue diagnosis, and emphasized on improvement in sensitivity of RDTs.

Smartphone-assisted Hepatitis C detection assay based on magnetic levitation.

This work describes development of smartphone-assisted magnetic levitation assay for Point-of-Care (PoC) applications. Magnetic levitation is a technique that detects and separates particles based on their density differences in a magnetic field. Observation of the levitated micro-particles is mainly performed by light microscope or additional optical components, which mostly limits applicability of the magnetic levitation technique for PoC diagnostics. In this paper, we demonstrated the capability of the smartphone assisted-magnetic levitation platform for Hepatitis C (HCV) detection assay. This method utilizes microsensor beads (MS beads) that are functionalized with anti-HCV NS3 antibody. First, the magnetic levitation platform was optimized via density marker polyethylene beads (DMB); then HCV NS3 protein was successfully detected based on levitation height differences of MS beads caused by density changes. The capability of the magnetic levitation platform for HCV detection was determined as almost 10-fold sensitive compared to conventional techniques such as enzyme-linked immunosorbent assay (ELISA). The imaging capability and resolution of the setup was improved over previously used configurations, and the developed platform enabled visualization of micro-scale objects only by smartphone assistance. This method requires no power, it is an easy-to-use and cost effective, therefore it could be easily adaptable to varied sensing assays as PoC tool.

1768. Dengue Virus Infection in Solid-Organ Transplant Recipients: Case Series and Literature Review

BackgroundDengue fever is the most prevalent arbovirus among humans, its incidence has increased since the re-emergence, and Colombia is a hyperendemic country for this infection. The number of solid-organ transplant (SOT) recipients, at risk of acquiring dengue virus infection, is constantly increasing, and there are few data regarding the clinical course and outcomes of dengue infection among this population. The aim of this study was to describe dengue virus infection in SOT recipients in Cali, Colombia.MethodsWe present a case series of SOT recipients with dengue virus infection, diagnosed by World Health Organization criteria and a positive NS1 and/or IgM dengue antibodies, which were attended at the FVL from 2001 to 2018. Furthermore, we performed a literature review regarding dengue infection in SOT recipients.ResultsA total of 20 patients were included: 17 kidney and 3 liver recipients. The median age was 50.5 years (IQR = 31–63.5), 65% were female. The median time from transplant to dengue was 27.6 months (IQR = 3.82–59.12), and 3 patients had the infection in the first month after the transplant. The most common symptoms were fever (95%), myalgia, headache, and abdominal pain. Warning signs were present in 75% of patients, thrombocytopenia and hemorrhagic manifestations were present in 30% and 15%, respectively. 35% of patients were classified as severe dengue, and 45% were managed at the intensive care unit. Regarding laboratory findings, six patients had transaminases elevation more than three times the upper limit and 7 had serum creatinine elevation, which returned to normal levels. All patients were discharged and none of them had alterations in the graft function. To date, there are approximately 180 reported cases of dengue in SOT recipients (Table 2).ConclusionDengue represents a threat among SOT recipients. Unlike other reports, all patients in this series had a full recovery after the infection, suggesting that timely and effective management of patients and the access to high complexity services could prevent fatal cases. Disclosures All authors: No reported disclosures.

2805. Kinetics of Anti-Zika Virus (ZIKV) Antibodies after Acute Infection in Pregnant Women

BackgroundThe kinetics and specificity of anti-ZIKV antibodies after acute ZIKV infection is not well known, especially in areas where different flaviviruses circulate. The objective of this study was to describe the kinetics of anti-ZIKV antibodies in pregnant women in whom an acute ZIKV infection was identified during pregnancy.MethodsWithin a cohort of pregnant women living in Guadeloupe and exposed to ZIKV during the 2016 zika outbreak, we identified 65 women who presented with an acute, symptomatic PCR-confirmed ZIKV infection at various times of their pregnancy, with a known date of first Zika symptom. Anti-ZIKV neutralizing antibodies (using a Virus Neutralisation Test (VNT)) and anti-ZIKV NS1 antibodies (using IgM and IgG ELISA Euroimmun® kits) were searched for on frozen serum samples obtained from blood drawn at the time of delivery in all women and at various times between acute infection and delivery in 23 women.ResultsPatients’ mean age was 30 years and ZIKV infection had occurred during the first, second, and third trimester of pregnancy in 14 (21%), 35 (54%), and 16 (25%) women, respectively. ZIKV serology on delivery samples was positive in 65/65 (100%; one-sided 97.5% CI: 94.4%–100%) women by both VNT and IgG ELISA assays and in 5/65 (8%) women by IgM ELISA assay. In these 5 cases, median time between first symptom and sampling date was 36 days. Results of ELISA assays on the intermediate samples were as follows: IgG antibodies were negative in all 5 samples that had been drawn within 7 days of first symptom and positive in the 18 samples that had been drawn afterwards; IgM antibodies were positive in 10 of the 19 samples that had been drawn within 3 months of first symptom and negative in 2 of the 4 samples that had been drawn afterwards.ConclusionAfter acute ZIKV infection, IgG antibodies developed and remained detectable until delivery by a commercially available ELISA assay in all women tested. These antibodies were specific of ZIKV, with concomitantly positive VNT results. From these findings, the absence of ZIKV antibodies at delivery would strongly indicate the absence of infection during pregnancy.Disclosures All authors: No reported disclosures.

High correlation between Zika virus NS1 antibodies and neutralizing antibodies in selected serum samples from normal healthy Thais

Despite the widespread presence of the mosquito transmitted Zika virus (ZIKV) over much of Southeast Asia, the number of reported cases remains low. One possibility is that residents in Southeast Asia are immunologically protected, although the nature of any such protection remains unclear. This study sought to investigate the presence of antibodies directed to ZIKV NS1 protein in a selected sub-set of samples from a well characterized cohort of serum samples from normal, healthy Thais that had been previously characterized for the presence of neutralizing antibodies to ZIKV, DENV 1-4, and JEV. Because of similarities in molecular weight between the flavivirus E and NS1 proteins, an immunoblot system was established in which the NS1 antigen was not denatured, allowing detection of the dimer form of NS1, distinctly clear from the migration position of the E and NS1 monomer proteins. The results showed that antibodies to ZIKV NS1 protein were only detected in samples with ZIKV neutralizing antibodies (27/30 samples), and no sample (0/30) with a ZIKV plaque reduction neutralization test (PRNT)90 < 20 showed evidence of anti-ZIKV NS1 antibodies. The high correlation between the presence of ZIKV NS1 antibodies and ZIKV PRNT suggests that immunological protection against ZIKV infection in Thailand arises from prior exposure to ZIKV, and not through cross neutralization.

Evaluation of currently available bovine viral diarrhoea virus (BVDV) and HoBi-like pestivirus (HoBiPeV) specific diagnostic tests in detection of highly divergent HoBiPeVs in cattle

The emergence of novel and divergent HoBi-like pestivirus (HoBiPeV) strains in cattle in Asia recently has raised concerns with regard to their reliable and accurate diagnosis. Hence, the aim of this study was to evaluate currently available BVDV diagnostic tests and HoBiPeV-specific diagnostic tests in detection of genetically divergent strains of HoBiPeV. One strain each of HoBiPeV-c and d were subjected to two BVDV diagnostic RT-PCR tests, one HoBiPeV specific RT-PCR test, three BVDV diagnostic qRT-PCR tests, one HoBiPeV specific qRT-PCR test and two BVDV antigen capture ELISAs. Archived cattle sera (n = 41) from farms with reports of HoBiPeV natural infection were assessed for detection of HoBiPeV antibodies by VNT and two commercial BVD antibody ELISA kits. BVDV diagnostic qRT-PCR tests had better sensitivity than BVDV diagnostic RT-PCR tests, while majority of them except a commercial kit showed a lower sensitivity for HoBiPeV-d strain. The HoBiPeV specific qRT-PCR test was found more sensitive than HoBiPeV specific RT-PCR but both had lower sensitivity for HoBiPeV-d strain, as displayed by primer/probe sequence mismatches. The BVDV Erns antigen ELISA detected both the strains of HoBiPeV, but with a lower sensitivity for HoBiPeV-d strain, whereas BVDV NS3 antigen ELISA failed to detect them even at a high HoBiPeV titre. Compared to VNT, commercial BVDV antibody ELISA showed low to moderate sensitivity in detection of HoBiPeV antibodies, with a failure rate of 31.25% for the whole virus antigen based ELISA and a failure rate of 56.25% for NS3 antibody ELISA. The present study demonstrated new challenges in HoBiPeV diagnosis indicating a need in improvement of both HoBiPeV specific diagnostic RT-PCR and qRT-PCR for better utility in HoBiPeV epidemiology and biological product safety. Although more studies are required, this study reinforces that combined use of BVDV Erns and NS3 antigen ELISA may have some utility in preliminary differentiation between HoBiPeV and BVDV infection in PI cattle. Additionally, we show that the comparative VNT has a better sensitivity in detection of HoBiPeV exposure and there is a need of robust antibody ELISA for reliable detection of antibodies against this emerging bovine pestivirus.

Development and application of a monoclonal-antibody-based blocking ELISA for detection of Japanese encephalitis virus NS1 antibodies in swine.

Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted by Culex mosquitoes and is the leading cause of viral encephalitis in humans. JEV infection of swine, which are the main amplifying hosts for JEV, can cause reproductive failure in sows; in boars it can cause testitis and infertility. The prevalence of JEV in swine is a continuous threat to human health. A practical diagnostic method for monitoring JEV infection in swine herds is essential for control of the disease in both swine and humans. Here, we have identified a high-affinity anti-JEV NS1 monoclonal antibody (mAb) by indirect ELISA and utilized it for the development of a blocking ELISA (bELISA). The optimal NS1 protein coating concentration (2 μg/mL) and mAb working concentration (1 μg/mL) were determined by checkerboard titration. One hundred ten JEV-antibody-negative serum samples were used to establish 34.03% inhibition as the cutoff value for a negative result. By the bELISA, seroconversion in 80% of newly JEV-vaccinated pigs was detected by 7 days post-immunization, while by the commercial envelope-protein-based iELISA, seroconversion was detected in 20% of the newly vaccinated pigs. We found 98.7% agreement between the bELISA and the commercial iELISA when we tested 157 field samples using both methods. From an epidemiological survey of swine serum collected between 2014 and 2016, we found that the average JEV seropositive rate in unvaccinated commodity pigs was 8.1%, and in vaccinated boars and sows, it was 67.6%.

Role of NS1 antibodies in the pathogenesis of acute secondary dengue infection

The role of NS1-specific antibodies in the pathogenesis of dengue virus infection is poorly understood. Here we investigate the immunoglobulin responses of patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) to NS1. Antibody responses to recombinant-NS1 are assessed in serum samples throughout illness of patients with acute secondary DENV1 and DENV2 infection by ELISA. NS1 antibody titres are significantly higher in patients with DHF compared to those with DF for both serotypes, during the critical phase of illness. Furthermore, during both acute secondary DENV1 and DENV2 infection, the antibody repertoire of DF and DHF patients is directed towards distinct regions of the NS1 protein. In addition, healthy individuals, with past non-severe dengue infection have a similar antibody repertoire as those with mild acute infection (DF). Therefore, antibodies that target specific NS1 epitopes could predict disease severity and be of potential benefit in aiding vaccine and treatment design.