Detection Of Viral RNA Research Articles

Representative honey bee viruses do not replicate in the small hive beetle, Aethina tumida Murray

The small hive beetle (SHB), Aethina tumida Murray, is an invasive pest of the honey bee and causes significant damage through the consumption of colony resources and brood. Two assumptions related to honey bee virus transmission have been made about SHB: first, that SHB vectors honey bee viruses and second, that these viruses replicate in SHB based on the detection of both positive and negative strand viral genomic RNA within SHB. To clarify the role of SHB in virus transmission, we sought to address whether selected honey bee viruses replicate in SHB. Sequences derived from five honey bee viruses were identified in the transcriptomes of field-caught SHB from the U.S., but not in those of lab-reared SHB, suggesting that these viruses do not replicate in SHB. To elucidate whether the representative viruses, Israeli acute paralysis virus (IAPV; Dicistroviridae) and Deformed wing virus (DWV; Iflaviridae) replicate in SHB, we tested for replication in vitro in an SHB-derived cell line (BCIRL-AtumEN-1129-D6). Following treatment of the cell line with viral particles or viral RNA, the number of virus genomes was monitored by reverse transcription quantitative PCR (RT-qPCR). In contrast to the positive control, IAPV and DWV RNA levels steadily decreased over a period of 8 days. Collectively, these results from bioinformatic observations and in vitro experiments indicate that IAPV and DWV do not replicate in SHB. These results are consistent with the host specificity of most insect viruses within a single insect order and indicate that while SHB may serve as a mechanical vector of honey bee viruses within and between hives, this insect does not serve as a biological vector for these honey bee viruses.

Low detection of H5N1 virus in commercial chickens with a low-level of vaccination coverage against H5N1 virus infection in Bangladesh

BackgroundBangladesh has reported > 560 H5N1 outbreaks in poultry and eight human cases since 2007. Commercial chicken farms were mostly affected. Commercial chicken farms across the country use imported vaccines against H5N1 virus; however, these vaccines did not use local circulatory isolates of H5N1virus. Vaccination may have limited effectiveness in chicken because of the mismatch in terms of subtypes and clades. To test this, we conducted a mixed-method study to assess the impact of ongoing vaccination against H5N1 virus on H5N1 viral shedding through freshly dropped feces of chickens raised in commercial farms that exclusively vaccinated or did not vaccinate their chickens.MethodsInitially, we collected vaccination coverage data from all active farms in a subdistrict of each of eight division. In each district, 25 vaccinated and 25 non-vaccinated chicken farms were selected randomly for sample collection. All samples were tested to detect avian influenza viruses using rRT-PCR.ResultsA total of 5092 poultry farms were surveyed; among them 1284 (25%) chicken farms administered vaccine against H5N1 virus. In total 21 of 400 tested farms (5%) had chicken feces samples that tested positive for AIVs; of these three were positive for H5N1 subtype of clade 2.3.2.1. Out of three H5N1 positive farms, 1 (33%) was vaccinated and 2 (67%) were unvaccinated. The chicken farms that administered vaccine against H5N1 was found protective for the detection of H5N1 viral RNA (aOR 0.39, 95% CI: 0.32–0.48). The H5N1 isolates of clade 2.3.2.1 sequenced in this study formed a cluster with the vaccine strain A/duck/Guangdong/S1322/2010 (H5N1) [Re-6].ConclusionsThe overall low vaccination coverage with low detection of H5N1 virus in commercial chickens makes it difficult to assess the effectiveness of the vaccine in reducing H5N1 viral shedding.

A DNA Aptamer for 2-Aminopurine: Binding-Induced Fluorescence Quenching.

2-Aminopurine (2AP) is a fluorescent analog of adenine, and its unique properties make it valuable in various biochemical and biotechnological applications. Its fluorescence property probes local dynamics in DNA and RNA because stacking with the surrounding bases quench its fluorescence. 2AP-labeled DNA or RNA sequences have been used for the detection of genetic mutations, viral RNA, or other nucleic acid-based markers associated with diseases like cancer and infectious diseases. In this study, we isolated aptamers for 2AP using the library immobilization capture-SELEX technique. A dominating aptamer family was isolated after 15 rounds of selection. The Kd values for the most abundant 2AP1 aptamer are 209 nM in a fluorescence assay and 72 nM in an isothermal titration calorimetry test. A 32 nM 2AP limit of detection was tested based on its intrinsic fluorescence change upon aptamer binding. Additionally, we conducted some mutation analysis. Furthermore, we tested the selectivity of this aptamer and discovered that it can bind adenine and adenosine with approximately 100-fold lower affinity than 2AP.

Immunogenicity and Efficacy of Combined mRNA Vaccine Against Influenza and SARS-CoV-2 in Mice Animal Models

Background. The combined or multivalent vaccines are actively used in pediatric practice and offer a series of advantages, including a reduced number of injections and visits to the doctor, simplicity of the vaccination schedule and minimization of side effects, easier vaccine monitoring and storage, and lower vaccination costs. The practice of widespread use of the combined vaccines has shown the potential to increase vaccination coverage against single infections. The mRNA platform has been shown to be effective against the COVID-19 pandemic and enables the development of combined vaccines. There are currently no mRNA-based combined vaccines approved for use in humans. Some studies have shown that different mRNA components in a vaccine can interact to increase or decrease the immunogenicity and efficacy of the combined vaccine. Objectives. In the present study, we investigated the possibility of combining the mRNA vaccines, encoding seasonal influenza and SARS-CoV-2 antigens. In our previous works, both vaccine candidates have shown excellent immunogenicity and efficacy profiles in mice. Methods. The mRNA-LNPs were prepared by microfluidic mixing, immunogenicity in mice was assessed by hemagglutination inhibition assay, enzyme-linked immunoassay and virus neutralization assay. Immunological efficacy was assessed in a mouse viral challenge model. Results. In this work, we demonstrated that the individual mRNA components of the combined vaccine did not affect the immunogenicity level of each other. The combined vaccine demonstrated excellent protective efficacy, providing a 100% survival rate when mice were infected with the H1N1 influenza virus and reducing the viral load in the lungs. Four days after the challenge with SARS-CoV-2 EG.5.1.1., no viable virus and low levels of detectable viral RNA were observed in the lungs of vaccinated mice. Conclusions. The combination does not lead to mutual interference between the individual vaccines. We believe that such a combined mRNA-based vaccine could be a good alternative to separated human vaccinations for the prevention of COVID-19 and influenza.

Crimean-Congo hemorrhagic fever in Herat province of Afghanistan, 2007-2021

Crimean-Congo hemorrhagic fever (CCHF) is a geographically widespread tick-borne hemorrhagic diseasecaused by the Crimean-Congo hemorrhagic fever virus (CCHFV). In Afghanistan, the first case of CCHF wasdetected in Takhar province in 1998, after which no additional cases were reported for ten years. The aim ofthis study is to report the analyses of the clinical and epidemiological features of confirmed CCHF cases thatwere recorded in Herat province between 2007 and 2021. This prospective case series study was conducted atHerat Regional Hospital between January 2007 and December 2021. Demographic and clinical data werecollected for each patient; all patients were hospitalized. Biochemical and hematological laboratoryexamination, and the detection of CCHV IgM and viral RNA was performed for all patients. Statistical analyseswere performed in IBM SPSS Statistics (version 26). A total of 252 confirmed CCHF cases including 160(63.5%) males and 91 (36.5%) females, with a mean age of 33.37±14.1 years were included in the study. Themajority of patients were in 11-50 age category, 32.9% were housewife, 38.4% were farmers, shephard, andbutchers. The most prevalent clinical presentations were fever (100%), headache (98.8%), body pain (96.4%),Ecchymosis (48.0%), epistaxis (44.8%) and gum bleeding (33.3%). The majority of cases occurred betweenApril and October. The case fatality rate of CCHF in this study was 2.8%. Considering the significant social,economic and health burden CCHF places on the community, enhanced public health measures are necessaryto control CCHF in Herat province and neighboring regions.

Comparative pathogenesis of Ethiopia/Habru/2014 Lineage-IV peste des petits ruminants virus in goats and cattle

BackgroundPeste des Petits Ruminants (PPR) is a highly contagious viral disease primarily affecting goats and sheep, with clinical manifestations ranging from peracute disease to subclinical infection, particularly in atypical hosts such as cattle. The role of atypical hosts such as cattle to the spread of PPR remains controversial, with conflicting reports in the literature. Despite its worldwide significance, considerable knowledge gaps exist regarding the pathogenesis and clinical progression in both primary and atypical hosts. This study aimed to elucidate the tissue tropism, pathogenesis, virus shedding, clinical progression, and pathology associated with experimental PPR virus infection in indigenous goats and cattle. To this end, 32 animals—16 goats and 16 cattle—were intranasally inoculated with the Ethiopia/Habru/2014 Lineage-IV strain of the PPR virus followed by detailed clinical evaluations and systematic sampling at pre-established intervals to assess serological conversion, viral shedding, and the pathogenesis of the infection across both species.ResultsThe results show that goats exhibited typical clinical signs 4 days post-inoculation, with seroconversion by day 6 and early detection of viral RNA in swabs and tissues by day 3 and virus isolation starting day 4. In contrast, cattle exhibited minimal clinical signs, with seroconversion occurring at day 8 with viral RNA detected in tissue samples at day 4 and virus isolation starting day 6 in tissues and in a single nasal swab at day 8. Clinical scores and tissue positivity rates significantly differed between goats and cattle (P = 0.007 and P < 0.001, respectively). While goats exhibited expected gross and histopathological lesions, cattle showed only nonspecific lesions.ConclusionsTogether, our findings highlight the importance of comparative pathology studies for better understanding virus dynamics and transmission pathways that may help inform more effective PPR control programs. Future research should explore the pathogenesis of different PPRV lineages in cattle, assessing variations in disease progression and potential for epidemiological impact.

Characterizing best practices for tonsil-oral-scrubbing (TOSc) collection for PRRSV RNA detection in sows

BackgroundA Tonsil-Oral-Scrubbing (TOSc) method was developed to sample the sow’s oropharyngeal and tonsillar area without snaring and has shown comparable porcine reproductive and respiratory syndrome virus (PRRSV) RNA detection rates with tonsil scraping in infected sows. This study investigated the effect of specific TOSc collection factors on the PRRSV RT-rtPCR results (detection rates and Ct values). Those factors include whether the sow was snared or not snared at TOSc collection (“snared” vs. “not snared”); whether the sow was laying down or standing at collection (“laying down” vs. “standing”); and type of collectors used for TOSc collection (“TOSc prototype” vs. “Spiral-headed AI catheter (SHAC)”). Volume of fluid was compared between “snared” and “not snared” groups, and collection time was compared between “laying down” and “standing” groups as well.ResultsThe effect for each factor was assessed in three independent studies following the same design: TOSc was collected twice from each studied sow, once with the baseline level for a factor (“not snared”, or “standing”, or “TOSc prototype”), and another time followed by the other level of the paired factor (“snared”, “laying down”, or “SHAC”, correspondingly). Results showed that “not snared” TOSc had numerically higher PRRSV RNA detection rate (60.7% vs. 52.5%, p = 0.11), significantly lower median Ct values (31.9 vs. 32.3, p < 0.01), and significantly higher volume of fluid than “snared” samples (1.8 mL vs. 1.2 mL, p < 0.01); “laying down” TOSc samples did not differ statistically (60.7% vs. 60.7%) in the PRRSV RNA detection rate, obtained numerically lower median Ct values (30.9 vs. 31.3, p = 0.19), but took 40% less collection time compared to “standing” TOSc samples; samples collected using the “TOSc prototype” had numerically higher PRRSV RNA detection rate (91.7% vs. 88.3%, p = 0.27) and significantly lower median Ct values (32.8 vs. 34.5, p < 0.01) than that from “SHAC”.ConclusionsUnder the conditions of this study best practices for TOSc collection aiming higher detection rate of PRRSV RNA while minimizing time for collection were suggested to be sampling TOSc without snaring, when sows are laying down, and using a prototype TOSc collector.

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.

CRISPR/Cas13a-mediated triple signal amplification strategy for viral RNA detection

CRISPR/Cas13a system has been combined with different isothermal amplification strategies for RNA analysis. Nevertheless, expensive enzymes are usually indispensable in these isothermal amplification methods, which increases the detection cost. In this study, a CRISPR/Cas13a-mediated triple signal amplification strategy has been designed for selective, sensitive and inexpensive detection of viral RNA. SARS-CoV-2 S RNA activated CRISPR/Cas13a to cut hairpin DNA, triggering catalytic hairpin assembly (CHA) and subsequent hybridization chain reaction (HCR) to form a long-stranded Y-type DNA. G-quadruplexes were distributed on the branch of the long-stranded DNA and bound with N-methyl mesoporphyrin IX (NMM) to enhance the fluorescence signal. SARS-CoV-2 S RNA was specifically detected with a limit of detection (LOD) of 285 fM. Notably, no additional expensive enzymes were required in the entire detection process. The CRISPR/Cas13a-mediated triple signal amplification strategy holds great potential for specific and sensitive viral RNA detection with low cost.

Location, Age, and Antibodies Predict Avian Influenza Virus Shedding in Ring-Billed and Franklin’s Gulls in Minnesota

Influenza A virus (IAV) is a multi-host pathogen maintained in water birds and capable of spillover into humans, wildlife, and livestock. Prior research has focused on dabbling ducks as a known IAV reservoir species, yet our understanding of influenza dynamics in other water birds, including gulls, is lacking. Here, we quantify morphological and environmental drivers of serological (antibody detection by ELISA) and virological (viral RNA detection by PCR) prevalence in two gull species: ring-billed (Larus delawarensis) and Franklin’s (Leucophaeus pipixcan) gulls. Across 12 months and 10 locations, we tested over 1500 gulls for influenza viral RNA, and additionally tested antibody levels in nearly 1000 of these. We find substantial virus prevalence and a large, nonoverlapping seroprevalence, with significant differences across age and species classifications. The body condition index had minimal explanatory power to predict (sero)positivity, and the effect of the surrounding environment was idiosyncratic. Our results hint at a nontrivial relationship between virus and seropositivity, highlighting serological surveillance as a valuable counterpoint to PCR. By providing indication of both past infections and susceptibility to future infections, serosurveillance can help inform the distribution of limited resources to maximize surveillance effectiveness for a disease of high human, wildlife, and livestock concern.

Development and Validation of a Highly Sensitive RT-qLAMP Assay for Rapid Detection of SARS-CoV-2: Methodological Aspects.

Specific and reliable diagnostic methods are becoming increasingly essential to identify patients in light of the high transmission rate and the recent appearance of the new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For the specific detection of SARS-CoV-2, our quantitative reverse transcription loop-mediated isothermal amplification (RT-qLAMP) assay implementation demonstrates how flexible it can be with two readouts: visualized colorimetric and real-time fluorescence. Different factors were optimized to improve the reaction conditions, including temperature (60°C), assay runtime (60min), primers, MgSO4 (6mM), dNTPs (1mM), LAMP Buffer (1.2mM Tris-HCl), KCl (50mM), pH (8), and phenol red (10mM) concentrations. Regarding analytical sensitivity, the colorimetric RT-LAMP method detected samples with Ct values up to 29, while the RT-qLAMP assay identified up to Ct = 31. RT-qLAMP was evaluated on 40 clinical samples (25 positives and 15 negatives) for viral RNA detection. All negative samples were found negative through fluorescent reading in RT-qLAMP and quantitative reverse transcription PCR (RT-qPCR) assays. Twenty-three clinically positive samples demonstrated a positive RT-qLAMP reaction (up to Ct ≤ 31) with 92% clinical sensitivity, 100% clinical specificity, 100% positive predictive value (PPV), 88.24% negative predictive values (NPV), and 95% accuracy.

A numbers game: mosquito-based arbovirus surveillance in two distinct geographic regions of Latin America.

Aedes mosquitoes, as vectors of medically important arthropod-borne viruses (arboviruses), constitute a major public health threat that requires entomological and epidemiological surveillance to guide vector control programs to prevent and reduce disease transmission. In this study, we present the collaborative effort of 1 year of Aedes ageypti (Linnaeus, 1762) mosquito-based arbovirus surveillance in 2 geographically distinct regions of Latin America (Nicaragua and Ecuador). Adult female mosquitoes were collected using backpack aspirators in over 2,800 randomly selected households (Nicaragua, Ecuador) and 100 key sites (Nicaragua) from 8 distinct communities (Nicaragua: 2, Ecuador: 6). A total of 1,358 mosquito female pools were processed for RNA extraction and viral RNA detection using real-time reverse transcription-polymerase chain reaction. Ten positive dengue virus (DENV) pools were detected (3 in Nicaragua and 7 in Ecuador), all of which were found during the rainy season and matched the serotypes found in humans (Nicaragua: DENV-1 and DENV-4; Ecuador: DENV-2). Infection rates ranged from 1.13 to 23.13, with the Nicaraguan communities having the lowest infection rates. Our results demonstrate the feasibility of detecting DENV-infected Aedes mosquitoes in low-resource settings and underscore the need for targeted mosquito arbovirus sampling and testing, providing valuable insights for future surveillance programs in the Latin American region.

An Exploration of the Viral Coverage of Mosquito Viromes Using Meta-Viromic Sequencing: A Systematic Review and Meta-Analysis.

The aim of this review was to delve into the extent of mosquito virome coverage (proportion of viral reads) via meta-viromic sequencing and uncover potential factors of heterogeneity that could impact this coverage. Data sources were PubMed, Web of Science, Embase, Scopus, Science-Direct, Google Scholar, and the China National Knowledge Infrastructure. Pooled coverage was estimated using random-effects modeling, and subgroup analyses further reveal potential heterogeneous factors. Within the three mosquito genera studied, Culex exhibited the highest pooled viral coverage of mosquito viromes at 7.09% (95% CI: 3.44-11.91%), followed by Anopheles at 5.28% (95% CI: 0.45-14.93%), and Aedes at 2.11% (95% CI: 0.58-7.66%). Subgroup analyses showed that multiple processing methods significantly affected the viral coverage of mosquito viromes, especially pre-treatment of mosquito samples with saline buffer/medium and antibiotics prior to DNase/RNase treatment and removal of the host genome prior to RNA library construction. In conclusion, the results of this study demonstrate that the viral coverage of mosquito viromes varies between mosquito genera and that pre-treatment of mosquito samples with saline buffer/medium and antibiotics before DNase/RNase treatment and removing host genomes prior to RNA library construction are critical for the detection of RNA viruses in mosquito vectors using meta-viromic sequencing.

Microfluidic-free droplet digital RAA/RT-RAA assisted CRISPR/Cas12a system for the detection of DNA/RNA viruses

Digital PCR (dPCR) is the most sensitive method for virus detection by accurate quantifying the nucleic acids. However, the requirement of sophisticated and expensive instrumentations greatly limits its application in clinical diagnosis particularly in poorly resourced settings. Herein, we developed a simple digital assay based on polydisperse droplet digital recombinase-aided amplification/reverse transcription recombinase-aided amplification dependent CRISPR/Cas12a system (PddRaaCas/PddRRaaCas) for viral DNA/RNA detection with minimal instruments. This assay does not require any microfluidic chip for droplet generation and can be completed about 1 hour. We found that the PddRaaCas and PddRRaaCas enabled to detect 0.015 fM target DNA and 10 fM target RNA respectively. In addition, we applied this method to detect human papillomavirus type 18 (HPV18) and severe acute respiratory syndrome coronavirus (SARS-CoV-2) in clinical samples, which represented the detection of DNA virus and RNA virus respectively. The sensitivity and specificity were 100 % and 100 % for both DNA and RNA virus detection respectively. Taken together, these results highlight that PddRaaCas/PddRRaaCas is a sensitive, rapid, and easy-to-use strategy for the detection of DNA/RNA viruses, which has great potential application for the control of infectious diseases.

Comparison of Extraction Methods for the Detection of Avian Influenza Virus RNA in Cattle Milk.

Since early 2024, a multistate outbreak of highly pathogenic avian influenza H5N1 has been affecting dairy cattle in the USA. The influenza viral RNA concentrations in milk make it an ideal matrix for surveillance purposes. However, viral RNA detection in multi-component fluids such as milk can be complex, and optimization of influenza detection methods is thus required. Raw bulk tank milk and mastitis milk samples were artificially contaminated with an avian influenza strain and subjected to five extraction methods. HCoV-229E and synthetic RNA were included as exogenous internal process controls. Given the high viral load usually observed in individual raw milk samples, four out of five tested methods would enable influenza detection in milk with normal texture, over a time window of at least 2 weeks post-onset of clinical signs. Nevertheless, sample dilution 1:3 in molecular transport medium prior to RNA extraction provided the best results for dilution of inhibitory substances and a good recovery rate of influenza RNA, that reached 12.5 ± 1.2% and 10.4 ± 3.8% in two independent experiments in bulk milk and 11.2 ± 3.6% and 10.0 ± 2.9% on two cohorts of mastitis milk samples. We have also shown compatibility of an influenza RT-qPCR system with synthetic RNA detection for simultaneous validation of the RNA extraction and RT-qPCR processes.

SARS-CoV-2 Variants from Long-Term, Persistently Infected Immunocompromised Patients Have Altered Syncytia Formation, Temperature-Dependent Replication, and Serum Neutralizing Antibody Escape.

SARS-CoV-2 infection of immunocompromised individuals often leads to prolonged detection of viral RNA and infectious virus in nasal specimens, presumably due to the lack of induction of an appropriate adaptive immune response. Mutations identified in virus sequences obtained from persistently infected patients bear signatures of immune evasion and have some overlap with sequences present in variants of concern. We characterized virus isolates obtained greater than 100 days after the initial COVID-19 diagnosis from two COVID-19 patients undergoing immunosuppressive cancer therapy, wand compared them to an isolate from the start of the infection. Isolates from an individual who never mounted an antibody response specific to SARS-CoV-2 despite the administration of convalescent plasma showed slight reductions in plaque size and some showed temperature-dependent replication attenuation on human nasal epithelial cell culture compared to the virus that initiated infection. An isolate from another patient-who did mount a SARS-CoV-2 IgM response-showed temperature-dependent changes in plaque size as well as increased syncytia formation and escape from serum-neutralizing antibodies. Our results indicate that not all virus isolates from immunocompromised COVID-19 patients display clear signs of phenotypic change, but increased attention should be paid to monitoring virus evolution in this patient population.

An attachment glycoprotein nanoparticle elicits broadly neutralizing antibodies and protects against lethal Nipah virus infection

Nipah virus (NiV) is a zoonotic emergent paramyxovirus that can cause severe encephalitis and respiratory infections in humans, with a high fatality rate ranging from 40% to 75%. Currently, there are no approved human vaccines or antiviral drugs against NiV. Here, we designed a ferritin-based self-assembling nanoparticle displaying the NiV G head domain on the surface (NiV G-ferritin) and assessed immune responses elicited by the soluble NiV G head domain (NiV sG) or NiV G-ferritin. Immunization with NiV G-ferritin or NiV sG conferred complete protection against lethal NiV challenge without detection of viral RNA in Syrian golden hamsters. Compared to NiV sG, NiV G-ferritin induced significantly faster, broader, and higher serum neutralizing responses against three pathogenic henipaviruses (NiV-Malaysia, NiV-Bangladesh, and Hendra virus). Moreover, NiV G-ferritin induced a durable neutralizing immunity in mice as antisera potently inhibited NiV infection even after six months of the third immunization. Additionally, we isolated a panel of 27 NiV G-binding monoclonal antibodies (mAbs) from NiV G-ferritin immunized mice and found that these mAbs targeted four distinct antigenic sites on NiV G head domain with two sites that have not been defined previously. Notably, 25 isolated mAbs have potent neutralizing activity with 50% inhibitory concentrations less than 10 ng/mL against NiV pseudovirus. Collectively, these findings provide new insights into the immunogenicity of NiV G protein and reveal that NiV G-ferritin is a safe and highly effective vaccine candidate against Nipah virus infection.

An interlaboratory proficiency test using metagenomic sequencing as a diagnostic tool for the detection of RNA viruses in swine fecal material.

Metagenomic shotgun sequencing (mNGS) is a generic molecular diagnostic method, involving laboratory preparation of samples, sequencing, bioinformatic analysis of millions of short sequences, and interpretation of the results. In this paper, we investigated the performance of mNGS on the detection of porcine astroviruses, a model for RNA viruses in a pig fecal material, among six European veterinary and public health laboratories. We showed that different methods for data generation affect mNGS performance among participants and that the selection of reference genomes is crucial for read classification. Follow-up investigation remains a critical factor for the diagnostic interpretation of mNGS results. The paper contributes to potential improvements of mNGS as a diagnostic tool in clinical settings.

Viral shedding and environmental dispersion of two clade 2.3.4.4b H5 high pathogenicity avian influenza viruses in experimentally infected mule ducks: implications for environmental sampling

High pathogenicity avian influenza viruses (HPAIVs) have caused major epizootics in recent years, with devastating consequences for poultry and wildlife worldwide. Domestic and wild ducks can be highly susceptible to HPAIVs, and infection leads to efficient viral replication and massive shedding (i.e., high titres for an extended time), contributing to widespread viral dissemination. Importantly, ducks are known to shed high amounts of virus in the earliest phase of infection, but the dynamics and impact of environmental contamination on the epidemiology of HPAIV outbreaks are poorly understood. In this study, we monitored mule ducks experimentally infected with two H5N8 clade 2.3.4.4b goose/Guangdong HPAIVs sampled in France in 2016–2017 and 2020–2021 epizootics. We investigated viral shedding dynamics in the oropharynx, cloaca, conjunctiva, and feathers; bird-to-bird viral transmission; and the role of the environment in viral spread and as a source of samples for early detection and surveillance. Our findings showed that viral shedding started before the onset of clinical signs, i.e., as early as 1 day post-inoculation (dpi) or post-contact exposure, peaked at 4 dpi, and lasted for up to 14 dpi. The detection of viral RNA in aerosols, dust, and water samples mirrored viral shedding dynamics, and viral isolation from these environmental samples was successful throughout the experiment. Our results confirm that mule ducks can shed high HPAIV titres through the four excretion routes tested (oropharyngeal, cloacal, conjunctival, and feather) while being asymptomatic and that environmental sampling could be a non-invasive tool for early viral RNA detection in HPAIV-infected farms.

Development of a Lateral Flow Assay for the Detection of the Hepatitis C Virus Core Antigen.

Hepatitis C virus (HCV) infection remains a global health challenge, with millions of people affected annually. Current diagnostic methods, reliant on antibody screening and viral RNA detection, are complex, costly, and often inaccessible, particularly in resource-limited settings. Development of a lateral flow immunochromatography-based assay for detecting the highly conserved hepatitis C core antigen (HCVcAg). The assay relies on the interaction of four highly specific and cross-reactive monoclonal antibodies with recombinant HCVcAg from five different genotypes in a double antibody sandwich format. Latex and colloidal gold were evaluated as detector nanoparticles. Extensive evaluation of 32 antibody combinations led to identifying the most sensitive antibody pairs. The chosen assay, named LN17, demonstrated a target sensitivity of 10 ng/strip, with potential clinical implications for detecting HCV. Furthermore, the study examined matrix effects in serum samples, providing valuable insights for future clinical application. The developed assay holds promise as a rapid, cost-effective, and user-friendly tool to enhance accessibility to hepatitis C screening, especially in high-risk populations and resource-limited environments.