Distribution and Clinical Associations of Ljungan Virus (Parechovirus B)

Among rodent-borne viruses, those belonging to the genera Mammarenavirus, Orthohantavirus and Orthopoxvirus are a particular focus of study both in humans and animals, since they represent some of the most widespread rodent-borne disease-causing pathogens. More recently, the interest in parechoviruses has been increasing because some are known to cause diseases in humans, while others are carried by rodents, although the zoonotic potential of rodent-borne parechoviruses has not been established. Ljungan virus (LV), which belongs to the species Parechovirus B, was first isolated from bank voles in Sweden in 1998. It belongs to the Picornaviridae family. Currently, there is little information about LV host range and epidemiology, but a few reports suggest an association between LV and human disease. The main aims of this doctoral thesis were to establish the symptoms associated with LV in humans, to investigate the association of LV with human central nervous system (CNS) disease, and to determine the prevalence and distribution of LV in human and other animal populations in Europe. LV-associated symptoms were investigated in two human cohorts. Serum samples from Finnish patients hospitalized for suspected nephropathia epidemica (NE) caused by the Orthohantavirus Puumala virus (PUUV) were screened for the presence of lymphocytic choriomeningitis virus (LCMV, Arenavirus), cowpox virus (CPXV, Orthopoxvirus) and LV, in order to compare the disease outcomes in these patients and to establish if the co-existence of viruses could lead to an increase in the severity of symptoms. However, no unusual or additional manifestations between PUUV cases and PUUV-LV/LCMV/CPXV cases were detected. To determine if LV (together with the rodent-borne virus LCMV) could be one of the causes of neurological symptoms in Finnish patients with suspected CNS disease, anti-LV and LCMV antibodies were analyzed from serum and cerebrospinal fluid samples. LV- and LCMV-specific nucleic acids were also analyzed from the patient samples. However, no association between LV or LCMV antibodies or nucleic acids and the neurological manifestations in the patient cohort was detected. In order to improve the knowledge of the host and geographical distribution of LV, tissues from multiple rodent and insectivore species from ten European countries were screened for LV nucleic acids. We confirmed that LV is widespread geographically, having been detected in at least one host species in nine out of ten countries involved in the study. Seventeen out of 21 species screened were LV PCR-positive. Results indicated that bank voles are the main rodent host for LV. Male and subadult bank voles are significantly more likely to be LV-positive, and the prevalence has a temporal pattern (higher in autumn compared to spring and summer), possibly due to adult bank voles clearing the infection.Sekä ihmis- että eläinperäisiä mammarena-, orthohanta- ja orthopoxviruksia on tutkittu laajimmin, koska niitä esiintyy maailmanlaajuisesti ja aiheuttavat lisäksi jyrsijävälitteisiä zoonooseja. Nykyään kiinnostuksen kohteina ovat lisäksi jyrsijöissä esiintyvät parechovirukset. Ihmisvälitteisten parechovirusten tiedetään olevan patogeenisia ja helposti leviäviä mutta vielä ei ole osoitettu jyrsijöiden parechovirusten aiheuttavan zoonooseja. Parechovirus B –lajiin kuuluva Ljungan virus (LV) löydettiin ensimmäisen kerran vuonna 1998 ruotsalaisesta metsämyyrästä. Ljungan virus kuuluu muiden parechovirusten kanssa suureen pikornavirusperheeseen. Ljungan virus on jyrsijävirus mutta muuten tästä viruksesta tiedetään vähän ja esimerkiksi sen isäntäjoukon laajuutta tai epidemiologiaa ei ole tarkkaan selvitetty. Löytyy vain harvoja tutkimuksia, joissa Ljungan viruksen seroprevalenssia ihmisillä on selvitetty tai ehdotettu yhteyttä ihmistauteihin. Tämän väitöskirjatyön päätavoitteina oli tutkia ja selvittää Ljungan viruksen assosiaatiota ihmistauteihin ja näistä varsinkin keskushermostoinfektioihin, sekä määrittää viruksen prevalenssia ja levinneisyyttä Euroopan laajuisesti ihmisissä ja jyrsijöissä. Ljungan viruksen assosiaatiota ihmistauteihin tutkittiin kahdessa eri potilaskohortissa. Toinen potilaskohortti koostui suomalaisista sairaalahoitoisista myyräkuumepotilaista. Myyräkuumeen aiheuttaa jyrsijävälitteinen orthohantaviruksiin kuuluva Puumala-virus (PUUV). Ljungan viruksen lisäksi tästä potilaskohortista tutkittiin myös lymfosyyttisen koriomeningiittiviruksen (LCMV) sekä lehmärokkoviruksen (CPXV, Orthopoxvirus) aiheuttamia serokonversioita. Tuloksia ja potilaiden oirekuvia verrattiin toisiinsa. Lopputuloksena voitiin todeta, että myyräkuumeen taudinkuvassa ei huomattu eroa riippumatta siitä oliko potilaalla vain PUUV-infektio vai mahdollinen LV/LCMV/CXPV-yhteisinfektio. Toinen potilaskohortti koostui potilaista, joilla epäiltiin keskushermostoinfektiota. Tässä kohortista tutkittiin potilasnaytteistä niin Ljungan viruksen kuin LCMV:n seerumivasta-aineita sekä etsittiin selkäydinnestenäytteistä näiden virusten nukleiinihappoa. Aineistosta ei löydetty yhtään LV- tai LCMV-nukleiinihappopositiivista selkäydinnäytettä. Ljungan viruksen jyrsijäisäntäkirjoa ja maantieteellistä levinneisyyttä tutkittiin eri jyrsijälajien kudosnäytteiden avulla. Näitä näytteitä kerättiin yhdeksästä eri Euroopan maasta ja näytteistä tutkittiin Ljungan viruksen nukleiinihappoja. Pystyimme osoittamaan että LV on maantieteellisesti erittäin laajalle levinnyt. Jokaisesta tutkitusta yhdeksästä Euroopan maasta pystyttiin osoittamaan Ljungan viruksen nukleiinihappoa ainakin yhdestä jyrsijälajista. Testatuista 21 jyrsijälajista 17 lajissa osoitettiin LV nukeliinihappoa. Tulosten perusteella metsämyyrä toimii kuitenkin Ljungan viruksen pääisäntänä. Urokset sekä nuoret aikuiset metsämyyrät olivat merkittävästi useammin Ljungan virus positiivisia ja temporaalisen prevalenssin mukaan syksyllä esiintyvyys oli korkeampi kuin keväällä tai kesällä. Temporaalinen vaihtelu viittaisi siihen, että aikuisilla metsämyyrillä infektio on mahdollisesti itsestäänrajoittuva

Lymphocytic choriomeningitis, Ljungan and orthopoxvirus seroconversions in patients hospitalized due to acute Puumala hantavirus infection

Background: The emergence and re-emergence of zoonotic and vector-borne diseases are increasing in Europe. Prominent rodent-borne zoonotic viruses include Puumala hantavirus (PUUV; the causative agent of nephropathia epidemica, NE), lymphocytic choriomeningitis virus (LCMV), and orthopoxviruses (OPV). In addition, Ljungan virus (LV) is considered a potentially zoonotic virus. Objective: The aim of this study was to compare clinical picture between acute PUUV patients with and without additional rodent-borne viral infections, to investigate if concurrent infections influence disease severity. Study design: We evaluated seroprevalence of and seroconversions to LCMV, LV and OPV in 116 patients hospitalized for NE. Clinical and laboratory variables were closely monitored during hospital care. Results: A total of five LCMV, 15 LV, and one OPV seroconversions occurred. NE patients with LCMV seroconversions were younger, and had lower plasma creatinine concentrations and platelet counts than patients without LCMV seroconversions. No differences occurred in clinical or laboratory findings between patients with and without seroconversions to LV and OPV. We report, for the first time, LCMV seroprevalence in Finland, with 8.5% of NE patients seropositive for this virus. Seroprevalences for LV and OPV were 47.8% and 32.4%, respectively. Conclusion: Cases with LCMV seroconversions were statistically younger, had milder acute kidney injury and more severe thrombocytopenia than patients without LCMV. However, the low number of seroconversion cases precludes firm conclusions. Concurrent LV or OPV infections do not appear to influence clinical picture for NE patients. (C) 2016 Elsevier B.V. All rights reserved.Peer reviewe

Evolutionary Relationships of Ljungan Virus Variants Circulating in Multi-Host Systems across Europe

The picornavirus named ‘Ljungan virus’ (LV, species Parechovirus B) has been detected in a dozen small mammal species from across Europe, but detailed information on its genetic diversity and host specificity is lacking. Here, we analyze the evolutionary relationships of LV variants circulating in free-living mammal populations by comparing the phylogenetics of the VP1 region (encoding the capsid protein and associated with LV serotype) and the 3Dpol region (encoding the RNA polymerase) from 24 LV RNA-positive animals and a fragment of the 5′ untranslated region (UTR) sequence (used for defining strains) in sympatric small mammals. We define three new VP1 genotypes: two in bank voles (Myodes glareolus) (genotype 8 from Finland, Sweden, France, and Italy, and genotype 9 from France and Italy) and one in field voles (Microtus arvalis) (genotype 7 from Finland). There are several other indications that LV variants are host-specific, at least in parts of their range. Our results suggest that LV evolution is rapid, ongoing and affected by genetic drift, purifying selection, spillover and host evolutionary history. Although recent studies suggest that LV does not have zoonotic potential, its widespread geographical and host distribution in natural populations of well-characterized small mammals could make it useful as a model for studying RNA virus evolution and transmission

Evolutionary Relationships of Ljungan Virus Variants Circulating in Multi-Host Systems across Europe

The picornavirus named ‘Ljungan virus’ (LV, species Parechovirus B) has been detected in a dozen small mammal species from across Europe, but detailed information on its genetic diversity and host specificity is lacking. Here, we analyze the evolutionary relationships of LV variants circulating in free-living mammal populations by comparing the phylogenetics of the VP1 region (encoding the capsid protein and associated with LV serotype) and the 3Dpol region (encoding the RNA polymerase) from 24 LV RNA-positive animals and a fragment of the 5′ untranslated region (UTR) sequence (used for defining strains) in sympatric small mammals. We define three new VP1 genotypes: two in bank voles (Myodes glareolus) (genotype 8 from Finland, Sweden, France, and Italy, and genotype 9 from France and Italy) and one in field voles (Microtus arvalis) (genotype 7 from Finland). There are several other indications that LV variants are host-specific, at least in parts of their range. Our results suggest that LV evolution is rapid, ongoing and affected by genetic drift, purifying selection, spillover and host evolutionary history. Although recent studies suggest that LV does not have zoonotic potential, its widespread geographical and host distribution in natural populations of well-characterized small mammals could make it useful as a model for studying RNA virus evolution and transmission

Geographical Distribution and Genetic Diversity of Bank Vole Hepaciviruses in Europe

The development of new diagnostic methods resulted in the discovery of novel hepaciviruses in wild populations of the bank vole (Myodes glareolus, syn. Clethrionomys glareolus). The naturally infected voles demonstrate signs of hepatitis similar to those induced by hepatitis C virus (HCV) in humans. The aim of the present research was to investigate the geographical distribution of bank vole-associated hepaciviruses (BvHVs) and their genetic diversity in Europe. Real-time reverse transcription polymerase chain reaction (RT-qPCR) screening revealed BvHV RNA in 442 out of 1838 (24.0%) bank voles from nine European countries and in one of seven northern red-backed voles (Myodes rutilus, syn. Clethrionomys rutilus). BvHV RNA was not found in any other small mammal species (n = 23) tested here. Phylogenetic and isolation-by-distance analyses confirmed the occurrence of both BvHV species (Hepacivirus F and Hepacivirus J) and their sympatric occurrence at several trapping sites in two countries. The broad geographical distribution of BvHVs across Europe was associated with their presence in bank voles of different evolutionary lineages. The extensive geographical distribution and high levels of genetic diversity of BvHVs, as well as the high population fluctuations of bank voles and occasional commensalism in some parts of Europe warrant future studies on the zoonotic potential of BvHVs.Peer reviewe

Finding a partner in the ocean: molecular and evolutionary bases of the response to sexual cues in a planktonic diatom

Microalgae play a major role as primary producers in aquatic ecosystems. Cell signalling regulates their interactions with the environment and other organisms, yet this process in phytoplankton is poorly defined. Using the marine planktonic diatom Pseudo-nitzschia multistriata, we investigated the cell response to cues released during sexual reproduction, an event that demands strong regulatory mechanisms and impacts on population dynamics. We sequenced the genome of P. multistriata and performed phylogenomic and transcriptomic analyses, which allowed the definition of gene gains and losses, horizontal gene transfers, conservation and evolutionary rate of sex-related genes. We also identified a small number of conserved noncoding elements. Sexual reproduction impacted on cell cycle progression and induced an asymmetric response of the opposite mating types. G protein-coupled receptors and cyclic guanosine monophosphate (cGMP) are implicated in the response to sexual cues, which overall entails a modulation of cell cycle, meiosis-related and nutrient transporter genes, suggesting a fine control of nutrient uptake even under nutrient-replete conditions. The controllable life cycle and the genome sequence of P. multistriata allow the reconstruction of changes occurring in diatoms in a key phase of their life cycle, providing hints on the evolution and putative function of their genes and empowering studies on sexual reproduction

Finding a partner in the ocean: molecular and evolutionary bases of the response to sexual cues in a planktonic diatom

Microalgae play a major role as primary producers in aquatic ecosystems. Cell signalling regulates their interactions with the environment and other organisms, yet this process in phytoplankton is poorly defined. Using the marine planktonic diatom Pseudo-nitzschia multistriata, we investigated the cell response to cues released during sexual reproduction, an event that demands strong regulatory mechanisms and impacts on population dynamics. We sequenced the genome of P. multistriata and performed phylogenomic and transcriptomic analyses, which allowed the definition of gene gains and losses, horizontal gene transfers, conservation and evolutionary rate of sex-related genes. We also identified a small number of conserved noncoding elements. Sexual reproduction impacted on cell cycle progression and induced an asymmetric response of the opposite mating types. G protein-coupled receptors and cyclic guanosine monophosphate (cGMP) are implicated in the response to sexual cues, which overall entails a modulation of cell cycle, meiosis-related and nutrient transporter genes, suggesting a fine control of nutrient uptake even under nutrient-replete conditions. The controllable life cycle and the genome sequence of P. multistriata allow the reconstruction of changes occurring in diatoms in a key phase of their life cycle, providing hints on the evolution and putative function of their genes and empowering studies on sexual reproduction

Is rodent-borne Ljungan virus responsible for mortality in migrating Norwegian lemmings (Lemmus lemmus)?

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PCR prevalence of rodent-borne Ljungan virus across Europe

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Comparison between Gibson–Cooke and Macroduct Methods in the Cystic Fibrosis Neonatal Screening Program and in Subjects Who Are Cystic Fibrosis Screen-Positive with an Inconclusive Diagnosis

The sweat test (ST) is the current diagnostic gold standard for cystic fibrosis (CF). Many CF centres have switched from the Gibson–Cooke method to the Macroduct system-based method. We used these methods simultaneously to compare CF screening outcomes. STs using both methods were performed simultaneously between March and December 2022 at CF Centre in Florence. We included newborns who underwent newborn bloodspot screening (NBS), newborns undergoing transfusion immediately after birth, and children with CF screen-positive, inconclusive diagnosis (CFSPID). We assessed 72 subjects (median age 4.4 months; range 0–76.7): 30 (41.7%) NBS-positive, 18 (25.0%) newborns who underwent transfusion, and 24 (33.3%) children with CFSPID. No significant differences were found between valid sample numbers, by patient ages and groups (p = 0.10) and between chloride concentrations (p = 0.13), except for sweat chloride (SC) measured by the Gibson–Cooke and Macroduct methods in CFSPID group (29.0, IQR: 20.0–48.0 and 22.5, IQR: 15.5–30.8, respectively; p = 0.01). The Macroduct and Gibson–Cooke methods showed substantial agreement with the SC values, except for CFSPID, whose result may depend on the method of sweat collection. In case of invalid values with Macroduct, the test should be repeated with Gibson–Cooke method