Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively

Genetic Adaptation of Coxsackievirus B1 during Persistent Infection in Pancreatic Cells

Coxsackie B (CVB) viruses have been associated with type 1 diabetes. We have recently observed that CVB1 was linked to the initiation of the autoimmune process leading to type 1 diabetes in Finnish children. Viral persistency in the pancreas is currently considered as one possible mechanism. In the current study persistent infection was established in pancreatic ductal and beta cell lines (PANC-1 and 1.1B4) using four different CVB1 strains, including the prototype strain and three clinical isolates. We sequenced 5′ untranslated region (UTR) and regions coding for structural and non-structural proteins and the second single open reading frame (ORF) protein of all persisting CVB1 strains using next generation sequencing to identify mutations that are common for all of these strains. One mutation, K257R in VP1, was found from all persisting CVB1 strains. The mutations were mainly accumulated in viral structural proteins, especially at BC, DE, EF loops and C-terminus of viral capsid protein 1 (VP1), the puff region of VP2, the knob region of VP3 and infection-enhancing epitope of VP4. This showed that the capsid region of the viruses sustains various changes during persistency some of which could be hallmark(s) of persistency

Application of digital PCR for public health-related water quality monitoring

Digital polymerase chain reaction (dPCR) is emerging as a reliable platform for quantifying microorganisms in the field of water microbiology. This paper reviews the fundamental principles of dPCR and its application for health-related water microbiology. The relevant literature indicates increasing adoption of dPCR for measuring fecal indicator bacteria, microbial source tracking marker genes, and pathogens in various aquatic environments. The adoption of dPCR has accelerated recently due to increasing use for wastewater surveillance of Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) -the virus that causes Coronavirus Disease 2019 (COVID-19). The collective experience in the scientific literature indicates that well-optimized dPCR assays can quantify genetic material from microorganisms without the need for a calibration curve and often with superior analytical performance (i.e., greater sensitivity, precision, and reproducibility) than quantitative polymerase chain reaction (qPCR). Nonetheless, dPCR should not be viewed as a panacea for the fundamental uncertainties and limitations associated with measuring microorganisms in water microbiology. With dPCR platforms, the sample analysis cost and processing time are typically greater than qPCR. However, if improved analytical performance (i.e., sensitivity and accuracy) is critical, dPCR can be an alternative option for quantifying microorganisms, including pathogens, in aquatic environments.Peer reviewe

Jätevesien mikrobianalytiikka avaa uusia mahdollisuuksia tartuntatautien seurantaan väestötasolla

Jätevesinäytteitä tutkimalla voidaan osoittaa erilaisten tartuntatautien esiintyminen viemäriverkoston kokooma-alueen väestön keskuudessa. Toisaalta jätevesi voi osoittaa, että tutkittavaa tartuntatautia ei todennäköisesti esiinny tässä väestössä. Suomessa on tutkittu jätevesistä kansallisesti kattavasti poliovirusta jo 60 vuoden ajan ja uutta SARS-CoV-2-koronavirusta lähes koko COVID-19-pandemian ajan, keväästä 2020 alkaen. Useat tutkimukset osoittavat jäteveden taudinaiheuttajalukumäärillä ja raportoitujen tartuntojen määrällä alueen väestössä olevan selkeän yhteyden. Korona-aikana jätevesiseurannasta on muodostunut varsin näkyvä epidemian tilannekuvan mittari, jonka tuottamat tulokset ovat riippumattomia esimerkiksi kulloinkin voimassa olevasta testausstrategiasta. Nämä havainnot ovat vauhdittaneet jätevesiseurannan kehittämistä.Peer reviewe

Human enterovirus and rhinovirus infections are associated with otitis media in a prospective birth cohort study

BACKGROUND: Human enteroviruses (HEVs) and rhinoviruses (HRVs) have been linked to acute otitis media (AOM). OBJECTIVES:The present study evaluates the aforementioned association in a birth cohort setting. STUDY DESIGN: The cohort included 286 healthy infants (191 boys) followed from birth up to the age of 2 years in the Type 1 Diabetes Prediction and Prevention study in Finland. Stool samples were collected monthly and analyzed for the presence of HRV and HEV RNA using RT-PCR. Clinical symptoms were recorded by a questionnaire every 3-6 months. RESULTS:Altogether 610 AOM episodes were reported during the follow-up. 9.8% of the stool samples were positive for HRV and 6.8% for HEV. HRV positivity peaked at the age of 3-6 months declining gradually after this age, whereas HEV positivity peaked later, at the age of 12-24 months. The risk of AOM was increased in children who were HEV positive at least once at the age of 6-12 months (OR 2.2 [95%CI 1.1-4.2], P=0.023) or who were HRV positive at least once at the age of 18-24 months (OR 2.3 [95%CI 1.0-5.2], P=0.042). Having an older sibling, short breast-feeding and maternal smoking during pregnancy were also significantly associated with AOM. CONCLUSIONS: HRV and HEV infections are frequent during the first months of life. The observed trend for increased risk of AOM in HRV and HEV positive children is in line with the results from hospital series suggesting that these viruses may play an independent role in the pathogenesis of AOM. (C) 2016 Elsevier B.V. All rights reserved.Peer reviewe

Infections and systemic inflammation are associated with lower plasma concentration of insulin-like growth factor I among Malawian children

Background: Insulin-like growth factor I (IGF-I) is the most important hormonal promoter of linear growth in infants and young children. Objectives: The objectives of this study were to compare plasma IGF-I concentration in a low- compared with a high-income country and characterize biological pathways leading to reduced IGF-I concentration in children in a low-income setting. Methods: We analyzed plasma IGF-I concentration from 716 Malawian and 80 Finnish children at 6-36 mo of age. In the Malawian children, we studied the association between IGF-I concentration and their environmental exposures; nutritional status; systemic and intestinal inflammation; malaria parasitemia and viral, bacterial, and parasitic enteric infections; as well as growth at 18 mo of age. We then conducted a pathway analysis to identify direct and indirect associations between these predictors and IGF-I concentration. Results: The mean IGF-I concentrations were similar in Malawi and Finland among 6-mo-old infants. At age 18 mo. the mean +/- SD concentration was almost double among the Finns compared with the Malawians [24.2 +/- 11.3 compared with 12.5 +/- 7.7 ng/mL., age- and sex-adjusted difference in mean (95% CI): 11.8 (9.9. 13.7) ng/mL; P <0.01]. Among 18-mo-old Malawians, plasma IGF-I concentration was inversely associated with systemic inflammation, malaria parasitemia, and intestinal Shigella. Campylobacter, and enterovirus infection and positively associated with the children's weight-for-length z score (WLZ), female sex, maternal height, mother's education, and dry season. Seasonally, mean plasma IGF-I concentration was highest in June and July and lowest in December and January, coinciding with changes in children's length gain and preceded by similar to 2 mo by the changes in their WLZ. Conclusions: The mean plasma IGF-I concentrations are similar in Malawi and Finland among 6-mo-old infants. Thereafter, mean concentrations rise markedly in Finland but not in Malawi. Systemic inflammation and clinically nonapparent infections are strongly associated with lower plasma IGF-I concentrations in Malawi through direct and indirect pathways.Peer reviewe

Enhancing and neutralizing anti-coxsackievirus activities in serum samples from patients prior to development of type 1 diabetes

Abstract Studies in prospective cohorts have suggested that enterovirus infections are associated with the appearance of islet autoantibodies that precede later appearance of type 1 diabetes (T1D). It was shown that in addition to an antibody-mediated anti-coxsackievirus (CV)-B neutralizing activity of serum from patients with T1D, there was also enhancing anti-CV-B activity in vitro. In this study the patterns of enhancing and neutralizing anti-CV activities were analyzed from consecutive serum samples collected from children who were followed from birth until they developed T1D in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study and compared to those in non-diabetic control children. The titers of serum neutralizing activity were analyzed against those CVs which were detected in the stools in these children (CV-B3, CV-B5 or CV-A4) using plaque assay. The enhancing activity of these serum samples was analysed by measuring interferon-alpha (INF-α) production in cultures of peripheral blood mononuclear cells (PBMC) inoculated with a mixture of these viruses and diluted serum. A sustained anti-CV enhancing activity was observed in consecutive serum samples in patients with T1D. The pattern of responses differed between children who developed T1D and control children. In patients, the anti-CV enhancing activity was predominant or even exclusive over the neutralizing activity, whereas in controls the enhancing and neutralizing activities were more balanced or the neutralizing activity was largely predominant. In conclusion, evaluating the anti-enterovirus neutralizing and enhancing activity of serum samples can be useful to investigate further the relationship between enteroviruses and the development of T1D. This article is protected by copyright. All rights reserved.Peer reviewe

Enhancing and neutralizing anti-coxsackievirus activities in serum samples from patients prior to development of type 1 diabetes

Abstract Studies in prospective cohorts have suggested that enterovirus infections are associated with the appearance of islet autoantibodies that precede later appearance of type 1 diabetes (T1D). It was shown that in addition to an antibody-mediated anti-coxsackievirus (CV)-B neutralizing activity of serum from patients with T1D, there was also enhancing anti-CV-B activity in vitro. In this study the patterns of enhancing and neutralizing anti-CV activities were analyzed from consecutive serum samples collected from children who were followed from birth until they developed T1D in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study and compared to those in non-diabetic control children. The titers of serum neutralizing activity were analyzed against those CVs which were detected in the stools in these children (CV-B3, CV-B5 or CV-A4) using plaque assay. The enhancing activity of these serum samples was analysed by measuring interferon-alpha (INF-α) production in cultures of peripheral blood mononuclear cells (PBMC) inoculated with a mixture of these viruses and diluted serum. A sustained anti-CV enhancing activity was observed in consecutive serum samples in patients with T1D. The pattern of responses differed between children who developed T1D and control children. In patients, the anti-CV enhancing activity was predominant or even exclusive over the neutralizing activity, whereas in controls the enhancing and neutralizing activities were more balanced or the neutralizing activity was largely predominant. In conclusion, evaluating the anti-enterovirus neutralizing and enhancing activity of serum samples can be useful to investigate further the relationship between enteroviruses and the development of T1D. This article is protected by copyright. All rights reserved.Peer reviewe