Median Tissue Culture Infective Dose Research Articles

Late gestation maternal infection with bovine viral diarrhea virus impacts offspring feedlot performance, digestion, blood parameters, and hot carcass weights.

Fetal infection with bovine viral diarrhea virus (BVDV) after 150 d results in transient fetal infections (TI). Twenty-five unvaccinated, yearling Hereford heifers, seronegative for antibodies to BVDV1 and BVDV2, were bred by artificial insemination with X chromosome-bearing sperm from one Angus sire to examine the impact of TI on postnatal growth, estimated dry matter digestibility, blood parameters, and carcass characteristics. On d 175 of pregnancy, dams were intranasally inoculated with either sham control or 4.0 log median tissue culture infectious dose noncytopathic type2 BVDV to generate control or TI offspring, respectively. All control dams remained seronegative and all BVDV-inoculated dams seroconverted by d 14 post-inoculation. All control offspring were seronegative and all TI offspring were seropositive for antibodies to type 2 BVDV at birth. All offspring were raised on pasture until weaning. At weaning, all calves were transported to our research feedlot facility, housed in three pens, and transitioned to a high-energy concentrate-based diet. Heifer body weights (BW) and jugular blood samples were collected every 28 d. On d 84 of the feeding period, titanium dioxide was added to the diet of 12, age-paired, individually fed, heifers (6 control and 6 TI heifers; approximately 1 yr. of age) for 28 d and used to estimate dry matter digestibility. On d 105 and 240 ruminal fluid (approximately 900ml) was collected from every animal using a stomach pump and analyzed for short chain fatty acids (SCFA). After approximately 287 d on feed, heifers were transported to a USDA-inspected abattoir and harvested. TI heifers had lighter final BW (P < 0.04) when compared to control heifers. Average daily gain was greater (P < 0.01) in control compared to TI heifers. TI heifers had a 2.2% lesser (P < 0.05) apparent dry matter digestibility, lighter (P < 0.01) hot carcass weights, but similar ruminal SCFA compared to controls. Blood glucose concentrations were similar (P > 0.8) between control and TI heifers. Ceruloplasmin activity (P < 0.03) and the oxidized form of glutathione (GSSG; P < 0.01), indicators of chronic inflammation, were increased in plasma from TI heifers compared to controls. Other indicators of oxidative stress were not impacted (P > 0.10) by TI status. These data suggest that fetal BVDV transient infection negatively impacts growth throughout the feeding period, possibly by impacting gastrointestinal tract function and increasing systemic inflammation.

Antiviral activity of Nigella sativa oil against two SARS-CoV-2surrogates in vitro as a dietary product

Background: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is primarily transmitted via aerosol and droplets, but environmental surfaces or potentially contaminated foods may contribute to further viral transmission. Nigella sativa seed (black cumin seed) oil possesses various biological activities against bacteria, fungi, parasites, orviruses. Objectives: This study aimed to explore black cumin seed oil (BSO) with anti-SARS-CoV-2 activities using bovine coronavirus (BCoV) and human coronavirus (HCoV) OC43 as surrogates. Methods and Results: BSO was tested at 1-100 mL/L concentrations for cytotoxicity towards human rectal tumor (HRT-18G) cells and 0.1-10 mL/L for efficacy against BCoV and HCoV OC43 in suspension. Two coronaviruses were separately mixed with BSO suspension and incubated for various times (0, 15, 30, 45, and 60 min) at 4, 23, and 37°C. The virus titerreduction was determined by median tissue culture infectious dose (TCID50) assay with HRT-18G cells as the host. BSO concentrations of 1-20 mL/L exhibited low cytotoxicity (&lt;10%) towards HRT-18G cells. The inactivation of coronaviruses by BSO was concentration-, temperature-, and exposure time-dependent. At 37°C, BSO reduced 2.0 logs of BCoV and3.0 logs of HCoV OC43 in 60 min, respectively. Specifically, half maximal effective concentration (EC50) of BSO at 4, 23, and 37°C after the 1-h exposure was 4.28, 3.06, and 0.87 mL/L against BCoV, respectively, and 0.61, 0.61 and 0.48 mL/L against HCoV OC43, respectively. Moreover, BSO didn’t affect the attachment of BCoV and HCoV OC43 into host cells. Conclusion: Our findings on the inhibition activity of BSO against SARS-CoV-2 surrogates suggested the potential use of this natural product in mitigating SARS-CoV-2 infection. Our results also suggest that HCoV OC43 may be a more appropriate surrogate for SARS-CoV-2 to screen antiviral dietary products. Keywords: Nigella sativa, SARS-CoV-2 surrogates, antiviral activity, quantitative suspension test, dietary supplement

Antiviral Effects of Ozonated Water for Handwashing Against SARS-CoV-2 Omicron Variant Using the E1052-20 Test Method of the American Society of Testing Materials International

ABSTRACT Alcohol-based hand rubs are generally recommended for infection control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hand hygiene agents, including ozonated water (OW), have been used as an alternative because alcohol-based products may cause skin damage. A few reports are available on the virucidal activity of OW against SARS-CoV-2; therefore, this study examined the inhibitory effect of 0.4, 1.0, and 4.0 ppm OW on the Omicron variant infection to VeroE6 cells expressing transmembrane protease serine 2 by calculating the median tissue culture infectious dose (TCID50) using the E1052–20 test method of the American Society of Testing Materials International. Additionally, viral RNA copies were measured in culture supernatants of control or OW. Each OW concentration demonstrated ≥5.6 log10 TCID50 reduction, corresponding to ≥99.999% reduction of infectious virus, while the control phosphate-buffered saline showed no inactivation effect. More than 5 log10 copies/mL reduction of viral RNA was observed in the culture supernatant of each OW compared with the control. These findings suggest that exposure of SARS-CoV-2 to at least 0.4 ppm OW for 5 s is sufficient for viral inactivation and that OW can be used as an alternative hand hygiene agent to prevent SARS-CoV-2 infection.

A recombinant pseudorabies virus surface - displaying the classical swine fever E2 protein induces specific antibodies rapidly

Pseudorabies virus (PRV) and classical swine fever virus (CSFV) are both economically important pathogens threatening the pig industry in many countries. The triple-gene-deleted variant of PRV, herein referred to as rPRVTJ-delgE/gI/TK, has exhibited pronounced efficacy and safety profiles. This underscores its viability as a prospective vaccine vector. However, the generation of specific anti-E2 antibodies necessitates elevated immunization doses and extended durations when the extracellular domain of the E2 protein of CSFV is secreted via the recombinant rPRVTJ-delgE/gI/TK vector. To enhance the presentation of exogenous antigens by antigen-presenting cells (APCs), we engineered the E2 protein expressed on the surface of PRV particles in this study. The recombinant virus expressing the E2 protein with a heterogonous transmembrane domain was generated in the backbone of rPRVTJ-delgE/gI/TK and designated as rPRVTJ-UL44-E2. The E2 gene was fused to the 3′ terminus of the UL44 gene utilizing P2A, a self-cleaving peptide sequence. The electron microscopy showed that the E2 protein was anchored on the surface of the viral particles of rPRVTJ-delgE/gI/TK-E2. The insertion of the E2 gene did not alter the native biological characteristics of the viral vector. Rabbits immunized with 107 median tissue culture infective doses (TCID50) of rPRVTJ-UL44-E2 exhibited a rapid seroconversion to anti-E2 specific antibodies within 7 days post-immunization (dpi). All the rabbits immunized with the rPRVTJ-UL44-E2 had generated antibodies specific to E2 prior to the administration of the booster immunization. However, the immunized rabbits were not protected from the CSFV C-strain challenge. Nevertheless, this strategy has notably achieved rapid induction of E2-specific non-neutralizing antibodies. These findings provide insights that the design of rPRVTJ-UL44-E2 requires optimization, thereby indicating a promising avenue for augmenting vaccine-induced immune responses.

Antiviral Efficacy Testing of a Rechargeable Textile.

In light of the COVID-19 (Coronovirus Disease 2019) pandemic, the use of personal protective equipment has become essential to reduce viral transmission and maintain public health. Viruses, particularly human coronavirus and influenza, pose significant challenges because of their various transmission routes. UMF Corporation's innovation, Micrillon, aims to address these challenges by creating durable, antiviral technology for textiles without harmful chemicals, reducing viral transmission risks. The study followed ISO Standard 18184:2019, testing Micrillon textiles against Human Coronavirus OC43 and H1N1 Influenza A virus using MDCK and HCT-8 cell lines. Cell propagation, viral application, TCID50 (Median Tissue Culture Infectious Dose) testing, and maintenance protocols were rigorously implemented to assess antiviral efficacy. Micrillon gloves, fabrics, and fibers exhibited high antiviral efficacy against both viruses across various contact times. Gloves demonstrated exceptional antiviral activity against H1N1 (99.88%) and OC43 (99.67%) at 120 minutes. Rolled fabrics showed strong efficacy against H1N1 (99.42% at 30 minutes) and OC43 (>97%) at all time points. Bundled fibers displayed substantial efficacy against H1N1 (99.17% at 120 minutes) and OC43 (>98%) at all time points. The study demonstrates that Micrillon technology effectively inhibits viral activity, particularly in gloves, fabrics, and fibers. The innovation not only shows high antiviral efficacy against both Human Coronavirus and Influenza but also promises a reusable, sustainable solution, mitigating environmental impact and reducing the use of harmful chemicals in personal protective equipment. The technology holds promise for widespread use in health care and hospitality, offering a layer of protection while being environmentally conscious. Further development of such technologies can significantly reduce infection risks while minimizing environmental harm.

The flavonoid quercetin decreases ACE2 and TMPRSS2 expression but not SARS-CoV-2 infection in cultured human lung cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to angiotensin-converting enzyme 2 (ACE2) on host cells, via its spike protein, and transmembrane protease, serine 2 (TMPRSS2) cleaves the spike-ACE2 complex to facilitate virus entry. As rate-limiting steps for virus entry, modulation of ACE2 and/or TMPRSS2 may decrease SARS-CoV-2 infectivity and COVID-19 severity. In silico modeling suggested the natural bioactive flavonoid quercetin can bind to ACE2 and a recent randomized clinical trial demonstrated that oral supplementation with quercetin increased COVID-19 recovery. A range of cultured human cells were assessed for co-expression of ACE2 and TMPRSS2. Immortalized Calu-3 lung cells, cultured and matured at an air-liquid interface (Calu-3-ALIs), were established as the most appropriate. Primary bronchial epithelial cells (PBECs) were obtained from healthy adult males (N = 6) and cultured under submerged conditions to corroborate the outcomes. Upon maturation or reaching 80% confluence, respectively, the Calu-3-ALIs and PBECs were treated with quercetin, and mRNA and protein expression were assessed by droplet digital PCR and ELISA, respectively. SARS-CoV-2 infectivity, and the effects of pre- and co-treatment with quercetin, was assessed by median tissue culture infectious dose assay. Quercetin dose-dependently decreased ACE2 and TMPRSS2 mRNA and protein in both Calu-3-ALIs and PBECs after 4 h, while TMPRSS2 remained suppressed in response to prolonged treatment with lower doses (twice daily for 3 days). Quercetin also acutely decreased ADAM17 mRNA, but not ACE, in Calu-3-ALIs, and this warrants further investigation. Calu-3-ALIs, but not PBECs, were successfully infected with SARS-CoV-2; however, quercetin had no antiviral effect, neither directly nor indirectly through downregulation of ACE2 and TMPRSS2. Calu-3-ALIs were reaffirmed to be an optimal cell model for research into the regulation of ACE2 and TMPRSS2, without the need for prior genetic modification, and will prove valuable in future coronavirus and respiratory infectious disease work. However, our data demonstrate that a significant decrease in the expression of ACE2 and TMPRSS2 by a promising prophylactic candidate may not translate to infection prevention.

Performance evaluation of influenza a rapid antigen test and PCR among nasopharyngeal and oropharyngeal samples

ObjectivesRapid antigen test (RAT) and polymerase chain reaction (PCR) using nasopharyngeal (NP) or oropharyngeal (OP) swab specimens are the two main testing techniques used for laboratory diagnosis of influenza in clinical practice. However, performance variations have been observed not only between techniques, but also between different specimens. This study evaluated the differences in performance between specimens and testing techniques to identify the best combination in clinical practice. MethodsBoth NP and OP samples from suspected influenza patients collected in the 2023/4–2023/5 Flu-season in Xiamen, China, were tested for RAT and quantitative PCR. The testing performance of the different specimens and testing techniques were recorded and evaluated. ResultsCompared to PCR, RAT showed 58.9 % and 10.3 % sensitivity for NP and OP swabs, respectively. The Limit of Detection (LoD) was 28.71 the Median Tissue Culture Infectious Dose (TCID50)/mL. Compared with PCR using NP swabs, PCR with OP swabs showed 89.5 % sensitivity and 95.4 % specificity. ConclusionsThere were no significant differences in performance between the specimens when PCR was used to test for influenza. However, a decrease in sensitivity was observed when the RAT was used, regardless of the specimen type. Therefore, to avoid false-negative results, PCR may be a better choice when OP swabs are used as specimens. In contrast, NP swabs should be the recommended specimens for RAT.

Comparison of the analytical sensitivity of COVID-19 rapid antigen tests in Australia and Canada

Rapid testing has become an indispensable strategy to identify the most infectious individuals and prevent the transmission of SARS-CoV-2 in vulnerable populations. As such, COVID-19 rapid antigen tests (RATs) are being manufactured faster than ever yet lack relevant comparative analyses required to inform on absolute analytical sensitivity and performance, limiting end-user ability to accurately compare brands for decision making. To date, more than 1000 different COVID-19 RATs are commercially available in the world, most of which detect the viral nucleocapsid protein (NP). Here, we examine and compare the analytical sensitivity of 26 RATs that are readily available in Canada and/or Australia using two NP reference materials (RMs) – a fluorescent NP-GFP expressed in bacterial cells and NCAP-1 produced in a mammalian expression system. Both RMs generate highly comparable results within each RAT, indicating minimal bias due to differing expression systems and final buffer compositions. However, we demonstrate orders of magnitude differences in analytical sensitivities among distinct RATs, and find little correlation with the median tissue culture infectious dose (TCID50) assay values reported by manufacturers. In addition, two COVID-19/Influenza A&B combination RATs were evaluated with influenza A NP-GFP. Finally, important logistics considerations are discussed regarding the robustness, ease of international shipping and safe use of these reference proteins. Taken together, our data highlight the need for and practicality of readily available, reliable reference proteins for end-users that will ensure that manufacturers maintain batch-to-batch quality and accuracy of RATs. They will aid international public health and government agencies, as well as health and aged care facilities to reliably benchmark and select the best RATs to curb transmission of future SARS-CoV-2 and influenza outbreaks.

Cordycepin Inhibits Enterovirus A71 Replication and Protects Host Cell from Virus-Induced Cytotoxicity through Adenosine Action Pathway.

Enterovirus A71 (EV-A71) infection typically causes mild illnesses, such as hand-foot-and-mouth disease (HFMD), but occasionally leads to severe or fatal neurological complications in infants and young children. Currently, there is no specific antiviral treatment available for EV-A71 infection. Thus, the development of an effective anti-EV-A71 drug is required urgently. Cordycepin, a major bioactive compound found in Cordyceps fungus, has been reported to possess antiviral activity. However, its specific activity against EV-A71 is unknown. In this study, the potency and role of cordycepin treatment on EV-A71 infection were investigated. Results demonstrated that cordycepin treatment significantly reduced the viral load and viral ribonucleic acid (RNA) level in EV-A71-infected Vero cells. In addition, EV-A71-mediated cytotoxicity was significantly inhibited in the presence of cordycepin in a dose-dependent manner. The protective effect can also be extended to Caco-2 intestinal cells, as evidenced by the higher median tissue culture infectious dose (TCID50) values in the cordycepin-treated groups. Furthermore, cordycepin inhibited EV-A71 replication by acting on the adenosine pathway at the post-infection stage. Taken together, our findings reveal that cordycepin could be a potential antiviral candidate for the treatment of EV-A71 infection.

Bivalent Hemagglutinin Cleavage-Site Peptide Vaccines Protect Chickens from Lethal Infections with Highly Pathogenic H5N1 and H5N6 Avian Influenza Viruses.

Outbreaks of highly pathogenic avian influenza viruses cause huge economic losses to the poultry industry worldwide. Vaccines that can protect chickens from infections caused by various variants of highly pathogenic H5Nx avian influenza viruses are needed owing to the continuous emergence of new variants. We previously showed that vaccines containing the H5 cleavage-site peptide from clade 2.3.4.4. H5N6 avian influenza virus protects chickens from infection with homologous clade 2.3.4.4. H5N6 avian influenza virus, but not from infection with the heterologous clade 1 H5N1 avian influenza virus. Therefore, we developed bivalent peptide vaccines containing H5 cleavage sites of viruses from both clades to protect chickens from both H5N1 and H5N6 avian influenza viruses. Chickens were vaccinated with two doses of a combined peptide vaccine containing cleavage-site peptides from clade 1 and clade 2.3.4.4. highly pathogenic H5N1 and H5N6 avian influenza viruses and then challenged with both viruses. The infected chickens were monitored for survival and their tracheae and cloacae were sampled to check for viral shedding based on the median tissue culture infectious dose of 50 (log10TCID50/mL) in Madin-Darby canine kidney cells. Antibody production was induced at similar levels in the sera of chickens immunized with two doses of the combined peptide vaccines containing cleavage-site peptides from highly pathogenic H5N1 and H5N6 avian influenza viruses. The immunized chickens were protected from infection with both H5N1 and H5N6 avian influenza viruses without viral shedding in the tracheae and cloacae. Dual-peptide vaccines containing cleavage-site peptides of both clades can protect chickens from highly pathogenic avian influenza virus infections.

Isolation and identification of BRV G6P[1] strain in Heilongjiang province, Northeast China.

Bovine rotavirus (BRV) is the main cause of acute gastroenteritis in calves, resulting in significant economic losses to the cattle industry worldwide. Additionally, BRV has multiple genotypes, which could enable cross-species transmission, thereby posing a significant risk to public health. However, there is a problem of multiple genotypes coexisting in BRV, and the cross-protection effect between different genotypes of rotavirus strains is not effective enough. Therefore, mastering clinical epidemic genotypes and using epidemic genotype strains for vaccine preparation is an effective means of preventing and controlling BRV. In this study, BRV strain DQ2020 in MA104 cells was identified by transmission electron microscopy (TEM), reverse transcription polymerase chain reaction (RT-PCR), and colloidal gold immunochromatographic test strips. The whole genome of BRV strain DQ2020 was sequenced and pathogenicity in suckling mice was assessed. The results showed that after 10 passages in MA104 cells, BRV strain DQ2020 induced cytopathic effects. Wheel-shaped virus particles (diameter, ~80 nm) were observed by TEM. A target band of 382 bp was detected by RT-PCR, a positive band was detected with the colloidal gold immunochromatographic test strips, and significant green fluorescence was observed by indirect immunofluorescence (IFA). The highest median tissue culture infectious dose of strain DQ2020 after 9 passages in MA104 cells was 10-4.81 viral particles/0.1 mL. Based on phylogenetic analysis of 11 gene fragments, the genotype of BRV strain DQ2020 was G6-P[1]-I2-R2-C2-M2-A11-N2-T6-E2-H3, confirming transmission of the G6-P[1] genotype in Chinese cattle herds. Further analysis showed that the isolated strain was a reassortant of bovine (VP7, VP6, NSP3, and NSP5), human (VP4, VP1, VP2, VP3, NSP2, and NSP4), and ovine (NSP1) rotaviruses. BRV strain DQ2020 caused damage to the intestinal villi of suckling mice and diarrhea, confirming pathogenicity. In summary, this study identified a reassortant strain of bovine, human, and ovine rotavirus that is pathogenic to lactating mice, and conducted whole genome sequence analysis, providing valuable insights for the genetic evolution of the virus and the development of vaccines.

Recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) designed for rapid detection of canine distemper virus.

In the present study, recombinase polymerase amplification (RPA) was combined with the colloidal gold lateral flow dipstick (LFD) method to establish a new, stable, and efficient assay for the detection of canine distemper virus (CDV). We designed a set of specific primers labeled with biotin and a specific probe labeled with dSpacer and C3 spacer, according to the conserved region in the N-terminal gene sequence of CDV. The reaction conditions and systems were then optimized, and the sensitivity and specificity were analyzed for potential clinical application. The results showed that the RPA-LFD assay for CDV detection was successfully established. We also found that the temperature in a closed fist (35°C) is optimal for the RPA reaction. The optimal ratio of primer to probe was 2:1. The minimum detection limit of the RPA-LFD assay was 1 × 101 the median tissue culture infective dose (TCID50)/mL. Using this assay with samples from experimentally infected dogs, CDV was detected in nasal secretions, eye secretions, and blood on the fourth day post infection. In summary, this novel RPA-LFD assay for CDV detection is simple to use, and preliminary findings indicate its high specificity and sensitivity.

Median Tissue Culture Infectious Dose 50 (TCID50) Assay to Determine Infectivity of Cytopathic Viruses.

The emergence of zoonotic viruses like severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 have significantly impacted global health and economy. The discovery of other viruses in wildlife reservoir species present a threatforfuture emergence in humans and animals. Therefore, assays that are less reliant on virus-specific information, such as neutralization assays, are crucial to rapidlydevelop diagnostics, understand virus replication and pathogenicity, and assess the efficacy of therapeutics against newly emerging viruses. Here, we describe the discontinuous median tissue culture infectious dose 50 (TCID50) assay to quantitatively determine the titer of any virus that can produce a visible cytopathic effect in infected cells.

Multivalent nanobody-based sandwich enzyme-linked immunosorbent assay for sensitive detection of porcine reproductive and respiratory syndrome virus

The pandemic of the porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses and continues to threaten the swine industry worldwide. Nucleocapsid protein (N protein) is the primary antigen of PRRSV for development of sensitive diagnostic assays. Two high affinity nanobodies against N protein, Nb12 and Nb35, were selected and employed to develop a sandwich ELISA. Further we improved the ELISA method to obtain greater sensitivity, a trivalent nanobody (3 × Nb35) and a bivalent nanobody-HRP fusion protein (2 × Nb12-HRP) were expressed and used. This modified ELISA was found to have high sensitivity for detecting PRRSV, with a detection limit of 10 TCID50/ml (median tissue culture infectious dose), which was approximately 200-fold greater than the single-copy nanobody-based sandwich ELISA. The developed assay shows high specificity and can detect almost all circulating lineages of PRRSV-2 in China. This study provides suggestions for reforming nanobodies and for the further development of multivalent nanobody-based ELISAs for other various viruses.

A Pilot Study to Investigate the Antimicrobial Activity of Pulsed UVA and UVC

UV irradiation has shown potential in reducing bacterial and viral loadings. This is a pilot study aimed at investigating the antimicrobial effect of a novel pulsed UVA and UVC technology on bacteria and human coronavirus 229E. The selection of these microorganisms is based on their relevance and significance in real-world scenarios. This study consists of independent experiments for the assessment of antibacterial and antiviral activities by using a lawn plate approach, measuring levels of adenine triphosphate (ATP) in three bacterial strains, Escherichia coli, Staphylococcus epidermidis and Bacillus subtilis, and performing Median Tissue Culture Infectious Dose (TCID50) of HCoV-229E on MRC-5 human lung fibroblast cell line. The results demonstrated the ability of UVA and UVC irradiation to reduce levels of adenine triphosphate (ATP) over a 12 h exposure period in all three bacterial strains, comparative to dark and artificial/natural light conditions using non-pulsing experiments. In addition to this, there was a reduction in colonies exposed to UVA and UVC pulsing experiments for E. coli K12 and S. epidermis compared to bacteria stored in artificial/natural and dark conditions. Furthermore, using dose-dependent modelling, it was demonstrated that the cross-contamination risk was reduced by 50% using E. coli as a typical model. Regarding the antiviral assay, the results showed that TCID50 of HCoV-229E was reduced after the first cycle of UV engagement. No cytopathic effect (CPE) was detected after three cycles using Protocol 1. The findings showed that UVA and UVC were effective under the conditions outlined in this paper for a reduction in the number of bacteria with additional applications to viruses.

Feeding Saccharomyces cerevisiae fermentation postbiotic products alters immune function and the lung transcriptome of preweaning calves with an experimental viral-bacterial coinfection

Bovine respiratory disease causes morbidity and mortality in cattle of all ages. Supplementing with postbiotic products from Saccharomyces cerevisiae fermentation (SCFP) has been reported to improve growth and provide metabolic support required for immune activation in calves. The objective of this study was to determine effects of SCFP supplementation on the transcriptional response to coinfection with bovine respiratory syncytial virus (BRSV) and Pasteurella multocida in the lung using RNA sequencing. Twenty-three calves were enrolled and assigned to 2 treatment groups: control (n = 12) or SCFP-treated (n = 11, fed 1 g/d SmartCare in milk and 5 g/d NutriTek on starter grain; both from Diamond V Mills Inc.). Calves were infected with ∼104 median tissue culture infectious dose per milliliter of BRSV, followed 6 d later by intratracheal inoculation with ∼1010 cfu of Pasteurella multocida (strain P1062). Calves were euthanized on d 10 after viral infection. Blood cells were collected and assayed on d 0 and 10 after viral infection. Bronchoalveolar lavage (BAL) cells were collected and assayed on d 14 of the feeding period (preinfection) and d 10 after viral infection. Blood and BAL cells were assayed for proinflammatory cytokine production in response to stimulation with lipopolysaccharide (LPS) or a combination of polyinosinic:polycytidylic acid and imiquimod, and BAL cells were evaluated for phagocytic and reactive oxygen species production capacity. Antemortem and postmortem BAL and lesioned and nonlesioned lung tissue samples collected at necropsy were subjected to RNA extraction and sequencing. Sequencing reads were aligned to the bovine reference genome (UMD3.1) and edgeR version 3.32.1 used for differential gene expression analysis. Supplementation with SCFP did not affect the respiratory burst activity or phagocytic activity of either lung or blood immune cells. Immune cells from the peripheral blood of SCFP-supplemented calves produced increased quantities of IL-6 in response to toll-like receptor stimulation, whereas cells from the BAL of SCFP-treated calves secreted fewer proinflammatory cytokines and less tumor necrosis factor-α (TNF-α) and IL-6 in response to the same stimuli. Transcriptional responses in lung tissues and BAL samples from SCFP-fed calves differed from the control group. The top enriched pathways in SCFP-treated lungs were associated with decreased expression of inflammatory responses and increased expression of plasminogen and genes involved in glutathione metabolism, supporting effective lung repair. Our results indicate that supplementing with SCFP postbiotics modulates both systemic and mucosal immune responses, leading to increased resistance to bovine respiratory disease.

Virucidal activity of trehalose 6-monolaurate against dengue virus in vitro.

Dengue fever is an acute febrile disease caused by dengue virus (DENV) infection. Over the past 60 years, DENV has spread throughout tropical regions and currently affects more than 50% of the world's population; however, there are as of yet no effective anti-DENV drugs for clinical treatment. A number of research teams have investigated derivatives of glycolipids as possible agents for the inhibition of DENV. Our objective in this research was to study the antiviral effects of trehalose 6-caprate (TMC), trehalose 6-monolaurate (TML), and trehalose 6-monooleate (TMO), based on reports that the corresponding glycosyl, trehalose, reduces the transmission of Zika virus (ZIKV). We also sought to elucidate the molecular mechanisms underlying inhibition using the RNA isolation and reverse transcription-quantitative polymerase chain reaction, western blot analysis, median tissue culture infectious dose (TCID50 ) assay, and immunofluorescence assayand immunochemistrystaining, in vitro. This is the first study to demonstrate the TML-induced inhibition of DENV serotype 2 (DENV-2) in a dose-dependent manner. The inhibitory effects of TML in the pretreated, cotreated, and full-treated groups were confirmed using time of addition assays. We determined that TML restricted viral binding, entry, replication, and release. We also confirmed the efficacy of TML against three clinical isolates of DENV serotypes 1, 3, and 4 (DENV-1, DENV-3, and DENV-4). The findings obtained in this study identify TML as a promising candidate for the development of drugs to treat DENV infection.

The WUR0000125 PRRS resilience SNP had no apparent effect on pigs' infectivity and susceptibility in a novel transmission trial.

Porcine reproductive and respiratory syndrome (PRRS) remains one of the most important infectious diseases for the pig industry. A novel small-scale transmission experiment was designed to assess whether the WUR0000125 (WUR for Wageningen University and Research) PRRS resilience single nucleotide polymorphism (SNP) confers lower susceptibility and infectivity to pigs under natural porcine reproductive and respiratory syndrome virus (PRRSV-2) transmission. Commercial full- and half-sib piglets (n = 164) were assigned as either Inoculation, Shedder, or Contact pigs. Pigs were grouped according to their relatedness structure and WUR genotype, with R- and R+ referring to pigs with zero and one copy of the dominant WUR resilience allele, respectively. Barcoding of the PRRSV-2 strain (SD09-200) was applied to track pig genotype-specific transmission. Blood and nasal swab samples were collected and concentrations of PRRSV-2 were determined by quantitative (q)-PCR and cell culture and expressed in units of median tissue culture infectious dose (TCID50). The Log10TCID50 at each sampling event, derived infection status, and area under the curve (AUC) were response variables in linear and generalized linear mixed models to infer WUR genotype differences in Contact pig susceptibility and Shedder pig infectivity. All Shedder and Contact pigs, except one, became infected through natural transmission. There was no significant (p > 0.05) effect of Contact pig genotype on any virus measures that would indicate WUR genotype differences in susceptibility. Contact pigs tended to have higher serum AUC (p = 0.017) and log10TCID50 (p = 0.034) when infected by an R+ shedder, potentially due to more infectious R+ shedders at the early stages of the transmission trial. However, no significant Shedder genotype effect was found in serum (p = 0.274) or nasal secretion (p = 0.951) that would indicate genotype differences in infectivity. The novel design demonstrated that it is possible to estimate genotype effects on Shedder pig infectivity and Contact pig susceptibility that are not confounded by family effects. The study, however, provided no supportive evidence that genetic selection on WUR genotype would affect PRRSV-2 transmission. The results of this study need to be independently validated in a larger trial using different PRRSV strains before dismissing the effects of the WUR marker or the previously detected GBP5 gene on PRRSV transmission.

A culture-free method for rapidly and accurately quantifying active SARS-CoV-2.

Determining the quantity of active virus is the most important basis to judge the risk of virus infection, which usually relies on the virus median tissue culture infectious dose (TCID50) assay performed in a biosafety level 3 laboratory within 5-7 days. We have developed a culture-free method for rapid and accurate quantification of active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by targeting subgenomic RNA (sgRNA) based on reverse transcription digital PCR (RT-dPCR). The dynamic range of quantitative assays for sgRNA-N and sgRNA-E by RT-dPCR was investigated, and the result showed that the limits of detection (LoD) and quantification (LoQ) were 2 copies/reaction and 10 copies/reaction, respectively. The delta strain (NMDC60042793) of SARS-CoV-2 was cultured at an average titer of 106.13 TCID50/mL and used to evaluate the developed quantification method. Copy number concentrations of the cultured SARS-CoV-2 sgRNA and genomic RNA (gRNA) gave excellent linearity (R2 = 0.9999) with SARS-CoV-2 titers in the range from 500 to 105 TCID50/mL. Validation of 63 positive clinical samples further proves that thequantification of sgRNA-N by RT-dPCR is more sensitive for active virus quantitative detection. It is notable that we can infer the active virus titer through quantification of SARS-CoV-2 sgRNA based on the linear relationship in a biosafety level 2 laboratory within 3 h. It can be used to timely and effectively identify infectious patients and reduce unnecessary isolation especially when a large number of COVID-19 infected people impose a burden on medical resources.

Comparative characterization of Crimean-Congo hemorrhagic fever virus cell culture systems with application to propagation and titration methods

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is a biosafety level 4 and World Health Organization top priority pathogen. Infection leads to an often fatal hemorrhagic fever disease in humans. The tick-borne virus is endemic in countries across Asia, Europe and Africa, with signs of spreading into new regions. Despite the severity of disease and the potential of CCHFV geographic expansion to cause widespread outbreaks, no approved vaccine or treatment is currently available. Critical for basic research and the development of diagnostics or medical countermeasures, CCHFV viral stocks are commonly produced in Vero E6 and SW-13 cell lines. While a variety of in-house methods are being used across different laboratories, there has been no clear, specific consensus on a standard, optimal system for CCHFV growth and titration. In this study, we perform a systematic, side-by-side characterization of Vero E6 and SW-13 cell lines concerning the replication kinetics of CCHFV under different culture conditions. SW-13 cells are typically cultured in a CO2-free condition (SW-13 CO2−) according to the American Type Culture Collection. However, we identify a CO2-compatible culture condition (SW-13 CO2+) that demonstrates the highest viral load (RNA concentration) and titer (infectious virus concentration) in the culture supernatants, in comparison to SW-13 CO2− and Vero E6 cultures. This optimal viral propagation system also leads to the development of two titration methods: an immunostaining-based plaque assay using a commercial CCHFV antibody and a colorimetric readout, and an antibody staining-free, cytopathic effect-based median tissue culture infectious dose assay using a simple excel calculator. These are anticipated to serve as a basis for a reproducible, standardized and user-friendly platform for CCHFV propagation and titration.