Academic literature on the topic 'Norovirus genome'

Noroviruses are small, positive-sense RNA viruses within the family Caliciviridae, and are now accepted widely as a major cause of acute gastroenteritis in both developed and developing countries. Despite their impact, our understanding of the life cycle of noroviruses has lagged behind that of other RNA viruses due to the inability to culture human noroviruses (HuNVs). Our knowledge of norovirus biology has improved significantly over the past decade as a result of numerous technological advances. The use of a HuNV replicon, improved biochemical and cell-based assays, combined with the discovery of a murine norovirus capable of replication in cell culture, has improved greatly our understanding of the molecular mechanisms of norovirus genome translation and replication, as well as the interaction with host cell processes. In this review, the current state of knowledge of the intracellular life of noroviruses is discussed with particular emphasis on the mechanisms of viral gene expression and viral genome replication.

Norovirus is a major cause of acute gastroenteritis worldwide. Over 30 different genotypes, mostly from genogroup I (GI) and II (GII), have been shown to infect humans. Despite three decades of genome sequencing, our understanding of the role of genomic diversification across continents and time is incomplete. To close the spatiotemporal gap of genomic information of human noroviruses, we conducted a large-scale genome-wide analyses that included the nearly full-length sequencing of 281 archival viruses circulating since the 1970s in over 10 countries from four continents, with a major emphasis on norovirus genotypes that are currently underrepresented in public genome databases. We provided new genome information for 24 distinct genotypes, including the oldest genome information from 12 norovirus genotypes. Analyses of this new genomic information, together with those publicly available, showed that (i) noroviruses evolve at similar rates across genomic regions and genotypes; (ii) emerging viruses evolved from transiently-circulating intermediate viruses; (iii) diversifying selection on the VP1 protein was recorded in genotypes with multiple variants; (iv) non-structural proteins showed a similar branching on their phylogenetic trees; and (v) contrary to the current understanding, there are restrictions on the ability to recombine different genomic regions, which results in co-circulating populations of viruses evolving independently in human communities. This study provides a comprehensive genetic analysis of diverse norovirus genotypes and the role of non-structural proteins on viral diversification, shedding new light on the mechanisms of norovirus evolution and transmission.

Noroviruses are non-enveloped viruses with a positive-sense single-stranded RNA (ssRNA) genome belonging to the genus Norovirus, from the family Caliciviridae, which are accountable for acute gastroenteritis in humans. The Norovirus genus is subdivided into seven genogroups, i.e., (GI-GVII); among these, the genogroup II and genotype 4 (GII.4) strains caused global outbreaks of human norovirus (HuNov) disease. The viral genome comprises three open reading frames (ORFs). ORF1 encodes the nonstructural polyprotein that is cleaved into six nonstructural proteins, which include 3C-like cysteine protease (3CLpro) and a viral RNA-dependent RNA polymerase. ORF2 and ORF3 encode the proteins VP1 and VP2. The RNA-dependent RNA polymerase (RdRp) from noroviruses is one of the multipurpose enzymes of RNA viruses vital for replicating and transcribing the viral genome, making the virally encoded enzyme one of the critical targets for the development of novel anti-norovirus agents. In the quest for a new antiviral agent that could combat HuNov, high throughput virtual screening (HTVS), combined with e-pharmacophore screening, was applied to screen compounds from the PubChem database. CMX521 molecule was selected as a prototype for a similarity search in the PubChem online database. Molecular dynamics simulations were employed to identify different compounds that may inhibit HuNov. The results predicted that compound CID-57930781 and CID-44396095 formed stable complexes with MNV-RdRp within 50 ns; hence, they may signify as promising human norovirus inhibitors.

ABSTRACT Noroviruses (Caliciviridae) are RNA viruses with a single-stranded, positive-oriented polyadenylated genome. To date, little is known about the replication strategy of norovirus, a so-far noncultivable virus. We have examined the initiation of replication of the norovirus genome in vitro, using the active norovirus RNA-dependent RNA polymerase (3Dpol), homopolymeric templates, and synthetic subgenomic or antisubgenomic RNA. Initiation of RNA synthesis on homopolymeric templates as well as replication of subgenomic polyadenylated RNA was strictly primer dependent. In this context and as observed for other enteric RNA viruses, i.e., poliovirus, a protein-primed initiation of RNA synthesis after elongation of the VPg by norovirus 3Dpol was postulated. To address this question, norovirus VPg was expressed in Escherichia coli and purified. Incubation of VPg with norovirus 3Dpol generated VPg-poly(U), which primed the replication of subgenomic polyadenylated RNA. In contrast, replication of antisubgenomic RNA was not primer dependent, nor did it depend on a leader sequence, as evidenced by deletion analysis of the 3′ termini of subgenomic and antisubgenomic RNA. On nonpolyadenylated RNA, i.e., antisubgenomic RNA, norovirus 3Dpol initiated RNA synthesis de novo and terminated RNA synthesis by a poly(C) stretch. Interestingly, on poly(C) RNA templates, norovirus 3Dpol initiated RNA synthesis de novo in the presence of high concentrations of GTP. We propose a novel model for initiation of replication of the norovirus genome by 3Dpol, with a VPg-protein-primed initiation of replication of polyadenylated genomic RNA and a de novo initiation of replication of antigenomic RNA.

Noroviruses are highly diverse viruses that are the major viral cause of acute gastroenteritis in humans. Although these viruses can infect multiple mammalian species, their potential for zoonosis is not well understood, especially within Genogroup IV (GIV), which contains viruses that infect humans, canines, and felines. The study of GIV viruses has been, in part, hindered by the limited number of complete genomes. Here, we developed a full-genome amplicon-based platform that facilitated the sequencing of canine noroviruses circulating in the United States. Eight novel nearly full-length canine norovirus genomes and two nearly complete VP1 sequences, including four GIV.2, three GVI.1, and three GVI.2 viruses, were successfully obtained. Only animal strains exhibited GVI/GIV chimeric viruses, demonstrating restrictions in norovirus recombination. Using genomic, phylogenetic, and structural analyses, we show that differences within the major capsid protein and the non-structural proteins of GIV and GVI noroviruses could potentially limit cross-species transmission between humans, canines, and felines.

Norovirus is the leading cause of acute gastroenteritis worldwide. Since the discovery of human norovirus (HuNoV), an efficient and reproducible norovirus replication system has not been established in cultured cells. Although limited amounts of virus particles can be produced when the HuNoV genome is directly transfected into cells, the HuNoV cycle of infection has not been successfully reproduced in any currently available cell-culture system. Those results imply that the identification of a functional cell-surface receptor for norovirus might be the key to establishing a norovirus culture system. Using a genome-wide CRISPR/Cas9 guide RNA library, we identified murine CD300lf and CD300ld as functional receptors for murine norovirus (MNV). The treatment of susceptible cells with polyclonal antibody against CD300lf significantly reduced the production of viral progeny. Additionally, ectopic CD300lf expression in nonsusceptible cell lines derived from other animal species enabled MNV infection and progeny production, suggesting that CD300lf has potential for dictating MNV host tropism. Furthermore, CD300ld, which has an amino acid sequence in the N-terminal region of its extracellular domain that is highly homologous to that of CD300lf, also functions as a receptor for MNV. Our results indicate that direct interaction of MNV with two cell-surface molecules, CD300lf and CD300ld, dictates permissive noroviral infection.

Norovirus full-genome sequencing is challenging due to sequence heterogeneity among genomes. Previous methods have relied on PCR amplification, which is problematic due to primer design, and transcriptome sequencing (RNA-Seq), which nonspecifically sequences all RNA, including host and bacterial RNA, in stool specimens. Target enrichment uses a panel of custom-designed 120-mer RNA baits that are complementary to all publicly available norovirus sequences, with multiple baits targeting each position of the genome, which overcomes the challenge of primer design. Norovirus genomes are enriched from stool RNA extracts to minimize the sequencing of nontarget RNA. SureSelect target enrichment and Illumina sequencing were used to sequence full genomes from 507 norovirus-positive stool samples with reverse transcription–real-time PCR cycle threshold (CT) values of 10 to 43. Sequencing on an Illumina MiSeq system in batches of 48 generated, on average, 81% on-target reads per sample and 100% genome coverage with >12,000-fold read depth. Samples included genotypes GI.1, GI.2, GI.3, GI.6, GI.7, GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.13, GII.14, and GII.17. When outliers were accounted for, we generated >80% genome coverage for all positive samples, regardless ofCTvalues. A total of 164 samples were tested in parallel with conventional PCR genotyping of the capsid shell domain; 164/164 samples were successfully sequenced, compared to 158/164 samples that were amplified by PCR. Four of the samples that failed capsid PCR analysis had low titers, which suggests that target enrichment is more sensitive than gel-based PCR. Two samples failed PCR due to primer mismatches; target enrichment uses multiple baits targeting each position, thus accommodating sequence heterogeneity among norovirus genomes.

ABSTRACTHuman noroviruses (HuNoV) are a significant cause of acute gastroenteritis in the developed world, and yet our understanding of the molecular pathways involved in norovirus replication and pathogenesis has been limited by the inability to efficiently culture these viruses in the laboratory. Using the murine norovirus (MNV) model, we have recently identified a network of host factors that interact with the 5′ and 3′ extremities of the norovirus RNA genome. In addition to a number of well-known cellular RNA binding proteins, the molecular chaperone Hsp90 was identified as a component of the ribonucleoprotein complex. Here, we show that the inhibition of Hsp90 activity negatively impacts norovirus replication in cell culture. Small-molecule-mediated inhibition of Hsp90 activity using 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) revealed that Hsp90 plays a pleiotropic role in the norovirus life cycle but that the stability of the viral capsid protein is integrally linked to Hsp90 activity. Furthermore, we demonstrate that both the MNV-1 and the HuNoV capsid proteins require Hsp90 activity for their stability and that targeting Hsp90in vivocan significantly reduce virus replication. In summary, we demonstrate that targeting cellular proteostasis can inhibit norovirus replication, identifying a potential novel therapeutic target for the treatment of norovirus infections.IMPORTANCEHuNoV are a major cause of acute gastroenteritis around the world. RNA viruses, including noroviruses, rely heavily on host cell proteins and pathways for all aspects of their life cycle. Here, we identify one such protein, the molecular chaperone Hsp90, as an important factor required during the norovirus life cycle. We demonstrate that both murine and human noroviruses require the activity of Hsp90 for the stability of their capsid proteins. Furthermore, we demonstrate that targeting Hsp90 activityin vivousing small molecule inhibitors also reduces infectious virus production. Given the considerable interest in the development of Hsp90 inhibitors for use in cancer therapeutics, we identify here a new target that could be explored for the development of antiviral strategies to control norovirus outbreaks and treat chronic norovirus infection in immunosuppressed patients.

ABSTRACT A generally accepted view of norovirus replication is that capsid expression requires production of a subgenomic transcript, the presence of capsid often being used as a surrogate marker to indicate the occurrence of viral replication. Using a polymerase II-based baculovirus delivery system, we observed capsid expression following introduction of a full-length genogroup 3 norovirus genome into HepG2 cells. However, capsid expression occurred as a result of a novel translation termination/reinitiation event between the nonstructural-protein and capsid open reading frames, a feature that may be unique to genogroup 3 noroviruses.

Noroviruses infect a wide range of mammals and are the major cause of gastroenteritis in humans. Recombination at the junction of ORF1 encoding nonstructural proteins and ORF2 encoding major capsid protein VP1 is a well-known feature of noroviruses. Using all available complete norovirus sequences, we systematically analyzed patterns of natural recombination in the genus Norovirus both throughout the genome and across the genogroups. Recombination events between nonstructural (ORF1) and structural genomic regions (ORF2 and ORF3) were found in all analyzed genogroups of noroviruses, although recombination was most prominent between members of GII, the most common genogroup that infects humans. The half-life times of recombinant forms (clades without evidence of recombination) of human GI and GII noroviruses were 10.4 and 8.4–11.3 years, respectively. There was evidence of many recent recombination events, and most noroviruses that differed by more than 18% of nucleotide sequence were recombinant relative to each other. However, there were no distinct recombination events between viruses that differed by over 42% in ORF2/3, consistent with the absence of systematic recombination between different genogroups. The few inter-genogroup recombination events most likely occurred between ancient viruses before they diverged into contemporary genogroups. The recombination events within ORF1 or between ORF2/3 were generally rare. Thus, noroviruses routinely exchange full structural and nonstructural blocks of the genome, providing a modular evolution.

Norovirus is the commonest cause of viral gastroenteritis, affecting all age groups worldwide. Outbreaks frequently occur in semi-closed communities such as schools, cruise ships, prisons and hospitals. Within the healthcare environment, the economic and logistical burdens and the inconvenience caused by norovirus is significant, since ward closure remains central to infection control. The aim of this study was to investigate norovirus transmission dynamics during hospital outbreaks. The ultimate goal was to provide information that could, in future, lead to the development of novel, less disruptive approaches to curtailing the spread of infection. The study explored the application of 'next generation' high throughput DNA sequencing technologies to the determination of large numbers of norovirus genomes. Whole genome sequences provide the highest possible level of discrimination among viruses, information which is essential to the identification of linked and independent cases of infection. The approach exploits the high norovirus mutation rate, which is typical of RNA viruses. Consequently, viruses within a single ward which differ by more than a few SNVs can be considered to represent independent introductions, rather than a single outbreak. Whole genome sequence data (determined for noroviruses collected between 2009 and 2013) were combined with epidemiological data, providing further insights into transmission dynamics. These data identified multiple independent virus introductions during single ward outbreaks. The possible origin of such outbreaks in Oxfordshire hospitals were investigated using viruses originating in the local community, and in other healthcare environments distributed throughout the UK. Whole genome sequences of noroviruses from consecutive years were genetically divergent, confirming the rapid evolution of the virus over time and excluding the possibility of prolonged environmental contamination as a reservoir of infection. Such detailed information on norovirus transmission within the healthcare environment could inform alternative future approaches to optimising infection control within the healthcare setting.

Caliciviruses are single-stranded positive-sense RNA viruses, most commonly associated with outbreaks of gastroenteritis in humans. In addition to the three open reading frames (ORFs) typical of caliciviruses a highly conserved fourth overlapping ORF within the murine norovirus (MNV) genome was identified and characterised during this project. ORF4 overlaps and is contained within the capsid encoding ORF2 in a +1 frame. Once a suitable antibody had been generated, immunoblotting was used to show that the ORF4 protein is produced during MNV infection. Although ORF4 mutant viruses were viable and replicated to wild-type MNV levels in tissue culture, pressure to restore ORF4 expression upon serial passage under specific conditions was demonstrated. Importantly, the ORF4 knockout virus was significantly attenuated in STAT1-/- mice. Proteomic analysis and a commercial yeast two-hybrid screen were used to identify host cell factors which interact with the ORF4 protein and confocal imaging was employed to examine cellular localisation. These approaches indicated that the ORF4 protein localises to the mitochondria and in vitro assays were subsequently used to demonstrate an involvement of the ORF4 protein in MNV induced apoptosis. As cells infected with the knockout virus showed an earlier and higher degree of apoptosis induction compared to wild-type infected cells, it is possible that the ORF4 protein may function to delay the onset of apoptosis during MNV infection. Whether or not the ORF4 protein has antiapoptotic activity or whether the difference in apoptotic phenotype is an indirect consequence of ORF4 protein function remains to be investigated. These data indicate that the ORF4 protein represents a novel, previously uncharacterised virulence determinant in MNV.

Genome-scale ordered RNA structure (GORS) was first identified in 2004. It refers to the presence of secondary structure throughout the length of the RNA genomes of certain genera of RNA virus families, as predicted by bioinformatic analysis. It was also observed that the viruses containing GORS were able to establish persistent infections in their natural hosts, raising the possibility that the presence of GORS could play a role in viral avoidance of the innate immune system. This thesis describes the first study of GORS and its possible role in persistence. Two GORS viruses have been studied, equine rhinitis A virus (ERAV) and murine norovirus (MNV). A 55% seroprevalence of ERAV has been determined in a cohort of Scottish horses indicating a wide exposure to the virus. Equine faecal samples were screened for ERAV by PCR with the intention of identifying a virus, possibly from a persistently infected animal, which would not have undergone any cell culture adaptations as laboratory strains have. Newly identified viruses would then be sequenced, their secondary structures predicted and further studies carried out. Unfortunately, none of the 50 faecal samples screened were positive and clinical isolates of ERAV provided by the Animal Health Trust were sequenced but were identical to laboratory strains, so the study then focussed on MNV. Prevalence of MNV in laboratory mice was determined by PCR of faecal samples to be 67%. MNV was also discovered in the faeces of a pet shop mouse and a wild wood mouse (Apodemus sylvaticus). The complete genomes of 4 laboratory mouse MNVs, the pet shop mouse and wood mouse MNVs were sequenced. Phylogenetic analysis showed the wood mouse MNV had a p distance of 23% from other MNVs, although the laboratory mice and pet shop mouse were closely related to other MNVs. Structural analysis of the genomes of 6 sequenced MNVs, including the wood mouse virus, showed all were GORS viruses. A laboratory strain of MNV, MNV-3, was serially passaged in RAW 264.7 cells to test the hypothesis that in an animal with an intact immune system, there is a pressure for GORS viruses to maintain their genomic RNA structure as a means of immune avoidance, and that cell culture adaptation would attenuate the degree of secondary structure. The complete genome of passage 33 was sequenced, which revealed 7 base mutations, a mutation rate of 0.1 %, which was not considered significant enough to have affected the degree of secondary structure. In order to assess if structured and unstructured RNA behaved differently in cells, replication deficient RNA transcripts were made from the infectious clones of a panel of GORS and non-GORS viruses. These transcripts were electroporated into cells and their rate of decay measured, but there was no difference between the GORS and non-GORS transcripts. The full length and 4 kilobase transcripts were transfected into NIH3T3 cells and the degree of interferon-β induction measured by quantitative PCR and a luciferase reporter assay. The IFN-β response differed across the panel of viruses, and although none of the GORS viruses induced strongly, the non-GORS viruses were variable in their ability to induce an IFN-β response, some inducing strongly, other not at all. This result indicates that during exposure of viral genomes in the cytoplasm during infection, GORS-virus RNAs are unlikely to induce an interferon response, possibly contributing to their ability to persist. It is unclear why some non-GORS-viruses failed to induce IFN and there are likely to be other contributory factors.

Uvod: Virusni gastrointestinalni sindrom je aktuelni zdravstveni problem u celom svetu. To važi kako u razvijenim zemljama, tako i u zemljama u razvoju, a posebno u nerazvijenim zemljama, gde je drugi po redu uzrok mortaliteta. Nagli početak bolesti, praćen pojavom velikog broja tečnih stolica, mukom, povraćanjem, bolovima u stomaku, temperaturom, malaksalošću, ima za posledicu dehidrataciju. U svim starosnim grupama obolelih, a naročito kod sasvim male dece, starih i imunodeficitarnih osoba može da dođe do smrtnog ishoda, ukoliko se brzo ne postavi tačna etiološka dijagnoza bolesti i ne pristupi se odmah nadoknadi vode i elektrolita, kao i primeni svih ostalih mera simptomatske terapije. Brzo postavljena tačna dijagnoza, što se najbolje postiže real-time PCR testom, sprečava pojavu komplikacija, pa i fatalnog ishoda bolesti. Istovremeno, omogućava primenu odgovarajućih epidemioloških mera da se spreči nastanak epidemija i njihovo širenje. Cilj ovog istraživanja bio je da se tačno utvrdi incidenca virusnog gastrointestinalnog sindroma u Vojvodini i učestalost pojave epidemijskog i sporadičnog javljanja ove bolesti. Cilj je bio i postavljanje algoritma za primenu real-time PCR testa u dijagnostici virusnog gastrointestinalnog sindroma u budućem radu. Isto tako, cilj je bio da se molekularnom analizom, sekvenciranjem delova genoma pozitivnih uzoraka stolice, izvrši genetska tipizacija i odredi filogenetska pripadnost virusa. Materijal i metode: Tokom petogodišnjeg istraživanja molekularnim real-time PCR testom pregledane su 1003 obolele osobe sa simptomima virusnog dijarealnog sindroma, starosti od mesec dana do preko 90 godina. Pregledani su na rota, noro, astro i enterične adenoviruse. Na osnovu podataka iz anketnih upitnika i istorija bolesti, detaljno su analizirani svi klinički pokazatelji (javljanje bolesti tokom godine, trajanje bolesti, simptomi). Procena težine kliničke slike vršena je prema Vesikari skali. Svi podaci su upoređivani prema vrsti virusnog uzročnika, prema starosti obolelih, godinama trajanja istraživanja i epidemijskom i sporadičnom javljanju oboljenja. Dobijeni podaci su statistički obrađeni, tabelarno i grafički prikazani. Rezultati: U petogodišnjem periodu real-time PCR testom pregledan je uzorak od 1003 obolele osobe različite starosti na 4 virusna uzročnika dijarealnog sindroma (rota, noro, astro i enterične adenoviruse). Virusni dijarealni sindrom dokazan je kod 709 obolelih (70,69%). Najčešće su dokazane rotavirusne infekcije u 28,81%. Statistički značajno najčešće rotavirusi su bili utvrđeni kod dece do 5 godina (38,90%), ali u visokom procentu i kod dece uzrasta 6 do 14 godina (24,83%). Deca mlađa od 5 godina imala su statistički značajno najtežu kliničku sliku, bila su češće hospitalizovana i imala su statistički značajno višu temperaturu. Pored više temperature kod obolelih od rotavirusa, klinička slika je kod ovih bolesnika bila teža i bolest je duže trajala nego kod obolelih od drugih virusa. Norovirusna infekcija je dokazana u 23,03% obolelih i to statistički značajno češće kod odraslih osoba, starijih od 20 godina. Od kliničkih simptoma kod ovih bolesnika statistički značajno češće su dokazani muka, povraćanje i bolovi u stomaku, nego kod obolelih od drugih virusa. Norovirusi su značajno češće bili uzročnici epidemijskog javljanja bolesti. Astrovirus je dokazan kod znatno manjeg broja obolelih (u 2,29%) i to samo kod dece do 5 godina i dece uzrasta 6 do 14 godina. Infekcija izazvana enteričnim adenovirusima dokazana je kod 13,36% bolesnika. Njačešće je utvrđena kod dece uzrsta do 5 godina i 6 do 14 godina. Oboleli od adenovirusa imali su statistički značajno blažu kliničku sliku bolesti. Dva virusna uzročnika u uzorku stolice dokazana su u 3,19% osoba, obično u toku epidemijskog javljanja bolesti. Ovi bolesnici su imali bitno težu kliničku sliku. Najviše obolelih od dijarealnog sindroma bilo je u hladnim mesecima, mada su dijagnostikovani i tokom cele godine. U petogodišnjem periodu utvrđene su 22 epidemije u kolektivima i 9 porodičnih epidemija. Epidemijsko javljanje bolesti bilo je statistički značajno najčešće kod najstarijih bolesnika (starijih od 50 godina), a sporadično javljanje bilo je statističko značajno najčešće kod dece. U cilju potvrde tačnosti dijagnostike virusa u ispitivanim uzorcima real-time PCR testom, genotipizacije, kao i detaljnije molekularne analize, izabrani su reprezentativni uzorci pozitivni na rota, noro, astro ili adenoviruse. Delovi genoma ovih uzoraka su amplifikovani, a zatim sekvencirani. Sekvencirani izolati rotavirusa pripadali su grupi A i tipovima G1P[8], G2P[4], G3P[8] i G9P[8]. Sekvencirani izolati norovirusa pripadali su genogrupi I tipu 2, zatim genogrupi II tipovima 1, 2, 4 i 17. Sekvencirani izolati astrovirusa pripadali su grupi klasičnih astrovirusa i tipovima 1, 4 i 5. Sekvencirani izolati adenovirusa pripadali su grupi F i tipovima 40 i 41, kao i grupi C tipu 2. Pripadnost dobijenih sekvenci u ovom istraživanju, dodatno je potvrđena izradom filogenetskog stabla za sekvence pozitivne na rota, noro, astro ili adenoviruse. Zaključak: Incidenca virusnog dijarealnog sindroma u Vojvodini (70,69%) vrlo je visoka i viša je nego što je bilo pretpostavljeno prilikom prijave teze (u hipotezi). Real-time PCR test treba da bude redovno korišćen u budućem dijagnostičkom radu, jer dovodi do brze dijagnostike, čak i ako su virusi prisutni u malom broju u uzorcima tečnih stolica, što je utvrđeno tokom ovog dijagnostičkog rada. Ispitivani virusi treba da budu redovno dijagnostikovani kod obolelih od dijarealnog sindroma i to u svim starosnim grupama, tokom epidemijskog i sporadičnog javljanja oboljenja.
Introduction: Viral gastrointestinal syndrome is a current ongoing health problem worldwide. This is true of both developed and developing countries, especially underdeveloped ones where it is the second leading cause of mortality. Sudden onset of the disease—accompanied by the occurrence of large numbers of liquid stools, nausea, vomiting, abdominal pain, fever, and exhaustion—leads to dehydration. A fatal outcome can occur in all age groups of patients, especially very young children, the elderly, and the immuno-deficient, unless an accurate etiological diagnosis of the disease is quickly established, followed by a prompt institution of fluid and electrolyte placement, and implementation of other symptomatic therapy measures. Quick establishment of an accurate diagnosis, which is best achieved using the real-time PCR test, prevents the onset of complications, including a potentially fatal outcome of the disease. Simultaneously, it enables the implementation of appropriate epidemiological measures to prevent epidemic outbreaks and their spread. The aim of this study was to accurately determine the incidence of viral gastrointestinal syndrome in Vojvodina and the frequency of epidemic and sporadic occurrence of this disease. The aim was also to set up an algorithm for the application of the real-time PCR test in diagnostics of viral gastrointestinal syndrome in future work. Likewise, the aim was to carry out genetic typing and determine phylogenetic affiliation of the virus using molecular analysis and sequencing of parts of genomes from positive stool samples. Material and Methods: During a five-year study, 1003 patients with symptoms of viral diarrheal syndrome, aged from one month to more than 90 years old, were examined using molecular real-time PCR test. They were screened for rota, noro, astro, and enteric adenoviruses. Based on the data from survey questionnaires and medical case history, all clinical indicators were meticulously analyzed (disease occurrence during the year, disease duration, symptoms). The assessment of the clinical severity was carried out according to the Vesikari Clinical Severity Scoring scale. All data were compared according to the type of the viral causing agent, age of the patients, duration of research in years, and epidemic and sporadic occurrence of the disease. Obtained data were statistically analyzed, tabulated, and graphically displayed. Results: In a five-year period, a sample of 1003 patients of different ages was screened for four different viral causing agents of diarrheal syndrome (rota, noro, astro, and enteric adenoviruses) using the real-time PCR test. Viral diarrheal syndrome was confirmed in 709 patients (70.69%). The most commonly found were rotavirus infections in 28.81% of the cases. Rotaviruses were statistically significantly most common in children younger than 5 years old (38.90%), but were also found in high percentage in children aged 6-14 years old (24.83%). Children under 5 years of age had statistically significantly highest clinical severity and fever, and were more frequently hospitalized. In addition to higher fever in patients with rotavirus, clinical severity in these patients was also higher, and the disease lasted longer than in patients with other viruses. Norovirus infections were reported in 23.03% of the subjects, statistically significantly more frequently in adults over 20 years of age. Regarding the clinical symptoms in these patients, nausea, vomiting, and abdominal pain were statistically significantly more common than in patients with other viruses. Noroviruses were significantly more common as causing agents of epidemic disease outbreaks. Astrovirus was found in a significantly smaller number of patients (in 2.29%), and only in children under 5 years of age and children aged 6-14 years old. Enteric adenovirus infections were reported in 13.36% of the subjects. They were most commonly found in children younger than 5, and those aged 6- 14 years old. Adenovirus sufferers had statistically significantly milder clinical disease. Two viral causing agents in the stool sample were found in 3.19% of the subjects, usually during an epidemic disease outbreak. These patients had a significantly more severe clinical disease. Highest numbers of sufferers from diarrheal syndrome occurred during the cold months, although they were diagnosed throughout the year. In a five-year period, 22epidemics in collective groups and 9 family epidemics were identified. Epidemic outbreaks of the disease were statistically significantly most frequent in the elderly patients (older than 50), while sporadic occurrences were statistically significantly most frequent in children. Representative samples positive for rota, noro, astro, or adenoviruses were selected in order to confirm the accuracy of virus diagnostics in samples tested by the real-time PCR test, and perform genotyping as well as more detailed molecular analyses. Parts of the genomes of these samples were amplified and then sequenced. Sequenced rotavirus isolates belonged to group A and types G1P[8], G2P[4], G3P[8], and G9P[8]. Sequenced norovirus isolates belonged to genogroup I type 2, and genogroup II types 1, 2, 4, and 17. Sequenced astrovirus isolates belonged to the group of classical astroviruses and types 1, 4, and 5. Sequenced adenovirus isolates belonged to group F and types 40 and 41, as well as group C type 2. The affiliation of the obtained sequences in this study was further confirmed by creating a phylogenetic tree for sequences positive for rota, noro, astro, or adenoviruses. Conclusion: The incidence of viral diarrheal syndrome in Vojvodina (70.69%) is very high—higher than what was assumed at the time of the thesis submission (in the hypothesis). The real-time PCR test should be regularly used in future diagnostic work, since it leads to rapid diagnostics even if viruses are present in small numbers in liquid stool samples, as determined in the course of this diagnostic study. The investigated viruses should be regularly tested in patients with diarrheal syndrome belonging to all age groups during both epidemic and sporadic occurrences of the disease.

Understanding the transmission dynamics of infectious diseases is important to well-informed public health policy, responsive infection control and individual patient management. The on-going revolution in whole-genome sequencing provides unprecedented resolution for detecting evidence of recent transmission and characterising population-level transmission dynamics. In this thesis, I develop and apply evolutionary approaches to investigating transmission, focusing on three globally important pathogens. Hepatitis C virus (HCV) is a major cause of liver disease affecting 150 million people and killing 350,000 annually. I conducted a meta-analysis of twentieth-century HCV epidemics, finding that the age of the epidemic can be predicted by genetic diversity. Using the coalescent, I fitted classic susceptible-infected (SI), susceptible-infected-susceptible (SIS) and susceptible-infected-recovered (SIR) epidemiological models. Most epidemics showed signatures of SI dynamics, but three, from Argentina, Hong Kong and Thailand, revealed complex SIR dynamics. Norovirus is the leading viral cause of diarrhoea, estimated to cost the NHS around £115 million annually. I analysed whole norovirus genomes via a stochastic transmission model, finding that up to 86% of hospital infection was attributable to transmission from another patient in the hospital. In contrast, the rate of new introductions to hospital by infected patients was extremely low (<0.0001%), underlining the importance of ward management during outbreaks. Campylobacter is the most commonly identified cause of bacterial gastroenteritis worldwide. I developed a zoonotic transmission model based on phylogeography approaches to test whether three strains previously associated with multiple host species were in fact aggregates of strongly host-restricted sub-strains, or genuine generalists. Members of the same strain isolated from different host species were often more closely related than those isolated from the same host species. I estimated 419, 389 and 31 zoonotic transmissions in ST-21, ST-45 and ST-828 respectively, strongly supporting the hypothesis that these strains are adapted to a generalist lifestyle.

Les masses d’eau douces sont sujettes à des contaminations fécales d’origines diverses. Parmi ces contaminants, les virus entériques, dont les norovirus (NoV), sont responsables de nombreuses épidémies à gastro entérites chaque année dans le monde. Les indicateurs de contamination fécale actuels (i.e. E. coli) recommandés par différentes réglementations s’avèrent peu fiables pour estimer le risque viral dans l’eau. D’autres indicateurs, ayant des caractéristiques proches des virus entériques, tels que les bactériophages ARN F-spécifiques (FRNAPH), sont proposés pour évaluer ce risque viral. Cependant, l’analyse des FRNAPH infectieux dans les eaux se heurte à certaines limites, notamment liées aux caractéristiques hydrodynamiques des milieux aquatiques. Afin de lever ces limites, une solution serait de réaliser les analyses au sein de capteurs accumulateurs et intégrateurs de ces cibles. Dans ce contexte, l’objectif de ce travail est de tester l’intérêt d’un mollusque bivalve dulcicole, la dreissène (Dreissena polymorpha), largement utilisé pour la surveillance chimique et écotoxique des masses d’eau, comme capteur biologique pour évaluer et suivre la contamination virale des masses d’eau. Pour ceci la stratégie suivie a consisté à i) caractériser la cinétique d’accumulation et de dépuration des FRNAPH infectieux chez la dreissène en conditions contrôlées de laboratoire et in situ, ii) définir un modèle toxico-cinétique pour formaliser la relation entre la concentration en FRNAPH infectieux dans la dreissène et le niveau d’exposition (concentration dans l’eau), iii) évaluer la contamination virale de masses d’eau sur une large échelle géographique et enfin iv) évaluer le couplage biocapteur-FRNAPH infectieux pour représenter la contamination des masses d’eau par le génome de NoV.Les données obtenues en laboratoire et in situ soulignent l’accumulation très rapide des FRNAPH infectieux par la dreissène avec une mise en équilibre avec son milieu en moins de 48h. De plus, les accumulations sont proportionnelles au niveau d’exposition sur une très large gamme de concentration et le signal en FRNAPH infectieux reste au sein des tissus de la dreissène plusieurs jours après l’exposition. Ainsi l’ensemble de ces données souligne l’intérêt de la dreissène comme système accumulateur et intégrateur. La définition d’un modèle toxico-cinétique à un compartiment, sur la base de ce qui est connu pour les contaminants chimiques, a permis de définir des facteurs de bioaccumulation in situ particulièrement intéressants (BCF ≈ 1 000) et autorisant un réel apport in situ. A l’aide d’une approche active (encagement d’organismes calibrés), le projet a validé l’apport de la dreissène comme biocapteur pour évaluer les concentrations en FRNAPH infectieux de nombreuses masses d’eau ainsi que son apport pour l’évaluation du risque viral vis à vis de la présence du génome de NoV
Freshwater bodies are subject to fecal contamination from a variety of sources. Among these contaminants, enteric viruses, including Noroviruses, are responsible for numerous gastroenteritis epidemics worldwide every year. The current fecal contamination indicators (i.e., E. coli) recommended by various regulations are proving unreliable for estimating the viral risk in water. Other indicators, with characteristics close to those of enteric viruses, such as specific-F RNA bacteriophages (FRNAPH), have been proposed to assess this viral risk. However, the analysis of infectious FRANPH in water comes up against certain limitations, notably linked to the hydrodynamic characteristics of aquatic environments. In order to overcome these limitations, one solution would be to carry out analyses using sensors that accumulate and integrate these targets. In this context, the aim of this work is to test the interest of a freshwater bivalve mollusc, the zebra mussel (Dreissena polymorpha), widely used for chemical and ecotoxic monitoring of water bodies, as a biological sensor for assessing and monitoring viral contamination of water bodies. The strategy followed consisted in i) characterizing the kinetics of accumulation and depuration of infectious FRNAPH in mussels under controlled laboratory and in situ conditions, ii) defining a toxico-kinetic model to formalize the relationship between the concentration of infectious FRNAPH in mussels and the level of exposure (concentration in water), iii) assess viral contamination of water bodies on a broad geographical scale, and finally iv) evaluate biosensor-infectious FRNAPH coupling to represent contamination of water bodies by the NoV genome.Data obtained in the laboratory and in situ underline the very rapid accumulation of infectious FRANPH by mussels, with equilibration with its environment in less than 48 hours. What's more, accumulations are proportional to the level of exposure over a very wide concentration range, and the infectious FRANPH signal remains in mussel tissues for several days after exposure. All these data underline the interest of D. polymorpha as an accumulator and integrator system. The definition of a single compartment toxicokinetic model, based on what is known for chemical contaminants, has enabled us to define particularly interesting in situ bioaccumulation factors (BCF ≈ 1,000) and authorizing a real in situ contribution. Using an active approach (caging of calibrated organisms), the project validated the contribution of zebra mussel as a biosensor for assessing infectious FRNAPH concentrations in numerous water bodies, as well as its contribution to viral risk assessment vis à vis the presence of the NoV genome

碩士
臺灣大學
醫學檢驗暨生物技術學研究所
95
Human norovirus (NoV) causes gastroenteritis in humans in all age groups. NoV is transmitted through fecal contamination of food and water, and secondary person-to-person spread is common. Annual epidemics cause severe morbidity and even mortality, especially in developing countries. In recent years several naturally occurring recombinant NoVs have been reported around the world. Considering the fact that recombination occur much more often than it was thought to be, further study of the diversity and ongoing recombination of NoV may bring tremendous assistance for epidemiological studies and more importantly bring medical and healthcare attention for diminishing worldwide epidemics. In this study the full-length genome of nine norovirus recombinants, isolated from stool specimens from 2002, 2003, and 2006 in Taiwan. Three PCR fragments covering the full-length genome were obtained for eight of the isolates. After the nucleotide sequences were determined, the relationships among different noroviruses (NoVs) in different genes were studied by phylogenetic analysis. The recombination break points were revealed by SimPlot analysis. According to the polymerase gene-based phylogenetic analysis, five isolates Taipei/0212/2003/TW, Taipei/2339/2002/TW, Taipei/0089/2003/TW, Taipei/0510/2002/TW, and Taipei/1969/2002/TW belonged to the recombinants with the GIIb polymerase gene. By capsid gene-based phylogenetic analysis, the above described GIIb polymerase recombinants clustered onto two distinct genotypes, Taipei/0089/2003/TW Taipei/0510/2002/TW, Taipei/1969/2002/TW, and Taipei/0212/2003/TW clustered with GII/3 strains, Taipei/2339/2002/TW clustered with GII/2 strains. Three isolates Taipei/0760/2002/TW, Taipei/1047/2006/TW, and Taipei/0898/2006/TW, clustered with GII/12 and GII/10 strains and shared 98% identity in polymerase gene. According to the capsid gene-based analysis, Taipei/0760/2002/TW, Taipei/1047/2006/TW, and Taipei/0898/2006/TW clustered with GII/10, GII/12, and GII/4 strains, respectively. Taken together, these results confirmed the existence of naturally occurring recombinant NoVs in Taiwan and this is the first evidence of naturally occurring recombinant NoV infection in Taiwan.

The genomes of positive strand RNA viruses often contain more than one open reading frame. Some of these viruses have evolved novel mechanisms to regulate the synthesis of the other open reading frames that in some cases involved the production of a subgenomic RNA or RNAs. Very often, the presence of the subgenomic RNA is used as indicator for active viral genome replication. Norovirus, a major cause for gastroenteritis as well as with all other caliciviruses follow a typical positive strand RNA viruses genome replication strategy. In addition, noroviruses also produce a subgenomic RNA during their replication in infected cells. Efficient and adequate synthesis of norovirus subgenomic RNA is crucial for successful viral replication and productive infection leading to the generation of infectious viral progeny. This chapter will dissect the significant findings on mechanisms involved in norovirus genome replication as well as focusing on subgenomic RNA production.

Norovirus are a major cause of acute gastroenteritis worldwide. Diarrheal disease is now the fourth common cause of mortality children under the age of 5 years but remain the 2nd most cause of morbidity. NoV are associated with 18% diarrheal diseases worldwide where rotavirus vaccinations has been successfully introduced. NoV has become major cause of gastroenteritis in children. NoV belong to family caliciviridae. They are non-enveloped, single stranded positive sense RNA Viruses. The genome consists of 3 Open reading frames, ORF-1 codes for non-structural protein, ORF-2 codes for major capsid protein VP1 and ORF-3 for minor capsid protein VP2. Based on sequence difference of the capsid gene (VP1), NoV have been classified in to seven genogroup GI-GVII with over 30 genotypes. Genogroups I, II, IV are associated with human infection. Despite this extensive diversity a single genotype GII.4 has been alone to be the more prevalent. Basic epidemiological disease burden data are generated from developing countries. NoV are considered fast evolving viruses and present an extensive diversity that is driven by acquisition of point mutations and recombinations. Immunity is strain or genotype specific with little or no protection conferred across genogroups. Majority of outbreaks and sporadic norovirus cases worldwide are associated with a single genotype, GII.4 which was responsible for 62% of reported NoV outbreaks in 5 continents from 2001 to 2007. GII.4 variants have been reported as major cause of global gastroenteritis pandemics starting in 1995 frequent emergence of novel GII.4 variants is known to be due to rapid evolution and antigenic variation in response to herd immunity. Novel GII.4 variants appear almost every 2 years. Recent GII.4 variant reported include Lordsdale 1996, Farmington Hills 2002, Hunter 2004, Yerseke 2006a, Den Haag 2006b, Apeldoon 2007, New Orleans 2009,most recently Sydney 2012. Detailed molecular epidemiologic investigation of NoV is associated for understanding the genetic diversity of NoV strain and emergence of novel NoV variants. However, reports have revealed that not all individuals develop symptoms and a significant proportion remains asymptomatic after NoV infections.

This chapter investigates caliciviruses. These are global in prevalence and depend on faecal–oral spread coupled with poor hygiene. They are notorious in causing serious gastroenteritis outbreaks on cruise ships and in restaurants, hospitals, schools, and military camps. Other members of the calicivirus family infect cattle, pigs, mice, and cats. The chapter talks about the clinical impact of caliciviruses on the immunosuppressed patients of developing countries. Developing countries experience 200,000 deaths among children under the age of five from noroviruses per year, but these viruses continue to be an under-investigated family due to the lack of cell-culture systems. The chapter covers the classification, morphology, genome, replication, and clinical and epidemiological aspects of caliciviruses.