World Organization for Animal Health has listed bluetongue (BT) under notifiable diseases. The BT is an arboviral infectious disease of domestic and wild ruminants caused by the bluetongue virus (BTV). Southern states of India had remained the point of attention for BT since first presence in 1964 in Maharashtra. Recently, northern states of India have also been reported positive for BTV in small ruminants. The present study reported the dual infection of BTV serotypes, BTV-12 and -16 in sheep population from Sirsa district of Haryana in the year 2016. After detection and serotyping with Seg-2 specific real time polymerase chain reaction (PCR), the Seg-2 and Seg-6 of BTV were PCR amplified and sequenced. On phylogenetic analysis it was detected to be clustered in nucleotype G and nucleotype B specific for BTV-12 and BTV-16, respectively. This was the first report of BTV-16 from Haryana. The results signified the co-infection of two different serotypes in an animal from a single outbreak.

The rocketing number of COVID-19 cases highlighted the critical role that diagnostic tests play in medical and public health decision-making to contain and mitigate the SARS-CoV-2 pandemic. This study reports the evaluation and implementation of different tests for the molecular detection of SARS-CoV-2 in the central region of Argentina. We evaluated 3 real time RT-PCR kits (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection Kit and WGene SARS-CoV-2 RT Detection), 2 nucleic acid extraction methods [MagaBio plus Virus DNA/RNA Purification Kit II (BioFlux), 35-min vs. 9-min], a pre-analytical reagent (FlashPrep®) and 2 isothermal amplification tests (Neokit Plus and ELA CHEMSTRIP®). The order according to the best performance of the 3 real-time RT-PCR kits evaluated was: DisCoVery>GeneFinderTM>WGene. The 2 RNA extraction methods showed similar good results: MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min was selected due to its faster performance. FlashPrep® reagent showed excellent results to perform direct RNA detection. Isothermal amplification assays showed acceptable sensitivity and specificity values (>80%), except in samples with Ct>30. Our data show optimal real time RT-PCR kits and alternative molecular methods for SARS-CoV-2 diagnostic. These alternative assays proved to be acceptable for their use in adverse contexts, decentralization, and different epidemiological scenarios, for rapid and accurate SARS-CoV-2 detection.

UNASSIGNED: Bovine coronavirus (BCoV) is a causative agent of enteric and respiratory diseases in cattle. Despite its importance for animal health, no data is available on its prevalence in Poland. The aim of the study was to determine the virus\' seroprevalence, identify risk factors of BCoV exposure in selected cattle farms and investigate the genetic variability of circulating strains.
UNASSIGNED: Serum and nasal swab samples were collected from 296 individuals from 51 cattle herds. Serum samples were tested with ELISA for the presence of BCoV-, bovine herpesvirus-1 (BoHV-1)- and bovine viral diarrhoea virus (BVDV)-specific antibodies. The presence of those viruses in nasal swabs was tested by real-time PCR assays. Phylogenetic analysis was performed using fragments of the BCoV S gene.
UNASSIGNED: Antibodies specific to BCoV were found in 215 (72.6%) animals. Seropositivity for BCoV was more frequent (P>0.05) in calves under 6 months of age, animals with respiratory signs coinfected with BoHV-1 and BVDV and increased with herd size. In the final model, age and herd size were established as risk factors for BCoV-seropositivity. Genetic material of BCoV was found in 31 (10.5%) animals. The probability of BCoV detection was the highest in medium-sized herds. Polish BCoVs showed high genetic homology (98.3-100%) and close relatedness to European strains.
UNASSIGNED: Infections with BCoV were more common than infections with BoHV-1 and BVDV. Bovine coronavirus exposure and shedding show age- and herd density-dependence.

SARS-CoV-2 can be shed in feces and can enter sewage systems. In order to implement effective control measures and identify new channels of transmission, it is essential to identify the presence of infectious virus particles in feces and sewage. In this study, we attempt to utilize Molecular techniques, cell cultures and animal models to find out the infectivity of SARS-CoV-2 in the feces of COVID-19 patients. Our findings exclude the presence of infectious virus particles, suggesting that fecal-oral transmission may not be the main mode of transmission. Larger-scale initiatives are nevertheless required, particularly considering the emergence of new viral strains.

SARS-CoV-2 variants of concern (VOC) and interest (VOI) present mutations in reference to the original virus, being more transmissible. We implemented a rapid strategy for the screening of SARS-CoV-2 VOC/VOIs using real time RT-PCR and performed monitoring and surveillance of the variants in our region. Consecutive real-time RT-PCRs for detection of the relevant mutations/deletions present in the Spike protein in VOC/VOIs (TaqMan™ SARS-CoV-2 Mutation Panel, Applied Biosystems) were implemented. A total of 6,640 SARS-CoV-2 RNA samples (Cts < 30) from infected individuals in Central Argentina during 2021 were analyzed using different algorithms that were gradually adapted to the changing scenarios of local variant circulation. The strategy developed allowed the early detection and the identification of VOC/VOIs that circulated through the year, with a 100% of concordance with the WGS. The analyses of the samples showed introductions of VOCs Alpha and Gamma in February and March 2021, respectively. Gamma showed an exponential increase, with a peak of detection in July (72%), being responsible of the second wave of COVID19 in Argentina. Since VOC Delta entered into the region, it increased gradually, together with VOI Lambda, replacing VOC Gamma, until being the main variant (84.9%) on November. By December, these variants were replaced by the emergent VOC Omicron in a term of 2 weeks, producing the third wave. We report a useful tool for VOC/VOI detection, capable to quickly and cost-effectively monitor currently recognized variants in resource-limited settings, which allowed to track the recent expansion of Omicron in our region, and contributed to the implementation of public health measures to control the disease spread.

The main route of the transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are through respiratory pathways and close contact of human-to-human. While information about other modes of transmission is comparatively less, some published literature supporting the likelihood of a fecal-oral mode of transmission has been accumulating. The diagnosis of SARS-COV-2 infected cases is based on the real-time reverse transcription-PCR (RT-PCR). The fecal excretion of SARS-COV-2 has been reported frequently, however, the role of fecal viral load with the severity of disease is not yet clear. Our study focused on the investigation of SARS-CoV-2 shedding in the fecal samples of patients with coronavirus disease 2019 (COVID-19). A total of 280 RT-PCR-positive patients were enrolled, among them 15.4% had gastrointestinal (GI) symptoms. It was shown that 62% of the patients were positive for SARS-CoV-2 RNA in fecal specimens. This positivity was not related to the presence of GI symptoms and the severity of disease. The next generation sequencing [NGS] of SARS-CoV-2 from fecal samples of patients was performed to analyze mutational variations. Findings from this study not only emphasized the potential presence of SARS-CoV-2 in feces, but also its continuing mutational changes and its possible role in fecal-oral transmission.

METHODS: Dietary fat composition is an important modulator of vascular function. Non-esterified fatty acids (NEFA) enriched in saturated fatty acids (SFA) are thought to reduce vascular reactivity by attenuating insulin signalling via vasodilator pathways (phosphoinositide 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS)) and enhancing signalling via pro-inflammatory pathways.
METHODS: To examine the effects of fatty acids on these pathways, human aortic endothelial cells were incubated with single fatty acids, and mixtures of these fatty acids to mimic typical NEFA composition and concentrations achieved in our previous human study. RNA was extracted to determine gene expression using real-time RT-PCR and cell lysates prepared to assess protein phosphorylation by Western blotting.
RESULTS: Oleic acid (OA, 100 µM) was shown to down regulate expression of the insulin receptor, PTEN and a PI3K catalytic (p110β) and regulatory (p85α) subunit compared to palmitic, linoleic and stearic acids (P < 0.04), and promote greater eNOS phosphorylation at Ser1177. Both concentration and composition of the SFA and SFA plus n-3 polyunsaturated fatty acids (PUFA) mixtures had significant effects on genes involved in the PI3K/Akt pathway. Greater up-regulation was found with 800 than 400 µM concentration (respective of concentrations in insulin resistant and normal individuals), whereas greater down-regulation was evident with SFA plus n-3 PUFA than SFA mixture alone.
CONCLUSIONS: Our findings provide novel insights into the modulation of the PI3K/Akt/eNOS pathway by single fatty acids and fatty acid mixtures. In particular, OA appears to promote signalling via this pathway, with further work required to determine the primary molecular site(s) of action.

The gold standard for diagnosis of SARS-CoV-2 infection has been nucleic acid amplification tests (NAAT). However, rapid antigen detection kits (Ag-RDTs), may offer advantages over NAAT in mass screening, generating results in minutes, both as laboratory-based test or point-of-care (POC) use for clinicians, at a lower cost. We assessed two different POC Ag-RDTs in mass screening versus NAAT for SARS-CoV-2 in a cohort of pediatric patients admitted to the Pediatric Emergency Unit of IRCCS-Polyclinic of Sant\'Orsola, Bologna (from November 2020 to April 2021). All patients were screened with nasopharyngeal swabs for the detection of SARS-CoV-2-RNA and for antigen tests. Results were obtained from 1146 patients. The COVID-19 Ag FIA kit showed a baseline sensitivity of 53.8% (CI 35.4-71.4%), baseline specificity 99.7% (CI 98.4-100%) and overall accuracy of 80% (95% CI 0.68-0.91); the AFIAS COVID-19 Ag kit, baseline sensitivity of 86.4% (CI 75.0-93.9%), baseline specificity 98.3% (CI 97.1-99.1%) and overall accuracy of 95.3% (95% CI 0.92-0.99). In both tests, some samples showed very low viral load and negative Ag-RDT. This disagreement may reflect the positive inability of Ag-RDTs of detecting antigen in late phase of infection. Among all cases with positive molecular test and negative antigen test, none showed viral loads > 106 copies/mL. Finally, we found one false Ag-RDTs negative result (low cycle thresholds; 9 × 105 copies/mL). Our results suggest that both Ag-RDTs showed good performances in detection of high viral load samples, making it a feasible and effective tool for mass screening in actively infected children.

SARS-CoV-2 infection was monitored in 1898 health care workers (HCWs) after receiving full vaccination with BNT162b2. Untill 30 June 2021, 10 HCWs tested positive for SARS-CoV-2 using real time RT-PCR, resulting in a 4-month cumulative incidence of 0.005%. The infection was mildly symptomatic in six (60%) and asymptomatic in four (40%) individuals. Among the infected HCWs, eight consenting individuals provided paired NPS and saliva during the course of infection, for the purpose of the analysis performed in the present study. Genomic and subgenomic viral RNAs were investigated using real-time RT-PCR in both biological specimens. The temporal profile of viral load was measured using ddPCR. Viral mutations were also analysed. Subgenomic viral RNA was detected in 8/8 (100%) NPS and in 6/8 (75%) saliva specimens at the baseline. The expression of subgenomic RNA was observed for up to 7 days in 3/8 (38%) symptomatic cases. Moreover, concordance was observed between NPS and saliva in the detection of viral mutations, and both N501Y and 69/70del (associated with the B.1.1.7 variant) were detected in the majority 6/8 (75%) of subjects, while the K417T mutation (associated with the P.1-type variants) was detected in 2/8 (25%) individuals. Overall, our findings report a low frequency of infected HCWs after full vaccination. It is, therefore, important to monitor the vaccinees in order to identify asymptomatic infected individuals. Saliva can be a surrogate for SARS-CoV-2 surveillance, particularly in social settings such as hospitals.

To evaluate the expression of genes encoding cytokines, grow factors and cell cycle regulators in the proliferative endometrium of women with chronic endometritis (CE) compared to controls. We performed a case-control study on seven women with CE as diagnosed by hysteroscopy and histology (Cases) compared to six women without CE (Controls). All women underwent diagnostic hysteroscopy plus endometrial biopsy during the mid-proliferative phase of the menstrual cycle. Endometrial samples were divided into two different aliquots for histological and molecular analyses. The endometrial expression profile of 16 genes encoding proteins involved in the inflammatory process, proliferation and cell cycle regulation/apoptosis was assessed by using high-throughput qPCR. Study endpoints were between-group differences in the expression of VEGF A, VEGF B, VEGF C, EGF, TNF, TGF B1, IFNG, TP73, TP73L, BAXva, CDC2, CDC2va, CCND3, CCNB1, BAX and IL12. RESULTS: VEGF A, VEGF B, VEGF C, EGF, TNF, TGF B1, IFNG, TP73, TP73L, BAXva, CDC2, CDC2va, CCND3, CCNB1 were significantly overexpressed in women with CE compared to controls, while BAX and IL12 had similar expression between groups. In women with CE, we found an altered endometrial expression of genes involved in inflammatory, cell proliferation, and apoptosis processes. The dominance of proliferative and anti-apoptotic activity in CE may potentially promote the development of polyps and hyperplastic lesions.