Amount Of Virus Research Articles

Quantification of human enteric viruses as alternative indicators of fecal pollution to evaluate wastewater treatment processes.

We investigated the potential use and quantification of human enteric viruses in municipal wastewater samples of Winnipeg (Manitoba, Canada) as alternative indicators of contamination and evaluated the processing stages of the wastewater treatment plant. During the fall 2019 and winter 2020 seasons, samples of raw sewage, activated sludge, effluents, and biosolids (sludge cake) were collected from the North End Sewage Treatment Plant (NESTP), which is the largest wastewater treatment plant in the City of Winnipeg. DNA (Adenovirus and crAssphage) and RNA enteric viruses (Pepper mild mottle virus, Norovirus genogroups GI and GII, Rotavirus Astrovirus, and Sapovirus) as well as the uidA gene found in Escherichia coli were targeted in the samples collected from the NESTP. Total nucleic acids from each wastewater treatment sample were extracted using a commercial spin-column kit. Enteric viruses were quantified in the extracted samples via quantitative PCR using TaqMan assays. Overall, the average gene copies assessed in the raw sewage were not significantly different (p-values ranged between 0.1023 and 0.9921) than the average gene copies assessed in the effluents for DNA and RNA viruses and uidA in terms of both volume and biomass. A significant reduction (p-value ≤ 0.0438) of Adenovirus and Noroviruses genogroups GI and GII was observed in activated sludge samples compared with those for raw sewage per volume. Higher GCNs of enteric viruses were observed in dewatered sludge samples compared to liquid samples in terms of volume (g of sample) and biomass (ng of nucleic acids). Enteric viruses found in gene copy numbers were at least one order of magnitude higher than the E. coli marker uidA, indicating that enteric viruses may survive the wastewater treatment process and viral-like particles are being released into the aquatic environment. Viruses such as Noroviruses genogroups GI and GII, and Rotavirus were detected during colder months. Our results suggest that Adenovirus, crAssphage, and Pepper mild mottle virus can be used confidently as complementary viral indicators of human fecal pollution.

Detection of Enteric Viruses from Wastewater and River Water in Botswana.

Waterborne diseases remain a public health concern in developing countries where many lack access to safe water. Water testing mainly uses bacterial indicators to assess water quality, which may not fully indicate the threat from other non-bacterial pathogens like enteric viruses. This study was done to ascertain and establish the viral load, the temporal and spatial distribution of rotavirus A and norovirus (GI and GII) in sewage and river water samples. A total of 45 samples of raw and treated sewage, and surface water, were collected from a sludge activated wastewater treatment plant in Gaborone, and after treatment from the Notwane River, Botswana, over a period of 9months (February 2016 to October 2016). Viruses were concentrated using polyethylene glycol/NaCl precipitation. Virus detection was performed using real-time polymerase chain reaction (RT-PCR). Rotavirus A was the most prevalent (84.4% positive samples), followed by Norovirus GI (48.9% positive samples), and Norovirus GII 46.7% positive samples). Detected viral loads went up to 104 genome copies per liter (copies/L) for all the viruses. The enteric viruses were detected in all the study sites with highest detection from site S1 (inlet). There was no significant association between physicochemical parameters and viral loads, except for pH which showed significant relationship with rotavirus and norovirus GII (p ≤ 0.05). This is the first study in Botswana to highlight the occurrence and quantification of the enteric viruses in treated and untreated wastewater, as well as surface water.

Longitudinal Monitoring of DNA Viral Loads in Transplant Patients Using Quantitative Metagenomic Next-Generation Sequencing.

Introduction: Immunocompromised patients are prone to reactivations and (re-)infections of multiple DNA viruses. Viral load monitoring by single-target quantitative PCRs (qPCR) is the current cornerstone for virus quantification. In this study, a metagenomic next-generation sequencing (mNGS) approach was used for the identification and load monitoring of transplantation-related DNA viruses. Methods: Longitudinal plasma samples from six patients that were qPCR-positive for cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyomavirus (BKV), adenovirus (ADV), parvovirus B19 (B19V), and torque teno-virus (TTV) were sequenced using the quantitative metagenomic Galileo Viral Panel Solution (Arc Bio, LLC, Cambridge, MA, USA) reagents and bioinformatics pipeline combination. Qualitative and quantitative performance was analysed with a focus on viral load ranges relevant for clinical decision making. Results: All pathogens identified by qPCR were also identified by mNGS. BKV, CMV, and HHV6B were additionally detected by mNGS, and could be confirmed by qPCR or auxiliary bioinformatic analysis. Viral loads determined by mNGS correlated with the qPCR results, with inter-method differences in viral load per virus ranging from 0.19 log10 IU/mL for EBV to 0.90 log10 copies/mL for ADV. TTV, analysed by mNGS in a semi-quantitative way, demonstrated a mean difference of 3.0 log10 copies/mL. Trends over time in viral load determined by mNGS and qPCR were comparable, and clinical thresholds for initiation of treatment were equally identified by mNGS. Conclusions: The Galileo Viral Panel for quantitative mNGS performed comparably to qPCR concerning detection and viral load determination, within clinically relevant ranges of patient management algorithms.

Life and death of smallpox

Smallpox is an ancient scourge known since the Antiquity. It is caused by a highly contagious airborne poxvirus. This strictly human disease exists in two forms: variola major (Asian smallpox) with mortality of 20–45%, and an attenuated form called variola minor or alatrim with mortality of 1–2%, which only recently appeared in Europe and America towards the end of the 19th century. The first smallpox pandemic was the "Antonine plague", which swept through the Roman Empire in the 2nd century AD, after which smallpox became endemic in the Old World, causing seasonal and regional epidemics in Europe, affecting mostly young children until the 19th century. The discovery of the New World in 1492 and the opening of the African slave trade favored in 1518 the contamination by smallpox of the native Amerindian populations, who were massively decimated during the following centuries. In the absence of any effective treatment, preventive methods were developed from the 18th century. First, variolation was used, a dangerous procedure that consists in inoculating intradermally a small quantity of virus from convalescent patients. In the early 19th century, Edward Jenner popularized the practice of inoculating cowpox, a mild cow disease. This procedure proved to be very effective and relatively safe, leading to the decline of smallpox during the 19th century. In the 20th century, a ten-year WHO vaccination campaign led to the total eradication of smallpox in 1977. During that century, smallpox caused an estimated 300–500 million deaths worldwide. Using molecular approach, it has been discovered that the smallpox virus emerged 3000–4000 years ago in East Africa and is closely related to the taterapox virus from African gerbils and to the camelpox virus, which causes variola in camelids. Today, smallpox virus strains are stored in freezers at the CDC in Atlanta and at the Vector Center in Koltsovo, Siberia. That is why smallpox remains a potential threat to the highly susceptible human species, as a result of an accident or malicious use of the virus as a biological weapon.

Effects of Buprenorphine Treatment on Influenza Pathogenesis in the Ferret (Mustela putorius furo).

Ferrets are the gold-standard model for influenza A virus (IAV) research due to their natural susceptibility to human and zoonotic IAV, comparable respiratory anatomy and physiology to humans, and development of clinical signs similar to those seen in infected people. Because the presence and progression of clinical signs can be useful in infectious disease research, uncertainty in how analgesics alter research outcomes or compromise characteristics of disease progression have outweighed the concern regarding animal discomfort from these symptoms. Nonetheless, the principles of animal research require consideration of refinements for this important model for IAV research. Opioids offer a possible refinement option that would not directly affect the inflammatory cascade involved in IAV infection. Mirroring pathogenicity studies that use ferrets, 12 ferrets were inoculated intranasally with the A(H3N2) IAV A/Panama/2007/1999 and divided into 3 treatment groups ( n = 4 each), of which 2 groups received buprenorphine treatments on different schedules and the third received a saline control. The duration and location of viral replication, lymphohematopoietic changes, and clinical signs were comparable across all groups at all time points. High quantities of infectious virus in nasal wash specimens were detected in ferrets from all groups through day 5 after inoculation, and peak viral titers from the upper respiratory tract did not differ between ferrets receiving buprenorphine treatments on either schedule. Compared with the saline group, ferrets receiving buprenorphine exhibited transient weight loss and pyrexia, but all groups ultimately achieved similar peaks in both of these measurements. Collectively, these findings support the continued evaluation of buprenorphine as a refinement for IAV-challenged ferrets.

Flow Virometry for Characterizing the Size, Concentration, and Surface Antigens of Viruses.

Application of flow cytometry principles for the analysis of viruses has been referred to as flow virometry (FVM). FVM is a multiparametric, high-throughput, and sensitive technique that allows viral particles to be detected, quantified, and characterized based on the biophysical properties of the virus and the expression of proteins on their surface. More specifically, by calibrating the flow cytometer with reference materials, it is possible to measure the concentration of intact viral particles in a sample, the abundance of a target antigen on the surface of the virus, and the relative diameter of the virus. Here, we describe a comprehensive overview of procedures used to stain, detect, and quantify viral and host-derived proteins located on the surface of retroviruses. These outlined techniques can be applied for the rapid phenotypic characterization of retroviruses, other enveloped viruses, and generally most viruses at the single-particle level through the direct staining of viruses collected from the supernatant of infected cells, without the need for enrichment or purification. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Virus production Basic Protocol 2: Instrument setup, standardization, and quality control for fluorescence quantification Basic Protocol 3: Flow virometry analysis Basic Protocol 4: Viral surface antigen staining and fluorescence quantification Support Protocol: Determination of the optimal antibody concentration for virus staining Basic Protocol 5: Gain configuration optimization.

Dried blood spot sampling for hepatitis B virus quantification, sequencing and mutation detection

Hepatitis B virus (HBV) diagnosis is performed on serum samples, but the access to this diagnosis is difficult in low-income regions. The use of dried blood spot (DBS) samples does not require special structure for collection, storage or transport. This study evaluates the use of DBS for detection, quantification and sequencing of HBV DNA using in-house techniques. Two study groups were included: 92 HBsAg + individuals and 49 negative controls. Serum and DBS samples were submitted to quantitative and qualitative in-house PCR for S/pol genes, sequencing and phylogenetic analyses. Total of 84 serum samples were successfully amplified. Of them, 63 paired DBS were also positive in qualitative PCR. Qualitative PCR in DBS presented a sensitivity of 75% and specificity of 100% (Kappa = 0.689). Quantitative PCR in DBS presented a detection limit of 852.5 copies/mL (250 IU/mL), sensitivity of 77.63% and specificity of 100% (Kappa = 0.731). A total of 63 serum samples and 36 DBS samples were submitted to sequencing, revealing the circulation of genotypes A (65.08%), D (4.8%), E (3.2%) and F (27%) with 100% of correspondence between serum and DBS. All sequenced samples displayed polymorphisms in HBsAg gene. An HIV-coinfected patient presented the rtM204V/I-rtL180M double resistance mutation in serum and DBS. In conclusion, DBS is an alternative to detect, quantify and characterize HBV DNA, being a possibility of increasing diagnosis in low-income settings, closing gaps in HBV control.

Phenomic analysis of the honey bee pathogen-web and its dynamics on colony productivity, health and social immunity behaviors.

Many pathogens and parasites have evolved to overwhelm and suppress their host’s immune system. Nevertheless, the interactive effects of these agents on colony productivity and wintering success have been relatively unexplored, particularly in large-scale phenomic studies. As a defense mechanism, honey bees have evolved remarkable social behaviors to defend against pathogen and parasite challenges, which reduce the impact of disease and improve colony health. To investigate the complex role of pathogens, parasites and social immunity behaviors in relation to colony productivity and outcomes, we extensively studied colonies at several locations across Canada for two years. In 2016 and 2017, colonies founded with 1-year-old queens of diverse genetic origin were evaluated, which represented a generalized subset of the Canadian bee population. During each experimental year (May through April), we collected phenotypic data and sampled colonies for pathogen analysis in a standardized manner. Measures included: colony size and productivity (colony weight, cluster size, honey production, and sealed brood population), social immunity traits (hygienic behavior, instantaneous mite population growth rate, and grooming behavior), as well as quantification of gut parasites (Nosema spp., and Lotmaria passim), viruses (DWV-A, DWV-B, BQCV and SBV) and external parasites (Varroa destructor). Our goal was to examine: 1) correlations between pathogens and colony phenotypes; 2) the dynamics of pathogens and parasites on colony phenotypes and productivity traits; and 3) the effects of social immunity behaviors on colony pathogen load. Our results show that colonies expressing high levels of some social immunity behaviors were associated with low levels of pathogens/parasites, including viruses, Nosema spp., and V. destructor. In addition, we determined that elevated viral and Nosema spp. levels were associated with low levels of colony productivity, and that five out of six pathogenic factors measured were negatively associated with colony size and weight in both fall and spring periods. Finally, this study also provides information about the incidence and abundance of pathogens, colony phenotypes, and further disentangles their inter-correlation, so as to better understand drivers of honey bee colony health and productivity.

Genomic and Virological Characterization of SARS-CoV-2 Variants in a Subset of Unvaccinated and Vaccinated U.S. Military Personnel.

The emergence of SARS-CoV-2 variants complicates efforts to control the COVID-19 pandemic. Increasing genomic surveillance of SARS-CoV-2 is imperative for early detection of emerging variants, to trace the movement of variants, and to monitor effectiveness of countermeasures. Additionally, determining the amount of viable virus present in clinical samples is helpful to better understand the impact these variants have on viral shedding. In this study, we analyzed nasal swab samples collected between March 2020 and early November 2021 from a cohort of United States (U.S.) military personnel and healthcare system beneficiaries stationed worldwide as a part of the Defense Health Agency's (DHA) Global Emerging Infections Surveillance (GEIS) program. SARS-CoV-2 quantitative real time reverse-transcription PCR (qRT-PCR) positive samples were characterized by next-generation sequencing and a subset was analyzed for isolation and quantification of viable virus. Not surprisingly, we found that the Delta variant is the predominant strain circulating among U.S. military personnel beginning in July 2021 and primarily represents cases of vaccine breakthrough infections (VBIs). Among VBIs, we found a 50-fold increase in viable virus in nasal swab samples from Delta variant cases when compared to cases involving other variants. Notably, we found a 40-fold increase in viable virus in nasal swab samples from VBIs involving Delta as compared to unvaccinated personnel infected with other variants prior to the availability of approved vaccines. This study provides important insight about the genomic and virological characterization of SARS-CoV-2 isolates from a unique study population with a global presence.

OsciDrop: A Versatile Deterministic Droplet Generator.

This paper describes OsciDrop, a versatile chip-free droplet generator used to produce size-tunable droplets on demand. Droplet generation is fundamental to miniaturized analysis. We designed OsciDrop to segment the fluid flowing out of the orifice of a disposable pipette tip into droplets by oscillating its distal end underneath an immiscible continuous phase. We described the theoretical model and investigated the effect of flow rate, oscillating amplitude, frequency, and waveform on droplet generation. Our study revealed a previously underexplored Weber number-dominated regime that leverages inertial force instead of viscous force to generate droplets. The same pipette tip allowed robust and deterministic generation of monodisperse droplets with programmable sizes ranging from 200 pL to 2 μL by asymmetrical oscillation. We validated this platform with two droplet-based nucleic acid amplification tests: a digital loop-mediated isothermal amplification assay for absolute quantification of African swine fever virus and a multi-volume digital polymerase chain reaction assay for the high dynamic range measurement of human genomic DNA. The OsciDrop method opens a facile avenue to miniaturization, integration, and automation, exhibiting full accessibility for digital molecular diagnostics.

SARS-CoV-2 Remains Infectious on Refrigerated Deli Food, Meats, and Fresh Produce for up to 21 Days.

SARS-CoV-2, the virus that causes COVID-19, has been detected on foods and food packaging and the virus can infect oral cavity and intestinal cells, suggesting that infection could potentially occur following ingestion of virus-contaminated foods. To determine the relative risk of infection from different types of foods, we assessed survival of SARS-CoV-2 on refrigerated ready-to-eat deli items, fresh produce, and meats (including seafood). Deli items and meats with high protein, fat, and moisture maintained infectivity of SARS-CoV-2 for up to 21 days. However, processed meat, such as salami, and some fresh produce exhibited antiviral effects. SARS-CoV-2 also remained infectious in ground beef cooked rare or medium, but not well-done. Although infectious SARS-CoV-2 was inactivated on the foods over time, viral RNA was not degraded in similar trends, regardless of food type; thus, PCR-based assays for detection of pathogens on foods only indicate the presence of viral RNA, but do not correlate with presence or quantity of infectious virus. The survival and high recovery of SARS-CoV-2 on certain foods support the possibility that food contaminated with SARS-CoV-2 could potentially be a source of infection, highlighting the importance of proper food handling and cooking to inactivate any contaminating virus prior to consumption.

Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques.

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, we used the rhesus macaque model of SARS-CoV-2 infection. Eight older and eight younger macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at predefined time points in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication were limited. Transcriptional signatures of inflammation-associated genes in bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both cohorts. However, age-specific divergence of immune responses emerged during the post-acute phase. Older animals exhibited sustained local inflammatory innate responses, whereas local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, and persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling suggests that age may delay or impair antiviral cellular immune responses and delay efficient return to immune homeostasis.

Experimental Susceptibility of North American Raccoons (Procyon lotor) and Striped Skunks (Mephitis mephitis) to SARS-CoV-2.

Recent spillback events of SARS-CoV-2 from humans to animals has raised concerns about it becoming endemic in wildlife. A sylvatic cycle of SARS-CoV-2 could present multiple opportunities for repeated spillback into human populations and other susceptible wildlife. Based on their taxonomy and natural history, two native North American wildlife species —the striped skunk (Mephitis mephitis) and the raccoon (Procyon lotor) —represent a high likelihood of susceptibility and ecological opportunity of becoming infected with SARS-CoV-2. Eight skunks and raccoons were each intranasally inoculated with one of two doses of the virus (103 PFU and 105 PFU) and housed in pairs. To evaluate direct transmission, a naïve animal was added to each inoculated pair 48 h post-inoculation. Four control animals of each species were handled like the experimental groups. At predetermined intervals, we collected nasal and rectal swabs to quantify virus shed via virus isolation and detect viral RNA via rRT-PCR and blood for serum neutralization. Lastly, animals were euthanized at staggered intervals to describe disease progression through histopathology and immunohistochemistry. No animals developed clinical disease. All intranasally inoculated animals seroconverted, suggesting both species are susceptible to SARS-CoV-2 infection. The highest titers in skunks and raccoons were 1:128 and 1:64, respectively. Low quantities of virus were isolated from 2/8 inoculated skunks for up to day 5 post-inoculation, however no virus was isolated from inoculated raccoons or direct contacts of either species. Neither species had gross lesions, but recovering mild chronic pneumonia consistent with viral insult was recorded histologically in 5/8 inoculated skunks. Unlike another SARS-CoV-2 infection trial in these species, we detected neutralizing antibodies in inoculated raccoons; thus, future wildlife serologic surveillance results must be interpreted with caution. Due to the inability to isolate virus from raccoons, the lack of evidence of direct transmission between both species, and low amount of virus shed by skunks, it seems unlikely for SARS-CoV-2 to become established in raccoon and skunk populations and for virus to spillback into humans. Continued outbreaks in non-domestic species, wild and captive, highlight that additional research on the susceptibility of SARS-CoV-2 in wildlife, especially musteloidea, and of conservation concern, is needed.

Microbiological characterization of stormwater in a high-income neighborhood in Rio de Janeiro, Brazil.

Stormwater harvesting and reuse in the urban environment is emerging as an alternative water source, despite human pathogens in the stormwater may represent a hazard to public health. This study presents the results of 1-year monitoring to evaluate the quality of stormwater obtained in a high-income neighborhood in Rio de Janeiro for a set of microbiological parameters as total coliforms, Escherichia coli (E. coli), human adenovirus (HAdV), human JC polyomavirus (JCPyV), Group A rotavirus (RVA), and norovirus GI and GII. Forty-eight stormwater samples obtained from two multiplex units presented total coliforms and E. coli in 91.7% (n = 44) and 58.3% (n = 28) of samples, while HAdV and JCPyV were detected in 20.8% (n = 10) and 12.5% (n = 6), respectively. Viral quantification ranged from 103 to 104 genomic copies/liter (GC/L) for HAdV and from 101 to 104 GC/L for JCPyV. Neither RVA nor norovirus GI and GII was detected. Fifteen out of sixteen (93.8%) samples containing viruses were compliant as per fecal indicator bacteria (FIB) according to Brazilian standards for rainwater reuse and US EPA Guidelines for Water Reuse, suggesting that viruses monitoring should complement the study of bacterial indicators.

Optimized Protocols for the Propagation and Quantification of Infectious Murine Hepatitis Virus (MHV-A59) Using NCTC Clone 1469 and 929 Cells.

Murine hepatitis virus (MHV) is a non-human pathogen betacoronavirus that is evolutionarily and structurally related to the human pathogenic viruses SARS-CoV, MERS-CoV, and SARS-CoV-2. However, unlike the human SARS and MERS viruses, MHV requires a biosafety level 2 laboratory for propagating and safe handling, making it a potentially suitable surrogate virus. Despite this utility, few papers discussed the propagation and quantification of MHV using cell lines readily available in biorepositories making their implementations not easily reproducible. This article provides protocols for propagating and quantifying MHV-A59 using the recommended NCTC clone 1469 and clone 929 cell lines from American Type Culture Collection (ATCC). More specifically, the methods detail reviving cells, routine cell passaging, preparing freeze stocks, infection of NCTC clone 1469 with MHV and subsequent harvesting, and plaque assay quantification of MHV using NCTC clone 929 cells. Using these protocols, a BSL-2 laboratory equipped for cell culture work would generate at least 6.0 log plaque-forming units (PFU) per mL of MHV lysate and provide an optimized overlay assay using either methylcellulose or agarose as overlays for the titration of infectious virus particles. The protocols described here are intended to be utilized for persistence and inactivation studies of coronaviruses.

Evaluating a Dry vs. Wet Disinfection in Boot Baths on Detection of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus RNA

Maintaining biosecurity between swine barns is challenging, and boot baths are an easily implementable option some utilize to limit pathogen spread. However, there are concerns regarding their efficacy, especially when comparing wet or dry disinfectants. The objective of this study was to evaluate the efficacy of boot baths in reducing the quantity of detectable porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) genetic material using wet or dry disinfectants. Treatments included 1) control; 2) dry chlorine powder (Traffic C.O.P., PSP, LLC, Rainsville, AL); and 3) wet quaternary ammonium/glutaraldehyde liquid (1:256 Synergize, Neogen, Lexington, KY). Prior to disinfection, rubber boots were inoculated with 1 mL of co-inoculants of PRRSV (1×105TCID50/mL) and PEDV (1×105 TCID50/mL) and dried for 15 min. After the drying period, a researcher placed the boot on the right foot and stepped directly on a stainless steel coupon (control). Alternatively, the researcher stepped first into a boot bath containing either the wet or dry sanitizer, stood for 3 s, and then stepped onto a steel coupon. After one min, an environmental swab was then collected and processed from each boot and steel coupon. The procedure was replicated 12 times per disinfectant treatment. Samples were analyzed using a duplex qPCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values, which indicate the presence or absence of the inoculants and their relative concentrations when present, were analyzed using SAS GLIMMIX (v. 9.4, SAS Institute, Inc., Cary, NC). There was no evidence of a disinfectant × surface × virus interaction (P > 0.10). An interaction between disinfectant × surface impacted (P < 0.05) the quantity of detectable viral RNA. As expected, the quantity of the viruses on the coupon were greatest in the control, indicating that a contaminated boot has the ability to transfer viruses from a contaminated surface to a clean surface. Comparatively, the dry disinfectant treatment resulted in no detectable viral RNA on either the boot or subsequent coupon. The wet disinfectant treatment had statistically similar (P > 0.05) viral contamination to the control on the boot, but less viral contamination compared to the control on the metal coupon. In this experiment, a boot bath with dry powder was the most efficacious in reducing the detectable viral RNA on both boots and subsequent surfaces.

Evaluating a Dry vs. Wet Disinfection in Boot Baths on Detection of Porcine Epidemic Diarrhea Virus and Porcine Reproductive and Respiratory Syndrome Virus RNA

Maintaining biosecurity between swine barns is challenging, and boot baths are an easily implementable option some utilize to limit pathogen spread. However, there are concerns regarding their efficacy, especially when comparing wet or dry disinfectants. The objective of this study was to evaluate the efficacy of boot baths in reducing the quantity of detectable porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV) genetic material using wet or dry disinfectants. Treatments included 1) control; 2) dry chlorine powder (Traffic C.O.P., PSP, LLC, Rainsville, AL); and 3) wet quaternary ammonium/glutaraldehyde liquid (1:256 Synergize, Neogen, Lexington, KY). Prior to disinfection, rubber boots were inoculated with 1 mL of co-inoculants of PRRSV (1×105TCID50/mL) and PEDV (1×105 TCID50/mL) and dried for 15 min. After the drying period, a researcher placed the boot on the right foot and stepped directly on a stainless steel coupon (control). Alternatively, the researcher stepped first into a boot bath containing either the wet or dry sanitizer, stood for 3 s, and then stepped onto a steel coupon. After one min, an environmental swab was then collected and processed from each boot and steel coupon. The procedure was replicated 12 times per disinfectant treatment. Samples were analyzed using a duplex qPCR at the Kansas State Veterinary Diagnostic Laboratory. Cycle threshold values, which indicate the presence or absence of the inoculants and their relative concentrations when present, were analyzed using SAS GLIMMIX (v. 9.4, SAS Institute, Inc., Cary, NC). There was no evidence of a disinfectant × surface × virus interaction (P > 0.10). An interaction between disinfectant × surface impacted (P < 0.05) the quantity of detectable viral RNA. As expected, the quantity of the viruses on the coupon were greatest in the control, indicating that a contaminated boot has the ability to transfer viruses from a contaminated surface to a clean surface. Comparatively, the dry disinfectant treatment resulted in no detectable viral RNA on either the boot or subsequent coupon. The wet disinfectant treatment had statistically similar (P > 0.05) viral contamination to the control on the boot, but less viral contamination compared to the control on the metal coupon. In this experiment, a boot bath with dry powder was the most efficacious in reducing the detectable viral RNA on both boots and subsequent surfaces.

Propagation and Quantification of SARS-CoV-2.

In late 2019, the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. Since its emergence, SARS-CoV-2 has been responsible for a world-wide pandemic resulting in over 80 million infections and over 1.8 million deaths. The severity of the pandemic has prompted widespread research efforts to more fully understand SARS-CoV-2 and the disease it causes, COVID-19. Research into this novel virus will be facilitated by the availability of clearly described and effective protocols that enable the propagation and quantification of infectious virus. Here, we describe protocols for the propagation of SARS-CoV-2 in Vero E6 cells as well as two human cells lines, the intestinal epithelial Caco-2 cell line and the respiratory epithelial Calu-3 cell line. Additionally, we provide protocols for the quantification of SARS-CoV-2 by plaque assays and immunofocus forming assays in Vero E6 cells utilizing liquid overlays. These protocols provide a foundation for laboratories acquiring the ability to study SARS-CoV-2 to address this ongoing pandemic.

Detection and Quantification of SARS-CoV-2 by Real-Time RT-PCR Assay.

The pandemic coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread and high mortality rate. Detection and quantification of the (+) ssRNA virus, which has a genome size of 29,903 nucleotides, is commonly performed via reverse transcription quantitative polymerase chain reaction (RT-qPCR) targeting conserved sequences. Here, we describe a one-step RT-qPCR protocol for the quantitative detection of SARS-CoV-2 genomic RNA targeting M and RdRP genes, respectively, as well as active virus replication detecting subgenomic RNAs (sgRNA 4 and 8) that are formed by discontinuous transcription of the viral genome. Concomitantly, an input control targeting the human RNaseP gene (RPP30) was used in multiplex PCR to monitor the input of human nucleic acids. In vitro-transcribed RNA harboring the amplicon regions for M and RdRP regions served to set up a standard curve for absolute quantification.In conclusion, the method described here allows for the detection and quantification of SARS-CoV-2 RNA isoforms for research by both using a probe-based or SYBR Green-based approach, but is also suitable for diagnostic purposes.

Molecular detection and quantification of bluetongue virus in small ruminant and Culicoides vectors in Gujarat, India

The present study was carried out to detect bluetongue virus (BTV) in Culicoides vectors and the biological samples of small ruminants with the use of PCR based molecular methods. The vectors were collected using the UV LED CDC traps, placed near animal sheds and then identified by morphological and PCR based methods. Primers specific to genus Culicoides and species Culicoides oxystoma were used in PCR reaction. The end point RT-PCR protocols were used for tentative and confirmatory diagnosis of BTV and its two serotypes viz. BTV-1 and BTV-16, whereas, the real time RT-PCR was used for confirmation of BTV and quantization of viral RNA load. Out of a total of 132 blood samples and 55 tissue samples viz., spleen, liver, kidney, thymus and heart blood of aborted fetuses and 09 pooled samples of Culicoides, total 21 (6.93%) samples i.e., 11 blood samples, 08 spleens and 02 pooled Culicoides samples were detected positive for BTV by NS1 gene specific RT-PCR. In nested PCR, 8 additional samples were found positive for BTV. Among these 29 positive samples, 6 were collected from spleens of aborted fetuses which was indicative of transplacental transmission of BTV among small ruminants. All the positive samples were of either of BTV-1 and BTV-16 serotypes as confirmed by serotype specific PCR. In real time PCR, plasmid vector based positive control was prepared, which confirmed the 21 samples to be BTV positive, whereas, quantization of viral RNA, showed that out of all clinical samples tested, highest and lowest RNA copy numbers i.e. 3566.41 and 81.79 were found in blood sample and spleen of sheep, respectively.