Nasopharyngeal and Peripheral Blood Type II Interferon Signature Evaluation in Infants during Respiratory Syncytial Virus Infection.

Background and Objectives: In this study, we applied one-step real time rt-PCR technology type II INF signature to blood and nasopharyngeal (NPS) swabs of acute early recovery children < 1 years hospitalized for bronchiolitis with laboratory-confirmed RSV infection. Materials and Methods: A prospective observational case-control study was conducted in 2021-2022. The study took place in Children Hospital "Regina Margherita", Torino Italy. The study included 66 infants, of which 30 patients were hospitalized for bronchiolitis due to RSV infection and 36 age-matched controls. Inclusion criteria included a positive RSV test for infants with bronchiolitis. We collected peripheral blood and nasopharyngeal swabs for relative quantification of type II Interferon signature by One-Step Multiplex PCR real time. Results: IFN levels were downregulated in the peripheral blood of bronchiolitis patients; these data were not confirmed in the nasopharyngeal swab. There was no correlation between NPS and the type II IFN score in peripheral blood. Conclusions: our study shows for the first time that type II IFN score was significant reduced in peripheral blood of infants with bronchiolitis by RSV compared to age-matched healthy controls; in the NPS swab this resulted downregulation was not statistically significant and the type II IFN score in the NPS swab can be used as marker of resolution of infection or improvement of clinical conditions.

Enhanced Expression of Human Endogenous Retroviruses, TRIM28 and SETDB1 in Autism Spectrum Disorder.

Human endogenous retroviruses (HERVs) are relics of ancestral infections and represent 8% of the human genome. They are no longer infectious, but their activation has been associated with several disorders, including neuropsychiatric conditions. Enhanced expression of HERV-K and HERV-H envelope genes has been found in the blood of autism spectrum disorder (ASD) patients, but no information is available on syncytin 1 (SYN1), SYN2, and multiple sclerosis-associated retrovirus (MSRV), which are thought to be implicated in brain development and immune responses. HERV activation is regulated by TRIM28 and SETDB1, which are part of the epigenetic mechanisms that organize the chromatin architecture in response to external stimuli and are involved in neural cell differentiation and brain inflammation. We assessed, through a PCR realtime Taqman amplification assay, the transcription levels of pol genes of HERV-H, -K, and -W families, of env genes of SYN1, SYN2, and MSRV, as well as of TRIM28 and SETDB1 in the blood of 33 ASD children (28 males, median 3.8 years, 25-75% interquartile range 3.0-6.0 y) and healthy controls (HC). Significantly higher expressions of TRIM28 and SETDB1, as well as of all the HERV genes tested, except for HERV-W-pol, were found in ASD, as compared with HC. Positive correlations were observed between the mRNA levels of TRIM28 or SETDB1 and every HERV gene in ASD patients, but not in HC. Overexpression of TRIM28/SETDB1 and several HERVs in children with ASD and the positive correlations between their transcriptional levels suggest that these may be main players in pathogenetic mechanisms leading to ASD.

Bufavirus, Cosavirus, and Salivirus in Diarrheal Italian Infants.

Three newly discovered viruses have been recently described in diarrheal patients: Cosavirus (CosV) and Salivirus (SalV), 2 picornaviruses, and bufavirus (BuV), a parvovirus. The detection rate and the role of these viruses remain to be established in acute gastroenteritis (AGE) in diarrheal Italian infants. From November 2016 to November 2017, stool samples were collected from 160 children <5 years old suffering from AGE and attending the Children's Hospital in Turin, Italy. During the study period, 1 (0.5%) sample was positive for 1 of the 3 investigated viruses: 0 (0%) CosV, 1 (0.5%) SalV, and 0 (0%) BuV, whereas 42 (26.0%) children were infected with rotavirus and 2 (1%) with adenovirus. No mixed infections involving the 3 viruses were found. Although these viruses are suspected to be responsible for AGE in children, our data showed that this association was uncertain. Therefore, further studies with large cohorts of healthy and diarrheal children will be needed to evaluate their clinical role in AGE.

TaqMan real time PCR for the Detection of the Gilbert's Syndrome Markers UGT1A1*28; UGT1A1*36 and UGT1A1*37.

Gilbert's syndrome is characterized by mild unconjugated hyperbilirubinemia. The key of this disease is a diminished activity of UDP-glucuronosyltransferase 1A1 (UGT1A1). TA insertion into the TATA box promoter region of the UGT1A1 gene on chromosome 2 is the genetic basis of Gilbert's syndrome (UGT1A1*28). An extra TA insert leads to eight (TA)8 repeats (UGT1A1*37) resulting in a further reduction of glucuronidation activity. A variant lacking one TA repeat (TA)5 (UGT1A1*36) has been identified. (TA)8 repeats (UGT1A1*37) and (TA)5 (UGT1A1*36) have been detected in Africans (frequency up to 0.07 and 0.08 respectively). We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR on 7500 and cfx96 Real-Time PCR System. We present a real time PCR method for genotyping the UGT1A1 (TA)n polymorphism (UGT1A1*28, UGT1A1*36, UGT1A1*37) using Taqman PCR. About clinical validation, all 53 samples collected from patients referred for suspected Gilbert's syndrome were analyzed. We found 21 on the 53 patients (39.6%) were homozygotes (UGT1A1-TATA (TA)6) and referred as wild-type, 13 on the 53 patients (24.5%) were homozygotes (UGT1A1-TATA (TA)7) and referred as mutated, 1 on the 53 patients (1.9%) were homozygotes (UGT1A1-TATA (TA)8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)7/8) and referred as mutated, 1 on the 53 patients (1.9%) were heterozygotes (UGT1A1-TATA (TA)5/6) and referred as mutated, and 16 on the 53 patients (30.2%) were heterozygotes (UGT1A1-TATA (TA)6/7). None were homozygotes UGT1A1-TATA (TA)5, homozygotes UGT1A1-TATA (TA)8, or heterozygotes with (TA)5 or (TA)8 alleles. The newly described technique represents a valid alternative method to sequencing, mainly due to its rapidity, easiness, and minor costs.

Overexpression of endogenous retroviruses in children with celiac disease.

Human endogenous retroviruses (HERVs) represent 8% of our genome. Although no longer infectious, they can regulate transcription of adjacent cellular genes, produce retroviral RNAs, and encode viral proteins that can modulate both innate and adaptive immune responses. Based on this, HERVs have been studied and proposed as contributing factors in various autoimmune disorders. Celiac disease (CD) is considered an autoimmune disease, but HERV expression has not been studied in celiac patients. The aim of this study is to assess the transcription levels of pol genes of HERV-H, -K, and -W and of their TRIM28 repressor in WBCs from celiac children and age-matched control subjects. A PCR real-time TaqMan amplification assay was used to evaluate HERV and TRIM28 transcripts with normalization of the results to glyceraldehyde-3-phosphate dehydrogenase. The RNA levels of pol genes of the three HERV families were significantly higher in WBCs from 38 celiac patients than from 51 control subjects. TRIM28 transcription was comparable between the two study populations.Conclusion: Present results show, for the first time, that pol genes of HERV-H, -K, and -W are overexpressed in patients with CD. Given their proinflammatory and autoimmune properties, this suggests that HERVs may contribute to the development of CD in susceptible individuals. What is Known: • Based on this, HERVs have been studied and proposed as contributing factors in various autoimmune disorders. What is New: • Present results show, for the first time, that pol genes of HERV-H, -K, and -W are overexpressed in patients with CD.

COVID-19 in Children: Expressions of Type I/II/III Interferons, TRIM28, SETDB1, and Endogenous Retroviruses in Mild and Severe Cases.

Children with the new coronavirus disease 2019 (COVID-19) have milder symptoms and a better prognosis than adult patients. Several investigations assessed type I, II, and III interferon (IFN) signatures in SARS-CoV-2 infected adults, however no data are available for pediatric patients. TRIM28 and SETDB1 regulate the transcription of multiple genes involved in the immune response as well as of human endogenous retroviruses (HERVs). Exogenous viral infections can trigger the activation of HERVs, which in turn can induce inflammatory and immune reactions. Despite the potential cross-talks between SARS-CoV-2 infection and TRIM28, SETDB1, and HERVs, information on their expressions in COVID-19 patients is lacking. We assessed, through a PCR real time Taqman amplification assay, the transcription levels of six IFN-I stimulated genes, IFN-II and three of its sensitive genes, three IFN-lIIs, as well as of TRIM28, SETDB1, pol genes of HERV-H, -K, and -W families, and of env genes of Syncytin (SYN)1, SYN2, and multiple sclerosis-associated retrovirus (MRSV) in peripheral blood from COVID-19 children and in control uninfected subjects. Higher expression levels of IFN-I and IFN-II inducible genes were observed in 36 COVID-19 children with mild or moderate disease as compared to uninfected controls, whereas their concentrations decreased in 17 children with severe disease and in 11 with multisystem inflammatory syndrome (MIS-C). Similar findings were found for the expression of TRIM-28, SETDB1, and every HERV gene. Positive correlations emerged between the transcriptional levels of type I and II IFNs, TRIM28, SETDB1, and HERVs in COVID-19 patients. IFN-III expressions were comparable in each group of subjects. This preserved induction of IFN-λs could contribute to the better control of the infection in children as compared to adults, in whom IFN-III deficiency has been reported. The upregulation of IFN-I, IFN-II, TRIM28, SETDB1, and HERVs in children with mild symptoms, their declines in severe cases or with MIS-C, and the positive correlations of their transcription in SARS-CoV-2-infected children suggest that they may play important roles in conditioning the evolution of the infection.

Chronic HCV Infection Is Associated with Overexpression of Human Endogenous Retroviruses that Persists after Drug-Induced Viral Clearance.

Chronic hepatitis C virus (HCV) infection is associated with several hepatic and extrahepatic complications, including cancers and autoimmune disorders, whose frequency is reduced but not abolished after drug-induced viral clearance. The causes of these complications and of their persistence are ill-defined. Human endogenous retroviruses (HERVs) are remnants of ancestral infections and constitute 8% of the human genome. Most HERV elements are inactive, but some are transcribed. HERV overexpression is associated with many cancers and autoimmune diseases with a putative pathogenetic role. Several viral infections trigger HERV activation, but there are no studies on HCV-infected subjects. We assessed, through a PCR real-time amplification assay, the transcription levels of the pol genes of HERV-H, -K, and -W, and of their repressor TRIM28 in white blood cells (WBCs) of vertically infected children, both before and after therapy with direct-acting antivirals (DAAs). The results documented significantly higher expressions of HERV-H-pol and HERV-K-pol, not of HERV-W-pol, in HCV-infected subjects as compared to age-matched controls. HERV RNA levels remained unchanged after DAA-driven viral clearance. No significant variations in transcription levels of TRIM28 were observed in infected subjects. Our findings demonstrate HERV-H-pol and HERV-K-pol overexpression in subjects with chronic HCV infection, without variations after a positive response to DAAs; this might justify their predisposition to cancers and autoimmune disorders that persist after a DAA-induced resolution of viremia.

Comparison of Quantitative Analysis of Methylated Alleles Real-Time PCR and Methylation-Specific MLPA for Molecular Diagnosis of Beckwith-Wiedemann Syndrome.

BACKGROUND/AIMS: Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth disorder predisposing to tumorigenesis caused by abnormal expression or function of imprinted genes of the chromosome 11p15.5 imprinting gene cluster. This real-time PCR-based assay determines the methylation status of a selected CpG island and has been proposed for use in high-throughput methylation analysis. METHODS: Here, we use quantitative analysis of methylated alleles (QAMA) for the detection of methylation status of the KCNQ10T1 gene, in a region immediately upstream of the transcription initiation site, and the CTCF binding site 6, located approximately 2 kb upstream of the SmaI site currently used for clinical laboratory testing. We assayed a series of controls and patients diagnosed with BWS at two different loci at 11p15.5 to assess the diagnostic yield of QAMA PCR for clinical laboratory testing. RESULTS: These results compare favorably with methylation-specific multiple ligation probe amplification (MS-MLPA) analysis at both differentially methylated region (DMR)1 and DMR2. There are several advantages of the QAMA PCR over MS-MLPA. The QAMA PCR is less labor-intensive and therefore more cost-effective and does not require dedicated analysis software. A second advantage is that the assay is amenable to high-throughput analysis. CONCLUSIONS: The small sample size reflects the rare nature of this epigenetic disorder, and the range of ages was quite wide, as was the degree of disease severity. Therefore, further validation with larger cohorts is warranted.

Peritoneal mesothelioma and asbestos exposure: a population-based case-control study in Lombardy, Italy.

OBJECTIVES: Asbestos is the main risk factor for peritoneal mesothelioma (PeM). However, due to its rarity, PeM has rarely been investigated in community-based studies. We examined the association between asbestos exposure and PeM risk in a general population in Lombardy, Italy. METHODS: From the regional mesothelioma registry, we selected PeM cases diagnosed in 2000-2015. Population controls (matched by area, gender and age) came from two case-control studies in Lombardy on lung cancer (2002-2004) and pleural mesothelioma (2014). Assessment of exposure to asbestos was performed through a quantitative job-exposure matrix (SYN-JEM) and expert evaluation based on a standardised questionnaire. We calculated period-specific and gender-specific OR and 90% CI using conditional logistic regression adjusted for age, province of residence and education. RESULTS: We selected 68 cases and 2116 controls (2000-2007) and 159 cases and 205 controls (2008-2015). The ORs for ever asbestos exposure (expert-based, 2008-2015 only) were 5.78 (90% CI 3.03 to 11.0) in men and 8.00 (2.56 to 25.0) in women; the ORs for definite occupational exposure were 12.3 (5.62 to 26.7) in men and 14.3 (3.16 to 65.0) in women. The ORs for ever versus never occupational asbestos exposure based on SYN-JEM (both periods) were 2.05 (90% CI 1.39 to 3.01) in men and 1.62 (0.79 to 3.27) in women. In men, clear positive associations were found for duration, cumulative exposure (OR 1.33 (1.19 to 1.48) per fibres/mL-years) and latency. CONCLUSIONS: Using two different methods of exposure assessment we provided evidence of a clear association between asbestos exposure and PeM risk in the general population.

Transcriptional Activity of Human Endogenous Retroviruses in Response to Prenatal Exposure of Maternal Cigarette Smoking.

OBJECTIVE: Transcription of human endogenous retrovirus (HERV) elements is usually suppressed by epigenetic factors such as DNA methylation and heterochromatin silencing by histone modifications. There is an association between maternal smoking during pregnancy and DNA methylation levels in placental tissue and in DNA from cord blood. STUDY DESIGN: We assessed the transcriptional activity of HERV-H, HERV-K, and HERV-W in umbilical cord blood from 47 term babies unexposed to tobacco smoke in utero and 23 term babies exposed to tobacco smoke in utero. RESULTS: In our population, the HERV-H, HERV-K, and HERV-W families were always transcriptionally active, and the levels of all HERVs (H, K, W) were significantly higher in unexposed than smoke-exposed babies. CONCLUSION: This study provides preliminary information about the transcriptional activity of HERV-H, HERV-K, and HERV-W families in human umbilical cord blood.

Is HERV-K and HERV-W Expression Regulated by miR-155 in Kidney Transplant Patients with Human Cytomegalovirus Infection?

According to the latest update, 2,578 unique mature micro-RNAs (miRNAs) are currently annotated in the human genome and participate in the regulation of multiple events, such as cellular proliferation or apoptosis. A previous study analyzing global miRNA expression patterns in GH cells (high human endogenous retrovirus, HERV, K vs. low) showed that 2 miRNAs (miR-663 and miR-638) are differentially regulated and exhibit expression parallel to that of HERV-K. The aim of this study was to evaluate HERV-K and -W pol gene and miR-155 expression in kidney transplant recipients and the possible relationship between them. The comparison between kidney transplant patients negative for human cytomegalovirus (HCMV) infection and positive patients showed a significant difference in terms of miR-155 expression (p = 0.0111). We demonstrated that HERV-K and -W pol gene expression was significantly higher in CMV-infected kidney transplant recipients versus those not infected as previously reported by our groups. Our correlation data suggest that miR-155 are not directly involved in regulating the HERV notwithstanding that we together observed increased expression of HERV-K and -W and diminished expression of miR-155 in HCMV-infected human kidney transplant recipients.

Prevalence and Clinical Profile of Human Salivirus in Children with Acute Gastroenteritis in Northern Italy, 2014-2015.

OBJECTIVE: Human Salivirus (SalV) has been associated with gastroenteritis on all continents. METHODS: This paper presents the real-time RT-PCR assay for the detection of SalV in clinical fecal samples collected from 192 hospitalized children with acute gastroenteritis in Piedmont, Italy. RESULTS: The most commonly detected virus was Norovirus genogroup II (GII) (33.8%), followed by Rotavirus (21.3%), Sapovirus (10.9%), Parechovirus (8%), Norovirus GI (6.7%), and Adenovirus (1%). PCR detected SalV in 1 (0.5%) subject. CONCLUSIONS: Our data show that the detection rate of SalV in diarrheal children (0.5%) is lower than that observed in other countries, where it is reported in diarrheal children in 8.6-1.2% of patients.

Characteristics of RNA Stabilizer RNApro for Peripheral Blood Collection.

Peripheral blood is the most practical tissue for human immune system gene expression profiling because it is easily accessible, whereas the site of primary infection in certain diseases may not be easily accessible. Due to the ex vivo instability of RNA transcripts, a key challenge in the gene expression analysis of blood samples is the rapid sample handling and stabilization of the mRNA by adding an RNA preservative (PAXgeneTM Blood RNA Tubes, TempusTM Blood RNA tubes, RNAlater Stabilization Reagent, RNAgard® Blood Tubes). BioMole (Turin, Italy) has developed a novel blood stabilizer, called RNApro, in which RNA is stabilized during phlebotomy and sample storage. In this study, RNApro performance intended as RNA yield, integrity, and stability was evaluated. Our results show that blood samples stored at -80 °C and re-extracted after 7 years show no differences in terms of quantity, quality, and amplificability. The samples in the RNAlater stabilization solution can be stored at room temperature for up to one week or at 4 °C for up to one month. Similar results can also be observed for PAXgene tubes, Tempus tubes, and RNAgard tubes. In agreement with these data, the RNApro stabilization solution preserves the RNA from degradation for up to 1 month at 4 °C and 1 week at room temperature. RNApro can be stored indifferently at -80, -20, 4 °C, or room temperature for up to 2 months after, and then could be stored at -80 °C for up to seven years. In summary, our study is the first to analyze the performance of an RNA stabilizer called RNApro. We can conclude that several studies have shown significant differences in gene expression analysis when the sample was preserved in different RNA stabilizers. Therefore, it is desirable to standardize the method of nucleic acid conservation when comparing data from transcriptomic analyses.

Prevalence of human Cosavirus and Saffold virus in young children with gastroenteritis in Northern Italy.

BACKGROUND: Gastroenteritis is a common disease in children, characterized by diarrhea, vomiting, abdominal pain, and fever. Human Cosavirus (HCoSV) and Saffold virus (SAFV) both have a worldwide distribution. Both viruses have been detected in the stools of patients with acute gastroenteritis in several countries. METHODS: In order to provide more insights into the epidemiology of enteric viruses that are not included usually in routine diagnostic tests, cases of childhood sporadic gastroenteritis of unknown etiology requiring hospital admission in Turin, Italy, during December 2014 to November 2015, were screened for HCoSV and SAFV. RESULTS: A total of 1 out of 164 (0.6%) episodes of acute gastroenteritis were associated with SAFV genomic detection. Among the 1 SAFV-positive cases, 1 were also positive for Adenovirus. The patient positive for SAFV do not present diarrheal episodes but vomiting. HCoSV was not detected in any of the samples. CONCLUSIONS: In conclusion, this study presents the current epidemiological data regarding the two viruses, HCoSV and SAFV, circulating in pediatric patients admitted to hospital with acute gastroenteritis in Turin, Italy.

Human Bocavirus in children with acute gastroenteritis in Piedmont, Italy.

BACKGROUND: Gastroenteritis is a common disease in children, characterized by diarrhea, vomiting, abdominal pain, and fever. Co-detection of human Bocavirus (HBoV) with other gastroenteric viruses was reported a lot in patients with acute gastroenteritis. METHODS: This paper presents the real-time RT-PCR Taqman assay for the detection and quantification of HBoV for clinical fecal samples collected from hospitalized children with acute gastroenteritis in Piedmont. RESULTS: All fecal specimens were tested for the presence of HBoV with specific primers and probe. A total of 17 out of 123 (13.92%) episodes of acute gastroenteritis were associated with HBoV genomic detection with median viral load 6864.75±19784.79 genomes/mg fecal specimens. Among the 17 HBoV-positive cases, 11 were also positive for other viral pathogens, including Rotavirus (N.=2), astrovirus (N.=1), norovirus GII (N.=6), norovirus GI (N.=2). Two cases were positive for more than one virus including norovirus GII and norovirus GI (N.=1) and Rotavirus, sapovirus and astrovirus (N.=1). A higher detection of HBoV infections was observed in winter, and peaking in February. CONCLUSIONS: Although HBoV is suspected to be responsible for gastroenteritis in children, our data showed that this association was uncertain since no difference was observed in term of viral load in the group with single infection of HBoV and group of coinfections with other viral agent.

Mir-155 expression is downregulated in hematopoietic stem cell transplantation patients with Epstein-Barr virus infection.

BACKGROUND: MicroRNAs (miRNAs) are a class of short length double strand genome encoded RNAs that are produced to repress post-transcriptionally the expression of cellular mRNAs. 2578 unique mature miRNAs are currently annotated in the human genome and participate in the regulation of multiple events, such as cellular proliferation or apoptosis. The over-expression of miR-155 of cellular origin might play a key role in the life cycle of EBV. In this study 24 pediatric patients undergoing HSCT seropositive and seronegative to EBV were enrolled. Thirty-one peripheral blood samples were collected from these patients. The mir-155 expression profile has been evaluated by a stem-loop Real Time PCR in all these conditions. METHODS: Of 24 patients, 4 were seronegative to EBV and EBV negative to PCR (Group I), 10 were seropositive to EBV and EBV negative to PCR (Group II) and 10 were seropositive to EBV and EBV positive to PCR (Group III). RESULTS: Based on relative quantification, the mir-155 expression was compared among the groups. The comparison between HSCT patients without EBV infection seronegative to EBV (Group I) showed higher levels of mir-155 expression than patients seropositive to EBV (P=0.1419). The mir-155 expression levels in seronegative to EBV were not significantly different compared with the patients seropositive to EBV (P=0.6504). The mir-155 expression levels in seropositive to EBV without and with EBV infection (positive viral load), were not significantly (P=0.7667). Also, when we evaluated the mir-155 expression levels comparing all EBV negative patients with an active EBV infection, we did not observe a statistically significant difference (P=0.9782). CONCLUSIONS: Our results are controversial, showing a higher production of mir-155 levels during EBV infection.

Quantification of fecal adenovirus viral load and correlation with Vesikari Score in children with acute gastroenteritis.

BACKGROUND: Human adenoviruses (HAdVs) are an important cause of acute respiratory tract infections, conjunctivitis, hemorrhagic cystitis, and gastroenteritis. In addition to enteric serotypes 40 and 41, some serotypes belonging to subgroups A, B, and C have also been implicated to be etiological agents of gastroenteritis among infants and young children. The Vesikari Scoring System (VSS) is the severity scale that was originally developed to evaluate the effectiveness and efficacy of rotavirus vaccines on 20 points. The aim of this study was to evaluate and compare the diagnostic value of the VSS with HAdVs genome quantification in fecal samples collected from hospitalized children with acute gastroenteritis. METHODS: A total of 137 fecal specimens (69 male and 68 female) were tested for HAdVs. The samples were collected from under-five-year-old children with acute gastroenteritis in pediatric Hospital Regina Margherita of Turin in Italy. RESULTS: A total of 69 out of 137 (50.3%) samples were associated with HAdV genomic detection with a mean viral load of 1.08×1011±9.02×1011 genomes/mg fecal specimens. The samples were grouped on the basis of Mild VSS and Moderate VSS and the HAdV viral load was calculated in the two groups. No statistical differences were observed between two groups (P=0.6123 calculated by Mann-Whitney Test). CONCLUSIONS: Our results did not show a difference in mean viral load between the group with mild VVS and moderate VVS.

Lack of detection of HPyV12 DNA using real-time PCR in Italian infants with diarrhea.

BACKGROUND: HPyV12 was found in organs of the digestive tract, in particular the liver but also in colon, rectum and feces. Until now, the prevalence of HPyV12 is not well characterized. METHODS: In this study, we investigate the presence of this novel polyomavirus DNA in stool specimens collected from under-five-year-old children with gastroenteritis compared to healthy infants. A total of 190 fecal specimens previously screened for rotavirus (RV) and adenovirus (ADV) and 80 fecal samples from healthy infants, were tested for HPyV12 DNA using a home-made real time PCR. All fecal specimens were tested for the presence of HPyV12 with specific primers and probes. RESULTS: None of 190 (0%) episodes of acute gastroenteritis was associated with HPyV12. We did not detect HPyV12 DNA in any of 80 control subjects, as well. CONCLUSIONS: Our study represents a pilot study aiming to clarify the current epidemiological pattern in pediatric Italian patients regarding the novel and rare HPyV12. Based on our negative data and the recent observations reported in literature, doubts remain on human tropism of the HPyV12 and epidemiology: these issues need further investigations.

Peripheral Blood and Nasopharyngeal Swab MiRNA-155 Expression in Infants with Respiratory Syncytial Virus Infection.

INTRODUCTION: MicroRNA (miR) 155 has been implicated in the regulation of innate and adaptive immunity as well as antiviral responses, but its role during respiratory syncytial virus (RSV) infections is not known. The objective of this study was to investigate the expression of miR-155 using pharyngeal swabs and peripheral blood in infants with RSV infection and uninfected controls. METHODS: A prospective age-matched study was conducted in primary care in Torino from 1 August 2018 to 31 January 2020. We enrolled 66 subjects, 29 of them patients with RSV infection and 37 age-matched uninfected controls, and collected pharyngeal swabs and peripheral blood in order to assess miR-155 expression with real-time stem-loop-TaqMan real-time PCR. RESULTS: The data show that there is no correlation between pharyngeal swabs and peripheral blood with respect to miR-155 expression. The 1/ΔCq miR-155 expression levels in throat swabs in RSV bronchiolitis patients and healthy controls were 0.19 ± 0.11 and 0.21 ± 0.09, respectively, and were not significantly different between healthy controls and bronchiolitis (p = 0.8414). In the peripheral blood, miR-155 levels were higher than those of healthy control subjects: 0.1 ± 0.013 and 0.09 ± 0.0007, respectively; p = 0.0002. DISCUSSION: Our data provide evidence that miR-155 expression is higher in peripheral blood during RSV infection but not in swabs. This difference in the timing of sample recruitment could explain the differences obtained in the results; miR-155 activation is probably only assessable in the very early stages of infection in the swab and remains visible for longer in the blood. New investigations are needed in order to clarify whether the miR-155 expression in swabs can be influenced by different stages of virus disease of infants.

Children with Chronic Immune Thrombocytopenia Exhibit High Expression of Human Endogenous Retroviruses TRIM28 and SETDB1.

Chronic immune thrombocytopenia (CITP) is an autoimmune disease whose underlying biologic mechanisms remain elusive. Human endogenous retroviruses (HERVs) derive from ancestral infections and constitute about 8% of our genome. A wealth of clinical and experimental studies highlights their pivotal pathogenetic role in autoimmune diseases. Epigenetic mechanisms, such as those modulated by TRIM28 and SETDB1, are involved in HERV activation and regulation of immune response. We assessed, through a polymerase chain reaction real-time Taqman amplification assay, the transcription levels of pol genes of HERV-H, HERV-K, and HERV-W; env genes of Syncytin (SYN)1, SYN2, and HERV-W; as well as TRIM28 and SETDB1 in whole blood from 34 children with CITP and age-matched healthy controls (HC). The transcriptional levels of all HERV sequences, with the exception of HERV-W-env, were significantly enhanced in children with CITP as compared to HC. Patients on eltrombopag treatment exhibited lower expression of SYN1, SYN2, and HERV-W-env as compared to untreated patients. The mRNA concentrations of TRIM28 and SETDB1 were significantly higher and were positively correlated with those of HERVs in CITP patients. The over-expressions of HERVs and TRIM28/SETDB1 and their positive correlations in patients with CITP are suggestive clues of their contribution to the pathogenesis of the disease and support innovative interventions to inhibit HERV and TRIM28/SETDB1 expressions in patients unresponsive to standard therapies.