Live enteroviruses, but not other viruses, detected in human pancreas at the onset of type 1 diabetes in the DiViD study.

AIMS/HYPOTHESIS: Enterovirus (EV) infection of pancreatic islet cells is one possible factor contributing to type 1 diabetes development. We have reported the presence of EV genome by PCR and of EV proteins by immunohistochemistry in pancreatic sections. Here we explore multiple human virus species in the Diabetes Virus Detection (DiViD) study cases using innovative methods, including virus passage in cell cultures. METHODS: Six recent-onset type 1 diabetes patients (age 24-35) were included in the DiViD study. Minimal pancreatic tail resection was performed under sterile conditions. Eleven live cases (age 43-83) of pancreatic carcinoma without diabetes served as control cases. In the present study, we used EV detection methods that combine virus growth in cell culture, gene amplification and detection of virus-coded proteins by immunofluorescence. Pancreas homogenates in cell culture medium were incubated with EV-susceptible cell lines for 3 days. Two to three blind passages were performed. DNA and RNA were extracted from both pancreas tissue and cell cultures. Real-time PCR was used for detecting 20 different viral agents other than EVs (six herpesviruses, human polyomavirus [BK virus and JC virus], parvovirus B19, hepatitis B virus, hepatitis C virus, hepatitis A virus, mumps, rubella, influenza A/B, parainfluenza 1-4, respiratory syncytial virus, astrovirus, norovirus, rotavirus). EV genomes were detected by endpoint PCR using five primer pairs targeting the partially conserved 5' untranslated region genome region of the A, B, C and D species. Amplicons were sequenced. The expression of EV capsid proteins was evaluated in cultured cells using a panel of EV antibodies. RESULTS: Samples from six of six individuals with type 1 diabetes (cases) and two of 11 individuals without diabetes (control cases) contained EV genomes (p<0.05). In contrast, genomes of 20 human viruses other than EVs could be detected only once in an individual with diabetes (Epstein-Barr virus) and once in an individual without diabetes (parvovirus B19). EV detection was confirmed by immunofluorescence of cultured cells incubated with pancreatic extracts: viral antigens were expressed in the cytoplasm of approximately 1% of cells. Notably, infection could be transmitted from EV-positive cell cultures to uninfected cell cultures using supernatants filtered through 100 nm membranes, indicating that infectious agents of less than 100 nm were present in pancreases. Due to the slow progression of infection in EV-carrying cell cultures, cytopathic effects were not observed by standard microscopy but were recognised by measuring cell viability. Sequences of 5' untranslated region amplicons were compatible with EVs of the B, A and C species. Compared with control cell cultures exposed to EV-negative pancreatic extracts, EV-carrying cell cultures produced significantly higher levels of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP1). CONCLUSIONS/INTERPRETATION: Sensitive assays confirm that the pancreases of all DiViD cases contain EVs but no other viruses. Analogous EV strains have been found in pancreases of two of 11 individuals without diabetes. The detected EV strains can be passaged in series from one cell culture to another in the form of poorly replicating live viruses encoding antigenic proteins recognised by multiple EV-specific antibodies. Thus, the early phase of type 1 diabetes is associated with a low-grade infection by EVs, but not by other viral agents.

The diabetes pandemic and associated infections: suggestions for clinical microbiology.

There are 425 million people with diabetes mellitus in the world. By 2045, this figure will grow to over 600 million. Diabetes mellitus is classified among noncommunicable diseases. Evidence points to a key role of microbes in diabetes mellitus, both as infectious agents associated with the diabetic status and as possible causative factors of diabetes mellitus. This review takes into account the different forms of diabetes mellitus, the genetic determinants that predispose to type 1 and type 2 diabetes mellitus (especially those with possible immunologic impact), the immune dysfunctions that have been documented in diabetes mellitus. Common infections occurring more frequently in diabetic vs. nondiabetic individuals are reviewed. Infectious agents that are suspected of playing an etiologic/triggering role in diabetes mellitus are presented, with emphasis on enteroviruses, the hygiene hypothesis, and the environment. Among biological agents possibly linked to diabetes mellitus, the gut microbiome, hepatitis C virus, and prion-like protein aggregates are discussed. Finally, preventive vaccines recommended in the management of diabetic patients are considered, including the bacillus calmette-Guerin vaccine that is being tested for type 1 diabetes mellitus. Evidence supports the notion that attenuation of immune defenses (both congenital and secondary to metabolic disturbances as well as to microangiopathy and neuropathy) makes diabetic people more prone to certain infections. Attentive microbiologic monitoring of diabetic patients is thus recommendable. As genetic predisposition cannot be changed, research needs to identify the biological agents that may have an etiologic role in diabetes mellitus, and to envisage curative and preventive ways to limit the diabetes pandemic.

Vitamin D status, enterovirus infection, and type 1 diabetes in Italian children/adolescents.

At the time of the clinical onset of type 1 diabetes (T1D), we investigated 82 pediatric cases in parallel with 117 non-diabetic controls matched by age, geographic area, and time of collection. The occurrence of an enteroviral infection was evaluated in peripheral blood using a sensitive method capable of detecting virtually all human enterovirus (EV) types. While non-diabetic controls were consistently EV-negative, 65% of T1D cases carried EVs in blood. The vitamin D status was assessed by measuring the concentration of 25-hydroxyvitamin D [25(OH)D] in serum. Levels of 25(OH)D were interpreted as deficiency (≤50 nmol/L), insufficiency (52.5-72.5 nmol/L), and sufficiency (75-250 nmol/L). In T1D cases, the median serum concentration of 25(OH)D was 54.4 ± 27.3 nmol/L vs 74.1 ± 28.5 nmol/L in controls (P = .0001). Diabetic children/adolescents showed deficient levels of vitamin D 25(OH)D (ie, 72.5 nmol/L) in 48.8% cases vs 17.9% in non-diabetic controls (P = .0001). Unexpectedly, the median vitamin D concentration was significantly reduced in virus-positive vs virus-negative diabetics (48.2 ± 22.5 vs 61.8 ± 31.2 nmol/L; P = .015), with deficient levels in 58.5% vs 31.0%, respectively. Thus, at the time of clinical onset, EV-positive cases had reduced vitamin D levels compared with EV-negative cases. This could indicate either that the virus-negative children/adolescents had been hit by a non-infectious T1D-triggering event, or that children/adolescents with proper levels of vitamin D had been able to rapidly clear the virus. Thus, it would be important to assess whether adequate vitamin D supplementation before or during the prediabetic phase of T1D may counteract the diabetogenic potential of infectious pathogens.

Tracking Nosocomial Klebsiella pneumoniae Infections and Outbreaks by Whole-Genome Analysis: Small-Scale Italian Scenario within a Single Hospital.

Multidrug-resistant (MDR) Klebsiella pneumoniae is one of the most important causes of nosocomial infections worldwide. After the spread of strains resistant to beta-lactams at the end of the previous century, the diffusion of isolates resistant to carbapenems and colistin is now reducing treatment options and the containment of infections. Carbapenem-resistant K. pneumoniae strains have spread rapidly among Italian hospitals, with four subclades of pandemic clonal group 258 (CG258). Here we show that a single Italian hospital has been invaded by three of these subclades within 27 months, thus replicating on a small scale the "Italian scenario." We identified a single clone responsible for an epidemic outbreak involving seven patients, and we reconstructed its star-like pattern of diffusion within the intensive care unit. This epidemiological picture was obtained through phylogenomic analysis of 16 carbapenem-resistant K. pneumoniae isolates collected in the hospital during a 27-month period, which were added to a database of 319 genomes representing the available global diversity of K. pneumoniae strains. Phenotypic and molecular assays did not reveal virulence or resistance determinants specific for the outbreak isolates. Other factors, rather than selective advantages, might have caused the outbreak. Finally, analyses allowed us to identify a major subclade of CG258 composed of strains bearing the yersiniabactin virulence factor. Our work demonstrates how the use of combined phenotypic, molecular, and whole-genome sequencing techniques can help to identify quickly and to characterize accurately the spread of MDR pathogens.

Performance of commonly used genotypic assays and comparison with phenotypic assays of HIV-1 coreceptor tropism in acutely HIV-1-infected patients.

OBJECTIVES: Although founder viruses in primary HIV-1 infections (PHIs) typically use the CCR5 coreceptor (R5-tropic), 3%-19% of subjects also harbour CXCR4-using viruses (X4-tropic), making tropism determination before CCR5 antagonist usage mandatory. Genotypic methods can be used to accurately determine HIV-1 tropism in chronically infected patients. METHODS: We compared the results of genotypic methods [geno2pheno, PSSMx4r5 including a novel nucleotide-input version (ntPSSM) and distant segments (ds)Kernel] to predict coreceptor usage in a cohort of 67 PHIs. Specimens with discrepant results were phenotypically tested after cloning the V3 gene region into proviral backbones. Recombinant viruses were used to infect U87 indicator cell lines bearing CD4 and either CCR5 or CXCR4. RESULTS: Geno2pheno10%, PSSMx4r5 and (ds)Kernel gave identical predictions in 85% of cases. Geno2pheno10% predicted the presence of CXCR4 viruses in 18% of patients. Two patients were predicted to carry X4-tropic viruses by all algorithms and X4-tropic viruses were detected in at least one of the recombinant AD8 or NL4-3 backbone-based assays. Ten samples resulted in discordant predictions with at least one algorithm. Full concordance between tropism prediction by using population sequencing and phenotypic assays was observed only with ntPSSM. Geno2pheno prediction and the phenotypic assay gave the same results in a minority of 'discordant' patients. CONCLUSIONS: Compared with both PSSMx4r5 versions, (ds)Kernel and our phenotypic assay, geno2pheno10% overestimated the frequency of X4-tropic viruses (18% versus 3%). ntPSSM was able to detect one additional X4 virus compared with (ds)Kernel that was confirmed with the phenotypic assay.

mRNA Technology and Mucosal Immunization.

Current mRNA vaccines are mainly administered via intramuscular injection, which induces good systemic immunity but limited mucosal immunity. Achieving mucosal immunity through mRNA vaccination could diminish pathogen replication at the entry site and reduce interhuman transmission. However, delivering mRNA vaccines to mucosae faces challenges like mRNA degradation, poor entry into cells, and reactogenicity. Encapsulating mRNA in extracellular vesicles may protect the mRNA and reduce reactogenicity, making mucosal mRNA vaccines possible. Plant-derived extracellular vesicles from edible fruits have been investigated as mRNA carriers. Studies in animals show that mRNA vehiculated in orange-derived extracellular vesicles can elicit both systemic and mucosal immune responses when administered by the oral, nasal, or intramuscular routes. Once lyophilized, these products show remarkable stability. The optimization of mRNA to improve translation efficiency, immunogenicity, reactogenicity, and stability can be obtained through adjustments of the 5'cap region, poly-A tail, codons selection, and the use of nucleoside analogues. Recent studies have also proposed self-amplifying RNA vaccines containing an RNA polymerase as well as circular mRNA constructs. Data from parenterally primed animals demonstrate the efficacy of nasal immunization with non-adjuvanted protein, and studies in humans indicate that the combination of a parenteral vaccine with the natural exposure of mucosae to the same antigen provides protection and reduces transmission. Hence, mucosal mRNA vaccination would be beneficial at least in organisms pre-treated with parenteral vaccines. This practice could have wide applications for the treatment of infectious diseases.

SARS-CoV-2 spread to endocrine organs is associated with obesity: an autopsy study of COVID-19 cases.

PURPOSE: SARS-CoV-2 infection may be limited to the respiratory tract or may spread to multiple organs. Besides disease severity, factors associated with virus spread within the host are elusive. Here, we tried to identify features associated with SARS-CoV-2 spread to endocrine organs. METHODS: In a retrospective autoptic cohort of 51 subjects who died because of COVID-19, we analyzed the severity and type of lung pathology, patients' features and the detection of virus in thyroid, testis, adrenal gland, pancreas, anterior pituitary, and the white adipose tissue (WAT). RESULTS: The SARS-CoV-2 genome was detected in endocrine organs of 30/51 cases. The anterior pituitary and WAT were most frequently positive for virus. While pathological features of lung were not associated with the presence of virus in endocrine organs, obesity (BMI > 30) was significantly associated to virus detection in pancreas (p = 0.01) and thyroid (p = 0.04). WAT infection was detected more frequently in males (p = 0.03). CONCLUSION: In subject with obesity dying of COVID-19, the virus frequently spreads to endocrine organs. The findings emphasize the need for optimal treatment of patients with obesity at the very onset of COVID-19. Since post-COVID conditions remain a major issue worldwide, a rigorous follow-up of endocrine function-especially of thyroid and pancreas-is advocated in subjects with obesity.

Intrafamilial spread of enterovirus infections at the clinical onset of type 1 diabetes.

BACKGROUND: At the clinical onset of type 1 diabetes mellitus (T1D), enterovirus (EV) infections are suspected to play a role. EVs in blood are seen as a possible biomarker of T1D. EV infections may occur in temporal and geographic clusters and may spread within families. OBJECTIVE: We checked whether EVs were present in the blood of newly diagnosed diabetic probands and of their consenting siblings and parents. We aimed at evaluating the frequency of EV infection, whether infections were spreading within families, and which EV species were involved. SUBJECTS AND METHODS: Blood was drawn from 24 newly diagnosed diabetic children/adolescents and their family members (20 siblings and 41 parents). Blood donors and non-diabetic children/adolescents diagnosed with overweight/short stature were used as controls. RNA was extracted from plasma/leukocytes. Reverse-transcription polymerase chain reaction assays capable of detecting virtually all EV types and of giving preliminary species identification were used. RESULTS AND CONCLUSIONS: EV genomes were found in the blood of 19 of 24 (79%) diabetics, 12 of 20 (60%) non-diabetic siblings, 26 of 41 (63%) parents, and 1 of 29 (3%) pediatric controls. EVs of the A, B, C, and D species were detected, with the B and C species more prevalent. Probands and virus-positive members of each family consistently shared the same EV species. During follow-up, 4 of 20 (20%) siblings of diabetic probands developed T1D with a latency of 3-25 months. In conclusion, infection by different EV species is highly prevalent at the clinical onset and extends to family members. EV may represent a precipitating factor of T1D. However, the disease only develops in a subset of infected individuals.

Transcriptional changes in multiple endocrine organs from lethal cases of COVID-19.

Altered circulating hormone and metabolite levels have been reported during and post-COVID-19. Yet, studies of gene expression at the tissue level capable of identifying the causes of endocrine dysfunctions are lacking. Transcript levels of endocrine-specific genes were analyzed in five endocrine organs of lethal COVID-19 cases. Overall, 116 autoptic specimens from 77 individuals (50 COVID-19 cases and 27 uninfected controls) were included. Samples were tested for the SARS-CoV-2 genome. The adrenals, pancreas, ovary, thyroid, and white adipose tissue (WAT) were investigated. Transcript levels of 42 endocrine-specific and 3 interferon-stimulated genes (ISGs) were measured and compared between COVID-19 cases (virus-positive and virus-negative in each tissue) and uninfected controls. ISG transcript levels were enhanced in SARS-CoV-2-positive tissues. Endocrine-specific genes (e.g., HSD3B2, INS, IAPP, TSHR, FOXE1, LEP, and CRYGD) were deregulated in COVID-19 cases in an organ-specific manner. Transcription of organ-specific genes was suppressed in virus-positive specimens of the ovary, pancreas, and thyroid but enhanced in the adrenals. In WAT of COVID-19 cases, transcription of ISGs and leptin was enhanced independently of virus detection in tissue. Though vaccination and prior infection have a protective role against acute and long-term effects of COVID-19, clinicians must be aware that endocrine manifestations can derive from virus-induced and/or stress-induced transcriptional changes of individual endocrine genes. KEY MESSAGES: • SARS-CoV-2 can infect adipose tissue, adrenals, ovary, pancreas and thyroid. • Infection of endocrine organs induces interferon response. • Interferon response is observed in adipose tissue independently of virus presence. • Endocrine-specific genes are deregulated in an organ-specific manner in COVID-19. • Transcription of crucial genes such as INS, TSHR and LEP is altered in COVID-19.

Autopsy Study of Testicles in COVID-19: Upregulation of Immune-Related Genes and Downregulation of Testis-Specific Genes.

CONTEXT: Infection by SARS-CoV-2 may be associated with testicular dysfunction that could affect male fertility. OBJECTIVE: Testicles of fatal COVID-19 cases were investigated to detect virus in tissue and to evaluate histopathological and transcriptomic changes. METHODS: Three groups were compared: (a) uninfected controls (subjects dying of trauma or sudden cardiac death; n = 10); (b) subjects dying of COVID-19 (virus-negative in testes; n = 15); (c) subjects dying of COVID-19 (virus-positive in testes; n = 9). SARS-CoV-2 genome and nucleocapsid antigen were probed using RT-PCR, in situ hybridization, and immunohistochemistry (IHC). Infiltrating leukocytes were typed by IHC. mRNA transcripts of immune-related and testis-specific genes were quantified using the nCounter method. RESULTS: SARS-CoV-2 was detected in testis tissue of 9/24 (37%) COVID-19 cases accompanied by scattered T-cell and macrophage infiltrates. Size of testicles and counts of spermatogenic cells were not significantly different among groups. Analysis of mRNA transcripts showed that in virus-positive testes immune processes were activated (interferon-alpha and -gamma pathways). By contrast, transcription of 12 testis-specific genes was downregulated, independently of virus positivity in tissue. By IHC, expression of the luteinizing hormone/choriogonadotropin receptor was enhanced in virus-positive compared to virus-negative testicles, while expression of receptors for androgens and the follicle-stimulating hormone were not significantly different among groups. CONCLUSION: In lethal COVID-19 cases, infection of testicular cells is not uncommon. Viral infection associates with activation of interferon pathways and downregulation of testis-specific genes involved in spermatogenesis. Due to the exceedingly high numbers of infected people in the pandemic, the impact of virus on fertility should be further investigated.

Treatment of severe post-traumatic bone defects with autologous stem cells loaded on allogeneic scaffolds.

Mesenchymal stem cells may differentiate into angiogenic and osteoprogenitor cells. The effectiveness of autologous pluripotent mesenchymal cells for treating bone defects has not been investigated in humans. We present a case series to evaluate the rationale of using nucleated cells from autologous bone marrow aspirates in the treatment of severe bone defects that failed to respond to traditional treatments. Ten adult patients (mean age, 49.6-years-old) with severe bone defects were included in this study. Lower limb bone defects were >or=5 cm3 in size, and upper limb defects .or=2 cm3. Before surgery, patients were tested for antibodies to common pathogens. Treatment consisted of bone allogeneic scaffold enriched with bone marrow nucleated cells harvested from the iliac crest and concentrated using an FDA-approved device. Postsurgery clinical and radiographic follow-up was performed at 1, 3, 6, and 12 months. To assess viability, morphology, and immunophenotype, bone marrow nucleated cells were cultured in vitro, tested for sterility, and assayed for the possible replication of adventitious (contaminating) viruses. In 9 of 10 patients, both clinical and radiographic healing of the bone defect along with bone graft integration were observed (mean time, 5.6 months); one patient failed to respond. No post-operative complications were observed. Bone marrow nucleated cells were enriched 4.49-fold by a single concentration step, and these enriched cells were free of microbial contamination. The immunophenotype of adherent cells was compatible with that of mesenchymal stem cells. We detected the replication of Epstein-Barr virus in 2/10 bone marrow cell cultures tested. Hepatitis B virus, cytomegalovirus, parvovirus B19, and endogenous retrovirus HERV-K replication were not detected. Overall, 470 to 1,150 million nucleated cells were grafted into each patient. This case series, with a mean follow-up of almost 2 years, demonstrates that an allogeneic bone scaffold enriched with concentrated autologous bone marrow cells obtained from the iliac crest provides orthopedic surgeons a novel option for treating important bone defects that are unresponsive to traditional therapies.

Higher seasonal temperature enhances the occurrence of methicillin resistance of Staphylococcus aureus in house flies (Musca domestica) under hospital and environmental settings.

Antimicrobial resistance (AMR) emergence in commensal and pathogenic bacteria is a global health issue. House flies (Musca domestica) are considered as biological and mechanical vectors for pathogens causing nosocomial infections, including methicillin-resistant Staphylococcus aureus (MRSA). However, the prevalence of antimicrobial resistance and the role of temperature on the occurrence of Staphylococcus aureus and MRSA in house flies in a hospital environment have not been studied. A total of 400 house flies were collected in winter and summer from four hospital-associated areas in Mymensingh, Bangladesh. Detection of S. aureus and MRSA in flies was done by culturing, staining, and PCR methods targeting nuc and mec genes (mecA and mecC), respectively. Disc diffusion test was used to detect resistance phenotype against six antimicrobials. Logistic regression models were constructed to assess the effect of temperature on the frequency of antimicrobial resistance, and on the presence of the nuc and mecA genes, and location of samples in and around a hospital environment. By PCR, S. aureus was detected in 208 (52%) samples. High frequencies of resistance (≥ 80% of isolates) to amoxicillin, azithromycin, and oxacillin were observed by disk diffusion test. Increase in temperature had a positive effect on the occurrence of S. aureus and MRSA isolates as well as on their resistance to individual and multiple antimicrobials. Among the study areas, hospital premises had increased odds of having S. aureus. Increased temperature of summer significantly increased the occurrence of MRSA in house flies in and around the hospital environment, which might pose a human and animal health risk.

High Prevalence of Common Human Viruses in Thyroid Tissue.

Introduction: Evidence points to viral infections as possible triggers of autoimmune thyroid disease (AITD), but little is known about the prevalence of common viruses in the thyroid gland. Using a novel approach based on virus enrichment in multiple cell lines followed by detection of the viral genome and visualization of viral proteins, we investigated the presence of multiple human viruses in thyroid tissue from AITD patients and controls. Methods: Thyroid tissue was collected by core needle biopsy or during thyroid surgery from 35 patients with AITD (20 Graves' disease and 15 Hashimoto's thyroiditis). Eighteen thyroid tissue specimens from patients undergoing neck surgery for reasons other than thyroid autoimmunity served as controls. Specimens were tested for the presence of ten different viruses. Enteroviruses and human herpesvirus 6 were enriched in cell culture before detection by PCR and immunofluorescence, while the remaining viruses were detected by PCR of biopsied tissue. Results: Forty of 53 cases (75%) carried an infectious virus. Notably, 43% of all cases had a single virus, whereas 32% were coinfected by two or more virus types. An enterovirus was found in 27/53 cases (51%), human herpesvirus 6 in 16/53 cases (30%) and parvovirus B19 in 12/53 cases (22%). Epstein-Barr virus and cytomegalovirus were found in a few cases only. Of five gastroenteric virus groups examined, only one was detected in a single specimen. Virus distribution was not statistically different between AITD cases and controls. Conclusion: Common human viruses are highly prevalent in the thyroid gland. This is the first study in which multiple viral agents have been explored in thyroid. It remains to be established whether the detected viruses represent causal agents, possible cofactors or simple bystanders.

Suppression of Pituitary Hormone Genes in Subjects Who Died From COVID-19 Independently of Virus Detection in the Gland.

CONTEXT: Involvement of the pituitary gland in SARS-CoV-2 infection has been clinically suggested by pituitary hormone deficiency in severe COVID-19 cases, by altered serum adrenocorticotropic hormone (ACTH) levels in hospitalized patients, and by cases of pituitary apoplexy. However, the direct viral infection of the gland has not been investigated. OBJECTIVE: To evaluate whether the SARS-CoV-2 genome and antigens could be present in pituitary glands of lethal cases of COVID-19, and to assess possible changes in the expression of immune-related and pituitary-specific genes. METHODS: SARS-CoV-2 genome and antigens were searched in the pituitary gland of 23 patients who died from COVID-19 and, as controls, in 12 subjects who died from trauma or sudden cardiac death. Real-time reverse transcription polymerase chain reaction (PCR), in situ hybridization, immunohistochemistry, and transmission electron microscopy were utilized. Levels of mRNA transcripts of immune-related and pituitary-specific genes were measured by the nCounter assay. RESULTS: The SARS-CoV-2 genome and antigens were detected in 14/23 (61%) pituitary glands of the COVID-19 group, not in controls. In SARS-CoV-2-positive pituitaries, the viral genome was consistently detected by PCR in the adeno- and the neurohypophysis. Immunohistochemistry, in situ hybridization, and transmission electron microscopy confirmed the presence of SARS-CoV-2 in the pituitary. Activation of type I interferon signaling and enhanced levels of neutrophil and cytotoxic cell scores were found in virus-positive glands. mRNA transcripts of pituitary hormones and pituitary developmental/regulatory genes were suppressed in all COVID-19 cases irrespective of virus positivity. CONCLUSION: Our study supports the tropism of SARS-CoV-2 for human pituitary and encourages exploration of pituitary dysfunction after COVID-19.

Immune Transcriptome of Cells Infected with Enterovirus Strains Obtained from Cases of Autoimmune Thyroid Disease.

BACKGROUND: Hashimoto's thyroiditis and Graves' disease are autoimmune thyroid disorders (AITD) of unknown origin. Enterovirus (EV) infection of thyroid cells has been implicated as a possible initiator of cell damage and of organ-specific autoimmunity. We asked whether persistent infection of human epithelial cells with EV strains obtained from thyroid tissue of AITD patients could be associated with transcriptional changes capable of fostering immunopathology. METHODS: EV isolates obtained from thyroid tissue of AITD cases were used to infect the AV3 epithelial cell line. AV3 cells incubated with a virus-free medium from thyroid tissue of subjects without evidence of thyroid autoimmunity were used as uninfected controls. Transcripts of immune-related genes were compared in infected vs. uninfected cells. RESULTS: The EV genome and antigens were detected only in the cells exposed to AITD-derived virus isolates, not in control cells. Persistent EV infection, while suppressing transcription of several type I IFN and cytokine determinants, was associated with enhanced transcription of NFKB1/RELA, IFNAR1, JAK1/STAT1, i.e., the determinants that play key immunologic roles. Infection also led to upregulation of the CCL2 chemokine and the IL-18 pro-inflammatory interleukin. CONCLUSION: As in the case of EV strains obtained from autoimmune diabetes, results show that the EV strains that are present in the thyroid of AITD cases do repress IFN and cytokine pathways. JAK1/STAT1 upregulation supports activation of TLR pathways and aberrant T cell signaling. In the early phases of AITD, our results highlight the potential benefit of interventions aimed at blocking the viral infection and easing the inflammatory response.

Activation of Type I and Type II Interferon Signaling in SARS-CoV-2-Positive Thyroid Tissue of Patients Dying from COVID-19.

Background: Thyroid dysfunctions have been reported after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. However, the biological mechanisms behind these conditions remain unexplored. Herein, we report on changes of the immune transcriptome in autoptic thyroid tissues of people who have died from coronavirus disease 2019 (COVID-19). Methods: Twenty-five autoptic thyroid specimens of subjects dying from COVID-19 were investigated. Eleven autoptic thyroid specimens of subjects dying from causes other than infectious conditions served as controls. RNA transcripts of 770 immune-related genes together with RNA genomes of multiple coronavirus types were measured by the nCounter system. Reverse transcription-polymerase chain reaction for two SARS-CoV-2 genes was used to assess virus positivity. Results were validated by immunohistochemistry. Results: The SARS-CoV-2 genome and antigens were detected in 9 of 25 (36%) thyroid specimens from the COVID-19 cohort. Virus-negative thyroid tissues from COVID-19 subject did not show changes of gene transcription nor significant numbers of infiltrating immune cells. Conversely, SARS-CoV-2-positive thyroid specimens showed marked upregulation of immune genes, especially those proper of the type I and type II interferon (IFN) pathways. In infected tissues, infiltrates of innate immune cells (macrophages and polymorphonuclear neutrophils) were prevalent. Conclusions: The thyroid gland can be directly infected by the SARS-CoV-2. Infection strongly activates IFN pathways. The direct viral insult combined with an intense immune response may trigger or worsen thyroid conditions in predisposed individuals.

Differential Detection of Encapsidated versus Unencapsidated Enterovirus RNA in Samples Containing Pancreatic Enzymes-Relevance for Diabetes Studies.

Using immunohistochemistry, enterovirus capsid proteins were demonstrated in pancreatic islets of patients with type 1 diabetes. Virus proteins are mainly located in beta cells, supporting the hypothesis that enterovirus infections may contribute to the pathogenesis of type 1 diabetes. In samples of pancreatic tissue, enterovirus RNA was also detected, but in extremely small quantities and in a smaller proportion of cases compared to the enteroviral protein. Difficulties in detecting viral RNA could be due to the very small number of infected cells, the possible activity of PCR inhibitors, and the presence-during persistent infection-of the viral genome in unencapsidated forms. The aim of this study was twofold: (a) to examine if enzymes or other compounds in pancreatic tissue could affect the molecular detection of encapsidated vs. unencapsidated enterovirus forms, and (b) to compare the sensitivity of RT-PCR methods used in different laboratories. Dilutions of encapsidated and unencapsidated virus were spiked into human pancreas homogenate and analyzed by RT-PCR. Incubation of pancreatic homogenate on wet ice for 20 h did not influence the detection of encapsidated virus. In contrast, a 15-min incubation on wet ice dramatically reduced detection of unencapsidated forms of virus. PCR inhibitors could not be found in pancreatic extract. The results show that components in the pancreas homogenate may selectively affect the detection of unencapsidated forms of enterovirus. This may lead to difficulties in diagnosing persisting enterovirus infection in the pancreas of patients with type 1 diabetes.

Is the COVID-19 pandemic masking dengue epidemic in Bangladesh?

COVID-19 is now a pandemic. Like other countries, Bangladesh is putting all its efforts to combat this pandemic. Dengue is a mosquito-borne viral infection causing a severe flu-like illness and, sometimes causing a potentially lethal complication called severe dengue. At this very crisis moment, there are reports on new cases of dengue in Bangladesh. More efforts now need to be taken for the control of dengue along with COVID-19 control measures.