Real-time RT Research Articles

ZAKGene Expression in Patients with Helicobacter pylori Infection.

ZAK protein is a member of the MLK family proteins defined as mediators in the cell cycle. A survey of ZAKgene expression in gastric antral epithelial cells (GAECs) of gastritis and gastric adenocarcinoma patients with Helicobacter pylori genotypes infection can elucidate carcinogenesis of H. pylori genotypes. In a case-control study, ZAKgene expression was evaluated in GAECs biopsy samples of gastritis and gastric adenocarcinoma patients with (n 23, 21) and without H. pylori infection (n 27, 32), respectively. Total RNA was extracted from each gastric antral biopsy samples and cDNA synthesized by using Takara kits. H. pylori virulence genes֝ cDNA were detected by traditional PCR and specific primers. The ZAKgene expression was measured using the relative Real-Time RT PCR. The prevalence of gastric adenocarcinoma was the highest in man and 61-85 aged groups (p < .05). There was no significant correlation between the prevalence of H. pylori infection and patients' demographic groups. This study showed that ZAKgene overexpression gradually increases with increasing age and tumor grade among gastric adenocarcinoma patients. The gastric antral biopsy samples with H. pylori vacA s1m2 genotype infection showed a weak correlation with ZAK gene overexpression (p < .1). ZAK gene expression was higher in GAECs of gastritis cancer than in gastric adenocarcinoma, indicating the protective effect of ZAK against gastric cancer (p < .005). Reducing ZAKgene expression shows the negative correlations with H. pylori infection and gastric adenocarcinoma.

Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification.

To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT—PCR (Reverse transcription—Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT—LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross‐reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called “COVIDISC” to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.

The real-time reproduction number, impact of interventions and prediction of the epidemic size of COVID-19 in the center of Iran.

Background:The monitoring of reproduction number over time provides feedback on the effectiveness of interventions and on the need to intensify control efforts. Hence, we aimed to compute basic (R0) and real-time (Rt) reproduction number and predict the trend and the size of the coronavirus disease 2019 (COVID-19) outbreak in the center of Iran.Materials and Methods:We used the 887 confirmed cases of COVID-19 from February 20, 2020, to April 17, 2020 in the center of Iran. We considered three scenarios for serial intervals (SIs) with gamma distribution. Rt was calculated by the sequential Bayesian and time-dependent methods. Based on a branching process using the Poisson distributed number of new cases per day, the daily incidence and cumulative incidence for the next 30 days were predicted. The analysis was applied in R packages 3.6.3 and STATA 12.0.Results:The model shows that the Rt of COVID-19 has been decreasing since the onset of the epidemic. According to three scenarios based on different distributions of SIs in the past 58 days from the epidemic, Rt has been 1.03 (0.94, 1.14), 1.05 (0.96, 1.15), and 1.08 (0.98, 1.18) and the cumulative incidence cases will be 360 (180, 603), 388 (238, 573), and 444 (249, 707) for the next 30 days, respectively.Conclusion:Based on the real-time data extracted from the center of Iran, Rt has been decreasing substantially since the beginning of the epidemic, and it is expected to remain almost constant or continue to decline slightly in the next 30 days, which is consequence of the schools and universities shutting down, reduction of working hours, mass screening, and social distancing.

Performance of Chip Based Real Time RT- PCR (TrueNat) and Conventional Real Time RT-PCR for Detection of SARS-CoV-2

Introduction: Coronavirus Disease-2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is ravaging the globe due to its rapid spread. Since providing fast results is of critical importance in a time of shortage of medical personnel and beds in isolation wards and to ensure timely treatment for patients, developing high quality rapid Point of Care (POC) diagnostics is essential. Aim: To compare the diagnostic performance of chip based real time Reverse Trancriptase Polymerase Chain Reaction (RT- PCR) (TrueNat) which has a shorter turnaround time compared to conventional real time RT-PCR in samples of suspected COVID-19 patients. Materials and Methods: The present cross-sectional observational study was carried out in a tertiary care hospital in New Delhi, India. Five hundred randomly selected Oropharyngeal (OP) swabs samples received from May-July 2020, were included in the study to compare the diagnostic performance of chip based real time RT- PCR (TrueNat) with conventional real time RT-PCR for diagnosis of SARS-CoV-2 infection. All statistical analysis was performed using STATA version 16.1 software (College station, Texas, USA). Results: The sensitivity of TrueNat test was 100% while the specificity was found to be 99.12% at 95% confidence intervals. The positive predictive value was 91.84% and the negative predictive value was 100%. Conclusion: The short turnaround time, good sensitivity and specificity makes TrueNat a reliable and affordable option to provide rapid results in cases requiring urgent interventions and to augment SARS-CoV-2 testing capacity at peripheral settings where sample load is less.

Statistical data driven approach of COVID-19 in Ecuador: and estimation via new method.

The growth of COVID-19 pandemic throughout more than 213 countries around the world have put a lot of pressures on governments and health services to try to stop the rapid expansion of the pandemic. During 2009, H1N1 Influenza pandemic, statistical and mathematical methods were used to track how the virus spreads around countries. Most of these models that were developed at the beginning of the XXI century are based on the classical susceptible–infected–recovered (SIR) model developed almost a hundred years ago. The evolution of this model allows us to forecast and compute basic and effective reproduction numbers (Rt and R0), measures that quantify the epidemic potential of a pathogen and estimates different scenarios.In this study, we present a traditional estimation technique for with statistical distributions by best fitting and a Bayesian approach based on continuous feed of prior distributions to obtain posterior distributions and computing real time . We use data from COVID-19 officially reported cases in Ecuador since the first confirmed case on February 29th. Because of the lack of data, in the case of we compare two methods for the estimation of these parameters below exponential growth and maximum likelihood estimation. We do not make any assumption about the evolution of cases due to limited information and we use previous methods to compare scenarios about and in the case of we used Bayesian inference to model uncertainty in contagious proposing a new modification to the well-known model of Bettencourt and Ribeiro based on a time window of days to improve estimations.Ecuadorian with exponential growth criteria was 3.45 and with the maximum likelihood estimation method was 2.93. The results show that Guayas, Pichincha and Manabí were the provinces with the highest number of cases due to COVID-19. Some reasons explain the increased transmissibility in these localities: massive events, population density, cities dispersion patterns, and the delayed time of public health actions to contain pandemic.In conclusion, this is a novel approach that allow us to measure infection dynamics and outbreak distribution when not enough detailed data is available. The use of this model can be used to predict pandemic distribution and to implement data-based effective measures.

Virological surveillance of SARS-CoV-2 in an Italian Northern area: differences in gender, age and Real Time RT PCR cycle threshold (Ct) values in three epidemic periods.

Background and aim of the work:Coronavirus Disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is a global public health emergency. The aim of this study was to investigate cases characteristics and Real Time RT PCR cycle threshold (Ct) values distribution of COVID-19 in an Italian Northern area during three periods: first period, February-May 2020; second period, June-August 2020; third period, September 2020-February 2021.Methods:Real Time RT PCR was used to detect SARS-CoV-2 in respiratory samples (oro/nasopharyngeal swabs).Results:A total of 254,744 samples were tested during the study period. Out of 20,188 positive samples (7.92%), 10,303 were females (51.04%) and 9,885 were males (48.96%). The percentage of positivity varied during the three different periods: 14.1% in the first period, 1.4% in the second and 9.2% in the third. The lowest Ct values were observed in the first phase of pandemic, with an overall average of 25.64. Overall average of the Ct values was lower in males than in females, 26.29 ± 6.04 and 26.84 ± 5.99 respectively. The oldest patients recorded lower Ct values.Conclusions:The findings of our study represent further evidence in support of the fact that male sex and older age showed lower Ct values, which means higher viral loads and higher infectious potential. These knowledges are useful to better understand the epidemiological aspects of COVID-19 and to perform effective Public Health Policies.

A household case evidences shorter shedding of SARS-CoV-2 in naturally infected cats compared to their human owners

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected in domestic and wild cats. However, little is known about natural viral infections of domestic cats, although their importance for modelling disease spread, informing strategies for managing positive human-animal relationships and disease prevention. Here, we describe the SARS-CoV-2 infection in a household of two human adults and sibling cats (one male and two females) using real-time RT–PCR, an ELISA test, viral sequencing, and virus isolation. On May 5th, 2020, the cat-owners tested positive for SARS-CoV-2. Two days later, the male cat showed mild respiratory symptoms and tested positive. Four days after the male cat, the two female cats became positive, asymptomatically. Also, one human and one cat showed antibodies against SARS-CoV-2. All cats excreted detectable SARS-CoV-2 RNA for a shorter duration than humans and viral sequences analysis confirmed human-to-cat transmission. We could not determine if cat-to-cat transmission also occurred.

In vitro and in vivo antiviral activity of a fluoronucleoside analog, NCC, against Coxsackie virus B3.

Coxsackie virus B3 (CVB3) is believed to be a major cause of viral myocarditis, with virus-induced apoptosis playing an important role in pathogenesis. The purpose of this study was to characterize the antiviral activity of a novel fluoronucleoside analogue, N-cyclopropyl-4'-azido-2'-deoxy-2'-fluoro-β-D-cytidine (NCC), against CVB3 in vitro and in vivo, and to establish whether NCC inhibits apoptosis in infected cells. In this study, HeLa cells infected with CVB3 were treated with NCC. Cell viability and apoptosis were examined. Caspase-3 and Bcl-2 levels were monitored by real-time RT PCR and Western blot analysis. For in vivo studies, BALB/c mice infected with CVB3 were treated with NCC daily. Serum markers of myocardial injury and histological studies were measured to examine myocardial injury on day 8 post-infection. To measure apoptosis, levels of Bcl-2 and caspase-3 were examined by immunohistochemistry and real-time RT-PCR. We found that NCC inhibited virus-mediated cytopathic effects in HeLa cells with an EC50 of 116.60 ± 0.32 μM. In infected mice, administration of NCC (2 mg/kg) decreased the activities of serum creatine kinase and lactic dehydrogenase, inhibited the replication of CVB3 and alleviated damage to the heart. Importantly, NCC suppressed CVB3-induced apoptosis in HeLa cells and affected the expression of apoptosis-related factors in infected mice. Together, our results demonstrate that NCC exerts significant antiviral activities against CVB3. We conclude that NCC is a potential therapeutic agent for the treatment of viral myocarditis. Keywords: coxsackie virus B3; viral myocarditis; N-cyclopropyl-4'-azido-2'-deoxy-2'-fluoro-β-D-cytidine; apoptosis.

754. Performance Characterization of a Real Time PCR Assay for Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid and Sputum

Background Pneumocystis jirovecii pneumonia (PJP) affects immunocompromised patients and contributes significantly to mortality. Outcomes depend on early treatment, making timely and accurate diagnosis critical. Typically, PJP diagnosis is through identification of trophozoite or cyst forms in bronchoalveolar lavage (BAL) fluid or sputum, a labor-intensive and insensitive process. Options for more accessible and sensitive molecular detection are limited. It is known that patients may be colonized, which can cast doubt on the clinical significance of low levels of DNA amplification in qualitative result reporting. In this study, we describe a real time (rt) PCR assay utilizing analyte specific reagent primers targeting the mtLSU gene of P. jirovecii and correlate amplification with morphological PJP identification.MethodsIUHPL Clinical Microbiology assessed sputum or BAL fluid from 109 patients with clinical concern for PJP microscopically via fungal stains (GMS, calcofluor white). Comparative rtPCR was conducted as follows. First, 2µL of residual specimen or control were mixed with an 8µL combination of rtPCR mastermix, control DNA, and primer pairs (Simplexa). No nucleic acid extraction was performed. Real time PCR was executed and analyzed on the LIAISON MDX (DiaSorin) platform. Qualitative amplification results and cycle threshold (CT) values were correlated with microscopic methods to establish performance. Chart review was performed to assess the clinical impact of this assay.Results P. jirovecii was microscopically detected in 26% (29/109) of samples, while 31.1% (34/109) exhibited amplification by rtPCR. Agreement between the two methods was 95.4%; rtPCR demonstrated 100% sensitivity and 93.8% specificity in comparison.ConclusionOur results indicate that this assay has exceptional negative predictive value (100%), and therefore may be valuable as a screening test. Considering this data alone, the positive predictive value is lower (85.3%). Further examination of the data, however, revealed that 80% (4/5) of discrepant results demonstrated CT values of >34, while the highest CT for a microscopically positive sample was 31.2. Further clinical correlation may establish a CT cutoff that will reduce false positive and potentially clinically insignificant cases.Disclosures All Authors: No reported disclosures

Host microRNAs regulate expression of hepatitis B virus genes during transmission from patients’ sperm to embryo

Human sperm nucleus contains diverse RNA populations. This study aimed to screen and identify host microRNAs (miRs) that regulate gene expression of hepatitis B virus (HBV) during transmission from patients’ sperm to sperm-derived embryos. Using microarrays, 336 miRs were found to be differentially expressed. After validation using real-time quantitative RT–PCR (RT-qPCR), four miRs were selected as targets. Using RT–qPCR and enzyme-linked immunosorbent assays, when patients’ sperm were treated with mimics (or inhibitors) specific for hsa-miR-19a-3p and hsa-miR-29c-3p, the S gene transcription in sperm and translation in sperm-derived embryos was downregulated (or upregulated). There were significant differences in transcriptional and translational levels of the S gene between the test and control groups. These findings suggest that hsa-miR-19a-3p and hsa-miR-29c-3p significantly suppressed expression of the S gene, offering potential therapeutic targets for treating patients with HBV infection, and further reducing the negative impact of HBV infection on sperm fertilizing capacity.

Characterization of mutations of Haemagglutinin (HA) and Neuraminidase (NA) genes of Influenza A H1N1pdm09 isolated during the 2018 outbreak in India

Background: The influenza virus is a segmented RNA virus, has higher propensity to undergo mutations, which carry major implications, both clinically and epidemiologically. Mutations in HA and NA are of particular importance. Changes in the HA region are associated with increased virulence, antigenicity and host specificity as it is involved in receptor binding and membrane fusion. Hence is a major target for vaccine development. The NA gene is involved in virus release as well as drug susceptibility. These changes can trigger the emergence of new strains, which could lead to epidemics and pandemics. Therefore, it is important to monitor and characterize the mutations closely. Thus, this study was undertaken to understand the genetic variation in Influenza H1N1 pdm09 for the outbreak in 2018. Methods and materials: This study conducted in Regional Influenza Laboratory, Puducherry on the isolates positive for Influenza A pdm09 by real time RT PCR. Conventional PCR was done using the WHO primers. The PCR positive products were then subjected to Sanger sequencing in ABI3500. The sequences were then analysed on the MEGA X software and Flusurver software for detection of the mutations in HA and NA. Results: Phylogenetic analysis was performed for both HA and NA gene along with the clade-specific reference strains. All our isolates clustered along with the clade 6B.1. In HA region, of the 28 mutations documented, the most common mutation was R240Q (100%) followed by S181T, S200P and S91R. The mutation of N173K was detected for the 1st time globally in the antigenic site. Of the 12 mutations in the HA stem region, 4 new mutations documented – K71Q, V466L, R467T and Q529R. The most common mutations in NA observed were G77R, I188T, S200G/V, F74S/L and V81A. We documented 4 new mutations that could be associated with drug resistance – W179G, R225K, V267L and R293K. Conclusion: Continuous surveillance of Influenza virus is important to monitor the changes in the HA genes for providing recommendations for vaccine strain. Further, as the treatment options for influenza is limited, it makes it all the more important to have continuous surveillance to track the antiviral resistance.

A Novel Molecule in Human Cyclic Endometrium: LncRNA TUNAR Is Involved in Embryo Implantation

Embryo implantation rate remains an inefficient process in in vitro fertilization and embryo transfer (IVF-ET) cycles. The role long non-coding RNA (lncRNA) plays in embryo implantation remains unclear. We aimed to investigate the expression pattern of lncRNA TCL1 upstream neural differentiation-associated RNA (TUNAR) in human cyclic endometrium and clarify the role of TUNAR in the development of endometrial receptivity. Endometrial biopsies were collected at the late proliferative phase, luteinizing hormone (LH) + 2 and LH + 7, from patients with or without recurrent implantation failure (RIF). Real-time RT PCR was performed to detect the level of lncRNAs. After pZW1-snoVector-TUNAR transfection, multiple function of TUNAR in endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) was investigated. The expression of TUNAR in endometrium was found down-regulated at LH + 7 and up-regulated in RIF patients. In proliferative phase, TUNAR was overwhelmingly more abundant in ESCs and regulated its proliferation. In LH + 7, the difference in the expression of TUNAR between ESCs and EECs was narrowed. Overexpression of TUNAR not only impaired spheroid attachment to EECs, but also inhibited decidualization of ESCs. TUNAR was found expressed in human endometrium for the first time, which might be involved in embryo implantation by modulating the blastocyst attachment to the endometrial epithelium and regulating the proliferation and decidualization of ESCs. Our study helps us to better understand the molecular mechanisms of embryo implantation and may provide a promising biomarker of endometrial receptivity.

Cell culture demonstrates superior sensitivity over one step real time RT PCR and nested VP1 amplification for Enteroviruses

This study evaluated and compared the sensitivity profile of routine cell culture, nested VP1 amplification and one step real time RT PCR for Enteroviruses. Serially diluted spiked samples of four model viruses (EV71, CVA16, CVB5 and PV1) and 32 true positive samples including Poliovirus (PV1 & PV3), Coxsackie virus (CVB5, CVB3, CVB1 & CVA4, 10, 16), Echovirus (Echo 6, 7, 11, 13, 18, 25 & 30) and Enterovirus 71 (E71), and 32 true negative stool samples were subjected to cell culture, nested RT PCR and one step real time RT PCR. The result of sensitivity test indicated superior sensitivity with one step real time RT PCR (75 %, 24/32) against cell culture (71.9 %, 23/32) and nested RT PCR (65.6 %, 21/32). The most specific test was cell culture (100 %, 32/32), followed by nested RT PCR (96.9 %, 31/32). Positive predictive values were 100 %: 23/23, 95.5 %; 21/22 and 88.9 %; 24/27, for cell culture, nested RT PCR and one step real time RT PCR, respectively, and one step real time RT PCR had the highest negative predictive value (78.4 %, 29/37). Overall result indicate relatively high analytical sensitivity with all the tests, suggesting superior performance by cell culture. Therefore, cell culture is the gold standard. However, considering intensive nature of cell cultures and prolong window for results, it is wise to consider one step real time RT PCR in routine diagnosis for its added advantages. Meanwhile, selecting a combination of tests can maximize detection, depending on the laboratory strength.

Sperm cryopreservation during the SARS-CoV-2 pandemic

PurposeSperm cryopreservation is fundamental in the management of patients undergoing gonadotoxic treatments. Concerns have risen in relation to SARS-CoV-2 and its potential for testicular involvement, since SARS-CoV-2-positive cryopreserved samples may have unknown effects on fertilization and embryo safety. This study therefore aimed to analyze the safety of sperm cryopreservation for cancer patients after the onset of the pandemic in Italy, through assessment of the risk of SARS-CoV-2 exposure and viral RNA testing of semen samples.MethodsWe recruited 10 cancer patients (mean age 30.5 ± 9.6 years) referred to our Sperm Bank during the Italian lockdown (from March 11th to May 4th 2020) who had not undergone a nasopharyngeal swab for SARS-CoV-2 testing. Patients were administered a questionnaire on their exposure to COVID-19, and semen samples were taken. Before cryopreservation, SARS-CoV-2 RNA was extracted from a 150 µl aliquot of seminal fluid in toto using QIAamp viral RNA kit (Qiagen) and amplified by a real time RT PCR system (RealStar SARS-CoV2 RT PCR, Altona Diagnostics) targeting the E and S genes.ResultsThe questionnaire and medical interview revealed that all patients were asymptomatic and had had no previous contact with COVID-19 infected patients. All semen samples were negative for SARS-CoV-2 RNA.ConclusionThis preliminary assessment suggests that a thorough evaluation (especially in the setting of a multidisciplinary team) and molecular confirmation of the absence of SARS-CoV-2 in seminal fluid from asymptomatic cancer patients may assist in ensuring the safety of sperm cryopreservation.

PARK7 Diminishes Oxidative Stress-Induced Mucosal Damage in Celiac Disease.

Coeliac disease (CD) is a chronic, immune-mediated small intestinal enteropathy, accompanied with gluten-triggered oxidative damage of duodenal mucosa. Previously, our research group reported an increased mucosal level of the antioxidant protein Parkinson's disease 7 (PARK7) in children with CD. In the present study, we investigated the role of increased PARK7 level on the epithelial cell and mucosal integrity of the small intestine. The presence of PARK7 was investigated using immunofluorescent staining on duodenal mucosa of children with CD and on FHs74Int duodenal epithelial cells. To investigate the role of oxidative stress, FHs74Int cells were treated with H2O2 in the absence or presence of Comp23, a PARK7-binding compound. Intracellular accumulation of reactive oxygen species (ROS) was determined by DCFDA-based assay. Cell viability was measured by MTT, LDH, and Annexin V apoptosis assays. Disruption of cytoskeleton and cell adhesion was investigated by immunofluorescence staining and by real-time RT PCR. Effect of PARK7 on mucosal permeability was investigated ex vivo using intestinal sacs derived from control and Comp-23-pretreated mice. Comp23 treatment reduced the H2O2-induced intracellular accumulation of ROS, thus preserving the integrity of the cytoskeleton and also the viability of the FHs74Int cells. Accordingly, Comp23 treatment increased the expression of antioxidants (NRF2, TRX1, GCLC, HMOX1, NQO1), cell-cycle regulators (TP53, CDKN1A, PCNA, BCL2, BAX), and cell adhesion molecules (ZO1, CDH1, VCL, ITGB5) of H2O2-treated cells. Pretreatment with Comp23 considerably decreased the small intestinal permeability. In this study, we demonstrate that PARK7-binding Comp23 reduces the oxidative damage of duodenal epithelial cells, via increased expression of NRF2- and P53-regulated genes. Our results suggest that PARK7 plays a significant role in the maintenance of mucosal integrity in CD.

No Significant Effect of COVID-19 on Immunological and Virological Parameters in Patients With HIV-1 Infection.

No Significant Effect of COVID-19 on Immunological and Virological Parameters in Patients With HIV-1 Infection.

EFFECT OF REGULAR EXERCISE ON EXPRESSION OF K ATP CHANNELS IN HEART OF DIABETIC RATS

There is abundant evidence that ATP sensitive potassium (KATP) channels play cytoprotective role in cardiac myocytes, allowing the cell to couple metabolic state to electrical activity of the cell membrane. In cardiac myocytes Kir6.2/SUR2A are the major subunits expressed. However, expression or function of KATP channels has been found to be impaired in the presence of persistent hyperglycemia in diabetes mellitus (DM). While regular exercise can improve hyperglycemic status in DM, its impact on the expression of KATP channels subunits in heart is unknown. PURPOSE: To assess the effect of regular exercise on expression of KATP channel subunits in heart of streptozotocin-induced diabetic rats. METHODS: Male Wistar rats (25 days old) were randomly divided into four groups, among them: sedentary control, trained control, sedentary diabetic, trained diabetic. Diabetes was induced by a single streptozotocin injection (100 mg/kg body weight), animals with fasting blood glucose levels ≥ 300 mg/dL were considered as diabetic. Groups with training program performed exercise on a treadmill (30 minutes daily, 5 days/week) for 8 weeks. At the end of the intervention, two subunits of cardiac KATP channel (SUR2A and Kir 6.2) were analyzed as indicators and quantitative analysis of these subunits was achieved with real-time RT–PCR. RESULTS: In control conditions, the regular exercise reduced Kir6.2 subunit mRNA levels significantly (76%; p=0.045) in heart. In diabetes, reduced Kir6.2 expression was also observed, and there was no difference in expression levels between sedentary diabetic and trained diabetic groups (P > 0.05). Otherwise, relative mRNA expression of the subunit SUR2A was increased in both sedentary diabetic and trained diabetic groups (80.33% and 86.08%, respectively). CONCLUSION: Collectively, our data demonstrate that the regular exercise modifies expression of KATP channel subunits of heart only in control conditions. However, the gene expression patterns of KATP channel subunits are different during diabetes, by increased SUR2A and decreased Kir6.2, which was not modified by exercise. These results may provide an opportunity to understand mechanisms leading to diabetic cardiomyopathy during stress and exercise in DM.

Protection following simultaneous vaccination with three or four different attenuated live vaccine types against infectious bronchitis virus

ABSTRACT Two or more different live attenuated infectious bronchitis virus (IBV) vaccine types are often given to broilers to induce homologous protection as well as to broaden protection against other IBV types in the field. However, the ability of broilers to respond to three or four different antigenic types of IBV vaccine has not been examined experimentally. In this study, we vaccinated one-day-old broiler chicks by eyedrop with three or four different IBV vaccine types simultaneously. The presence and relative amount of each vaccine was examined in all of the birds by IBV type-specific real-time RT–PCR at 5 days post-vaccination and each vaccine was detected in all of the birds given that vaccine. The birds were challenged at 28 days of age and protection was measured by clinical signs, virus detection and by ciliostasis. Birds vaccinated with three different IBV types (Ark, Mass and GA98) were protected against challenge with each of those IBV types and were partially protected against challenge with the GA08 virus. Birds vaccinated with four different IBV types (Ark, Mass, GA98 and GA08) were protected against challenge with each of those IBV types with the exception of Mass challenged birds which clearly had 3/11 birds not protected based on individual ciliostasis scores, but had an average ciliostasis score of >50% which is considered protected. The results are important for the control of IBV because they indicate that simultaneous vaccination with up to four different IBV vaccine types can provide adequate protection against challenge for each type.

Comparative Analysis of Early Dynamic Trends in Novel Coronavirus Outbreak: A Modeling Framework

Background: The 2019 coronavirus disease (COVID-19) represents a significant public health threat globally. Here we describe efforts to compare the dynamic transmissibility trends and epidemic size in China, as well as 9 countries in Asia, Europe, North America in the early epidemic phase. Methods: Using the time series of cases reported from January 20, 2020 to February 13, 2020 and transportation data from December 1, 2019 to January 23, 2020, we have built a novel time-varying growth model to predict the epidemic trend in China. We extended our method, using cases reported from January 26, 2020 - or the date of the first case reported, to March 15, 2020 to predict future epidemic trend and size in Japan, South Korea, Singapore, Iran, Italy, Germany, Spain, France, and the United States from January 26, 2020 to March 9, 2020. We compared the real-time effective reproduction number Rt of COVID-19 across countries. Findings: Here we predicted that the final epidemic size will be millions in Germany, Spain, and the United States, with more than 100,000 cases in Italy and France, and around 50,000 cases in Japan. The estimated epidemic would peak around April to July 2020. The duration will last on average 5-6 months in Europe and Asia, but longer in the United States at 10 months. The initial Rt at the onset of the epidemic is similar across all the countries (median 1.90 to 3.81) except the lowest in Singapore (median 0.53) and highest in Iran (10.96). Rt declined with time across all the countries, whereas it was reduced most rapidly in China with the lowest Rt in the fourth week (median 0.26 to 0.27). Effective intervention measures by reducing the infectious period in 2 days would result in a 4.0 to 49.3% reduction (median 43%) in the final epidemic size. Conclusions: A large-scale pandemic in Europe and the United States is inevitable. The heterogeneity in Rt reveals the impact of intervention measures in different countries. The control measures in South Korea, Singapore and China could lend further insight to other countries. Funding Statement: The research were partially supported by National Natural Science Foundation of China (Nos. 11931014 and 11829101) and Fundamental Research Funds for the Central Universities (No. JBK1806002) of China. Declaration of Interests: The authors declare no competing interests.

Geographical restriction of Hepatitis E virus circulation in wild boars (Sus scrofa) in Emilia-Romagna region, Northern Italy.

Hepatitis E virus (HEV) is a singlestrand RNA virus that causes an acute viral hepatitis in humans. Among its eight recognized genotypes, HEV-3 and HEV-4 are zoonotic, infecting humans, pigs and wild boars. Recently, HEV-3 has been also detected in red deer, which represents another reservoir of HEV. Consumption of raw pork products (mainly liver sausages), undercooked wild boar meat, raw wild boar liver and deer meat has been responsible for foodborne HEV human worldwide. From November 2018 to March 2019, liver samples collected from 97 wild boars hunted in Emilia-Romagna region (Northern Italy) were tested for HEV RNA. The hunting area included two territories for an extension of 33 km2, named A (about 13 km2,natural park, deciduous wood) and B (about 20 km2, cultivated fields in proximity of a river) areas. Distance between the two areas ranged between 8 to 10 km. A total of 73 wild boars were hunted in area A, and 24 in area B. HEV RNA was detected by Real-time RT– PCR in 23/73 liver samples of wild boars living in area A only (31.5% - 95% CI: 22.0-42.8%). The HEV sequences (n=13) clustered within genotype 3. The majority of positives belonged to animals < 12 months (12/25; 48%), followed by subadults (13-24 months) (7/16; 43.8%) and adults (4/32; 12.5%). This difference was found to be statistically significant (p=0.0024). In absence of pig farms, the restriction of HEV-positive animals to a well-defined territory of 13 km2 (Boschi di Carrega Regional Park) could hypothetically be related to the presence of red deer (Cervus elaphus), which lived in area A at the beginning of the hunting season. Further studies are needed to confirm or deny our hypothesis.