Auswahl der wissenschaftlichen Literatur zum Thema „Human Astrovirus“

Diarrhoea remains a significant cause of morbidity and mortality in developing countries where numerous cases remain without identified aetiology. Astroviruses are a recently identified cause of animal gastroenteritis which currently includes two species suspected of causing human diarrhoea. Using pan-astrovirus RT-PCR, we analysed human stool samples from different continents for astrovirus-related RNA sequences. We identified variants of the two known human astrovirus species plus, based on genetic distance criteria, three novel astrovirus species all distantly related to mink and ovine astroviruses, which we provisionally named HMOAstV species A–C. The complete genome of species A displayed all the conserved characteristics of mammalian astroviruses. Each of the now three groups of astroviruses found in human stool (HAstV, AstV-MLB and HMOAstV) were more closely related to animal astroviruses than to each other, indicating that human astroviruses may periodically emerge from zoonotic transmissions. Based on the pathogenic impact of their closest phylogenetic relatives in animals, further investigations of the role of HMOAstV, so far detected in Nigeria, Nepal and Pakistan, in human gastroenteritis are warranted.

Human astroviruses have been increasingly identified as important agents of diarrheal disease in children. However, the disease burden of astrovirus infection is still incompletely assessed. This paper reports results on the epidemiological and clinical characteristics of astrovirus-associated diarrhea, as well as the impact of astrovirus infection on the ambulatory setting at a Public Hospital in Córdoba city, Argentina. From February 2001 through January 2002, 97 randomly selected outpatient visits for diarrhea among children < 36 months old were enrolled. A single specimen of stool from each child was collected and tested for astrovirus antigen by enzyme immunoassay. Astroviruses were detected in 12.37% of the diarrheal episodes. All the positive cases occurred in children 4 to 18 months, but the highest rate was in children aged 4 to 6 months (23.80%). The clinical symptoms of astrovirus associated-diarrhea were fever 41.66%, vomiting 25.00% and dehydration 8.33%; overall 16.66% required hospitalization. Astrovirus was identified through the year and no seasonally pattern was detected (cool semester 15.21% versus warm semester 9.80% p > 0.05). According to our estimation about one out of seventy-four children in this cohort would be assisted annually for an astroviral-diarrheal episode in the Public Hospital and one out of eight diarrheal cases could be attributed to astrovirus infection. Astrovirus is a common symptomatic infection in pediatric outpatient visits in the public hospital in the study area, contributing 12.37% of the overall morbidity from diarrhea.

We have sequenced the genomic 3′-end, including the structural gene, of human astrovirus (HAstV) serotype 7 and morphologically related viruses infecting pig (PAstV), sheep (OAstV) and turkey (TAstV-1). These sequences were compared with corresponding astrovirus sequences available in the nucleic acid databases, including sequences of the seven other HAstV serotypes, two other avian astroviruses (TAstV-2 and avian nephritis virus) and astrovirus from cat (FAstV). A 35 nt stem–loop motif near the 3′-end of the genome, previously described as being highly conserved, was present in all of the astroviruses except TAstV-2. In the N-terminal half of the capsid precursor protein, there were several short conserved peptide motifs. Otherwise the capsid proteins of astroviruses infecting different hosts were highly divergent. Calculation of genetic distances revealed that the distance between FAstV and HAstV is comparable to the largest distances between different HAstV serotypes. Higher similarities between the HAstV, FAstV and PAstV capsid sequences suggest interspecies transmissions involving humans, cats and pigs relatively recently in the evolutionary history of astroviruses.

ABSTRACT Astrovirus infection in a variety of species results in an age-dependent diarrhea; however, the means by which astroviruses cause diarrhea remain unknown. Studies of astrovirus-infected humans and turkeys have demonstrated few histological changes and little inflammation during infection, suggesting that intestinal damage or an overzealous immune response is not the primary mediator of astrovirus diarrhea. An alternative contributor to diarrhea is increased intestinal barrier permeability. Here, we demonstrate that astrovirus increases barrier permeability in a Caco-2 cell culture model system following apical infection. Increased permeability correlated with disruption of the tight-junction protein occludin and decreased the number of actin stress fibers in the absence of cell death. Additionally, permeability was increased when monolayers were treated with UV-inactivated virus or purified recombinant human astrovirus serotype 1 capsid in the form of virus-like particles. Together, these results demonstrate that astrovirus-induced permeability occurs independently of viral replication and is modulated by the capsid protein, a property apparently unique to astroviruses. Based on these data, we propose that the capsid contributes to diarrhea in vivo.

Human astroviruses are one of the known pathogens of gastroenteritis in infants, children and rarely in elderly. It causes 4.2-7.3% of the sporadic gastroenteritis cases with diarrhea and vomiting in children. The etiological role of astrovirus has not been confirmed yet in outbreaks of gastroenteritis in Hungary. Aims: The first description of the detection and molecular epidemiology of astrovirus in outbreaks of gastroenteritis in Hungary. Materials and methods: Stool samples originated from Komárom-Esztergom County, from a day-care center (nursery) where a gastroenteritis outbreak occurred in June, 2010. Astrovirus was detected by RT-PCR methods. The nucleotide sequence of the nearly complete genome was sequenced. Clinical and epidemiological data were collected by epidemiological investigation. Results: Out of the 29 exposed persons (24 children and 5 adults) 7 (24.1%) children had gastroenteritis with diarrhea, and vomiting in one case, in the period of June 4-15, 2010. Bacterial pathogens, rotavirus, adenovirus and norovirus were not detected, but genotype 1 astrovirus could be identified in 3 (42.8%) stool samples (HQ398856). The nucleotide sequence of the astrovirus ORF1a/ORF1b/ORF2/3’UTR regions was determined. The source of the outbreak was presumably the firstly recognized ill child and the virus was spread by fecal-oral route with direct contact in the children community. Conclusions: Epidemiologic and clinical characteristics of the astrovirus outbreak in the nursery are described in details to prove that the possible etiological role of astroviruses in viral gastroenteritis which should not forget in order after rotaviruses, caliciviruses (norovirus and sapovirus) and enteric adenoviruses. Orv. Hetil., 2011, 152, 45–50.

Astroviruses are non-enveloped, single-stranded RNA viruses that infect mammalian and avian species. In humans, astrovirus infections are one of the most common causes of gastroenteritis in children. Infection has also been linked to serious neurological complications, especially in immunocompromised individuals. More extensive disease has also been characterized in non-human mammalian and avian species. To date, astroviruses have been detected in over 80 different avian and mammalian hosts. As the number of hosts continues to rise, the need to understand how astroviruses transmit within a given species as well as to new host species becomes increasingly important. Here, we review the current understanding of astrovirus transmission, the factors that influence viral spread, and the potential for cross-species transmission. Additionally, we highlight the current gaps in knowledge and areas of future research that will be key to understanding astrovirus transmission and zoonotic potential.

ABSTRACT Astroviruses are a new family of positive-stranded RNA viruses that cause gastroenteritis in a wide range of animals and in humans. Seven types of astrovirus, tentatively considered serotypes, have been distinguished by enzyme-linked immunosorbent assays (ELISA) or immunoelectron microscopy, but it is unclear whether the serotype designation is used properly. To test human sera for the presence of neutralizing antibodies and to type field strains, neutralization tests (NT) using CaCo2 tissue-culture-adapted astrovirus strains 1 to 7 and the corresponding rabbit reference sera were developed. In rabbits, neutralizing antibodies were predominantly serotype specific, with the exception of low-level cross-reactivity in astrovirus serotype 4 reference serum with astrovirus serotype 1 virus. Similarly, in humans, no evidence of cross-reactivity was found for the serotype combinations tested (all except the combination 1 and 7 and the combination 6 and 7). Typing by NT was concordant with typing by ELISA and genotyping, with one exception. The seroprevalence rates of neutralizing antibodies in an age-stratified sample of the population in Utrecht Province (n = 242) were 91% for astrovirus serotype 1, 69% for astrovirus serotype 3, 56% for astrovirus serotype 4, 36% for astrovirus serotype 5, 31% for astrovirus serotype 2, 16% for astrovirus serotype 6, and 10% for astrovirus serotype 7. Acquisition of antibodies was slower among persons seropositive for astrovirus serotype 5 than among those seropositive for astrovirus serotypes 1 to 4, suggesting that the epidemiology of serotype 5 astrovirus is different from that of astrovirus serotypes 1 to 4.

ABSTRACTHuman astroviruses (HAstVs) are nonenveloped, positive-sense, single-stranded RNA viruses that are a leading cause of viral gastroenteritis. HAstV particles display T=3 icosahedral symmetry formed by 180 copies of the capsid protein (CP), which undergoes proteolytic maturation to generate infectious HAstV particles. Little is known about the molecular features that govern HAstV particle assembly, maturation, infectivity, and immunogenicity. Here we report the crystal structures of the two main structural domains of the HAstV CP: the core domain at 2.60-Å resolution and the spike domain at 0.95-Å resolution. Fitting of these structures into the previously determined 25-Å-resolution electron cryomicroscopy density maps of HAstV allowed us to characterize the molecular features on the surfaces of immature and mature T=3 HAstV particles. The highly electropositive inner surface of HAstV supports a model in which interaction of the HAstV CP core with viral RNA is a driving force in T=3 HAstV particle formation. Additionally, mapping of conserved residues onto the HAstV CP core and spike domains in the context of the immature and mature HAstV particles revealed dramatic changes to the exposure of conserved residues during virus maturation. Indeed, we show that antibodies raised against mature HAstV have reactivity to both the HAstV CP core and spike domains, revealing for the first time that the CP core domain is antigenic. Together, these data provide new molecular insights into HAstV that have practical applications for the development of vaccines and antiviral therapies.IMPORTANCEAstroviruses are a leading cause of viral diarrhea in young children, immunocompromised individuals, and the elderly. Despite the prevalence of astroviruses, little is known at the molecular level about how the astrovirus particle assembles and is converted into an infectious, mature virus. In this paper, we describe the high-resolution structures of the two main astrovirus capsid proteins. Fitting these structures into previously determined low-resolution maps of astrovirus allowed us to characterize the molecular surfaces of immature and mature astroviruses. Our studies provide the first evidence that astroviruses undergo viral RNA-dependent assembly. We also provide new insight into the molecular mechanisms that lead to astrovirus maturation and infectivity. Finally, we show that both capsid proteins contribute to the adaptive immune response against astrovirus. Together, these studies will help to guide the development of vaccines and antiviral drugs targeting astrovirus.

ABSTRACT The persistence of human astroviruses dried on representative porous (paper) and nonporous (china) surfaces was investigated. Long-term astrovirus survival on fomites was monitored by an integrated cell culture-reverse transcription-PCR procedure. Viruses were applied to inanimate surfaces in the presence and absence of fecal material, and their survival was assayed at 4 and 20°C with high relative humidity. Astroviruses exhibited a notable persistence when dried on porous and nonporous materials, particularly at low temperature. Short-term survival of astroviruses on fomites was compared to that of other enteric viruses significant for health, such as rotavirus, adenovirus, poliovirus, and hepatitis A virus. Overall, astroviruses persisted better than poliovirus and adenovirus, although they exhibited a shorter survival than rotavirus and hepatitis A virus. Astroviruses show a high level of persistence at the desiccation step, which is of major significance in determining the chance of subsequent virus survival dried on fomites. Astroviruses are able to survive on inert surfaces long enough to suggest that fomites may play a relevant role in the secondary transmission of astrovirus diarrhea.

ABSTRACTLittle is known about intrinsic epithelial cell responses against astrovirus infection. Here we show that human astrovirus type 1 (HAstV-1) infection induces type I interferon (beta interferon [IFN-β]) production in differentiated Caco2 cells, which not only inhibits viral replication by blocking positive-strand viral RNA and capsid protein synthesis but also protects against HAstV-1-increased barrier permeability. Excitingly, we found similar resultsin vivousing a murine astrovirus (MuAstV) model, providing new evidence that virus-induced type I IFNs may protect against astrovirus replication and pathogenesisin vivo.IMPORTANCEHuman astroviruses are a major cause of pediatric diarrhea, yet little is known about the immune response. Here we show that type I interferon limits astrovirus infection and preserves barrier permeability bothin vitroandin vivo. Importantly, we characterized a new mouse model for studying astrovirus replication and pathogenesis.

The work described in this thesis identifies the structural polypeptide profile of human astrovirus type 1, grown in tissue culture in the presence of trypsin. Four major polypeptides were observed, of molecular weights: 34,000, 33.000, 26,500 and 6-8,000. A fifth of molecular weight 37,000 observed when the virus is prepared by PEG precipitation is considered to represent a precursor polypeptide. Of the four structural polypeptides, designated VP1, VP2, VP3 and VP4 respectively, VP1 and VP2 were found to be immunologically reactive with anti-astrovirus type 1 polyclonal antiserum. Astrovirus type 1 particles were found to have a buoyant density of 1.33-1.34 gm/ral in caesium chloride and a genome approximately 7,200 bases in length, corresponding to a molecular weight of 2.43 x 10° which is assumed to be RNA. This data suggests a relationship between astroviruses and picornaviruses, most especially the enterovirus subgroup, which have similar structural polypeptide and physico-chemical properties to those described above. However, the use of trypsin for the propagation of astrovirus in tissue culture means that further analysis of the structural polypeptides is required to determine whether those observed in this analysis represent four individual proteins, similar to the profile of picornaviruses, or whether some are cleavage products of the larger proteins due to the action of trypsin. The 3' terminal sequence of the astrovirus genome has been cloned and sequenced. It was found to have a poly (A) tail, stop codons in all three reading frames within the last 95 nucleotides and no polyadenylation signals. Several other astrovirus specific clones were obtained, but there is no suggestion as to which regions of the genome these represent. Comparisons between the astrovirus specific clones and the microgenie sequence databank has shown no significant homologies. The highest homology obtained for the 3' terminal sequence when compared to those of other picornaviruses was 47Z with coxsackie A21, this is not taken to confirm a relationship between the viruses. Assay systems were developed for astrovirus during the course of this study. An immunofluorescent end point titration, using type specific antisera, allowed the determination of the infectivity of a virus stock within 48 hours of infection. An immuno dot blot system was developed to assay for amounts of virus protein in samples, such as gradient fractions, and was used to determine the peak banding of virus in sucrose.

Novel human astrovirus (HAstV) are enteric virus belonging to the Astroviridae family and were discovered 10 years ago by high-throughput sequencing. They are divided in two different clades, the HAstV-MLB including 3 genotypes (MLB1-3) and HAstV-VA including 5 genotypes (VA1-5). While their role during gastroenteritis is debated, they have been reported as the sole agent identified in many cases of severe central nervous system infection, mainly in immunocompromised patients. This suggests that these emerging and highly divergent viruses can be associated with serious clinical manifestations, requiring additional basic and epidemiological studies to better understand their pathogenesis, prevalence and clinical correlation. We implemented several cell culture systems allowing the propagation of two distinct genotypes of novel HAstV from clinical stool samples, namely HAstV-MLB1 and HAstV-MLB2. Both strains could efficiently replicate in human HuH-7 hepatoma and A549 respiratory cell lines. In addition, both strains could establish a persistent infection over several cell passages in both cell lines, and HAstV-MLB1 could also establish a persistent infection in HuH-7.5 cells. In the latter, electron microscopy revealed a high production of capsid arrays and significant intracellular reorganization. Immunofluorescence assays revealed only a low proportion (5-10%) of infected cells. We explored the innate immune response to HAstV-MLB infection and observed that IFN expression was either abolished or delayed and diminished, depending on the cell line, during acute infection. During persistent infection, IFN expression was abolished in all cases, while when co-stimulated with poly I:C, IFN expression remained inhibited in a cell and genotype-dependent manner. Addition of exogenous IFN led to the cure (IFN-β) and relative inhibition (IFN-λ) of HAstV-MLB infection in HuH-7 cell line, while there was no effect in A549 infected cell line. At the epidemiological level, using a sensitive and specific real-time RT-PCR assay, we found that novel HAstVs could be identified in 6-10% of cases of undiagnosed gastroenteritis in Spanish pediatric children of < 5 years old. Together with a Japanese study, our prevalence is the highest observed to date. Children under 2 years old had a higher prevalence rate, compared to older ones. HAstV-MLB1 and HAstV-VA1 accounted for 31% and 26% of all novel HAstV observed in our cohort, while no HAstV-MLB3 were detected. Nevertheless, we found a high rate of co-infection with other enteric viruses (66%), precluding to assess a firm association between the presence of novel HAstV and the occurrence of gastroenteritis in such cases. We could not identify differences in the mean Cq values between mono- and co-infection episodes. We then performed a case-control study to assess the role of novel HAstV in gastroenteritis. We found a prevalence of 6.3% and 4% in symptomatic and asymptomatic children, respectively, without statistical difference between groups. However, we found that asymptomatic children had statistically significant higher HAstV-MLB viral load (median 6.52 log10 genomes/ml, IQR 4.52-6.84) compared to symptomatic children (median 2.35 log10 genomes/ml, IQR 2.13-3.76). Similarly, in symptomatic patients, we observed a higher viral load when novel HAstVs were found in coproculture-positive (median 5.19 log10 genomes/ml, IQR 4.24-6.22) compared to coproculture-negative (median 2.31 log10 genomes/ml, IQR 2.11-3.32) stool samples. These findings suggest that novel HAstV are not associated with gastroenteritis, but could modulate the gut microbiome and may confer protection to invading pathogens, although the mechanism remains to be elucidated.
Los astrovirus humanos (HAstV) no clásicos son virus entéricos emergentes que pertenecen a la familia de los Astroviridae, la cual incluye virus asociados a gastroenteritis principalmente en la población pediátrica. Se descubrieron por primera vez hace 10 años mediante secuenciación masiva, y hoy se dividen en dos grupos filogenéticos distintos: los HAstV-MLB (MLB1-3), y los HAstV-VA (VA1-5). Su asociación con gastroenteritis no está del todo confirmada, y también han sido identificados en casos de meningoencefalitis en pacientes inmunodeprimidos. En nuestro trabajo hemos implementado varios sistemas de cultivo celular permisivos para la replicación de dos genotipos de HAstV-MLB, HAstV-MLB1 y HAstV-MLB2, utilizando muestras clínicas. Ambos genotipos pueden replicar en las líneas celulares humanas HuH-7 y A549, de hepatoma y tejido respiratorio, respectivamente. Además, ambos pueden establecer una infección persistente en el cultivo, detectándose señal positiva por inmunofluorescencia en 5-10% de las células. La microscopía electrónica identifica una gran cantidad de cápsides víricas dentro de las células infectadas, y una importante reorganización intracelular. En los cultivos persistentemente infectados no se detecta inducción de la respuesta interferón (IFN), y la capacidad de los virus para bloquear la expresión de IFN inducida por poliI:C es distinta para cada tipo celular. La sensibilidad frente a un tratamiento exógeno tanto con IFN-β como con IFN-λ, es efectivo en las células HuH-7 pero nulo en las células A549. En nuestros estudios epidemiológicos en niños menores de 5 años con gastroenteritis no diagnosticada, se detectaron HAstV no clásicos en 6-10% de los casos. MLB1 y HAstV-VA1 representaron el 31% y el 26% de todos ellos, respectivamente. Se detectó co-infección con algún otro virus entérico en 66% de las muestras positivas, y no se observaron diferencias en los valores Cq entre casos de mono- y co-infección. En un estudio de casos y controles, su prevalencia fue similar en ambos grupos (6.3% versus 4%, respectivamente). No obstante, se observó que el promedio de carga vírica en los casos asintomáticos fue significativamente superior que en los niños enfermos, y en pacientes sintomáticos, se observó una carga viral mayor en aquellas heces que eran positivas para coprocultivo en comparación con las negativas.

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.

Abstract Background: Gastroenteritis is a common infection and the leading cause of morbidity worldwide and is mostly caused by viruses. Outbreaks appear in both developed and developing countries and result in large economic costs. Rapid detection is important for appropriate treatment, control and to prevent the spread of infection.  Objective: Evaluation and performance comparison between the BioFire®FilmArray® Torch System gastrointestinal panel and the Molecular BD MAXTMenteric viral panel to indicate a multiplex method for viral gastroenteritis diagnostic in a routine laboratory setting.  Material and methods: In this study, 58 different samples were used which consisted of selected stool specimens from patients who were tested and treated for gastroenteritis infection at Uppsala Academic Hospital and Norrlands University Hospital in Umeå during 2018-2021, samples from Quality control for molecular diagnostics viral gastroenteritis EQA pilot study during 2018-2019 and cultivated strains of different adenovirus species from 2018. All samples were analyzed with both systems for comparison of detected pathogens.  Results: Sensitivity and specificity values were 95% and 100% respectively for the BioFire®FilmArray®Torch System and 100% and 93.3% for the BD MAXTMSystem.   Conclusions: Bothsystems are rapid and adequate diagnostic tools. The BioFire®FilmArray®Torch System with greater coverage has the ability of detecting more pathogens and is more promising particularly in the occasional infection circumstance. The BD MAXTMSystem demonstrated almost the same results and seems to be a better option in times of an outbreak when the numbers of patients are significantly higher.

Los astrovirus humanos son virus de gastroenteritis infantiles y tienen un genoma RNA monocatenario de polaridad positiva organizado en tres pautas abiertas de lectura (ORF1a y ORF1b para las proteínas no estructurales, y ORF2 para las proteínas estructurales). Actualmente se desconoce la función de muchas de sus proteínas no estructurales. No obstante, en la región C-terminal de la poliproteína nsP1a codificada por el ORF1a (denominada nsP1a/4) se han descrito multitud de motivos que podrían tener una función biológica relevante para su ciclo replicativo. Entre otros, cabe destacar la presencia de una región hipervariable (HVR), que se ha relacionado con un posible papel en la replicación del RNA vírico, y una putativa región codificante para una proteína VPg. Bajo estas premisas los objetivos de esta tesis fueron profundizar en la caracterización de la proteína nsP1a/4 tanto a nivel de variabilidad genética como a nivel funcional y confirmar experimentalmente la existencia de una VPg unida covalentemente al genoma vírico. El análisis de la HVR de la proteína nsP1a/4 mostró un grado elevado de variabilidad, sobre todo a nivel de aminoácidos, con una buena tolerancia a las inserciones y deleciones y a los cambios no sinónimos. Por otro lado, el análisis filogenético de la HVR de 104 aislados permitió la identificación de 12 genotipos diferentes y el análisis de la carga vírica excretada en muestras clínicas correspondientes a distintos genotipos derivados de la HVR mostró una correlación significativa entre estos dos parámetros. Aprovechando la variabilidad de esta región del genoma de astrovirus, se desarrolló también un método de tipado molecular por amplificación por RT-PCR y análisis de polimorfismo de longitud de fragmentos de restricción (RFLP). Con el fin de profundizar en la caracterización de la proteína nsP1a/4 y establecer posibles diferencias funcionales entre genotipos, se clonaron y expresaron en el sistema de baculovirus varias formas de la proteína correspondientes a los genotipos IV, V, VI y XII. Así, para todos los genotipos estudiados, se observó que existen diferentes isoformas de la proteína, una isoforma no fosforilada y diferentes isoformas fosforiladas, y que las isoformas no fosforiladas interaccionan entre sí formando oligómeros. Además, dada la implicación de la proteína nsP1a/4 en el proceso de replicación del RNA, se estudió la potencial interacción de la proteína nsP1a/4 con la polimerasa vírica. Para todos los genotipos estudiados, se observó que la proteína nsP1a/4 interacciona con la polimerasa formando un heterodímero. En cuanto a la forma de la nsP1a/4 que interacciona con la polimerasa, los resultados mostraron que lo hacen tanto la isoforma no fosforilada como la fosforilada, si bien en este caso sí que se observaron diferencias entre los distintos genotipos. Concretamente, la isoforma fosforilada de la proteína nsP1a/4 correspondiente al genotipo VI presentó un patrón de interacción más fuerte con la polimerasa en comparación con la isoforma no fosforilada. En cuanto a la existencia de una proteína VPg, el análisis de las proteínas co-purificadas con el RNA de astrovirus reveló la presencia de una proteína con un peso molecular (13-15 kDa) compatible con el de la VPg predicha computacionalmente. La identificación de un péptido de 15 aminoácidos confirmó que la región codificante para la VPg se localiza dentro de la región codificante para la proteína nsP1a/4 y el tratamiento proteolítico del RNA de astrovirus con Proteinasa K demostró que dicha proteína resulta esencial para la infectividad del virus. Finalmente, el uso de distintas formas mutadas de la VPg permitió identificar el residuo Tyr693 como potencial residuo de unión al RNA. Así pues, la mejor caracterización de las proteínas nsP1a/4 y VPg de astrovirus proporciona las bases para su posible aplicación en el futuro como nuevas dianas antivíricas.
Human astroviruses are single-stranded (+) RNA viruses that frequently cause gastroenteritis in children. Upon infection, nonstructural proteins are translated as two polyproteins, nsP1a and nsP1ab, which are further proteolitically processed. The roles of their mature products are mostly unknown. However, several domains have been described in the nsP1a C-terminal end protein (also named nsP1a/4 protein), including a putative VPg protein and a hypervariable region (HVR). Moreover, recent data support the correlation between nsP1a/4 variability and higher replication properties. The main goals of this study were to characterize the genetic variability and functionality of nsP1a/4, and to experimentally prove the existence of a VPg protein. Sequencing the HVR of 104 isolates showed a significant degree of amino acid variability, composed mainly of insertions and deletions and non- synonymous mutations. Based on this variability, 12 genotypes were established, and a RFLP typing method was developed. Moreover, a significant correlation was observed between viral load in feaces and certain genotypes, confirming the influence of nsP1a/4 variability on the replication phenotype. To better understand the role of HVR-derived genotypes in viral replication, four HVR genotypes (IV, V, VI, and XII) of nsP1a/4 proteins, as well as the astrovirus polymerase, were expressed in the baculovirus system. A nonphosphorylated isoform and different phosphorylated isoforms of nsP1a/4 proteins were detected for all genotypes, and nonphosphorylated isoforms formed oligomers. Coexpression of the viral RNA polymerase resulted in the formation of heterodimers. This interaction involved both the unphosphorylated and the phosphorylated isoforms for all genotypes, but the phosphorylated isoforms of nsP1a/4 type VI showed a stronger interactive pattern with the polymerase than the nonphosphorylated isoform. Regarding the presence of a VPg protein, a protein of 13-15 kDa was identified after RNAse treatment of RNA purified from infected cells, and the protein sequence obtained confirmed its inclusion in the nsP1a/4 coding region. Proteolytic treatment of the genomic RNA lead to a loss of infectivity, showing that astrovirus VPg is essential for viral infectivity and mutagenesis of its Tyr-693 was lethal for HAstV replication, supporting its functional role in the covalent link with the viral RNA. The better characterization of nsP1a/4 and VPg proteins will allow us to assess their potential use as new antiviral targets.