Detection Of Virus Research Articles

Molecular detection of lumpy skin disease virus in naturally infected cattle and buffaloes: unveiling the role of tick vectors in disease spread.

Lumpy skin disease (LSD) is a viral disease that affects cattle and buffaloes in Egypt, causing considerable economic losses in the animal sector. This study aimed to investigate the recent outbreak of LSDV in cattle and buffaloes and evaluate the potential role of the hard tick Rhipicephalus annulatus in their transmission through isolation and molecular characterization by multiplex PCR (mPCR) and real-time quantitative PCR (rt-qPCR) assays. A total of 50 skin biopsies (cattle n = 30, buffaloes n = 20), 110 nasal swabs (cattle n = 76, buffaloes n = 44), and 129 blood samples (cattle n = 84, buffaloes n = 45) were collected. In addition, 145 hard ticks of different stages were collected from cattle and buffaloes of different breeds and ages in different governorates in Egypt from November 2021 to June 2022. Multiplex PCR and real-time quantitative PCR (rt-qPCR) assays based on SYBR Green and targets (P32, VP32, G protein, and viral fusion protein) were used. We identified positive results in 17 out of 30 cattle skin biopsies (56.6%), 1 out of 7 buffalo skin scabs (14.3%), and 5 out of 45 buffalo blood samples (11.11%) using mPCR and RT-qPCR methods. We successfully isolated LSDV from hard ticks and cattle infested with ticks and exhibited characteristic signs of LSD on the chorioallantois membrane (CAM) of specific pathogen-free embryonated chicken eggs (SPF-ECE). The isolates were confirmed by multiplex PCR and RT-qPCR. The cyclic threshold (Ct) with correlation-slandered curve values of rt-qPCR ranging from 10.2 to 36.5 showed the amount of LSDV-DNA in different samples. The study's findings demonstrated the widespread circulation of LSDV in both cattle and buffaloes in Egypt and provided strong evidence that hard ticks R. annulatus play a role in the transmission of LSDV in susceptible animals.

Detection and Quantification of Soil-Borne Wheat Mosaic Virus, Soil-Borne Cereal Mosaic Virus and Japanese Soil-Borne Wheat Mosaic Virus by ELISA and One-Step SYBR Green Real-Time Quantitative RT-PCR.

Furoviruses are bipartite viruses causing mosaic symptoms and stunting in cereals. Infection with these viruses can lead to severe crop losses. The virus species Furovirus tritici with soil-borne wheat mosaic virus (SBWMV), Furovirus cerealis with soil-borne cereal mosaic virus (SBCMV) and Furovirus japonicum with Japanese soil-borne wheat mosaic virus (JSBWMV) and French barley mosaic virus (FBMV) as members are biologically and genetically closely related. Here, we develop SYBR green-based real-time quantitative RT-PCR assays to detect and quantify the RNA1 and RNA2 of the three virus species. Using experimental data in combination with Tm-value prediction and analysis of primer and amplicon sequences, we determine the capacity of our method to discriminate between the different viruses and evaluate its genericity to detect different isolates within the same virus species. We demonstrate that our method is suitable for discriminating between the different virus species and allows for the detection of different virus isolates. However, JSBWMV RNA1 primers may amplify SBWMV samples, bearing a risk for false positive detection with this primer. We also test the efficiency of polyclonal antibodies to detect the different viruses by ELISA and suggest that ELISA may be applied as a first screening to identify the virus. The real-time qRT-PCR assays developed provide the possibility to screen for quantitative disease resistance against SBCMV, SBWMV and JSBWMV. Moreover, with our method, we hope to promote research to unravel yet unresolved questions with respect to furovirus-host interaction concerning host range and resistance as well as regarding the role of multipartite genomes.

HybridSA: GPU Acceleration of Multi-pattern Regex Matching using Bit Parallelism

Multi-pattern matching is widely used in modern software for applications requiring high throughput such as protein search, network traffic inspection, virus or spam detection. Graphics Processor Units (GPUs) excel at executing massively parallel workloads. Regular expression (regex) matching is typically performed by simulating the execution of deterministic finite automata (DFAs) or nondeterministic finite automata (NFAs). The natural implementations of these automata simulation algorithms on GPUs are highly inefficient because they give rise to irregular memory access patterns. This paper presents HybridSA, a heterogeneous CPU-GPU parallel engine for multi-pattern matching. HybridSA uses bit parallelism to efficiently simulate NFAs on GPUs, thus reducing the number of memory accesses and increasing the throughput. Our bit-parallel algorithms extend the classical shift-and algorithm for string matching to a large class of regular expressions and reduce automata simulation to a small number of bitwise operations. We have developed a compiler to translate regular expressions into bit masks, perform optimizations, and choose the best algorithms to run on the GPU. The majority of the regular expressions are accelerated on the GPU, while the patterns that exhibit random memory accesses are executed on the CPU in parallel. We evaluate HybridSA against state-of-the-art CPU and GPU engines, as well as a hybrid combination of the two. HybridSA achieves between 4 and 60 times higher throughput than the state-of-the-art CPU engine and between 4 and 233 times better than the state-of-the-art GPU engine across a collection of real-world benchmarks.

Common viral respiratory infections in children with cancer during the COVID-19 pandemic: a multicenter study from Türkiye.

Microbiologic confirmation of respiratory tract infections gained importance during the coronavirus disease 2019 (COVID-19) pandemic. This study retrospectively evaluated seasonal distribution, clinical presentation, and complications of respiratory viral infections (RVIs) other than COVID-19 in children with cancer during and after the pandemic lockdown. Two hundred and sixty-five inpatient and outpatient RVI episodes in 219 pediatric cancer patients confirmed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) panels from 13 centers were enrolled. Eighty-six (32.5%) of the total 265 episodes occurred in 16 months corresponding to the lockdowns in Türkiye, and the remaining 67.5% in 10 months thereafter. Human rhinovirus/enterovirus (hRE) (48.3%) was the most common agent detected during and after lockdown. Parainfluenza virus (PIV) (23.0%), influenza virus (9.8%), and respiratory syncytial virus (RSV) (9.1%) were the other common agents. The 28.7% of episodes were lower respiratory tract infections (LRTIs), and complications and mortality were higher than upper respiratory tract infections (URTIs) (25.0% vs 5.3%). Bacteremia was identified in 11.5% of culture-drawn episodes. Treatment delay in one-third and death within four weeks after RVI in 4.9% of episodes were observed. During the pandemic, fewer episodes of RVIs occurred during the lockdown period. Respiratory viruses may cause complications, delays in treatment, and even death in children with cancer. Therefore, increased awareness of RVIs and rapid detection of respiratory viruses will benefit the prevention and, in some cases, abrupt supportive and some antiviral treatment of RVI in children with cancer.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for In-Field Detection of American Plum Line Pattern Virus.

American plum line pattern virus (APLPV) is the most infrequently reported Ilarvirus infecting stone fruit trees and is of sufficient severity to be classified as an EPPO quarantine A1 pathogen. In late spring, yellow line pattern symptoms were observed on leaves in a few flowering cherries (Prunus serrulata Lindl.) grown in a public garden in Northwest Italy. RNA extracts from twenty flowering cherries were submitted to Ilarvirus multiplex and APLPV-specific RT-PCR assays already reported or developed in this study. One flowering cherry (T22) with mixed prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) infection also showed infection with APLPV. Blastn analysis of PCR products of the full coat protein (CP) and movement protein (MP) genes obtained from flowering cherry T22 showed 98.23% and 98.34% nucleotide identity with reference APLPV isolate NC_003453.1 from the USA. Then, a LAMP-specific assay was designed to facilitate the fast and low-cost identification of this virus either in the laboratory or directly in the field. The developed assay allowed not only the confirmation of APLPV (PSer22IT isolate) infection in the T22 flowering cherry but also the identification of APLPV in an asymptomatic flowering cherry tree (TL1). The LAMP assay successfully worked with crude flowering cherry extracts, obtained after manually shaking a single plant extract in the ELISA extraction buffer for 3-5 min. The developed rapid, specific and economic LAMP assay was able to detect APLPV using crude plant extracts rather that RNA preparation in less than 20 min, making it suitable for in-field detection. Moreover, the LAMP assay proved to be more sensitive in APLPV detection in flowering cherry compared to the specific one-step RT-PCR assay. The new LAMP assay will permit the estimation of APLPV geographic spread in the territory, paying particular attention to surrounding gardens and propagated flowering cherries in ornamental nurseries.

Fluorescence Polarization Assay for Infection Diagnostics: A Review

Rapid and specific diagnosis is necessary for both the treatment and prevention of infectious diseases. Bacteria and viruses that enter the bloodstream can trigger a strong immune response in infected animals and humans. The fluorescence polarization assay (FPA) is a rapid and accurate method for detecting specific antibodies in the blood that are produced in response to infection. One of the first examples of FPA is the non-competitive test for detecting brucellosis in animals, which was followed by the development of other protocols for detecting various infections. Fluorescently labeled polysaccharides (in the case of brucellosis and salmonellosis) or specific peptides (in the case of tuberculosis and salmonellosis, etc.) can be used as biorecognition elements for detecting infections. The availability of new laboratory equipment and mobile devices for fluorescence polarization measurements outside the laboratory has stimulated the development of new fluorescence polarization assays (FPAs) and the emergence of commercial kits on the market for the detection of brucellosis, tuberculosis, and equine infectious anemia viruses. It has been shown that, in addition to antibodies, the FPA method can detect both viruses and nucleic acids. The development of more specific and sensitive biomarkers is essential for the diagnosis of infections and therapy monitoring. This review summarizes studies published between 2003 and 2023 that focus on the detection of infections using FPA. Furthermore, it demonstrates the potential for using new biorecognition elements (e.g., aptamers, proteins, peptides) and the combined use of FPA with new technologies, such as PCR and CRISPR/Cas12a systems, for detecting various infectious agents.

Recent Developments and Prospects of Electromagnetically Active Metamaterials in Biosensing

This paper provides an overview of the progress of research on electromagnetically active metamaterials in the field of biosensing, especially the potential of terahertz metamaterials for biosensor applications. Electromagnetically active metamaterials exhibit a negative refractive index and perfect absorption through their special structure, and their electromagnetic behavior is affected by their structure and geometry, which is different from traditional materials. The article reviews the application of terahertz technology for the bio-detection of cancer cells and apoptotic processes using periodic metal arrays by terahertz biosensors, demonstrating the advantages of terahertz biosensors, such as high sensitivity and detection without labeling. This paper presents a comprehensive overview of the advancements in research concerning electromagnetically active metamaterials within the domain of biosensing, with a particular emphasis on the potential applications of terahertz metamaterials in biosensor technology. Electromagnetically active metamaterials are characterized by their negative refractive index and perfect absorption, which arise from their distinctive structural properties. The electromagnetic behavior of these materials is significantly influenced by their design and geometry, setting them apart from conventional materials. The article examines the utilization of terahertz technology for the bio-detection of cancer cells and apoptotic processes tunability, employing periodic metal arrays in terahertz biosensors. It underscores the advantages of terahertz biosensors, which include high sensitivity and the capability to detect biological entities without the necessity for labeling. Terahertz metamaterial biosensors are promising for protein, virus, and cancer cell detection. This paper also explores the design and application of chiral metamaterials, especially indium tin oxide-based mid-infrared chiral metamaterials, to solve the problem of the large size of traditional materials and investigates their circular dichroism. Looking ahead, electromagnetically active metamaterials, especially terahertz metamaterials, are expected to improve resolving power and sensitivity, reduce costs, and expand the applications of biosensors in the biomedical field. The applications and research of these sensors will continue to advance with the advancement of micro and nanoprocessing technologies.

Implementation and validation of MICaFVi: A highly efficient nanotechnology-based method for coronaviruses detection

Coronaviruses have emerged as a significant public health concern due to the global impact of Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which cause COVID-19. The development of sensitive and accurate detection methods is critical for early diagnosis, disease management, and outbreak control. In a previous study, we developed and optimized a nanobased detection methodology called Magnetic Immuno-Capture Followed by Flow Virometry (MICaFVi) using virus-mimicking silica nanoparticles and MERS-CoV/SARS-CoV-2 pseudoviral particles. In the present study, we have extended this methodology to evaluate its specificity and sensitivity for detecting wild-type MERS-CoV and SARS-CoV-2 in human and camel samples. Our results demonstrated that MICaFVi successfully detected MERS-CoV and SARS-CoV-2 viruses with high sensitivity and specificity, although it showed reduced performance for samples with Ct values of 30 or lower compared to qPCR. Despite some limitations in detection speed and sensitivity, MICaFVi represents a significant advancement in diagnostic methodologies by combining nanotechnology with flow cytometry. Additionally, we adapted the MICaFVi methodology to simultaneously detect MERS-CoV and SARS-CoV-2 in a single multiplex assay. The successful implementation of this advanced detection approach has important implications for improving early detection, surveillance, and control of both current and future viral or bacterial pandemics. Our results underscore the potential of MICaFVi as a valuable tool for monitoring the spread of these viruses and highlight its role in advancing diagnostic technologies. This extension of our earlier work offers new insights into the application of nanotechnology and flow cytometry for viral detection.

Applications of CRISPR/Cas as a Toolbox for Hepatitis B Virus Detection and Therapeutics.

Hepatitis B virus (HBV) infection remains a significant global health challenge, leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Covalently closed circular DNA (cccDNA) and integrated HBV DNA are pivotal in maintaining viral persistence. Recent advances in CRISPR/Cas technology offer innovative strategies to inhibit HBV by directly targeting both cccDNA and integrated HBV DNA or indirectly by degrading HBV RNAs or targeting host proteins. This review provides a comprehensive overview of the latest advancements in using CRISPR/Cas to inhibit HBV, with a special highlight on newer non-double-strand (non-DSB) break approaches. Beyond the canonical use of CRISPR/Cas for target inhibition, we discuss additional applications, including HBV diagnosis and developing models to understand cccDNA biology, highlighting the diverse use of this technology in the HBV field.

Development of an indirect ELISA for the serologic detection of bovine viral diarrhea virus based on E2 antigen sub-genotypes 1b, 1e, and 1d.

Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study, we developed a new indirect ELISA (rE2-ELISA) for serologic detection of BVDV. The assay considers three recombinant E2 protein subtypes as antigens, allowing serologic diagnosis of BVDV-1b (high prevalence worldwide), BVDV-1d and 1e (high prevalence in southern Chile) sub-genotypes. Recombinant E2 (rE2) proteins were successfully expressed in stably transfected CHO cells. Conditions for rE2 ELISAs were established after determining appropriate concentrations of antigen, blocking agent, secondary antibody, and serum dilutions to achieve maximum discrimination between positive and negative serum samples. The developed rE2-ELISA showed a sensitivity of 92.86% and a specificity of 98.33%. Clinical testing of 180 serum samples from herds in southern Chile showed high accuracy (kappa > 0.8) compared to the commercial BVDV Total Ab kit (IDEXX), with 95.37% positive and 87.5% negative predictive value. In addition, the rE2 ELISA has shown the capability to detect anti-BVDV antibodies from naturally infected animals with sub-genotypes 1b, 1e, or undetermined. These results indicate that the developed indirect ELISA could serve as a valid, and efficient alternative for identifying BVDV-infected animals, thus contributing to the success of disease control and eradication programs.

B-201 Clinical Performance Evaluation of IRON-qPCR™ RV, a Novel Molecular POCT system, for the Qualitative Detection of Respiratory Viruses in Nasopharyngeal Swab

Abstract Background The availability of Point-of-Care testing (POCT) based on molecular diagnostics to test simultaneously for multiple respiratory viruses is very limited though significant progress has been made in molecular testing for COVID-19 during the pandemic. Considering the similar symptoms presented by patients infected with various respiratory pathogens, it is crucial to have multiple POCT systems capable of detecting multiple respiratory pathogens while distinguishing SARS-CoV-2 from other pathogens. To meet the growing demand for multiplex testing for respiratory pathogens, a molecular POCT, IRON-qPCR™ RV has been developed. IRfON-qPCR™ RV is a multiplex real-time RT-PCR assay designed for the qualitative detection and differentiation of SARS-CoV-2, Influenza A, Influenza B, RSV-A, and RSV-B RNA from human nasopharyngeal swab specimens. The IRON-qPCR™ proceeds the entire process automatically from nucleic acid extraction to real-time RT-PCR within 40 minutes. The main goal of this study is to evaluate the clinical performance of IRON-qPCR™ RV in comparison with other assays and kits using nasopharyngeal swab samples. Methods To assess the clinical performance of the IRON-qPCR™ RV, specimen collection and evaluation were conducted at a clinical institution in France, utilizing a total of 980 nasopharyngeal swab samples. The comparators used were the Xpert® Xpress CoV-2/Flu/RSV plus Kit and the RealStar® RSV RT-PCR Kit 3.0. Since RSV subtypes are not distinguished by the Xpert kit, all RSV without subtype distinction were compared. Additionally, RealStar kit was used to compare the results of IRON-qPCR™ RV for RSV subtypes A and B. Clinical samples showing discrepancies between Xpert and RealStart, and the IRON-qPCR™ RV were further analyzed using Allplex™ SARS-CoV-2/FuA/FluB/RSV Assay and sequencing. Results Through the comparison of clinical performance of the IRON-qPCR™ RV with Xpert for SARS-CoV-2, Influenza A, and Influenza B in nasopharyngeal swab samples, the clinical sensitivity was analyzed to be over 95.5%, and the clinical specificity was over 99.6%. For RSV, compared to Xpert, the clinical sensitivity was analyzed to be 96.9%, and the clinical specificity was 98.9%. Furthermore, in comparison with RealStar, the clinical sensitivity and specificity of IRON-qPCR™ RV for RSV-A were determined to be 96.2% and 100%, respectively, and the clinical sensitivity and specificity for RSV-B were 96.0% and 100%. Additionally, discrepant samples in comparison with Xpert were further tested using the Allplex as a resolver kit. The result showed that the clinical sensitivity and specificity of IRON-qPCR™ RV for SARS-CoV-2, Influenza A, and Influenza B were over 98.3% and 100%. For RSV, the clinical sensitivity and specificity were 99.5% and 99.1%. Conclusions The clinical performance of IRON-qPCR™ RV was analyzed with the clinical sensitivity of over 95% and the clinical specificity of over 99%. Upon analysis including the results of the resolver kit for discrepant samples, increased clinical sensitivity and specificity were determined. The results of this study strongly support that the IRON-qPCR™ RV is a reliable POCT system for the simultaneous detection and differentiation of multiple respiratory viruses. Furthermore, it is applicable to district hospitals as well as clinical laboratories for accurate and timely testing.

B-215 Evaluating RT-qPCR for Dengue Virus Detection: Outcomes of a Brazilian External Quality Assessment Program

Abstract Background Dengue virus (DENV), responsible for a prevalent arthropod-borne viral illness, poses a substantial public health challenge worldwide. Clinically, DENV's manifestation often resembles that of other arboviruses, resulting in frequent healthcare visits and complicating the early diagnosis, which is critical for patient management. While virus isolation is a viable method, RT-qPCR assays yield significantly quicker results. Rapid immunochromatographic tests are available; yet, RT-qPCR not only matches their sensitivity but also provides distinct advantages in clinical laboratory settings. These include higher throughput and integrated, traceable quality control within each routine, offering less subjective and more dependable results. Maintaining high diagnostic standards for DENV is imperative. External quality assessment programs (EQAP) play a key role in enhancing laboratory diagnostic capabilities by monitoring and assessing diagnostic methods. This study evaluates the diagnostic efficacy of RT-qPCR for DENV using data from an EQAP conducted by a Brazilian provider, accredited by ABNT NBR ISO/IEC 17043:2011. Methods The quality control samples comprised lyophilized suspensions containing viable DENV particles, grown on vero cells (BCRJ 0245/ATCC CCL-81) under BSL-3 conditions. From February 2017 to November 2022, twenty-four EQAP survey rounds were carried out, each encompassing three samples, culminating in 1,153 data points. The evaluation metrics included participant numbers, adequacy percentage, sensitivity, specificity, and the incidence of false positives and negatives. The Mann-Kendall test assessed trends in these metrics over the survey rounds. The RT-qPCR kits employed by participants were classified either as in-vitro diagnostic (IVD) or as laboratory-developed tests (LDT). Kits that demonstrated high performance in terms of adequacy, sensitivity, and specificity were identified and listed. Results The number of laboratories participating has increased. Starting with four in 2017, the program expanded to 28 by 2022. Laboratory accuracy was high during the evaluation period, with a median of 99% (97.8% in 2017 and 99.2% in 2022). The Mann-Kendall test indicated a significant trend of increased accuracy (p<0.001). Specificity remained stable at 98.8%, while sensitivity improved, with a median of 99.4%—rising from 93.3% in 2017 to 100% in 2022 (p<0.001). Only three false negatives were reported over three separate rounds, and six false positives were reported across four rounds. Both LDT and IVD RT-qPCR kits demonstrated high accuracy (99.4% and 98.4%, respectively). Biomanguinhos ZDC, Viasure ZDC, and ZDC Multiplex kits all showed 100% adequacy, sensitivity, and specificity. Conclusions This study highlights the increasing adoption of RT-qPCR for DENV detection, as evidenced by the growing number of laboratories in the EQAP program and the consistently high adequacy rates from 2017 to 2022. Nonetheless, the number of participating laboratories remains relatively small for a vast tropical country like Brazil. The high sensitivity and specificity, along with the infrequency of false results, underscore its effectiveness. The superior performance of certain kits suggests possible advancements in DENV diagnosis, contributing to global public health initiatives against this arboviral disease.

B-048 Proposal of a new monitoring algorythm for patients with a HIV positive result with the Elecsys HIV DUO test

Abstract Background Worldwide HIV infections remain a public health problem, contributing significantly to morbidity. In Mexico we have had a progressive adaptation to the HIV epidemic providing free therapy and testing (4th generation tests) for the whole population in anbulatory centers for HIV/AIDS prevention and medical care. During 2022 Mexico reported 12,374 new cases. In the last decade there has been an increasing interest in the identification of patients HIV positives subgroups, based on disease progression and serological features. To date, 6 generations of tests for HIV detection have been developed, strongly reducing the window period for a positive result. Elecsys HIV Duo is a 5th generation immunological test that provides in a separate manner an antibody and antigen results for HIV detection. These results show a better insight of the serologycal status reducing the window period post infection. By using these tests we want to provide an early detection of new cases that gives a better prognosis for the patient and propose a new monitoring for patients with a positive antigen result but that have not yet produced antibodies. Such algorythm is currently lacking in the Mexican public health services. Methods A retrospective statystical analysis of HIV results of samples procesed with the Elecsys HIV DUO test during the january-april 2023 period in the National Reference Center of Salud Digna correlated with western blot results. Results The study population was 6405 patients, 6340 (98.98%) had a negative result in the test and 65 (1.02%) a positive test. Based in antibody/antigen results we categorized the positives results in three grupos: Group 1 Ab positive/Ag negative (16), group 2 Ab positive/Ag positive (32) and group 3 Ab negative/Ag positive (7). When correlated with the WB results all the patients from group 1 and 2 had a positive result while all patients in group 3 had a negative result. Conclusions Actually Mexican public health services do not have access to 5th generation HIV tests like the Elecsys HIV DUO test, therefore, public health profesional are not qualified to interprete these results and do not have a stated algorythm for patientes belonging to group 3. Therefore and based in our results we suggest 5th generation test (or superior if available) and propose a new patient monitoring algorythm with a molecular confirmatory test for viral detection. This proposal is strongly supported by the fact the we have a population that have not generated antibodys during the first weeks of post infection and will test negative in a serologycal confirmatory test like WB. Screening for patients in early infection stages is important because if we provide early therapy which may lead to infection reversal or a less impact in C4+ T cells numbers. Studies like this provide information for the generation of public politics in benefit of the population.

Avian influenza overview June-September 2024.

Between 15 June and 20 September 2024, 75 highly pathogenic avian influenza (HPAI) A(H5) and A(H7) virus detections were reported in domestic (16) and wild (59) birds across 11 countries in Europe. Although the overall number of detections in Europe continued to be low compared to previous epidemiological years, an increase in cases along the Atlantic, North Sea and Baltic coasts was notable, particularly an increase in the detection of HPAI viruses in colony-breeding seabirds. Besides EA-2022-BB and other circulating genotypes, these detections also included EA-2023-DT, a new genotype that may transmit more efficiently among gulls. In Germany, HPAI A(H7N5) virus emerged in a poultry establishment near the border with the Netherlands. No new HPAI virus detections in mammals were reported in Europe during this period, but the number of reportedly affected dairy cattle establishments in the United States of America (USA) rose to >230 in 14 states, and HPAI virus was identified in three new mammal species. Between 21 June and 20 September 2024, 19 new human cases with avian influenza virus infection were reported from the USA (six A(H5N1) cases and five A(H5) cases), Cambodia (five A(H5N1) cases, including one fatal), China (one fatal A(H5N6) case and one A(H9N2) case), and Ghana (one A(H9N2) case). Most of the human cases (90%, n = 17/19) had reported exposure to poultry, live poultry markets, or dairy cattle prior to avian influenza virus detection or onset of illness. Human infections with avian influenza viruses remain rare and no evidence of human-to-human transmission has been documented in the reporting period. The risk of infection with currently circulating avian A(H5) influenza viruses of clade 2.3.4.4b in Europe remains low for the general public in the European Union/European Economic Area (EU/EEA). The risk of infection remains low-to-moderate for those occupationally or otherwise exposed to infected animals or contaminated environments.

Protocol for detection of pathogenic enteric RNA viruses by regular monitoring of environmental samples from wastewater treatment plants using droplet digital PCR

BackgroundThe present comprehensive protocol is focused on the detection of pathogenic enteric RNA viruses, explicitly focusing on norovirus genogroup Ⅱ (GⅡ), astrovirus, rotavirus, Aichi virus, sapovirus, hepatitis A and E viruses in wastewater treatment plants through droplet digital PCR (ddPCR). Enteric viruses are of significant public health concern, as they are the leading cause of diseases like gastroenteritis. Regular monitoring of environmental samples, particularly from wastewater treatment plants, is crucial for early detection and control of these viruses. This research aims to improve the understanding of the prevalence and dynamics of enteric viruses in urban India and will serve as a model for similar studies in other regions. Our protocol objective is to establish a novel ddPCR-based methodology for the detection and molecular characterization of enteric viruses present in wastewater samples sourced from Bhopal, India. Our assay is capable of accurately quantifying virus concentrations without standard curves, minimizing extensive optimization, and enhancing sensitivity and precision, especially for low-abundance targets. MethodsThe study involves fortnightly collecting and analyzing samples from nine wastewater treatment plants over two years, ensuring comprehensive coverage and consistent data. Our study innovatively applies ddPCR to simultaneously detect and quantify enteric viruses in wastewater, a more advanced technique. Additionally, we will employ next-generation sequencing for detailed viral genome identification in samples tested positive for pathogenic viruses. ConclusionThis study will aid in understanding these viruses’ genetic diversity and mutation rates, which is crucial for developing tailored intervention strategies. The findings will be instrumental in shaping public health responses and improving epidemiological surveillance, especially in localities heaving sewage networks.

Development and validation of a new and rapid molecular diagnostic tool based on RT-LAMP for Hepatitis C virus detection at point-of-care

PurposeGlobally, it is estimated that 1.0 million individuals are newly infected by Hepatitis C virus (HCV) every year, and nearly 50 million people live with a chronic infection, according to World Health Organization. To overcome underdiagnosis of HCV infection among hard-to-reach populations, it is essential to develop new rapid and easy-to-use molecular diagnostic systems. In this work, we have developed a pangenotypic diagnostic tool based on Loop-Mediated Isothermal Amplification (LAMP), coupled to a direct sample lysis procedure for molecular detection of HCV at point-of-care (POC). MethodsProcedure validation was performed using 129 different samples from HCV infected patients (116 serum samples, and 13 fresh blood samples), 27 individuals who tested negative for HCV but positive for HIV, and 11 healthy donors. Serum was collected, lysed for 10 min at room temperature, and assayed by RT-LAMP. To achieve this, a set of 9 LAMP-primers was used for the first time. Parallel RT-qPCR assays were conducted for HCV to both validate the procedure and quantify viral loads. ResultsHCV was detected by RT-LAMP in 109/116 HCV positive serum samples, and in 11/13 positive blood samples in less than 40 min. Compared to RT-qPCR results, our RT-LAMP procedure showed a sensitivity of 94 %, 100 % specificity, and a limit of detection of 3.26 log10 IU/mL (10–20 copies per reaction). ConclusionsWe have developed an accurate system, more affordable than the current available rapid tests for HCV. Since no prior RNA purification step from capillary blood is required, we strongly recommend our RT-LAMP system as a valuable and rapid tool for the molecular detection of HCV at POC.

Risk factors for introduction of BVDV in the context of a mandatory control program in Dutch dairy herds.

Bovine Viral Diarrhea Virus (BVDV) is a common viral disease in cattle, causing economic losses in naive herds where the virus is introduced. In the Netherlands, a BVDV control program has been in place since 1998, evolving from voluntary to mandatory participation for dairy herds since April 2018. Participation in the BVDV control program is not mandatory for non-dairy farms. The aim of this study was to determine risk factors for new introduction of BVDV into certified BVDV-free herds in the context of a national BVDV control program in dairy herds. In this retrospective case-control study, survey data were collected from 149 dairy farms that lost their BVDV-free status (case herds) and 148 matched dairy farms that maintained their BVDV-free status (control herds) between 2018 and 2021. The survey captured information about potential risk factors and herd characteristics in the 2 years leading up to the loss of BVDV-free status (case herds, virus detection in at least one animal or when seroconversion was detected) or remaining BVDV-free (control herds). Descriptive statistics and logistic regression with a backward selection and elimination procedure were used to identify potential risk factors associated with losing BVDV-free status. Risk factors were quantified as an Odds Ratio (OR) with the associated 95% confidence interval (CI). The risk factor with the highest OR for losing BVDV-free status was purchasing cattle from herds without BVDV-free status (OR 1.25, CI 1.10-1.41), followed by the farmer having another profession that resulted in contact with other cattle (OR 1.25, CI 1.06-1.47), housing young calves and adult cows in the same barn (OR 1.22, CI 1.08-1.38), having a permanent employee on the farm (OR 1.17, CI 1.04-1.31), having a group calving pen (OR 1.16, CI 1.03-1.32), escaped cattle from other farms that mingled with own cattle (OR 1.16, CI 1.01-1.33), and nearest distance to a non-dairy farm (OR 1.15, CI 1.03-1.28). Although the BVDV status of most dairy herds can be checked in an open register, approximately half of the farmers indicated that they purchased cattle from BVDV-free herds while they were actually purchasing from non-BVDV-free farms. Farmers should be stimulated to actively check the true BVDV status of the herd from which cattle are purchased to further reduce the risk of introduction. In addition, indirect contact with cattle from other farms through either the farmer or other on-farm staff should be avoided. It is strongly advised to work in these situations with proper biosecurity measures such as changing boots and coveralls. The results can be used to improve BVDV control programs to further decrease the prevalence.

Performance of an envelope glycoprotein-based multiplex immunoassay for Ebola virus antibody detection in a cohort of Ebola virus disease survivors

Serological surveillance in animal and human hosts can be a cost-effective strategy for orthoebolavirus detection, but is challenged by accurate estimates of seroprevalence, potential pauci-symptomatic disease presentation, and antigenic cross-reactivity. Here, we describe the use of an envelope glycoprotein (GP)-based multiplex microsphere immunoassay, consisting of nine filovirus GP antigens, for the detection of anti-Ebola virus (EBOV) antibodies in a well-characterized cohort of Guinean Ebola virus disease (EVD) survivors and contacts from the 2013 – 2016 West African EVD outbreak. We examined sensitivity and specificity for the detection of anti-EBOV antibodies by GP expressed as recombinant trimeric ectodomains, yielding an assay performance of 95.96% sensitivity and 98.61% specificity. Additionally, agreement between the multiplex test and a whole virus ELISA and virus neutralization test showed strong correlations. The results demonstrate this filovirus multiplex test is a sensitive tool for high-throughput serosurveillance

Environmental sampling for the detection of capripox viruses and peste des petits ruminants virus in households and livestock markets in Plateau State, Nigeria.

Multiple transboundary animal diseases (TADs) circulate in Plateau State, Nigeria, where livestock keeping is common and contributes to both the physical and socio-economic well-being of a large proportion of the population. In this study, we explored the potential for environmental sampling to detect viruses causing TADs circulating in the region. Electrostatic dust cloths were used to swab areas of the environment likely to have contact with secretions and excretions from infected animals. Samples were collected monthly from five households, one transhumance site and one livestock market in two local government areas in Plateau State between March and October 2021. These were tested for the presence of peste des petits ruminants virus (PPRV) and capripox viruses using real-time PCR. Of the 458 samples collected, 2.4% (n = 11) were positive for PPRV RNA and 1.3 % (n = 6) were positive for capripox virus DNA. A capripox differentiation assay showed that these samples were positive for sheep pox virus (n = 2), goat pox virus (n = 2) and lumpy skin disease virus (n = 2). Our results demonstrate that environmental sampling could be used as part of TAD surveillance in the area. Environmental swabs require little technical knowledge to collect and can be used to detect multiple viruses from a single sample.

Molecular detection of Tomato leaf curl New Delhi virus infecting pumpkin plants in sub-Himalayan plains of West Bengal, India

Molecular detection of Tomato leaf curl New Delhi virus infecting pumpkin plants in sub-Himalayan plains of West Bengal, India