Viral Nucleic Acid Research Articles

From lab to field: Innovative RPA‒CRISPR/Cas12a platform for early short-beak and dwarfism syndrome virus nucleic acids detection.

From lab to field: Innovative RPA‒CRISPR/Cas12a platform for early short-beak and dwarfism syndrome virus nucleic acids detection.

West Nile Virus positivity amongst Canadian blood donors and effectiveness of off-season screening.

We sought to examine the prevalence of West Nile Virus (WNV) nucleic acid test (NAT) positivity over time in Canadian blood donors, examine the level of false-positive NAT screening, and understand the effectiveness of travel-related testing outside of WNV seasonality in Canada. All blood donations in Canada (except Québec) between 2018 and 2023 were tested by the Roche Cobas WNV-NAT assay during the summer-fall season by Canadian Blood Services. Testing for recent travel outside Canada was performed in the winter-spring season. All 2023 WNV-NAT-positives were confirmed by two independent WNV-specific polymerase chain reaction (PCRs). While WNV positivity varied by location and time, no increasing trend of positivity was observed over the 6-year study period. No WNV-NAT-positive donors with a history of recent travel outside of Canada in the winter-spring season were identified by donor screening (2018-2023). In total, 2,454,102 samples were tested during summer months and 267,988 in winter months. In 2023, WNV-specific PCR confirmed 10 of 13 (76.9%) screening WNV-NATs. Recent Japanese Encephalitis Virus (JEV) vaccination was identified as a potential false positive for initial WNV screening results. Between 2018 and 2023, WNV-NAT positivity amongst Canadian blood donors remained stable, with no evidence of an increasing positivity trend. The utility of off-season travel-related testing may be limited, as no donors were identified during the six-year study timeframe. Finally, confirmation testing using WNV-specific PCR is recommended to identify true cases and to limit misidentification of other genetically similar viruses, such as JEV.

Review of porcine circovirus 3-associated lesions in swine: Challenges and advances in diagnostics.

Porcine circovirus 3 (PCV3) is a recently identified pathogen in swine populations. It is considered a ubiquitous virus and is frequently associated with subclinical infections throughout various stages of production. PCV3 is detectable in diverse tissues, blood, and secretions, indicating systemic dissemination and potential for both vertical and horizontal transmission. PCV3 has been implicated in reproductive and postnatal diseases collectively named as PCV3-associated diseases (PCV3-AD). Clinically, PCV3-AD encompasses reproductive disorders such as mummified fetuses, stillbirths, and weak neonates, alongside postnatal manifestations including anorexia, weight loss, and progressive wasting. Histopathologically, PCV3-AD is primarily defined by systemic nonsuppurative periarteritis and arteritis that are observed across multiple tissues, particularly within the heart, mesenteric arterial plexus, and kidneys. Despite the broad tissue tropism and frequent detection of viral nucleic acids within affected vascular and parenchymal structures, the precise mechanisms underpinning PCV3 pathogenesis remain poorly understood. Diagnosis of PCV3-AD relies on the confluence of characteristic clinical signs, compatible histopathological findings, and the in situ detection of the virus within lesions. However, the true prevalence of PCV3-AD under field conditions is likely underestimated due to the limited availability and high costs associated with laboratory techniques for definitive viral detection. This review seeks to consolidate and interpret clinical and pathological evidence indicative of PCV3-AD while addressing the critical diagnostic challenges faced by veterinary pathologists. Enhanced understanding of the disease's clinical-pathological correlations and diagnostic approaches is essential to accurately assess its impact on swine health and production.

Evaluating bacterial and viral colonization of the upper respiratory tract of neonatal beef calves during the first 24 hours of life

Little is known regarding the timing of initial establishment of bovine respiratory disease complex (BRDC)-associated bacterial and viral pathogens. The objective was to evaluate the presence of BRDC-associated bacteria and viruses in the upper respiratory tract of newborn calves within the first 24h of life. Commercial beef cows (n = 26) were allocated to indi­vidual pens before calving. A left nasal swab (LNS), right na­sal swab (RNS) and vaginal swab were collected from each cow immediately following parturition (0h). Calf (n = 26) LNS and RNS sampling occurred at 0h, 6h, 12h and 24h post-calv­ing. Samples were submitted to a diagnostic lab for bacterial culture (n = 26 pairs) and real-time PCR (rtPCR; n = 10 pairs) to isolate and identify bacteria and to detect bacterial and viral nucleic acid. While we were unable to isolate bacteria of interest using culture, rtPCR yielded some success. At birth, Histophilus somni (Hs) was detected in 4/30 dam swabs and 1/20 calf swabs, Mannheimia haemolytica (Mh) in 1/30 dam swabs and 1/20 calf swabs, Mycoplasma bovis (Mb) in 4/30 dam swabs and 1/20 calf swabs, Pasteurella multocida (Pm) in 2/30 dam swabs, bovine herpes virus type-1 (BHV1) in 1/30 dam swabs, and bovine respiratory syncytial virus (BRSV) in 1/30 dam swabs. In 6h calf swabs, Hs was detected in 3/20, Mb in 1/20, and BRSV in 1/20. Associations of bacteria and viruses be­tween dam and offspring remain unclear as establishment of the neonatal microbiome appears complex and more sensitive methods may be needed.

Respiratory viral detection in children hospitalized with pneumonia during periods of major population disruptions in Nepal, 2014-2018.

Respiratory viruses commonly cause pneumonia in children. We aimed to identify respiratory viral nucleic acids in the nasopharynx of children admitted with pneumonia from 2014 to 2018, a period including a major earthquake (April 2015), PCV10 introduction (August 2015), and a fuel shortage (October 2015 to March 2016). Children 2 months to 14 years admitted to Patan Hospital between March 2014 and February 2018 with a clinical diagnosis of pneumonia had nasopharyngeal swabs collected and tested with a multiplex panel for the presence of genetic material from 23 respiratory pathogens. Of 1343 children with pneumonia, 974 (72.5%) had the nucleic acids of at least one respiratory virus in the nasopharynx. The median age of children with any viral genetic material detected was lower than those without (1.18, IQR: 0.59-2.39 years; versus 2.01 years, IQR: 0.81-4.34 years; p<0.001). Commonly detected viral nucleic acids included those of RSV (21.0%), rhino/enterovirus (30.8%), and parainfluenza (7.4%). The odds of detecting any respiratory viral genetic material in children with pneumonia increased by 1.88 (95% confidence interval: 1.15, 3.06) in the year after the earthquake, when there were several aftershocks and a fuel crisis, relative to other periods and accounting for other potential confounding factors. These findings highlight the importance of viral diagnostics in pediatric pneumonia and suggest that public health measures addressing environmental conditions during disasters might help reduce respiratory infections.

Lethal gastrointestinal manifestations of COVID-19 infection at autopsy.

COVID-associated acute mesenteric ischaemia is a rare event that has a high mortality rate, particularly in the elderly. Despite gastrointestinal symptoms being relatively common in COVID-19 infections, however, lethal gut complications are often not considered. Two cases with fatal COVID-associated acute mesenteric ischaemia are therefore reported. Case 1: A 74-year-old woman who died following a presentation with diarrhoea and abdominal pain was found at autopsy to have extensive ischaemic changes of her small and large intestines. The superior mesenteric artery and its major branches were widely patent. Histologically acute inflammation and necrosis of the mucosa and intramucosal haemorrhage were found with fibrin thrombi in intramucosal and submucosal vessels. Respiratory viral nucleic acid testing performed on a retronasal swab obtained at autopsy was positive for COVID-19 (SARS-CoV-2). Death was due to multi-organ failure complicating mesenteric ischaemia associated with COVID-19 infection. Case 2: A 94-year-old woman with COVID-19 infection died after developing abdominal pain with melaena and bright red rectal bleeding. At autopsy there was focal transmural acute inflammation of the small intestine with multiple fibrin microthrombi. Death was also due to multi-organ failure complicating mesenteric ischaemia associated with COVID-19 infection. In the absence of obstructive vascular lesions at autopsy in cases of intestinal ischaemia, COVID-19 should be considered with appropriate swabbing and careful histological sampling of the intestine and mesentery to check for microvascular fibrin thrombi.

Assessment of Risk Factors Related to Environmental Factors and Herd Management for Bovine Respiratory Syncytial Virus and Bovine Parainfluenza Virus‐3 Infections Frequently Observed in Beef and Dairy Cattle

ABSTRACTBackgroundBovine Respiratory Disease Complex (BRDC) represents a multifactorial infection that poses a significant threat to animal health, leading to severe and fatal pneumonia outbreaks in large herds.ObjectiveIn this study, the molecular diagnosis of Bovine Parainfluenza Virus Type 3 (BPIV3) and Bovine Respiratory Syncytial Virus (BRSV) was targeted in nasal swab samples collected from cattle with clinical signs of respiratory infections. Furthermore, this study aimed to identify possible environmental and herd management‐related risk factors associated with the occurrence of these infections in herds/farms where BPIV3 and BRSV were detected, using statistical methods.MethodsTherefore, a total of 200 nasal swab samples were randomly collected from 24 different cattle herds with respiratory infection outbreaks, representing 10% of each herd population.ResultsIn the study, which utilized the RT‐PCR technique, viral nucleic acid of BPIV3 was detected in 8% (16/200) of the samples, and BRSV was detected in 11.5% (23/200) of the samples. In addition, logistic regression models incorporating both fixed and multiple variables identified the following risk factors for BRSV infection: in the univariate regression analysis, quarantine status, air quality, the period of disease occurrence and previous occurrence of BRDC in the farm; and in the multivariate analysis, the disease period, herd size, bedding type and qualitative air quality were significant risk factors for the presence of infection in the sampled herds. For BPIV3, univariate regression analysis indicated that animal transport, housing type, ventilation and the duration from infection onset to sampling were risk factors, while in multivariate analysis, age, duration from infection onset to sampling, and air quality were identified as risk factors for infection in the sampled herds.ConclusionA statistical relationship was demonstrated between BRSV and BPIV3 infections in cattle and certain herd, environmental and infection‐related factors, identifying these factors as risk factors contributing to the occurrence of these infections.

Evaluation of a concentration method for the recovery of human adenovirus from mineral water, tap water and well water.

Evaluation of a concentration method for the recovery of human adenovirus from mineral water, tap water and well water.

Viral Metagenomics of the Bharal (Pseudois nayaur) within the Qinghai-Tibet Plateau Revealed Diverse Viruses

The Qinghai-Tibetan Plateau (QTP) provides a home to diverse flora and fauna, and its ecosystems are unique worldwide. The study focused on the bharal, an endemic species found in the QTP and adjacent regions. We applied viral metagenomics technology to extract samples from the feces of 10 wild bharal. Viral nucleic acids were isolated, enriched, and sequenced from these samples, revealing the presence of a novel strain of Astroviridae virus. Phylogenetic analysis and sequence comparison identified this virus as part of the Mamastro-virus, forming a cluster with other Mamastrovirus species. Recombination analysis confirmed a multiple recombination event, suggesting that the new strain may be a potential recombinant. Additionally, nearly complete genome sequences of viruses belonging to the family Circoviridae were characterized, and a phylogenetic tree was constructed based on genotyping and predicted amino acid sequence analysis of the rep protein. Overall, this study helps us better understand the viral communities in the gut microbiome of the rare bharal. Moreover, the new recombinant discovered in this study will provide insights into the origin, genetic diversity, and evolution of bharal from the QTP and play a crucial role in future research on its presence in the intestinal ecology of sheep.

Detection and prevalence of avian influenza epidemic in the southwest of Poyang Lake and analysis of the influence of meteorological factors.

Detection and prevalence of avian influenza epidemic in the southwest of Poyang Lake and analysis of the influence of meteorological factors.

Hepatitis C Nucleic Acid Test Positive (NAT+) Solid Organ Consent Rates Are Highest in Patients Listed for Liver Transplant and With an English Language Preference.

Transplantation of hepatitis C virus (HCV) nucleic acid (NAT) positive organs is associated with shorter time to transplant and decreased risk of death on the waiting list. Treatment for HCV post-transplant is well-tolerated, successful, and leads to similar transplant outcomes to patients transplanted with HCV NAT- organs. Despite these outcomes, not all patients consent to receive HCV NAT+ organs, and factors associated with consent are not well-known. This retrospective single-center study of adult patients listed for heart, liver, lung, and kidney transplant aimed to determine whether sociodemographic and organ-specific disparities exist in consent for HCV NAT+ donor organs. Of 2788 transplant candidates, 44% (N=1229) consented to receive an HCV NAT+ organ. Patients who designated English as their preferred language were more likely to consent compared to a non-English preference (45% vs. 19%, p<0.001). Consent rates were highest amongst patients listed for liver transplantation compared to kidney, heart, and lung transplants (67%, N=319 vs. 42%, N=602 vs. 38%, N=159 vs. 32%, N=149; p<0.001). Overall, more efforts are needed to ensure that all patients who may benefit from consenting for HCV NAT+ organs are appropriately educated in their language of choice on the risks and benefits.

Proliferation of Trionyx sinensis hemorrhagic syndrome virus using primary alveolar macrophages invitro.

Proliferation of Trionyx sinensis hemorrhagic syndrome virus using primary alveolar macrophages invitro.

Indoor air sampling for detection of viral nucleic acids

Indoor air sampling for detection of viral nucleic acids

Molecular detection of hrHPV-induced high-grade squamous intraepithelial lesions of the cervix through a targeted RNA next generation sequencing assay

BackgroundCervical cancer screening programs are increasingly relying on sensitive molecular approaches as primary tests to detect high-risk human papillomaviruses (hrHPV), the causative agents of cervix cancer. Although hrHPV infection is a pre-requisite for the development of most precancerous lesions, the mere detection of viral nucleic acids, also present in transient infections, is not specific of the underlying cellular state, resulting in poor positive predictive values (PPV) regarding lesional states. There is a need to increase the specificity of molecular tests for better stratifying individuals at risk of cancer and to adapt follow-up strategies.MethodsHPV-RNA-SEQ, a targeted RNA next generation sequencing assay allowing the detection of up to 16 hrHPV splice events and key human transcripts, has previously shown encouraging PPV for the detection of precancerous lesions. Herein, on 302 patients with normal cytology (NILM, n = 118), low-grade (LSIL, n = 104) or high-grade squamous intraepithelial lesions (HSIL, n = 80), machine learning-based model improvement was applied to reach 2-classes (NILM vs HSIL) or 3-classes (NILM, LSIL, HSIL) predictive models.ResultsLinear (elastic net) and nonlinear (random forest) approaches resulted in five 2-class models that detect HSIL vs NILM in a validation set with specificity up to 0.87, well within the range of PPV of other competing RNA-based tests in a screening population.ConclusionsHPV-RNA-SEQ improves the detection of HSIL lesions and has the potential to complete and eventually replace current molecular approaches as a first-line test. Further performance evaluation remains to be done on larger and prospective cohorts.

Seroprevalence, and molecular detection of Peste des Petits Ruminants in selected districts of Tigray Region, Ethiopia

Peste des Petits Ruminants is a contagious trans-boundary viral disease affecting domestic and wild small ruminants, causing high mortality and morbidity. A cross-sectional study was conducted from January to December 2024 in the Enderta and Kilteawulaelo districts of the Tigray region, Ethiopia. The study aimed to assess the seroprevalence, associated risk factors, and investigate the recent circulation of the Peste des Petits Ruminants virus strains in small ruminants. A total of 384 serum samples were collected from 133 sheep and 251 goats in the study districts using multistage sampling methods. In addition, 43 swab samples were obtained from animals suspected of having Peste des Petits Ruminants to detect the viral antigen. Peste des Petits Ruminants specific antibodies and viral nucleic acid were identified using the virus neutralization test and real-time PCR, respectively. The relationship between Peste des Petits Ruminants seroprevalence and the possible risk factors was examined using Pearson's chi-square and logistic regression analysis using STATA version 14. Of the 384 serum samples tested, 41.9% (n = 161) were positive for Peste des Petits Ruminants Virus antibodies. The seroprevalence in the districts of Enderta and Kilteawulaelo was 47.6% (n = 98) and 35% (n = 63), respectively, while at the species level, it was 36.8% (n = 49) in sheep and 44.6% (n = 112) in goats. The logistic regression model revealed that districts and flock size were significant risk factors of Peste des Petits Ruminants seropositivity in sheep and goats with P = 0.016 and P = 0.025 respectively. Out of 43 swab samples about 23.26% (n = 10) of the clinical samples were positive for viral nucleic acid via real-time Peste des Petits Ruminants. The serological and molecular results both confirm the presence of the Peste des Petits Ruminants virus circulating among sheep and goat populations in the study areas. Key risk factors include flock size and districts. Ongoing infections, indicated by viral nucleic acid detection, highlight the potential for wider spread. The study recommends routine vaccination, movement restrictions, continuous surveillance, farmer education, and further genetic research.

Assess the Variability and Robustness of an Aluminum-Based Adsorption–Precipitation Method for Virus Detection in Wastewater Samples

Wastewater-based molecular epidemiology enables the surveillance of both symptomatic and asymptomatic individuals in a non-invasive, cost-effective, rapid, and early-detection manner. The use of wastewater analysis to monitor the prevalence of viral pathogens in a given population has increased significantly since the COVID-19 pandemic. These studies typically involve three main steps: viral concentration, nucleic acid extraction, and DNA/RNA quantification. However, the absence of a standardized methodology remains a major limitation, hindering result comparability across studies. Among the available viral concentration techniques, aluminum-based adsorption–precipitation is one of the most commonly used due to its simplicity, efficiency, and low cost. This study evaluates the robustness and variability of the viral concentration and nucleic acid extraction steps by implementing different process controls in wastewater samples across 122 independent experiments. Additionally, correlations between viral recovery efficiencies and relevant physicochemical parameters were also analyzed (n = 600). The results indicate that, despite the overall robustness of the method, the concentration step exhibits the highest variability (CV = 53.82%), which accounted for 53.73% of the overall variability. In addition, our results show that, on average, 0.65 logarithmic units were lost during the viral concentration step. Furthermore, viral recovery rates were influenced by seasonality and sample characteristics, while no significant correlation was observed with pH or conductivity. These findings highlight the importance of process controls, confirming the robustness of the methodology, and identifying key parameters that should be considered in future studies for improved data interpretation.

Amplification-Free CRISPR/Cas12a-Based Electrochemical Biosensor with Enhanced Sensitivity for Viral Detection.

To detect contagious viral nucleic acids, traditional biosensors often require target amplification steps or use fluorescence and Raman probes tagged on nucleic acids, which are time-consuming, complex, and expensive. Recently, the CRISPR/Cas12a has received the attraction for development of nucleic acid biosensors, beyond its conventional role-like gene editing, but the enhancement of the sensitivity of CRISPR/Cas-based biosensors is still required to simplify the biosensing steps. Here, we develop a CRISPR/Cas12a-based electrochemical biosensor for the detection of viral nucleic acids in a simple manner. The novel mismatch Ag probe (MAP), as a sensing probe, and the highly conductive gold electrode on indium tin oxide with a nano array (GELITION) are introduced that enable the amplification-free and ultrasensitive detection of nucleic acids using a CRISPR/Cas12a system. The biosensing ability of the developed biosensor is validated using human papillomavirus type 16 and 18 viral DNAs (HPV16 and HPV18), achieving a limit of detection (LOD) of 1 fM without amplification and complex steps. Our developed biosensor is expected to be applicable in detecting various viruses and could contribute to the early detection of future pandemics.

Rapid visual detection of Monkeypox virus by one-step LAMP-CRISPR/Cas12b assay

BackgroundMonkeypox virus (MPXV) infection has garnered significant global attention due to its rising incidence and substantial public health implications. A rapid, sensitive, and accurate diagnostic method is urgently required to enable early intervention and effective management of MPXV outbreaks.ResultsIn this study, we developed a novel one-step assay that integrates loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12b in one-pot for the detection of MPXV. The entire detection process did not require opening the lid of the reaction tube and could be completed within 40 min using extracted viral nucleic acids, which is faster than real-time quantitative PCR (qPCR). And the results could be interpreted through either real-time fluorescence or naked-eye visualization. The limit of detection (LOD) of the assay was demonstrated to be 6.5 copies per reaction and no cross-reactivity with other pathogens such as HSV, EBV, CVA16, EV-A71, and MV was found. Furthermore, when evaluated using 113 clinical samples, the assay achieved 100% sensitivity (71/71) and 100% specificity (42/42) compared to the qPCR.ConclusionsIn resource-limited settings, our method requires only a portable heat block or water bath and a blue light or ultraviolet flashlight for visual detection of MPXV, making it highly accessible. The integration of LAMP and CRISPR/Cas12b provides a robust, user-friendly platform for point-of-care testing, with promising potential for the rapid molecular diagnosis of infectious diseases.

Evaluation of Discrepant Infectious Disease Results in Deceased Organ Donors: Insights from a Retrospective Analysis of Post-Policy Testing.

In 2021, a new policy was implemented by the Organ Procurement Transplant Network requiring Organ Procurement Organizations to draw a repeat blood sample for deceased organ donors if donation had not proceeded within 96-h after the initial blood sample for screening was obtained. We performed an analysis of over 2600 deceased donor test results, comparing initial results to repeated blood draw results for human immunodeficiency virus, Hepatitis B virus, and Hepatitis C virus serology and nucleic acid test (NAT) tests. This study reviews result discrepancies and explores investigations behind peculiar results. Infectious disease results from deceased organ donors were analyzed retrospectively for this study. Donor specimens were collected throughout the United States and tested at eleven laboratories. Food & Drug Administration-approved donor screening tests were used to determine donor eligibility. There was a 1.69% discrepancy rate comparing results from repeat blood draw specimens to original specimen results. Of these discrepancies, 0.75% of the donors had results (enzyme-linked immunoassay and/or NAT) that changed from non-reactive to reactive. 0.68% of donors had results that changed from reactive to non-reactive. 0.26% of results changed from Ultrio repeatedly reactive, non-discriminated to either non-reactive or reactive. This study represents that there is more than a 1% chance that discrepant results will be obtained. Despite the low incidence of discrepancies, these rare occurrences can complicate clinical decision-making, requiring case-by-case assessments. We present several cases in which variability in results can make clinical decisions complex with limited information and the inability to perform timely confirmatory testing using tests not required by Organ Procurement Transplant Network regulations.

Evaluating the impact of large-scale nucleic acid testing and home quarantine on a novel emerging infectious disease prevention and control: a dynamic modeling approach

IntroductionConducting large-scale viral nucleic acid testing and isolating SARS-CoV-2-infections were crucial strategies in China, which played a key role in successfully controlling multiple waves of the Omicron epidemic. To thoroughly analyze the mechanisms and value of these measures, including testing and isolation, in epidemic prevention and control, and to provide a theoretical basis for scientific epidemic prevention and precise strategies in the face of potential future outbreaks of novel respiratory infectious diseases.MethodsWe developed an individual-based computational model of infectious disease dynamics. The model simulates regular large-scale nucleic acid testing for community residents during an epidemic. When individuals tested positive, they and their household members, as close contacts, are subjected to home isolation. During home isolation, the virus is assumed not to spread outside the household, but the potential for transmission within the household remained. Isolation measures can be lifted once the testing results turned negative. Finally, sensitivity analysis was conducted to verify the scientific validity and reliability of the model.ResultsThe study found that the efficacy of testing and isolation in epidemic prevention is closely related to the speed of disease transmission. When the basic reproduction number (R0) is less than 3, these measures can significantly reduce the infection rate among the population and the speed of epidemic spread; otherwise, they fail to achieve the goal of controlling the epidemic.DiscussionReducing person-to-person contact is crucial for epidemic prevention and control. In addition to testing and isolation, comprehensive non-pharmaceutical interventions (NPIs) should also be implemented, such as increasing social distancing, restricting gatherings in public places, and promoting vaccination, to control the transmission of the epidemic.