The Rabies virus, a single-strand RNA virus with a negative-sense polarity, is responsible for causing encephalitis and is a zoonotic disease. If not promptly treated after infection, it has a close to 100% fatality rate. Similar to other negative-sense polarity single-strand RNA viruses, the Rabies virus requires the creation of a positive-strand RNA intermediate for replication. One approach to identify this replication activity is to detect the complementary strand of the viral RNA genome in suspected infected cells or tissues. The reported Rabies virus RT-qPCR detection methods are designed to detect total viral load in samples without distinguishing between positive- and negative-strand for RNA viruses. As such, in this study, a sensitive Taqman-based strand-specific RT-qPCR assay has been developed to quantitatively detect both the positive- and negative-strand of the Rabies virus. This method demonstrates good reproducibility across a wide dynamic range and exhibits linearity of 8 logs with a lower limit of detection of 10 copies/μL for the positive-strand and 9 logs with a lower limit of detection of 10 copies/μL for the negative-strand. Notably, it can accurately detect a specific viral RNA strand even in the presence of high levels of the opposite strand, confirming the method's specificity. In summary, a reliable strand-specific RT-qPCR assay has been developed and validated to differentiate replicating from non-replicating Rabies virus.

Virus concentration using hollow fibers is widely used for vaccine production to maintain viral infectivity with low shear stress and to allow for easier scale-up of production. However, research laboratories often have limited available viral materials at the early stage of vaccine development, making it difficult to find an optimal hollow-fiber. In addition, few research articles have reported on optimizing hollow fiber pore size and membrane structure for virus concentration. In this study, the previously established handheld hollow fiber virus concentration method was modified to reduce the sample volume required and to enable simple and easy hollow fiber screening. The handheld hollow fiber screening method for virus concentration was confirmed to be effective using Zika as a model virus.

CMV infection remains a well-recognized threat in immunocompromised patients and is a major cause of congenital infection. If plasma and whole blood are routinely used as clinical samples for detection of CMV infection and disease, CMV laboratory testing in non-blood samples is becoming more and more relevant to support the diagnosis, prognosis and management of CMV infection. Accurate CMV viral load assessment in various body fluids is therefore essential. We evaluated the performance of the Alinity m CMV assay for detecting and quantifying CMV in plasma, amniotic fluid, bronchoalveolar lavage, cerebrospinal fluid, saliva and urine. Using a commercially available CMV reference panel and CMV culture supernatant, we assessed the linearity and accuracy of the Alinity m CMV assay across the different sample types. Excellent linear correlations (r values > 0.98) and a good accuracy (bias < ± 0.50 Log10 IU/mL and SD < 0.23) were observed. In conclusion, the Alinity m CMV assay is suitable to detect and quantify CMV DNA in plasma but also in all non-plasma samples tested. Random and continuous access capabilities of the Alinity m enable rapid and efficient laboratory detection and quantification of CMV DNA.

Potato virus A belongs to the genus Potyvirus, a group of single-stranded positive sense RNA viruses infecting crops worldwide. To initiate infection in a host, its genome takes part in different activities, viz., translation, replication, encapsidation during the infection cycle. Extensive research has been carried out to scrutinize the stages of potyviral infection cycle and decipher the strategies it employs to cause disease. Nonetheless, the amount of viral RNA taking part in translation and virion formation, at a given time point, is missing. In this study, we quantified the percentage of viral RNA that exists as virions and those that associates with host polysome, relative to total viral RNA in infected plant tissue. We employed a revised version of immune-capture reverse transcription PCR and polysome profiling to address our queries. We tested three different coating antibody concentrations and further optimized the immuno-capture reverse transcription PCR protocol to address its limitation of binding and retaining viral particles. Our results indicate that most of the viral RNA (69%) exists as encapsidated genomes, while 3% of total viral RNA associates with host polysomes. These findings are crucial for correct interpretation of quantitative translational studies in which correlation must be made between the number of polysome-associated transcripts and the amount of protein synthesized.

Wastewater-based epidemiology (WBE) is a crucial tool for health and environmental monitoring, providing real-time data on public health indicators by analysis of sewage samples. Ensuring the integrity of these samples from collection to analysis is paramount. This study investigates the effects of different cold-storage conditions on the integrity of wastewater samples, focusing on both microbiological markers (such as extractable nucleic acids, SARS-CoV-2, and crAssphage) and physicochemical parameters (including ammonium, orthophosphate, pH, conductivity, and turbidity). Composite samples from the raw wastewater influent from five wastewater treatment works in South Wales, UK, were stored at 4°C, -20°C, and -80°C, and subjected to up to six freeze-thaw cycles over one year. The study found significant effects of storage temperature on the preservation of certain WBE markers, with the best yield most frequently seen in samples stored at -80°C. However, the majority of WBE markers showed no significant difference between storage at -80°C or at 4°C, demonstrating that it may not always be necessary to archive wastewater samples at ultra-low temperatures, thus reducing CO emissions and laboratory energy costs. These findings underscore the importance of optimized storage conditions to maintain sample integrity, while ensuring accurate and reliable WBE data for public health and environmental monitoring.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is the primary cause of the Coronavirus disease 2019 (COVID-19) pandemic, which affects millions of people worldwide with high levels of infectivity and mortality. However, the antibodies developed for COVID-19 research and diagnostics are still limited. Therefore, in this study, we developed polyclonal immunoglobulin (IgY) antibodies from chicken egg yolk targeting multi-epitope antigen of SARS-CoV-2. After immunizing hens with a SARS-CoV-2 multi-epitope peptide, IgY antibodies were isolated from chicken eggs and further characterized using SDS-PAGE and ELISA. The results showed that the IgY antibodies were successfully isolated from egg yolks. The sandwich ELISA results demonstrated that the isolated IgYs could bind to SARS-CoV-2 antigens, both the multi-epitope peptide and the trimeric Spike. Furthermore, the developed polyclonal antibodies could recognize SARS-CoV-2 in human nasopharyngeal swab samples, even at the lowest concentration (dilution at 1:10000). Thus, it can be concluded that the developed polyclonal IgYs were successfully produced and have the potential to be applied in the development of COVID-19 diagnostics.

Cytomegalovirus (CMV) can cause symptomatic CMV syndrome or tissue-invasive CMV disease in immunocompromised individuals, including solid-organ transplant and hematopoietic stem cell transplant recipients. In these populations, monitoring of CMV load is essential, assessing both risk of disease and response to antiviral therapy. High throughput commercial assays are currently available for CMV quantitation, but they are often evaluated independently, with few studies comparing these assays. This study evaluated CMV quantitative assays for use with the Roche cobas 6800, Abbott Alinity m and Hologic Panther platforms using stored patient plasma.

A descriptive study was carried out with health professionals in Sao Paulo city. Were included individuals vaccinated with 2 doses of the inactivated vaccine. Demographic, clinical and vaccination information was obtained from professionals with or without comorbidities. Two serological assays were used to identify the presence and quantity of anti-Spike IgG in serum samples. 433 healthy healthcare professionals were included and 58.9% completed the 4 clinical stages of serological assessment. Among adults and elderly people, 25.2% had chronic diseases (hypertension 50.5%, diabetes 10% and obesity 6.5%). Most individuals have 95% protection in the first 3 months after the second dose, and 67.68% protection after 6 months. Total antibodies ranged from 3 to 10 on the reactivity index, and the anti-RBD IgG levels were high. CoronaVac has a 94% seroconversion rate after 2 doses and can prevent serious cases and outbreaks of the disease, if used on a large scale.

A multiplexed, lentivirus-based pseudovirus neutralisation assay (pVNT) was developed for high-throughput measurement of neutralising antibodies (nAbs) against three distinct SARS-CoV-2 spike variants. Intra-assay variability was minimised by optimising the plate layout and determining an optimal percentage transduction for the pseudovirus inoculum. Comparison of EC titres between single and multiplexed pVNT assays showed no significant differences, indicating reliability of the multiplexed assay. Evaluation of convalescent human sera confirmed assay robustness, with consistent EC titres for variant pseudoviruses relative to the ancestral strain observed across single and multiplexed assays. This multiplexed pVNT provides a reliable tool for assessing nAb responses against SARS-CoV-2 variants and could be used to accelerate preclinical vaccine assessment in preparation for the next coronavirus pandemic.

Gammaretroviral vectors are widely used in cellular and gene therapy products because of the availability of stable vector producer cells. Accurately assessing vector copy number (VCN) is critical for selecting appropriate clones to avoid the risks of homologous recombination and complications in mutation detection. Traditional methods such as quantitative polymerase chain reaction (PCR) and Southern blotting have limitations in accuracy and throughput. This study presents a triplex droplet digital PCR (ddPCR) method for analyzing the VCN in gammaretroviral vector producer cells. We designed a universal primer- probe set targeting the packaging signal sequence common to murine leukemia virus- and murine stem cell virus- based gammaretroviral vectors. Two reference genes were selected after karyotyping the PG13 gammaretroviral vector packaging cell line to identify stable chromosomes. The triplex ddPCR assay was optimized and verified using PG13 cells transduced with constructs of different transgene and vector backbones. The assay showed high concordance with Southern blot. Using multiple reference genes ensures accurate and robust VCN assessment, especially in cell lines with chromosomal instability. This method improves the clone selection process for gammaretroviral vector producer cells, accelerates the development of novel cellular and gene therapy products, and ensures their rapid delivery to patients.

Vector competence studies in mosquitoes present valuable opportunities to explore arboviral transmission and virus-vector interactions. However, oral infection studies in mosquitoes can be challenging. An alternative approach is to infect mosquitoes during their aquatic larval stage, resulting in the emergence of infected adults. To investigate the potential of this method, Culex pipiens biotype molestus larvae were infected with Usutu virus (USUV, Orthoflavivirus usutuense). For this purpose, larvae were exposed to USUV-infected mammalian and mosquito cell cultures for 24 h before being reared to adults. Subsequent analysis via RT-qPCR revealed that the Culex larvae successfully acquired USUV from the infected cells and exhibited high susceptibility to infection. Immediately after emergence, 32.10 % (26/81) of male and 41.03 % (16/39) of female mosquitoes tested positive for USUV RNA. Notably, females that were incubated for 15 days post-emergence demonstrated even higher infection rates, reaching 100.00 % (23/23). In addition, viral RNA and infectious particles were detected in some saliva samples, indicating the potential for transmission. This experimental infection of mosquito larvae thus offers the opportunity to produce infected adult mosquitoes for studies enhancing our understanding of virus-vector interactions, co-infections, and transmission routes. Such research contributes to better public health strategies addressing arboviral diseases.

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are Culicoides-transmitted viruses, circulating in multiple serotypes, that cause two relevant WOAH-listed diseases of ruminants. Following its first identification in Tunisia in 2021, a novel EHDV strain belonging to serotype 8 has been detected in cattle showing BTV-like symptoms in Italy and Andalusia, Spain in 2022, and soon after in Portugal, and France. These are European regions with recurrent circulations of different BTV serotypes. Hence, in this study we describe the validation of a TaqMan RT-qPCR pan-BTV/pan-EHDV assay, based on well-established primers and probes sets, able to simultaneously detect and distinguish between BTV and EHDV. The implemented assay, characterized by high sensitivity and specificity and good reproducibility, can be successfully applied for the rapid and affordable diagnosis needed in the current epidemiological situation, and can be a powerful tool to be employed in surveillance and control strategies with a significant reduction of costs.

Global outbreaks of mosquito-transmitted arbovirus infections, such as dengue (DENV) and chikungunya (CHIKV), are increasing. Differentiating these infections is challenging due to non-specific symptoms and serology limitations. PCR-based approaches offer higher sensitivity and specificity. This study evaluated the performance of TaqMan™ Arbovirus Triplex Kit (ZIKV/DENV/CHIKV) (TaqMan™ Kit) to detect DENV and CHIKV in clinical samples from patients in south India.

When diagnosing viral infections in humans and animals, the presence of virus in a sample in trace amounts that are below the analytical sensitivity of the detection system may cause false negative results and inaccurate diagnosis. We previously reported the development of a simple virion concentration technique using 12 ml large-volume samples that can dramatically improve diagnostic sensitivity by increasing analytical sensitivity by 100-fold over conventional methods. The present study was conducted to further improve the simplicity and versatility of this method. We constructed a simple and highly sensitive method for the detection of SARS-CoV-2 in human saliva after concentration using a magnetic nanoparticle conjugated with polyethylene glycol (PEG).

Virus yellows disease (VY) is a major threat to sugar beet production in Europe. Beet chlorosis virus (BChV) and beet mild yellowing virus (BMYV) are of particular economic importance and are both persistently transmitted by the aphid vector Myzus persicae. As part of integrated pest management strategies, M. persicae influx into sugar beet fields is recorded weekly using yellow water pan traps. To date, only ELISA and RT-PCR assays have been described for BChV and BMYV detection in individual aphids. In this study, we describe for the first time two one-step TaqMan® RT-qPCR assays designed for the specific detection of BChV and BMYV in M. persicae after 7d incubation in water pan trap medium. Both viruses were reproducibly detected in individual aphids. After 7d incubation in trap medium, both viruses were reproducibly detected in individual aphids, as well as in one viruliferous aphid in a pool of 99 non-viruliferous aphids. Significant correlations can be shown between different mixing ratios of viruliferous to non-viruliferous aphids and Ct values of total RNA templates, allowing the percentage of viruliferous aphids in yellow water pan traps to be estimated using a standard curve. The described methodology provides a high sensitivity combined with a high sample throughput and can be used, after evaluation in the field, for practical monitoring, risk modelling and development of decision support systems for VY.

The purpose of this study was to optimize the infectivity of four different orthoflaviviruses in mice for evaluating antiviral drugs by using wild-type mice with intact interferon responses, type 1 interferon alpha/beta receptor knockout mice, or by injecting wild type C57BL/6 mice with varying doses of anti-type 1 interferon receptor antibody (MAR1-5A3) to optimize the infectivity and lethality. West Nile virus productively infected wild-type C57BL/6 mice to cause lethality, whereas Usutu virus required a complete absence of type 1 interferon receptor function. Deer tick virus (lineage 2 Powassan virus) and Japanese encephalitis viruses required a dampening of type 1 interferon responses by adjusting the doses of MAR1-5A3 antibody injections. Challenge dose-responsive mortality, weight loss, and viral titers of these two viruses were observed if the type 1 interferon responses were dampened with MAR1-5A3. Conversely, without MAR1-5A3 injections, these disease phenotypes were not viral challenge dose-responsive. From these different interferon-responsive models, the appropriate lethality was identified to determine that 7-deaza-2'-C-methyladenosine has high efficacy for West Nile and Usutu viruses, and low efficacy for deer tick and Japanese encephalitis viruses.

Mosquito-borne pathogens pose a significant threat to both human health and blood safety. The primary mosquito-borne viruses that present this threat are Zika virus, Chikungunya virus, and Dengue virus. At present, there are limited efficacious vaccines or therapeutic drugs for the prevention and treatment of these viral infections. Blood donors can remain asymptomatically infected and unfortunately, screening for these three viruses in Chinese blood donors are not mandatory, leaving the residual risk to transfusion recipients uncertain.

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 that this filovirus multiplex test is a sensitive tool for high-throughput serosurveillance.

ORFV of the family poxvirus，which produces a pustular dermatitis both in humans and animals.,Previous studies have found an fatal case caused by the infection of ORFV in the viscera. However, the mechanisms of ORFV how to infect the viscera remain unknown. Our sequencing results revealed that the CBP of the visceral infection strain lacked a 24-base pair segment at position 217 comparison to the oral infection strain. Subsequently, we successfully packaged the recombinant adenoviruses pAd-CBP-K and pAd-CBP-N in HEK-293A cells and carried it to infect lymphocytes. RT-PCR analysis showed that the CBP protein was expressed in lymphocytes, and pAd-CBP-N group exhibited a significantly higher CBP expression level compared to the pAd-CBP-K group. At 4, 8, and 12 hours post-infection, both pAd-CBP-K and pAd-CBP-N were found to downregulate the expression of MIP-1 and CCL-5 in the supernatant of lymphocytes. However, the expression of IL-2, IL-6, IL-8, IL-12, INF-γ, and TNF-α showed a significant up-regulation. Furthermore, the inflammatory factors relative expression levels of IL-2, IL-6, IL-8, IL-8, IL-12, IFN-γ and TNF-α were significantly up-regulated in the both group. Interestingly, a significant increase in the expression of IL-6, IL-8 and TNF-α were detected in the pAd-CBP-N group at both 8 and 12 hours compared to pAd-CBP-N. Taken together, these findings showed that CBP can regulate the expression of free chemokines and activate the expression of inflammatory factors, and provide a basis for follow-up research.

The culture-attenuated alcelaphine herpesvirus 1 (AlHV-1) C500 strain can be grown to high titre and has been used successfully as a candidate vaccine for wildebeest-associated malignant catarrhal fever (MCF). This vaccine virus was also used to develop an indirect ELISA to allow monitoring of virus-specific antibodies in vaccinated cattle. However the extraction method was expensive and time-consuming, and the resulting test was not suitable for use in sheep. Here we describe an improved antigen extraction method that also broadens the application of the assay, allowing its application to sheep samples. The updated assay was tested using control samples from cattle and sheep, and showed a high level of accuracy in both species. This novel assay should prove to be a useful tool in MCF diagnosis and in evaluation of MCF vaccine responses.