Pathogenic and Molecular Characteristics of Two Isolates of HEV-B Species

Enteroviruses (EVs) are small non-enveloped RNA viruses forming a large group of different serotypes. EVs belong to the family Picornaviridae. The primary replication site of an enterovirus is typically the epithelium of the respiratory tract and the gastrointestinal mucosa. Virus replication in the gastrointestinal mucosa may continue, often asymptomatically, for several weeks occasionally causing viremia. During the viremia the virus spreads through the lymphatic system and circulation. Organ-specific symptoms rise after viral replication in the secondary target tissues. Occasionally, cellular adaptation is required for a virus to initiate replication in the secondary target tissue(s). Adaptation is linked to mutation(s) which may lead to alteration in cellular tropism, e.g., recognition of new surface receptor molecules or other host cell constituents essential for virus entry and replication. However, the critical step may also occur later during in the interaction of the host cell and the replicating virus. In the present study, genetic changes responsible for altered phenotypic features were sought using two strains of Human enterovirus B (HEV-B) species. Firstly, a laboratory isolate of coxsackievirus B5 (CV-B5), strain DS, was passaged 15 times in mouse pancreas in vivo, which resulted in a diabetogenic mouse pancreas passaged virus strain (MPP). The concept of diabetogenic means the ability of the MPP strain to replicate, cause insulitis and dysregulation of the glucose metabolism in the mouse pancreas in vivo. The interaction between the MPP virus strain and insulin producing β-cells was further studied in cell culture using a mouse-derived insulinoma cell line, MIN-6 cells, as an experimental model. The replication of the MPP virus strain was clearly slower in the MIN-6 cells compared to the other tested cell lines. After three days of incubation, extensive replication of MPP was evident in MIN-6 cells and resulted in a MIN-6 cell-adapted virus strain (MCA). Secondly, the ability of the D207 virus strain, isolated from a type 1 diabetic patient, to replicate in a primary human β-cell culture was tested. D207 was initially serotyped as coxsackievirus A9 (CV-A9) in a virus-specific neutralization assay. The D207 virus strain was found to cause cytolysis in the primary human β-cells and, simultaneously, severe functional damage of the surviving β-cells. The genomes of the four virus strains DS, MPP, MCA and D207 were cloned and sequenced. The sequence comparison of three CV-B5 strains (DS, MPP, and MCA) revealed only limited changes, three capsid and two non-structural (NS) amino acid substitutions between MPP and DS, and two capsid and six NS amino acid substitutions between MCA and MPP. In order to determine which of the amino acid substitutions were responsible for the changed phenotype in vivo and in vitro, full-length infectious clones were constructed from the MPP virus and its parental DS virus. By using reverse mutagenesis and chimeric viruses (MPP/DS and DS/MPP), it was shown that a change from MPP to the MCA phenotype in MIN-6 cells was mediated by only a single amino acid at position 94 in VP1, while the in vivo adaptation of the DS virus strain to the inflammation-inducing MPP virus strain may require multiple genetic determinants in the virus capsid and probably also in the NS proteins. Sequence analyses of D207 revealed that the virus belonged to a genogroup D of E-11, but was also neutralized with monotypic antisera to CV-A9. The isolate D207 was found to be closely related to a specific E-11 strains known to cause uveitis. Uveitis-causing E-11 strains were also found to be well neutralized with both CV-A9- and E-11-specific antisera. In a further study, a wide range of E-11 isolates were included to test the observed dual neutralizibility among isolates belonging to the D genogroup. Five of the six studied strains belonging to genogroup D were also neutralized with antisera against coxsackievirus A9 Griggs. The peptide scanning technique was utilized to identify antigenic regions of the capsid proteins of the D207 strain responsible for the observed dual neutralization. Several regions in the capsid of D207 were found to cross-react with an antiserum raised against CV-A9. However, epitopes responsible for the cross-neutralization remained unidentified. In conclusion, these studies indicate that the specific location of mutation may affect the phenotype of an enterovirus more than the overall quantity of changes. In the experimental settings, radical changes in the viral phenotypic features occurred only after a few amino acid substitutions. The majority of the studied viruses in the genogroup D of E-11 maintained exceptional phenotypic property, the cross-neutralization with CV-A9 specific antiserum, despite their genetic divergence.Pikornaviruksiin kuuluvat ihmisen enterovirukset ovat pieniä, pyöreitä ja vaipattomia RNA-viruksia, joista tunnetaan suuri määrä erilaisia serotyyppejä. Enterovirusinfektiot alkavat yleensä hengitystien ja suoliston epiteelissä, jossa suurin osa viruksista kulkeutuu mahalaukun läpi suolistoon. Enterovirukset aiheuttavat pääasiallisesti systeemisiä yleisinfektioita, joiden oirekirjo määräytyy virusten toissijaisen lisääntymispaikan mukaan. Joskus enteroviruksen lisääntyminen toissijaisessa lisääntymispaikassa edellyttää sopeutumisprosessia. Sopeutuminen johtuu geneettisistä mutaatioista, jotka voivat johtaa uuden solunpintareseptorimolekyylin tai muun isäntäsolun molekyylin tunnistamiseen ja jotka ovat välttämättömiä virusten pääsylle soluun. On myös mahdollista, että sopeutumiselle kriittinen vaihe on myöhempi vuorovaikutus isäntäsolun ja lisääntyvän viruksen välillä. Tutkimuksen tarkoituksena oli tunnistaa fenotyyppisistä ominaisuuksista vastaavia virusgenomin osia kahdesta ihmisen enterovirus B -lajiin kuuluvasta viruksesta. Coxsackie B5 -viruskanta (DS) saatiin muuntumaan kasvattamalla sitä viisitoista kertaa hiiren haimassa in vivo. Muuntuneiden virusten joukosta löytyi ns. diabetogeeninen virus (diabetogeeninen CV-B, CV-B5-MPP), joka sai aikaan kroonisen tulehdusreaktion hiiren haimassa. Osalla hiiristä todettiin myös häiriötä sokerimetaboliassa. Seuraavaksi MPP-infektiota tutkittiin yksityiskohtaisemmin hiiren insulinomasolulinjassa (MIN-6). Tutkimuksessa havaittiin, että edellä kuvatun MPP-viruksen lisääntyminen on hyvin hidasta. Replikaatio kiihtyi vasta kolmen vuorokauden kuluttua. Tutkimuksessa tarkasteltiin myös tyypin 1 diabeetikolta (lapsi) eristetyn coxsackievirus A9:n (CV-A9) molekyyligeneettisiä ja biologisia ominaisuuksia. Kyseinen kanta (D207) oli eristetty Slovakiassa, jossa se tyypitettiin serologisin testein coxsackievirus A9:ksi. D207-kanta infektoi ihmisen primäärisiä insuliinia tuottavia β-soluja tehokkaasti aiheuttaen solutuhon sekä vakavan toiminnallisen häiriön eloonjääneissä β-soluissa. Neljän viruksen DS, MPP, MCA ja D207 genomit kloonattiin ja sekvensoitiin. Ensimmäisten kolmen viruksen sekvenssit analysoitiin ja havaittiin, että DS- ja MPP-kantojen välillä löytyi vain viisi aminohappoeroa, joista kolme sijaitsi kapsid- ja kaksi ei-rakenneproteiinissa. Vertailtaessa MPP- ja MCA-kantoja niissä havaittiin kahdeksan aminohappoeroa, joista kaksi kapsid- ja kuusi ei-rakenneproteiineissa. Kapsidialueelta löydettyjen aminohappomuutosten merkitys selvitettiin ns. kohdennetun mutageneesin avulla. Hiiren haimassa in vivo kasvatetun CV-B5-MPP-viruksen infektiivisen genomin mutaatiokohdat muutettiin yksitellen alkuperäiseen DS- tai MCA-viruskannan kaltaisiksi. Tutkimuksessa tehtiin myös kaksi rekombinanttivirusta liittämällä MPP:n viruskannan 5 -UTR-kapsidi-alue DS viruskannan ei-rakenneproteiinin 3 UTR-alueeseen ja vastaavasti päinvastoin (MPP/DS ja DS/MPP). Tulokset mutanteilla tehdyistä MIN-6-solujen infektiokokeista osoittivat selvästi, että kapsidiproteiinin VP1 aminohappo 94 (Ile) oli vastuussa viruksen lisääntymisestä insuliinia tuottavissa MIN-6-soluissa. Sen sijaan in vivo infektio oli monimutkaisempi ja vaati useampia aminohappomuutoksia kapsidissa ja todennäköisesti myös ei-rakenneproteiini-alueella. D207-viruksen sekvenssien analyysi muihin virussekvensseihin verrattuna osoitti, että D207 kuuluu echovirus 11:n (E-11) alaryhmään D. Tästä huolimatta viruksen havaittiin neutraloituvan hyvin CV-A9 spesifisellä neutraloivalla vasta-aineella. Kaksoisneutraloituvuus osoittautui olevan yhteinen ominaisuus suuremmalla osalla E-11-virusten D-alaryhmään kuuluvilla viruksilla. Jatkotutkimuksen tulokset osoittivat, että kaikki tutkitut kannat neutraloituivat odotetusti E-11:n prototyyppikannan Gregoryn antiseerumilla. Yllättävää oli se, että viisi kuudesta D-genoryhmään kuuluvasta viruskannasta neutraloitui myös CV-A9:n Griggs-prototyypin antiseerumilla. Tämä osoitti, että tutkituilla echoviruskannoilla oli yhteisiä antigeenisiä alueita CV-A9:n kanssa. Vaikka monia ristiinreagoivia antigeenisiä peptidejä on löydetty, tässä tutkimuksessa neutraloituvuudesta vastaavia epitooppeja ei yrityksistä huolimatta kyetty tunnistamaan. Nämä tutkimukset osoittivat, että mutaation sijainnilla on enemmän vaikutusta enteroviruksen fenotyyppiin kuin kokonaismutaatiomäärällä. Koejärjestelyissä vain muutamat aminohapposubstituutiot johtivat radikaaleihin muutoksiin virusfenotyypissä. Suuralla osalla E-11 D -alaryhmän viruksia oli poikkeuksellinen fenotyyppi omaisuus, ristiin neutraloituminen CV-A9 -spesifisellä antiseerumilla, vaikka virukset olivat geneettisesti hyvin erilaisia

Molecular surveillance of norovirus, 2005-16 : an epidemiological analysis of data collected from the NoroNet network

Background The development of a vaccine for norovirus requires a detailed understanding of global genetic diversity of noroviruses. We analysed their epidemiology and diversity using surveillance data from the NoroNet network. Methods We included genetic sequences of norovirus specimens obtained from outbreak investigations and sporadic gastroenteritis cases between 2005 and 2016 in Europe, Asia, Oceania, and Africa. We genotyped norovirus sequences and analysed sequences that overlapped at open reading frame (ORF) 1 and ORF2. Additionally, we assessed the sampling date and country of origin of the first reported sequence to assess when and where novel drift variants originated. Findings We analysed 16 635 norovirus sequences submitted between Jan 1, 2005, to Nov 17, 2016, of which 1372 (8.2%) sequences belonged to genotype GI, 15 256 (91.7%) to GII, and seven ( Interpretation Continuous changes in the global norovirus genetic diversity highlight the need for sustained global norovirus surveillance, including assessment of possible immune escape and evolution by recombination, to provide a full overview of norovirus epidemiology for future vaccine policy decisions.Peer reviewe

Enterovirus strain and type-specific differences in growth kinetics and virus-induced cell destruction in human pancreatic duct epithelial HPDE cells

Enterovirus infections have been suspected to be involved in the development of type 1 diabetes. However, the pathogenetic mechanism of enterovirus-induced type 1 diabetes is not known. Pancreatic ductal cells are closely associated with pancreatic islets. Therefore, enterovirus infections in ductal cells may also affect beta-cells and be involved in the induction of type 1 diabetes. The aim of this study was to assess the ability of different enterovirus strains to infect, replicate and produce cytopathic effect in human pancreatic ductal cells. Furthermore, the viral factors that affect these capabilities were studied. The pancreatic ductal cells were highly susceptible to enterovirus infections. Both viral growth and cytolysis were detected for several enterovirus serotypes. However, the viral growth and capability to induce cytopathic effect (cpe) did not correlate completely. Some of the virus strains replicated in ductal cells without apparent cpe. Furthermore, there were strain-specific differences in the growth kinetics and the ability to cause cpe within some serotypes. Viral adaptation experiments were carried out to study the potential genetic determinants behind these phenotypic differences. The blind-passage of non-lytic CV-B6-Schmitt strain in HPDE-cells resulted in lytic phenotype and increased progeny production. This was associated with the substitution of a single amino acid (K257E) in the virus capsid protein VP1 and the viral ability to use decay accelerating factor (DAF) as a receptor. This study demonstrates considerable plasticity in the cell tropism, receptor usage and cytolytic properties of enteroviruses and underlines the strong effect of single or few amino acid substitutions in cell tropism and lytic capabilities of a given enterovirus. Since ductal cells are anatomically close to pancreatic islets, the capability of enteroviruses to infect and destroy pancreatic ductal cells may also implicate in respect to enterovirus induced type 1 diabetes. In addition, the capability for rapid adaptation to different cell types suggests that, on occasion, enterovirus strains with different pathogenetic properties may arise from less pathogenic ancestors. (C) 2015 Elsevier B.V. All rights reserved.Peer reviewe

Two Drinking Water Outbreaks Caused by Wastewater Intrusion Including Sapovirus in Finland

Drinking water outbreaks occur worldwide and may be caused by several factors, including raw water contamination, treatment deficiencies, and distribution network failure. This study describes two drinking water outbreaks in Finland in 2016 (outbreak I) and 2018 (outbreak II). Both outbreaks caused approximately 450 illness cases and were due to drinking water pipe breakage and subsequent wastewater intrusion into the distribution system. In both outbreaks, the sapovirus was found in patient samples as the main causative agent. In addition, adenoviruses and Dientamoeba fragilis (outbreak I), and noroviruses, astroviruses, enterotoxigenic and enterohemorragic Escherichia coli (ETEC and EHEC, respectively) and Plesiomonas shigelloides (outbreak II) were detected in patient samples. Water samples were analyzed for the selected pathogens largely based on the results of patient samples. In addition, traditional fecal indicator bacteria and host-specific microbial source tracking (MST) markers (GenBac3 and HF183) were analyzed from water. In drinking water, sapovirus and enteropathogenic E. coli (EPEC) were found in outbreak II. The MST markers proved useful in the detection of contamination and to ensure the success of contaminant removal from the water distribution system. As mitigation actions, boil water advisory, alternative drinking water sources and chlorination were organized to restrict the outbreaks and to clean the contaminated distribution network. This study highlights the emerging role of sapoviruses as a waterborne pathogen and warrants the need for testing of multiple viruses during outbreak investigation

Two Drinking Water Outbreaks Caused by Wastewater Intrusion Including Sapovirus in Finland

Drinking water outbreaks occur worldwide and may be caused by several factors, including raw water contamination, treatment deficiencies, and distribution network failure. This study describes two drinking water outbreaks in Finland in 2016 (outbreak I) and 2018 (outbreak II). Both outbreaks caused approximately 450 illness cases and were due to drinking water pipe breakage and subsequent wastewater intrusion into the distribution system. In both outbreaks, the sapovirus was found in patient samples as the main causative agent. In addition, adenoviruses and Dientamoeba fragilis (outbreak I), and noroviruses, astroviruses, enterotoxigenic and enterohemorragic Escherichia coli (ETEC and EHEC, respectively) and Plesiomonas shigelloides (outbreak II) were detected in patient samples. Water samples were analyzed for the selected pathogens largely based on the results of patient samples. In addition, traditional fecal indicator bacteria and host-specific microbial source tracking (MST) markers (GenBac3 and HF183) were analyzed from water. In drinking water, sapovirus and enteropathogenic E. coli (EPEC) were found in outbreak II. The MST markers proved useful in the detection of contamination and to ensure the success of contaminant removal from the water distribution system. As mitigation actions, boil water advisory, alternative drinking water sources and chlorination were organized to restrict the outbreaks and to clean the contaminated distribution network. This study highlights the emerging role of sapoviruses as a waterborne pathogen and warrants the need for testing of multiple viruses during outbreak investigation

Detection and quantification of SARS-CoV-2 RNA in wastewater influent in relation to reported COVID-19 incidence in Finland

Wastewater-based surveillance is a cost-effective concept for monitoring COVID-19 pandemics at a population level. Here, SARS-CoV-2 RNA was monitored from a total of 693 wastewater (WW) influent samples from 28 wastewater treatment plants (WWTP, N = 21-42 samples per WWTP) in Finland from August 2020 to May 2021, covering WW of ca. 3.3 million inhabitants (~ 60% of the Finnish population). Quantity of SARS-CoV-2 RNA fragments in 24 h-composite samples was determined by using the ultrafiltration method followed by nucleic acid extraction and CDC N2 RT-qPCR assay. SARS-CoV-2 RNA signals at each WWTP were compared over time to the numbers of confirmed COVID-19 cases (14-day case incidence rate) in the sewer network area.& nbsp;Over the 10-month surveillance period with an extensive total number of samples, the detection rate of SARSCoV-2 RNA in WW was 79% (including 6% uncertain results, i.e., amplified only in one out of four, two original and two ten-fold diluted replicates), while only 24% of all samples exhibited gene copy numbers above the quantification limit. The range of the SARS-CoV-2 detection rate in WW varied from 33% (including 10% uncertain results) in Pietarsaari to 100% in Espoo. Only six out of 693 WW samples were positive with SARS-COV-2 RNA when the reported COVID-19 case number from the preceding 14 days was zero. Overall, the 14-day COVID19 incidence was 7.0, 18, and 36 cases per 100 000 persons within the sewer network area when the probability to detect SARS-CoV-2 RNA in wastewater samples was 50%, 75% and 95%, respectively. The quantification of SARS-CoV-2 RNA required significantly more COVID-19 cases: the quantification rate was 50%, 75%, and 95% when the 14-day incidence was 110, 152, and 223 COVID-19 cases, respectively, per 100 000 persons. Multiple linear regression confirmed the relationship between the COVID-19 incidence and the SARS-CoV-2 RNA quantified in WW at 15 out of 28 WWTPs (overall R2 = 0.36, p < 0.001). At four of the 13 WWTPs where a significant relationship was not found, the SARS-CoV-2 RNA remained below the quantification limit during the whole study period. In the five other WWTPs, the sewer coverage was less than 80% of the total population in the area and thus the COVID-19 cases may have been inhabitants from the areas not covered.& nbsp;Based on the results obtained, WW-based surveillance of SARS-CoV-2 could be used as an indicator for local and national COVID-19 incidence trends. Importantly, the determination of SARS-CoV-2 RNA fragments from WW is a powerful and non-invasive public health surveillance measure, independent of possible changes in the clinical testing strategies or in the willingness of individuals to be tested for COVID-19.Peer reviewe

Recombination in the Evolution of Enterovirus C Species Sub-Group that Contains Types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99

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