Serum RNA Research Articles

Macrophage Depletion Reactivates Fecal Virus Shedding following Resolution of Acute Hepatitis A in Ifnar1-/- Mice.

Although hepatitis A virus (HAV) is associated only with acute hepatitis in humans, HAV RNA persists within the liver for months following resolution of liver inflammation and cessation of fecal virus shedding in chimpanzees and murine models of hepatitis A. Here, we confirm striking differences in the kinetics of HAV RNA clearance from liver versus serum and feces in infected Ifnar1-/- mice and investigate the nature of viral RNA persisting in the liver following normalization of serum alanine aminotransferase (ALT) levels. Fecal shedding of virus produced in hepatocytes declined >3,000-fold between its peak at day 14 and day 126, whereas intrahepatic HAV RNA declined only 32-fold by day 154. Viral RNA was identified within hepatocytes 3 to 4 months after inoculation and was associated with membranes, banding between 1.07 and 1.14 g/cm3 in isopycnic iodixanol gradients. Gradient fractions containing HAV RNA demonstrated no infectivity when inoculated into naive mice but contained neutralizing anti-HAV antibody. Depleting CD4+ or CD8+ T cells at this late point in infection had no effect on viral RNA abundance in the liver, whereas clodronate-liposome depletion of macrophages between days 110 and 120 postinoculation resulted in a striking recrudescence of fecal virus shedding and the reappearance of viral RNA in serum coupled with reductions in intra-hepatic Ifnγ, Tnfα, Ccl5, and other chemokine transcripts. Our data suggest that replication-competent HAV RNA persists for months within the liver in the presence of neutralizing antibody following resolution of acute hepatitis in Ifnar1-/- mice and that macrophages play a key role in viral control late in infection. IMPORTANCE HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficient Ifnar1-/- mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA. Our study identifies an important role for macrophages in virus control following resolution of acute hepatitis A in Ifnar1-/- mice and may have relevance to relapsing hepatitis A in humans.

Manuscript Title: A 4-miRNAs Serum Panel for Obstructive Sleep Apnea Syndrome Screening.

Obstructive sleep apnea syndrome (OSAS) is a common chronic sleep disorder. OSAS is closely related to cardiovascular disease, metabolic disorders, cancer risk, and sudden death. This association has special significance in young people. Although it is known that OSAS has a great impact on physical health, it is estimated that 70-80% of patients with moderate-to-severe OSAS remain undiagnosed. Therefore, a new method for early diagnosis of the disease, the therapeutic effect of OSAS and prevention of complications to important. A total of 110 patients with moderate-to-severe OSAS diagnosed in the Sleep Disorders Diagnosis and Treatment Center of Peking University Shenzhen Hospital were selected. After excluding other diseases, 59 patients were finally selected as the OSAS group. In addition, 60 healthy people were selected as the control group. Serum RNA was then extracted. Eight RNA samples were randomly selected from the two groups for high-throughput miRNA sequencing. The 10 miRNAs with the greatest differences were selected as preselected markers from the results. Then, qRT-PCR was performed on the remaining RNA samples of the two groups to extract and verify the 10 miRNAs, and statistical analysis was performed between groups. A diagnostic panel was constructed by a stepwise logistic regression model combined with the expression data of miRNAs in the validation phase. A four-miRNA panel was identified to better predict OSAS, and the model was calculated using the following formula: Logit (P)= 0.77-1.65 × miR-486-5p - 4.56 × miR-148a-3p + 1.79 × miR-744-5p + 1.13 × let-7d-3p. The AUC for the four-miRNA panel was 0.955 (95% CI: 0.899 to 0.985; sensitivity = 91.38%, specificity = 91.38%). Gene Ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis was included in bioinformatic analysis. A 4-miRNAs panel as a diagnostic biomarker for OSAS screening is feasible.

Analytical and clinical validation of 3′ RACE RT-qPCR assay for detection and quantification of hepatitis B virus (HBV) serum RNA

BackgroundOver 296 million people worldwide are living with chronic Hepatitis B (CHB) infection who require monitoring of viral activity and disease progression. Serum HBV RNA is a promising new biomarker in CHB management. No standardized method for serum HBV RNA quantification has been established. ObjectivesTo develop and validate, both analytically and clinically, a 3′ RACE RT-qPCR assay for quantification of serum HBV RNA. Study designThe 3′ RACE RT-qPCR method was developed using published primers. The analytical limit of detection and quantification, linearity, inter- and intra-assay repeatability, and clinical specificity and sensitivity were evaluated using synthetic pre-genomic RNA and specimens from various patient populations. Intra- and inter-laboratory ring trials involving three laboratories were completed. ResultsThe 3′ RACE RT-qPCR assay dynamic range is 25 to 108 copies/µL of synthetic RNA. Theoretical and measured serum HBV RNA quantities from a serially diluted sample showed excellent linearity (R2=0.9795). The inter- and intra-assay repeatability were 94.91% and 95.12%, respectively. Clinical specificity and sensitivity were 100% and 90%, respectively, in treated patients. Inter-laboratory analysis demonstrated moderate to high agreement among participating laboratories (κ = 0.581 to 0.867). High agreement was also observed between both operators participating in the intra-laboratory evaluation (κ = 0.867). ConclusionsOur methodology for the quantification of serum HBV RNA is specific and repeatable and employs a biologically relevant RNA standard suitable for medium throughput laboratories. Method standardization is required to facilitate the comparison of studies and better understand the clinical role of this novel biomarker.

Feasibility of dried blood spot for hepatitis C diagnosis in vulnerable subjects and people living in remote areas from Brazil

BackgroundAgile, accessible and cheap diagnosis of hepatitis C virus (HCV) infection is essential to achieve the elimination of this infection, worldwide, as mandated by the World Health Organzation as part of its strategy for 2030. Dried blood spots (DBS) can be an attractive alternative for sample collection among people living in remote areas and vulnerable populations due to the less invasive collection, its biosafety, and storage & transportation of samples at room temperature.DesignThis study aims to estimate the usefulness of dried blood spot samples for the diagnosis and the assessment of HCV infection rates in three different settings in Brazil. Cross-sectional analysis of a sample collection from different populations, aiming to assess the performance of the testing algorithms and respective procedures among different populations with diverse background infection rates.MethodsWe reported the evaluation of DBS as alternative samples for detecting anti-HCV in different groups in real life conditions: (I) Vulnerable subjects living in remote areas of Southeast, North and Northeast Brazil (n = 1464); (II) Beauticians (n = 288); (III) People who use non-injectable drugs (n = 201); (IV) patients referred to outpatient care (n = 275).ResultsGeneral assay accuracy was 99%, with a weighted kappa value of 0.9, showing an excellent performance. Sensitivities ranged from 87.5% to 100.0% between groups and specificities were above 99.2%. A total of 194 individuals had HCV RNA in serum and concordance of anti-HCV detection in DBS was 98.4%.ConclusionsDBS samples could be used for anti-HCV detection in different populations recruited in real life conditions and ambulatory settings, with a high overall sensitivity and specificity.

Hepatitis D virus-induced interferon response and administered interferons control cell division-mediated virus spread.

Besides HBV-dependent de novo infection, cell division-mediated spread contributes to HDV persistence and dampens the effect of antivirals that abrogate de novo infection. Nonetheless, the combination of these antivirals with interferons (IFNs) showed strong synergism in recent clinical trials, implying a complementary mode-of-action of IFNs. Therefore, we investigated the effect of IFN response on cell division-mediated HDV spread. Cells infected with HDV were passaged to undergo cell division. The effect of the IFN response was evaluated by blocking HDV-induced IFN activation, by applying different IFN treatment regimens, and by adjusting HDV infection doses. Cell division-mediated HDV spread was highly efficient following infection of HuH7NTCP cells (defective in IFN production), but profoundly restricted in infected IFN-competent HepaRGNTCP cells. Treatment with IFN-α/-λ1 inhibited HDV spread in dividing HuH7NTCP cells, but exhibited a marginal effect on HDV replication in resting cells. Blocking the HDV-induced IFN response with the JAK1/2 inhibitor ruxolitinib or knocking down MDA5 augmented HDV spread in dividing HepaRGNTCP cells. The virus-induced IFN response also destabilized HDV RNA in dividing cells. Moreover, the effect of exogenous IFNs on cell division-mediated HDV spread was more pronounced at low multiplicities of infection with weak virus-induced IFN responses. Both HDV-induced IFN response and exogenous IFN treatment suppress cell division-mediated HDV spread, presumably through acceleration of HDV RNA decay. Our findings demonstrate a novel mode-of-action of IFN, explain the more pronounced effect of IFN therapy in patients with lower HDV serum RNA levels, and provide insights for the development of combination therapies. Chronic hepatitis D is a major health problem. The causative pathogen hepatitis D virus (HDV) can propagate through viral particle-mediated infection and the division of infected cells. Although viral particle-dependent infection can be blocked by recently developed drugs, therapies addressing the cell division route have not been reported. Taking advantage of relevant cell culture models, we demonstrate that the widely used immune modulator interferon can efficiently suppress HDV spread through cell division. This work unveils a new function of interferon and sheds light on potentially curative combination therapies.

Molecular detection and phylogenetic tree of echovirus in children at Thi-Qar province Iraq

The current study was conducted in Thi-Qar province, southern Iraq, A number of 100 blood samples collected from sick Children in Mohammed Al-Mousawi Hospital for Children (ages between 2 days - 3 years), males and females, during the period from November 2021 to February 2022.This study aimed to detects the presence of Echovirus RNA in patients serum by RT-PCR and compare sequences results of current study isolates with previously described isolates from other region of the world and then draw its phylogenetic tree. Based on the results recorded among 100 child have different symptoms only 9% of patients infected with Echovirus, the results also noted a significant difference in viral prevalence in babies patients. The results from the sequencing analysis indicated that three viral isolates belong to the human echovirus, out of nine positive samples for PCR analysis. The results of the viral sequencing showed that the Iraqi strain is identical to the American isolate, China's isolate and Russia's isolate ,all of them have 100% sequencing identical.

Presence of hepatitis E virus in testis of naturally infected wild boars.

The hepatitis E virus (HEV) is the main cause of viral acute hepatitis in the world, affecting more than 20 million people annually. During the acute phase of infection, HEV can be detected in various body fluids, which has a significant impact in terms of transmission, diagnosis or extrahepatic manifestations. Several studies have isolated HEV in the genitourinary tract of humans and animals, which could have important clinical and epidemiological implications. So, our main objective was to evaluate the presence of HEV in testis of naturally infected wild boars (Sus scrofa). For it, blood, liver, hepatic lymph node and testicle samples were collected from 191 male wild boars. The presence of HEV was evaluated in serum by PCR, as well as in tissues by PCR and immunohistochemistry. Four animals (2.09%; 95%CI: 0.82-5.26) showed detectable HEV RNA in serum, being confirmed the presence of HEV-3f genotype in three of them by phylogenetic analysis. HEV was also detected in liver and/or hepatic lymph nodes of the four animals by RT-PCR, as well as by immunohistochemistry analysis. Only one of these wild boars also showed detectable viral load in testis, observing HEV-specific labelling in a small number of fibroblasts and some Sertoli cells. Our results confirm the presence of HEV genotype 3 in naturally infected wild boar testis, although no associated tissue damage was evidenced. This study does not allow us to discard semen as a possible source of HEV transmission in suids. Future experimental studies are necessary to evaluate the impact of HEV genotype 3 on fertility and the possibility of transmission through sexual contact in this specie.

Insulin Resistance in Chronic Hepatitis C Non Diabetic Patients

Background: Patients with chronic hepatitis have impaired glucose metabolism with hyperinsulinemia and insulin resistance, this hyperinsulinemia has been shown to be due to decreased insulin catabolism rather than increased pancreatic insulin secretion. We aimed to evaluate insulin resistance in non-diabetic patients with chronic hepatitis C virus infection. Subjects and methods: The study was a case-control study conducted in Tropical Medicine and Gastroenterology Department King Abdullah University Hospital. 60 patients and 30 healthy controls were included in the study. The patients were classified into two groups: Group A: 30 patients with chronic hepatitis C infection were selected with positive HCV RNA in serum for at least 6 months; Patients were not receiving anti-viral therapy at the time of sampling. They showed no evidence of cirrhosis. Group B: 30 patients with HCV related liver cirrhosis. They were divided according to Child Pugh score; twenty patients with HCV related compensated liver cirrhosis (Child A). Ten patients with HCV related decompensated liver cirrhosis (Child B and C). Group C: The control group: included 30 healthy individuals. All patients and controls were subjected to the following: Liver function tests: Alanine transaminase (ALT), Aspartate transaminase (AST), total and direct bilirubin, total protein, serum albumin. Prothrombin time (PT) & international normalization ratio (INR). Renal function tests: Blood urea nitrogen (BUN), Na, K. Complete blood count. Alpha fetoprotein (αFP). Diagnosis of chronic hepatitis C infection was based on positive HCV by PCR, persistent elevation of liver enzymes more than 6 months and liver biopsy for some of the patients. Anti-hepatitis C virus antibody (HCV Ab) using third generation enzyme linked immune sorbant assay (ELISA) test., Hepatitis B virus (HBV): HBVsAg., Overnight fasting and two hours postprandial blood glucose level. Fasting serum insulin of everyone. Insulin resistance was determined via the Homeostasis Model assessment (HOMA-IR) by the following equation: - Insulin resistance: Fasting insulin (μu/ml) x Fasting glucose (mmol/L) 22.5 An index value of > 2.5 was defined as IR. This cutoff value was chosen because studies suggested that a HOMA-IR of 2.4-3.0 is probably suitable to define IR in CHC patients. Blood samples were collected after 12 hours of overnight fasting. Results: We found that out of 30 CHC and 30 LC (20 compensated LC, 10 de compensated LC) 8 (26.7%); 8 (40%) patients and 5(50%) respectively had HOMA-IR levels greater than 2.5, which is consistent with IR diagnosis. Decompensated cirrhotic patients showed higher frequency of IR compared to CHC and compensated cirrhotic patients. Conclusion: In chronic hepatitis C patients, HOMA-IR, fasting serum insulin and fasting blood glucose were significantly higher than healthy controls (p <0.0001).

Onset and Progression of Infection Based on Viral Loads in Rhesus Macaques Exposed to Zika Virus

Outbreaks of Zika virus (ZIKV) have resulted in a call by global health advocates for increased surveillance and research with aggressive measures to combat ZIKV infections. There is no licensed ZIKV vaccine yet available, but a number of vaccine candidates are in development. Advancement of promising vaccine candidates to licensure may rely upon the development and use of well-characterized preclinical models developed based on the essential elements of an animal model as outlined in the U.S. FDA “Product Development Under the Animal Rule: Guidance for Industry”. Further, in the absence of adequate clinical cases to support a more traditional approval pathway based on clinical efficacy, regulatory approval could be based upon human safety data and use of a well-characterized animal model to evaluate vaccine efficacy. This report summarizes a statistical analysis that characterizes the progression of ZIKV infection in Rhesus macaques (RMs) with respect to viral load using available data on twenty-six (26) RMs from three (3) studies that were exposed to ZIKV and were not immunized with a ZIKV vaccine. Progression of infection was characterized by time to detection of viral RNA in serum (RT-qPCR) or time to positive viremia (plaque assay). Viral RNA was detected via RT-qPCR as early as day 1 post-infection and was undetectable for all animals by day 7. Viremia also was indicated by plaque assay as early as day 1 and was undetectable for all animals by day 5. Viral RNA was detected in all animals following exposure, while viremia was not observed in all animals. No significant differences in viral loads measured by either RT-qPCR or plaque assay were observed across sex, age, or study. Neither sex nor age were significant predictors of either time to detection of viral RNA or time to positive viremia following exposure to ZIKV. Progression of viral load, which is studied since infection is largely asymptomatic in both RMs and humans, is similar in RMs and humans with positive presentation ranging from 1 to 7 days post-infection and clearance by day 10. Overall, due to consistency of the model across sexes and ages and similarity to the infection profile in humans, it is concluded that the RM model of ZIKV infection is a well-characterized model for use for evaluation of ZIKV countermeasures.

Transmission of yellow fever vaccine virus from breast feeding mothers to their infants: reporting of yellow fever virus (YFV) RNA detection in milk specimens.

Because of yellow fever's serious impact on health, vaccination is the principal strategy to control the disease. Administration of the yellow fever vaccine to breastfeeding women should be before they complete 9 months post-delivery, in order to prevent transmission of the yellow fever vaccine virus to their infants through breast feeding. This study aimed to confirm whether the excretion of yellow fever vaccine virus is in milk of vaccinated breastfeeding mothers and to confirm the probable transmission to their infants through breast milk. Samples were taken as follows: one serum specimen was taken 3-14 days after the date of the vaccination, and breast milk specimens were taken at four different time points between 3-4 days apart. Specimens were obtained from eight nursing mothers, who received the YVF vaccine (17DD). Mothers were asymptomatic before and after the vaccine administration but their infants developed symptoms after administration. Maternal serum samples were tested for YFV specific IgM antibodies through immuno-fluorescent assay (IFA). RNA was extracted from serum and breast milk specimens and YFV RNA screened using real-time polymerase chain reaction (RT-PCR). In total, five mothers (62.5%) were positive for YFV and two mothers (25%) had YFV RNA in serum. Among milk specimens, YFV RNA was detected during the four different mentioned collection times as follows (positive milk specimens/total milk specimens): 3/8 (37.5 %), 4/6 (66.6%) and 1/4(25%). RNA was completely undetectable in the last collection time. YFV transmission from mothers to their babies through breast-feeding was highly probable indicated by the temporal relationship to mother's YF vaccination.

Serum RNA biomarkers for predicting survival in non-human primates following thoracic radiation

In a mass radiation exposure, the healthcare system may rely on differential expression of miRNA to determine exposure and effectively allocate resources. To this end, miRNome analysis was performed on non-human primate serum after whole thorax photon beam irradiation of 9.8 or 10.7 Gy with dose rate 600 cGy/min. Serum was collected up to 270 days after irradiation and sequenced to determine immediate and delayed effects on miRNA expression. Elastic net based GLM methods were used to develop models that predicted the dose vs. controls at 81% accuracy at Day 15. A three-group model at Day 9 achieved 71% accuracy in determining if an animal would die in less than 90 days, between 90 and 269 days, or survive the length of the study. At Day 21, we achieved 100% accuracy in determining whether an animal would later develop pleural effusion. These results demonstrate the potential ability of miRNAs to determine thorax partial-body irradiation dose and forecast survival or complications early following whole thorax irradiation in large animal models. Future experiments incorporating additional doses and independent animal cohorts are warranted to validate these results. Development of a serum miRNA assay will facilitate the administration of medical countermeasures to increase survival and limit normal tissue damage following a mass exposure.

High prevalence of acute hepatitis E virus infection in pigs in Dutch slaughterhouses

Hepatitis E is caused by hepatitis E virus (HEV), one of the causes of acute viral hepatitis. Domestic pigs are considered as the main reservoir of HEV-3. The recently reported high prevalence of HEV in liver- and meat products on the Dutch market warranted a cross-sectional prevalence study on HEV infection among 5–6 months old pigs slaughtered in the Netherlands (n = 250). For this, liver, caecum content and blood samples were analyzed for the presence of genomic HEV RNA by RT-PCR. In addition, a serological test was performed to detect HEV IgG. Background information was retrieved on the corresponding farms to evaluate potential risk factors for HEV at pig slaughter age.HEV IgG was detected in sera from 167 pigs (67.6 %). HEV RNA was detected in 64 (25.6 %) caecum content samples, in 40 (16.1 %) serum samples and in 25 (11.0 %) liver samples. The average level of viral contamination in positive samples was log10 4.6 genome copies (gc)/g (range 3.0–8.2) in caecum content, log10 3.3 gc/ml (range 2.4–5.9) in serum and log10 3.2 gc/0.1 g (range 1.7–6.2) in liver samples. Sequence analyses revealed HEV-3c only. Ten times an identical strain was detected in two or three samples obtained from the same pig. Each animal in this study however appeared to be infected with a unique strain. The presence of sows and gilts and welfare rating at the farm of origin had a significant effect (p < 0.05) on the distribution over the four groups representing different stages of HEV infection based on IgG or RNA in caecum and/or serum. The observed proportion of tested pigs with viremia (16 %) was higher than in other reported studies and was interestingly often observed in combination with a high number of HEV genome copies in liver and caecum content as detected by RT-qPCR. Data provided will be useful for risk assessment for food safety of pork products, will provide baseline data for future monitoring of HEV infections in pigs and new thoughts for mitigation strategies.

SAT398 - Characterizing of hepatitis B virus serum RNA kinetics during TDF plus pegylated interferon alfa-2a with and without nucleic acid polymers

SAT398 - Characterizing of hepatitis B virus serum RNA kinetics during TDF plus pegylated interferon alfa-2a with and without nucleic acid polymers

Peripheral blood immune cell dynamics reflect antitumor immune responses and predict clinical response to immunotherapy

BackgroundDespite treatment advancements with immunotherapy, our understanding of response relies on tissue-based, static tumor features such as tumor mutation burden (TMB) and programmed death-ligand 1 (PD-L1) expression. These approaches are...

168 Asymptomatic dengue illness complicated by Guillain Barre syndrome

Arboviral infection Dengue can cause a spectrum of disease varying from asymptomatic illness to dengue fever to the severe dengue hemorrhagic fever. Neurological complications are rarely reported in the lit- erature (0.5–6%), mostly from dengue-endemic areas in tropics and subtropical regions. We report a case of a 59-year-old man who returned from a recent work-related travel from India. Three weeks following a mild viral prodrome, he developed bilateral lower motor neuron facial palsy with absent deep tendon reflexes. CSF showed albuminocytologic dissociation and electrophysiological studies showed demyeli- nating neuropathy. Routine GBS screen for common etiological viruses was negative and subsequently additional testing based on recent travel was performed which showed positive Dengue IgM and RNA in serum. The patient was treated with plasma exchange for 5 days and his facial weakness improved to a great extent.This case demonstrates the importance of considering arboviral infections such as Dengue in patients with GBS particularly in people living in or travelling from endemic areas. Additionally, like many other viral illnesses, there may not be clear clinical evidence of Dengue fever and the prodrome may go unnoticed and undiagnosed, hence a high index of suspicion is required.s_hasan_s@yahoo.com

Virological and Histopathological Findings in Boars Naturally Infected With Porcine Reproductive and Respiratory Syndrome Virus Type 1.

Major geographical transmission of porcine reproductive and respiratory syndrome virus (PRRSV) occurs via semen when a boar stud is infected. This happened in Denmark in 2019, providing an opportunity to compare previous experimental PRRSV boar studies with natural PRRSV-1 infection in boars. The aim of this study was to investigate the association between the presence of PRRSV RNA in serum, semen, testicles, and epididymis of boars naturally infected with PRRSV and to describe the histological lesions in the testes and epididymis combined with direct visualisation of PRRSV-infected cells by immunohistochemical staining (IHC). The exact timing of infection of each boar was not determined, but based on serology the boars were divided into two groups: acute and late infections. All boars included were sampled the same day. In this study, 35 boars and 10 healthy boars from another PRRSV-negative boar stud were included as histological controls. PRRSV RNA was found most often in serum (51%) and least frequently in semen (22%) and was more often detected in the reproductive tract in the acute phase of infection (p < 0.0001; RR: 2.58). Mononuclear cells and multinuclear giant cells were present in the adluminal compartment of the testis and epididymis in PRRSV-infected boars, but not in control boars (p < 0.05), which supports the hypothesis that macrophages are involved in the venereal spread of the virus.

Congested Heart Failure in Small Breeds Dogs

Mitral valve disease, commonly known as MVD, is the most common heart disease in dogs, accounting for nearly 75% of all dogs affected. The characteristic of this predominant disease includes collagen degradation, elastic fiber fragmentation and proteoglycan accumulation, leading to deterioration of the mitral valve and ultimately, congestive heart failure (CHF). Treatment to reverse or even delay the damaging effects of MVD has not yet been discovered due to a lack of awareness of the underlying processes. Furthermore, this disease in dogs has many similarities with the human MVD and may potentially serve as a translational model for studying MVD further. Therefore, it is imperative to identify the causes and underlying mechanisms associated with canine MVD in order to treat these patients more efficiently at the earliest detectable stage. MicroRNAs (miRNAs) are small, 21‐25 nucleotides in length, non‐coding RNAs that can down‐regulate the expression of protein coding genes by binding to the 3’UTR sites in their messenger RNAs (mRNA), causing either degradation or gene silencing. Prior studies have shown that one miRNA may regulate multiple mRNAs, subsequently effecting the downstream levels of gene expression. Furthermore, if miRNAs prove to be a useful biomarker of heart disease, not only may they serve as detectors of early disease onset, but they may also be useful as therapeutic agents. Given this recent data, we propose to test the hypothesis that circulating miRNAs may be useful biomarkers for detection of CHF, secondary to MVD. Liquid biopsies (blood samples) were collected in EDTA tubes from 7 healthy dogs with no prior history of heart disease and 7 dogs being treated for CHF secondary to MVD. Total serum RNA was isolated using miRNeasy Serum/Plasma Kit (Qiagen). Isolated RNA was then reverse transcribed using miScript II RT Kit (Qiagen) and lastly, miRNA was quantified using miScript SYBR Green PCR Kit, along with miScript miRNA PCR Arrays (Qiagen).

Prevalence of Occult Hepatitis C Virus Infection in Egyptian Patients with Lymphoma: A New Vision.

Occult hepatitis C virus infection (OCI) is the absence of HCV RNA in serum and the presence of actively replicating HCV RNA in hepatocytes and peripheral blood mononuclear cells (PBMCs), as evidenced by the presence of antigenomic negative sense single-stranded RNA. This study aimed to determine the prevalence of OCI in Egyptian lymphoma patients and assess changes in biochemical parameters in patients with confirmed OCI. The current study was conducted on 100 apparently healthy subjects as control group and 100 patients with lymphoma as a case group. HCV RNA was extracted and detected in both plasma and PBMCs using qRT-PCR. Total protein, albumin, ALT, AST, and total and direct bilirubin were measured in serum. OCI was detected in 6% of the patient group. OCI patients had lower levels of total protein and serum albumin and higher ALT and AST compared with lymphoma patients without OCI. Our study revealed that six out of 100 patients with lymphoma disorders had occult HCV infection (6%). Therefore, the possibility of this infection should be considered in patients with lymphoma.

Association Among the Gut Microbiome, the Serum Metabolomic Profile and RNA m6A Methylation in Sepsis-Associated Encephalopathy.

Objective: To investigate the relationship among the gut microbiome, serum metabolomic profile and RNA m6A methylation in patients with sepsis-associated encephalopathy (SAE), 16S rDNA technology, metabolomics and gene expression validation were applied. Methods: Serum and feces were collected from patients with and without (SAE group and non-SAE group, respectively, n = 20). The expression of serum markers and IL-6 was detected by enzyme-linked immunosorbent assay (ELISA), and blood clinical indicators were detected using a double antibody sandwich immunochemiluminescence method. The expression of RNA m6A regulator were checked by Q-RTPCR. The gut microbiome was analyzed by 16S rDNA sequencing and the metabolite profile was revealed by liquid chromatography-mass spectrometry (LC-MS/MS). Results: In the SAE group, the IL-6, ICAM-5 and METTL3 levels were significantly more than those in the non-SAE group, while the FTO levels were significantly decreased in the SAE group. The diversity was decreased in the SAE gut microbiome, as characterized by a profound increase in commensals of the Acinetobacter, Methanobrevibacter, and Syner-01 genera, a decrease in [Eubacterium]_hallii_group, while depletion of opportunistic organisms of the Anaerofilum, Catenibacterium, and Senegalimassilia genera were observed in both groups. The abundance of Acinetobacter was positively correlated with the expression of METTL3. The changes between the intestinal flora and the metabolite profile showed a significant correlation. Sphingorhabdus was negatively correlated with 2-ketobutyric acid, 9-decenoic acid, and l-leucine, and positively correlated with Glycyl-Valine [Eubacterium]_hallii_group was positively correlated with 2-methoxy-3-methylpyazine, acetaminophen, and synephrine acetonide. Conclusion: The gut microbiota diversity was decreased. The serum metabolites and expression of RNA m6A regulators in PBMC were significantly changed in the SAE group compared to the non-SAE group. The results revealed that serum and fecal biomarkers could be used for SAE screening.

Transmission of yellow fever vaccine virus from breast feeding mothers to their infants: reporting of yellow fever virus (YFV) RNA detection in milk specimens

Background: Because of yellow fever’s serious impact on health, vaccination is the principal strategy to control the disease. Administration of the yellow fever vaccine to breastfeeding women should be before they complete 9 months post-delivery, in order to prevent transmission of the yellow fever vaccine virus to their infants through breast feeding. This study aimed to confirm whether the excretion of yellow fever vaccine virus is in milk of vaccinated breastfeeding mothers and to confirm the probable transmission to their infants through breast milk. Methods: Samples were taken as follows: one serum specimen was taken 3-14 days after the date of the vaccination, and breast milk specimens were taken at four different time points between 3-4 days apart. Specimens were obtained from eight nursing mothers, who received the YVF vaccine (17DD). Mothers were asymptomatic before and after the vaccine administration but their infants developed symptoms after administration. Maternal serum samples were tested for YFV specific IgM antibodies through immuno-fluorescent assay (IFA). RNA was extracted from serum and breast milk specimens and YFV RNA screened using real-time polymerase chain reaction (RT-PCR). Results: In total, five mothers (62.5%) were positive for YFV IgM and two mothers (25%) had YFV RNA in serum. Among milk specimens, YFV RNA was detected during the four different mentioned collection times as follows (positive milk specimens/total milk specimens): 3/8 (37.5 %), 4/6 (66.6%) and 1/4(25%). RNA was completely undetectable in the last collection time. Conclusions: YFV transmission from mothers to their babies through breast-feeding was highly probable indicated by the temporal relationship to mother’s YF vaccination.