Detection Of Viral Antigens Research Articles

Neuroanatomical evidence of the transport of the rabies virus through the propriospinal tract in the spinal cord of mice

Introduction: Information about the neuroanatomical details of the ascendant transport of the rabies virus through the spinal cord is scarce. Objective: To identify the neuroanatomical route of dissemination of the rabies virus at each of the levels of the spinal cord of mice after being inoculated intramuscularly. Materials and methods: Mice were inoculated with the rabies virus in the hamstrings. After 24 hours post-inoculation, every eight hours, five animals were sacrificed by perfusion with paraformaldehyde. Then, the spinal cord was removed, and transverse cuts were made at the lumbosacral, thoracic, and cervical levels. These were processed by immunohistochemistry for the detection of viral antigens. Results: The first antigens of rabies were observed as aggregated particles in the lumbar spinal cord at 24 hours post-inoculation, within the ventral horn in the same side of the inoculated limb. At 32 hours post inoculation the first motoneurons immunoreactive to the virus became visible. At 40 hours postinoculation the first immunoreactive neurons were revealed in the thoracic level, located on lamina 8 and at 48 hours post-inoculation in the cervical cord, also on lamina 8. At 56 hours post-inoculation the virus had spread throughout the spinal cord, but the animals still did not show signs of the disease. Conclusion: In the mouse model we used, the rabies virus entered the spinal cord through the motoneurons and probably used the descending propriospinal pathway for its retrograde axonal transport to the encephalus.

A fatal case associated with respiratory syncytial virus infection in a young child

BackgroundRespiratory syncytial virus (RSV) is the most common viral cause of pediatric bronchiolitis and pneumonia worldwide. Risk factors for high mortality and prolonged morbidity after RSV infection include premature birth, bronchopulmonary dysplasia, congenital heart disease, and Down syndrome. However, some previously healthy, full-term children who are infected with RSV also require hospitalization and even experience severe sequelae or death.Case presentationIn this report, we present the case of an RSV-associated death of a child who was born at full-term and developed normally up to the age of 2 years old. Cardiopulmonary arrest occurred within 3 days after the onset of symptoms, which included cough and high fever. Complete brain edema was prominent, and encephalopathy was developing. Viral antigen detection and microbiome analyses of oral swab and nasopharyngeal aspirate specimens verified an RSV infection, while bacterial culture of blood specimens yielded negative results. The RSV strain detected in this patient was subtyped as RSVB9, and no mutation was found in the six antigenic sites for targeted drugs or vaccines.ConclusionsThe patient had a severe infection associated with RSV, which was very likely the cause of her central nervous system infection and acute neurological complications.

Evaluation of an immunochromatography rapid diagnosis kit for detection of chikungunya virus antigen in India, a dengue-endemic country

BackgroundChikungunya virus (CHIKV) and dengue virus (DENV) are arboviruses that share the same Aedes mosquito vector, and there is much overlap in endemic areas. In India, co-infection with both viruses is often reported. Clinical manifestations of Chikungunya fever is often confused with dengue fever because clinical symptoms of both infections are similar. It is, therefore, difficult to differentiate from those of other febrile illnesses, especially dengue fever. We previously developed a CHIKV antigen detection immunochromatography (IC) rapid diagnosis kit [1]. The current study examined the efficacy of previously mentioned IC kit in India, a dengue-endemic country.MethodsSera from 104 CHIKV-positive (by qRT-PCR) and/or IgM-positive (ELISA) subjects collected in 2016, were examined. Fifteen samples from individuals with CHIKV-negative/DENV-positive and 4 samples from healthy individuals were also examined. Of the 104 CHIKV-positive sera, 20 were co-infected with DENV.ResultsThe sensitivity, specificity and overall agreement of the IC assay were 93.7, 95.5 and 94.3%, respectively, using qRT-PCR as a gold standard. Also, there was a strong, statistically significant positive correlation between the IC kit device score and the CHIKV RNA copy number. The IC kit detected CHIKV antigen even in DENV-co-infected patient sera and did not cross-react with DENV NS1-positive/CHIKV-negative samples.ConclusionsThe results suggest that the IC kit is useful for rapid diagnosis of CHIKV in endemic areas in which both CHIKV and DENV are circulating.

Field validation of recombinant antigen immunoassays for diagnosis of Lassa fever

Lassa fever, a hemorrhagic fever caused by Lassa virus (LASV), is endemic in West Africa. It is difficult to distinguish febrile illnesses that are common in West Africa from Lassa fever based solely on a patient’s clinical presentation. The field performance of recombinant antigen-based Lassa fever immunoassays was compared to that of quantitative polymerase chain assays (qPCRs) using samples from subjects meeting the case definition of Lassa fever presenting to Kenema Government Hospital in Sierra Leone. The recombinant Lassa virus (ReLASV) enzyme-linked immunosorbant assay (ELISA) for detection of viral antigen in blood performed with 95% sensitivity and 97% specificity using a diagnostic standard that combined results of the immunoassays and qPCR. The ReLASV rapid diagnostic test (RDT), a lateral flow immunoassay based on paired monoclonal antibodies to the Josiah strain of LASV (lineage IV), performed with 90% sensitivity and 100% specificity. ReLASV immunoassays performed better than the most robust qPCR currently available, which had 82% sensitivity and 95% specificity. The performance characteristics of recombinant antigen-based Lassa virus immunoassays indicate that they can aid in the diagnosis of LASV Infection and inform the clinical management of Lassa fever patients.

Application and Comparative Evaluation of Fluorescent Antibody, Immunohistochemistry and Reverse Transcription Polymerase Chain Reaction Tests for the Detection of Rabies Virus Antigen or Nucleic Acid in Brain Samples of Animals Suspected of Rabies in India.

Accurate and early diagnosis of animal rabies is critical for undertaking public health measures. Whereas the direct fluorescent antibody (DFA) technique is the recommended test, the more convenient, direct rapid immunochemistry test (dRIT), as well as the more sensitive, reverse transcription polymerase chain reaction (RT-PCR), have recently been employed for the laboratory diagnosis of rabies. We compared the three methods on brain samples from domestic (dog, cat, cattle, buffalo, horse, pig and goat) and wild (leopard, wolf and jackal) animals from various parts of India. Of the 257 samples tested, 167 were positive by all the three tests; in addition, 35 of the 36 decomposed samples were positive by RT-PCR. This is the first study in which such large number of animal samples have been subjected to the three tests simultaneously. The results confirm 100% corroboration between DFA and dRIT, buttress the applicability of dRIT in the simple and rapid diagnosis of rabies in animals, and reaffirm the suitability of RT-PCR for samples unfit for testing either by DFA or dRIT.

Diagnosis of viral central nervous system infections using antipeptide antibody against viral antigen by ELISA

Viral infections of the central nervous system (CNS) occur sporadically and have been extensively studied because of the potential for permanent neurological damage or death. The neurotropic viruses have been reported to lead to various CNS infections. The objective of the present study is to develop an antigen detection ELISA protocol for detection and quantification of viral antigen in CNS infections by assessing the usefulness of antipeptide antibodies against potential peptides of cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV), Japanese encephalitis virus (JEV), dengue (DENV), West Nile virus (WNV) and Chandipura virus (CHPV). Atotal of 182 cerebrospinal fluid (CSF) samples from confirmed, suspected and non-viral infections of the CNS were evaluated using panels of antipeptide antibodies against synthetic peptides of viral proteins. The cases of confirmed and suspected viral infections of the CNS showed 77% and 11% positivity, respectively, for the detection of viral antigen using antipeptide against synthetic peptides of CMV, EBV, VZV and JEV. The concentration of viral antigen was also obtained by using antipeptide of respective viruses in CSF from both the groups. The viral antigen concentration was also correlated with viral load in confirmed cases of viral infection of the CNS. This study demonstrates the use of antipeptide against synthetic peptide derived from CMV, EBV, VZV and JEV in diagnostics of viral infections of the CNS using patients' CSF samples. Keywords: viral infection of the CNS; synthetic peptide; antipeptide antibody; viral load; antigen concentration.

Assessment of Biosecurity Measures Applied in Infected Broiler Farms with Avian Influenza

Questionnaire survey and observational study was conducted to collect data on biosecurity measures to investigate the relationship between the application of biosecurity measures and the prevalence of avian diseases (AI was an example) in broiler farms. A total of 100 small scale broiler farms located in Qaliobiya province were included in the current study where data were collected through direct observation and interviews made with owners and workers using a structured-questionnaire during the period extended from January to December 2016. Also, five cloacal swabs were obtained from each farm to be investigated for the presence of HPAI by application of rapid avian influenza virus antigen test kit. Finally, the application of different biosecurity measures and the prevalence of AI was related and analyzed statistically. Detection of avian influenza virus antigen was carried out by using rapid avian influenza virus antigen test kit (ANIGEN® Animal Genetics, Inc. Korea). It was recorded that the total prevalence of AI was 34%. Statistical analysis showed that there was a significant difference between the prevalence of AI in the five investigated districts of Qaliobiya province. The broiler farms located in Qaliob district scored the highest prevalence (50%) followed by broiler farms located in Toukh district (40%) then El Khanka (35%) then Banha (25%) and lastly El Qanater (20%). Chi-square analysis of the obtained results showed that there was a significant association between the application of traffic control measures and the prevalence of AI. Also, it was noticed that there was a significant association between the application of sanitation measures and the prevalence of AI. On contrary, there was non-significant association between isolation of the diseased houses and the prevalence of AI. Moreover, there was a significant association between the application of prophylactic measures and the prevalence of AI. At the end, it was observed that the prevalence of AI was higher in broiler farms showed relaxed biosecurity measures compared to other farms with strict biosecurity measures.

Diagnostic performances of two rapid tests for detection of feline leukemia virus antigen in sera of experimentally feline leukemia virus-infected cats.

ObjectivesThe objective of this study was to compare the diagnostic sensitivities and specificities of WITNESS FeLV-FIV (Zoetis) and SNAP FIV/FeLV Combo Test (IDEXX) for the detection of FeLV p27 antigen in the sera of experimentally feline leukemia virus (FeLV)-infected cats.MethodsDiagnostic sensitivities of WITNESS and SNAP were determined through testing of 47 serum samples collected from cats day 56 post-experimental infection with a virulent FeLV Rickard strain. Successful experimental infection was confirmed based on observation of FeLV antigen and proviral DNA in anti-coagulated (EDTA) whole-blood samples by immunofluorescent antibody (IFA) test and PCR, respectively. Diagnostic specificities of both tests were determined through testing of sera of 92 laboratory-housed, non-FeLV-exposed specific pathogen-free (SPF) cats.ResultsForty-one of 47 blood samples were IFA positive, whereas all 47 samples were PCR positive. All 92 non-FeLV-infected SPF cats were IFA and PCR negative. In comparison to IFA as the reference method, both WITNESS and SNAP tests yielded equivalent sensitivities and specificities of 100% and 97.8%, respectively. In comparison to PCR as the reference method, both WITNESS and SNAP tests likewise performed equivalently, with sensitivities and specificities of 91.5% and 100%, respectively.Conclusions and relevanceSensitivity and specificity of WITNESS FeLV-FIV for identifying FeLV p27 antigen in the sera of these experimentally FeLV-infected and non-FeLV-exposed SPF cats equaled those of the SNAP FIV/FeLV Combo Test. However, all positive results, regardless of the point-of-care test used, should be confirmed before making clinical decisions such as segregation from other cats or euthanasia.

Immunohistochemical Detection of Porcine Reproductive and Respiratory Syndrome Virus Antigen in Formalin-Fixed, Paraffin-Embedded Tissues with Correlation to Clinicopathologic Data

Immunohistochemistry (IHC) was used in this study to detect the presence of porcine reproductive and respiratory virus (PRRSV) antigen using monoclonal antibody (Mab SDOW17) in formalin-fixed, paraffin embedded tissues of lung, lymph node, heart, spleen, and kidney of pre- weaning to less than 90 day old pigs. Out of the 25 tissue samples examined from swine cases of the Philippine Animal Health Center (PAHC), Bureau of Animal Industry and College of Veterinary Medicine (CVM) Histopathology Laboratory, University of the Philippines-Los Baños (UPLB), 14 or 56% (14/25) were IHC positive for PRRSV antigen. Among the selected tissue samples, the PRRS virus was detected the most in the lymph nodes (64%) and lungs (40%), respectively, compared to other organs such as spleen (33%), kidney (28%), and the heart (17%). Only 43% (6/14) of cases coincided with the pathogenesis and clinicopathologic lesions of PRRS which are proliferative interstitial pneumonia and lymphoid follicular hyperplasia and necrosis. PRRS positive cases in IHC were consistently found to have co-infections with viral and bacterial diseases. Since PRRSV has tropism for macrophages and destruction of these cells leads to immunosuppression, affected animals are vulnerable to secondary infections. Keywords: formalin-fixed; immunohistochemistry; paraffin embedded tissue; piglets; Porcine Reproductive and Respiratory Syndrome; PRRS virus antigen; Mab SDOW17

EBOLA Ag K-SeT rapid test: field evaluation in Sierra Leone

ObjectivesEfficient interruption of Ebola virus disease (EVD) transmission chains critically depends on reliable and fast laboratory diagnosis. We evaluated the performance of the EBOLA Virus Antigen Detection K-SeT (EBOLA Ag K-SeT), a new rapid diagnostic antigen test in field settings. MethodsThe study was conducted in a field laboratory located in Freetown (Sierra Leone) by the Italian National Institute for Infectious Diseases ‘L. Spallanzani’ and the EMERGENCY Onlus NGO. The EBOLA Ag K-SeT was tested on 210 residual plasma samples (EVD prevalence 50%) from patients hospitalized at the EMERGENCY Ebola treatment center in Goderich (Freetown), comparing the results with quantitative real-time PCR. ResultsOverall, the sensitivity of EBOLA Ag K-SeT was 88.6% (95% confidence interval (CI), 82.5–94.7), and the corresponding specificity was 98.1% (95% CI, 95.5–100.7). The positive and negative predictive values were 97.9% (95% CI, 95.0–100.8) and 89.6% (95% CI, 84–95.2), respectively. The sensitivity strongly increased up to 98.7% (95% CI, 96.1–101.2) for those samples with high virus load (≥6.2 log RNA copies/mL). ConclusionsOur results suggest that EBOLA Ag K-SeT could represent a new effective diagnostic tool for EVD, meeting a need for resource-poor settings and rapid diagnosis for individuals with suspected EVD.

All Bite and No Bark: A Rare Case of Adult Croup Due to Influenza B

SESSION TITLE: Other Infections SESSION TYPE: Affiliate Case Report Poster PRESENTED ON: Tuesday, October 31, 2017 at 01:30 PM - 02:30 PM INTRODUCTION: The incidence of adult croup is rare. Reported cases demonstrate variable presentations and severity, often prompting additional laboratory testing and imaging not routinely ordered in pediatric croup. We describe a unique case of croup in an elderly female with positive influenza B viral PCR testing and CT imaging that revealed subglottic narrowing consistent with laryngotracheobronchitis. CASE PRESENTATION: 89 year-old female presented with 2 days of sore throat, generalized malaise and non-productive cough. She was hemodynamically stable, temperature 101.1°F, respiratory rate 16bpm, and SpO2 88% on room air. Patient’s lungs were clear to auscultation without retractions, wheezes, or stridor. CBC and BMP were unremarkable. Rapid influenza A/B screen was negative. A neck x-ray revealed the characteristic “steeple sign” (fig. 1), which was re-demonstrated on neck CT showing moderate gradual subglottic narrowing consistent with laryngotracheobronchitis (fig. 2). Initial therapies included supplemental oxygen, IV dexamethasone, albuterol/ipratropium nebulizer and IV ceftriaxone. Viral PCR panel was positive for influenza B. Antibiotics and supplemental oxygen were subsequently discontinued and the patient improved clinically and was discharged home with close follow-up. DISCUSSION: Acute laryngotracheobronchitis, more commonly known as croup, is an upper respiratory illness of the pediatric population. Croup is typically a viral etiology, most often parainfluenza, respiratory syncytial virus, and influenza A/B. Traditionally, the diagnosis of pediatric croup is made clinically based on the presence of a hallmark barking cough and stridor; however, this was not the situation in the illustrated case and further imaging of the neck was required to confirm the diagnosis. The standard treatment for viral croup is supplemental oxygen, steroids, and inhaled racemic epinephrine. Close monitoring in an advanced care unit is recommended given the risk of acute respiratory failure. Positive direct viral antigen detection from nasal washings allows for cessation of antibiotics. This patient was outside the window to benefit from oseltamivir and demonstrated significant improvement following 24 hours of supportive therapy. CONCLUSIONS: There have been only 15 previously documented cases of adult croup. The presentation of laryngotracheobronchitis is insidious in adult patients that lack the hallmark barking cough or stridor of pediatric croup. Therefore, unlike the clinical diagnosis in children, the work-up in adults warrants supplementary laboratory testing and imaging to confirm the diagnosis of adult croup. Curative therapies are ultimately the same in both demographics. Reference #1: Woo PCY, et al. Adult Croup: A Rare but More Severe Condition. Respiration. 2000;67:684-688. Reference #2: Tong MCF, et al. Adult Croup. CHEST. 1996;109:1659-62. Reference #3: Patel JJ, et al. A Narrowing Diagnosis: A Rare Cause of Adult Croup and Literature Review. Case Reports in Critical Care. 2017;2017:9870762. DISCLOSURE: The following authors have nothing to disclose: Amy Stacey, Josh Erlandsen, Marc Raslich No Product/Research Disclosure Information

Development of molecular confirmation tools for swift and easy rabies diagnostics

BackgroundAs rabies still represents a major public threat with tens of thousands of deaths per year, particularly in developing countries, adequate surveillance based on rapid and reliable rabies diagnosis for both humans and animals is essential. Rabies diagnosis relies on highly sensitive and specific laboratory tests for detection of viral antigens. Among those tests, at present the immunofluorescence antibody test is the “gold standard test” for rabies diagnosis, followed by virus isolation in either mice or cell culture. Because of the advantages of molecular assays in terms of sensitivity and applicability their approval as confirmatory diagnostic test by international organizations (OIE, WHO) is envisaged. Therefore, the objective was to develop and validate novel molecular assays and RNA extraction methods for rabies that reduce the turnaround time but remain highly sensitive and specific.MethodsHere, novel assays, i.e. HighSpeed RT-qPCR and isothermal recombinase polymerase amplification (RPA) were designed and tested. Furthermore, three magnetic bead-based rapid extraction methods for manual or automated extraction were validated and combined with the new downstream assays.ResultsWhile the conventional column based RNA extraction method showed the highest intra-run variations, all magnetic bead-based rapid extraction methods delivered nearly comparable sensitivity and efficiency of RNA recovery. All newly developed molecular tests were able to detect different rabies virus strains in a markedly reduced timeframe in comparison to the standard diagnostic assays. The observed detection limit for the HighSpeed RT-qPCR was 10 genome copies per reaction, and 1000 genome copies per reaction for the RPA assay.ConclusionMagnetic bead-based rapid RNA extraction methods are highly sensitive and show a high level of reproducibility and therefore, are particularly suitable for molecular diagnostic assays including rabies. In addition, with a detection limit of 10 genome copies per reaction, the HighSpeed RT-qPCR is suitable for rapid ante mortem rabies diagnosis in humans as well as confirmatory test in integrated bite management and subsequent post-exposure prophylaxis.

Progress and Challenges towards Point-of-Care Diagnostic Development for Dengue.

Dengue detection strategies involve viral RNA, antigen, and/or antibody detection. Each strategy has its advantages and disadvantages. Optimal, user-friendly, rapid diagnostic tests based on immunochromatographic assays are pragmatic point-of-care tests (POCTs) in regions where dengue is endemic where there are limited laboratory capabilities and optimal storage conditions. Increasingly, there is a greater public health significance for a multiplexing assay that differentiates dengue from Zika or pathogens with similar clinical presentations. Although there have been many assay/platform developments toward POCTs, independent validation and implementation remain very limited. This review highlights the current key progress and challenges toward the development of a dengue POCT.

Field-Effect Transistor Biosensor for Rapid Detection of Ebola Antigen

The Ebola virus transmits a highly contagious, frequently fatal human disease for which there is no specific antiviral treatment. Therefore, rapid, accurate, and early diagnosis of Ebola virus disease (EVD) is critical to public health containment efforts, particularly in developing countries where resources are few and EVD is endemic. We have developed a reduced graphene oxide-based field-effect transistor method for real-time detection of the Ebola virus antigen. This method uses the attractive semiconductor characteristics of graphene-based material, and instantaneously yields highly sensitive and specific detection of Ebola glycoprotein. The feasibility of this method for clinical application in point-of-care technology is evaluated using Ebola glycoprotein suspended in diluted PBS buffer, human serum, and plasma. These results demonstrate the successful fabrication of a promising field-effect transistor biosensor for EVD diagnosis.

Novel graphene-based biosensor for early detection of Zika virus infection

We have developed a cost-effective and portable graphene-enabled biosensor to detect Zika virus with a highly specific immobilized monoclonal antibody. Field Effect Biosensing (FEB) with monoclonal antibodies covalently linked to graphene enables real-time, quantitative detection of native Zika viral (ZIKV) antigens. The percent change in capacitance in response to doses of antigen (ZIKV NS1) coincides with levels of clinical significance with detection of antigen in buffer at concentrations as low as 450pM. Potential diagnostic applications were demonstrated by measuring Zika antigen in a simulated human serum. Selectivity was validated using Japanese Encephalitis NS1, a homologous and potentially cross-reactive viral antigen. Further, the graphene platform can simultaneously provide the advanced quantitative data of nonclinical biophysical kinetics tools, making it adaptable to both clinical research and possible diagnostic applications. The speed, sensitivity, and selectivity of this first-of-its-kind graphene-enabled Zika biosensor make it an ideal candidate for development as a medical diagnostic test.

First report of immunohistochemical detection of Peste des petit ruminants, parainfluenza 3 and respiratory syncytial viral antigens in lungs of Nigerian goats

ABSTRACTThis study determined the of involvement of PPR, PI3, and RS viruses in the pathology of caprine pneumonia across Nigeria. 150 goats were selected randomly. PI3 and RSV monoclonal antibodies and PPR polyclonal antibody were used for the immunolocalization of the antigens. Histologically, 61 of the goats had broncho-interstitial pneumonia, 25 had interstitial pneumonia, 42 had bronchopneumonia, 12 had bronchiolitis, and 10 were normal. PPR, PI3, and RS viral antigens were demonstrated in: intact and desquamated bronchial, bronchiolar epithelial cells, macrophages, leukocytes, pneumocytes, and giant cells. 23% of the caprine lungs had positive immuno-staining to PI3 viral antigen, 10% were positive for RSV antigen while 34% were positive for PPR viral antigen. 8% showed immunostaining for the two and or three respiratory viral antigens in the goats. PI3 and RSV antigens were more in the young goats, red sokoto breed and during the dry season. This is the first report of immunohistochemical detection of PPR, PI3 and RS viral antigens in caprine lungs in Nigeria. These findings underscore the importance of PI3 and RSV viruses in the control of caprine pneumonia in Nigeria.

Indirect competitive electrochemical immunosensor for hepatitis A virus antigen detection

Indirect competitive electrochemical immunosensor for hepatitis A virus antigen (HAV) detection was developed by immobilizing HAV on the surface of carbon nanopowder paste electrode. The monitoring of the target HAV antigen was performed using a secondary antibody labeled with peroxidase. The analytical signal corresponds to an amperometric current resulting from the addition of hydrogen peroxide and hydroquinone as a redox mediator. A calibration curve was obtained by plotting the intensity of the amperometric signal versus the concentrations of HAV antigens. A low detection limit for HAV antigen was attained and estimated to be 26·10−5IU·mL−1. The sensitivity was also investigated and compared with indirect competitive ELISA. Moreover, the immunosensor provides exact results over a broad linear range from 2·10−4 to 5·10−3IU·mL−1, (R2=0.998). The average relative standard deviation (RSD) of this method was less than 3% for the assays performed on the same day and 7% for the measurements made on different days. The immunosensor exhibits a good stability and high biological selectivity for HAV antigen detection and was successfully applied for the determination of HAV in water samples.

Development of an immunoenzyme test system for diagnosis if rabies

Rabies remains one of the most serious and pressing problems for veterinary science and medicine. According to the data of Rosselkhoznadzor Information and analytical center, the Russian Federation has been an area of natural focal endemicity for many years [13]. An important stage in the control and eradication of rabies in its laboratory diagnosis by detection of virus antigen in the pathological material. The aim of the study was to design an enzyme-linked immunosorbent assay (ELISA) test kit intended for the laboratory diagnosis of rabies in agricultural, domestic, and wild carnivores. Obtaining specific components for the kit, the GNKI “Sheep” master seed strain of rabies virus, the standard CVS strain of rabies virus, and white mice of the BALB line were used.In order to obtain high-activity of specific immunoglobulins (Igs ), the processes of antirabies serum production, immunoglobulin isolation, and purification have been improved as well as the conditions for immunoglobulin conjugation with peroxidase. It was found that the binding ratio should not to be lower than 0.45–0.6.A method of inactivation of rabies virus antigen has been developed, which eliminates the risk of being infected for laboratory workers and shows high specific activity in serological tests. The optimal concentrations of the reactants (immunoglobulin, antigen, conjugated) were determined; basic conditions for conducting the reaction were identified (pH of the carbonate/bicarbonate coating buffer, the optimum temperature, and duration of incubation) that ensure the required level of accuracy and sensitivity of the method.As a result of the conducted studies, a "Kit of preparations for the laboratory diagnosis of animal rabies in animals by ELISA method" was developed. The correlation between results obtained by the immunofluorescence (IF) method and the enzyme-linked immunoadsorbent assay (ELISA) methods was determined; the compliance coefficient constituted 98.8%, the sensitivity threshold of ELISA method being 3.3 Ig LD50 / ml. The sensitivity, specificity, and reproducibility of the developed test system were determined through laboratory and production tests.Â

Pathobiological investigation of naturally infected canine rabies cases from Sri Lanka

BackgroundThe recommended screening of rabies in ‘suspect’ animal cases involves testing fresh brain tissue. The preservation of fresh tissue however can be difficult under field conditions and formalin fixation provides a simple alternative that may allow a confirmatory diagnosis. The occurrence and location of histopathological changes and immunohistochemical (IHC) labelling for rabies in formalin fixed paraffin embedded (FFPE) canine brain is described in samples from 57 rabies suspect cases from Sri-Lanka. The presence of Negri bodies and immunohistochemical detection of rabies virus antigen were evaluated in the cortex, hippocampus, cerebellum and brainstem. The effect of autolysis and artefactual degeneration of the tissue was also assessed.ResultsRabies was confirmed in 53 of 57 (93%) cases by IHC. IHC labelling was statistically more abundant in the brainstem. Negri bodies were observed in 32 of 53 (60.4%) of the positive cases. Although tissue degradation had no effect on IHC diagnosis, it was associated with an inability to detect Negri bodies. In 13 cases, a confirmatory Polymerase chain reaction (PCR) testing for rabies virus RNA was undertaken by extracting RNA from fresh frozen tissue, and also attempted using FFPE samples. PCR detection using fresh frozen samples was in agreement with the IHC results. The PCR method from FFPE tissues was suitable for control material but unsuccessful in our field cases.ConclusionsHistopathological examination of the brain is essential to define the differential diagnoses of behaviour modifying conditions in rabies virus negative cases, but it is unreliable as the sole method for rabies diagnosis, particularly where artefactual change has occurred. Formalin fixation and paraffin embedding does not prevent detection of rabies virus via IHC labelling even where artefactual degeneration has occurred. This could represent a pragmatic secondary assay for rabies diagnosis in the field because formalin fixation can prevent sample degeneration. The brain stem was shown to be the site with most viral immunoreactivity; supporting recommended sampling protocols in favour of improved necropsy safety in the field. PCR testing of formalin fixed tissue may be successful in certain circumstances as an alternative test.

Squamous epitheliotropism of Enterovirus A71 in human epidermis and oral mucosa

Hand-foot-and-mouth disease is a self-limiting paediatric infectious disease commonly caused by Enterovirus A71 (Genus: Enterovirus, Family: Picornaviridae). Typical lesions in and around the hands, feet, oral cavity and other places may rarely be complicated by acute flaccid paralysis and acute encephalomyelitis. Although virus is readily cultured from skin vesicles and oral secretions, the cellular target/s of Enterovirus A71 in human skin and oral mucosa are unknown. In Enterovirus A71-infected human skin and oral mucosa organotypic cultures derived from the prepuce and lip biopsies, focal viral antigens and viral RNA were localized to cytoplasm of epidermal and mucosal squamous cells as early as 2 days post-infection. Viral antigens/RNA were associated with cytoplasmic vacuolation and cellular necrosis. Infected primary prepuce epidermal keratinocyte cultures showed cytopathic effects with concomitant detection of viral antigens from 2 days post-infection. Supernatant and/or tissue homogenates from prepuce skin organotypic cultures and primary prepuce keratinocyte cultures showed viral titres consistent with active viral replication. Our data strongly support Enterovirus A71 squamous epitheliotropism in the human epidermis and oral mucosa, and suggest that these organs are important primary and/or secondary viral replication sites that contribute significantly to oral and cutaneous viral shedding resulting in person-to-person transmission, and viraemia, which could lead to neuroinvasion.