Different norovirus genotypes in patients with gastroenteritis in Kuwait.

Norovirus is a leading cause of acute gastroenteritis worldwide. The importance of this virus infection in Kuwait is not known. Eight out of 100 stool samples (8.0%) from children up to 5 years of age with gastroenteritis studied during 2006-2007 from one hospital, and 6 out of 70 stool samples (8.5%) from similar children studied from another hospital during 2010-2011 were positive for norovirus by RT-PCR. Out of these 170 samples studied from both hospitals, 10 samples were positive for norovirus when tested by ELISA. Phylogenetic tree analysis of norovirus strains showed that 50% of the norovirus strains belonged to genotype GII.4, and the predominant strain was GII.4 2006b. Other detected genotypes were GII.12, GII.b, GII.3, GII.8, and GII.7. This study highlights the importance of screening for norovirus infection in acute gastroenteritis and having a reporting system to understand better the epidemiology of norovirus infection in Kuwait.

Novel NSP4 genotype in a camel G10P[15] rotavirus strain.

In this study a Kuwaiti camel rotavirus strain, RVA/Camel-wt/KUW/s21/2010/G10P[15], is characterized by sequencing and phylogenetic analysis. The strain had multiple genes with high nucleotide sequence similarities to ovine and bovine strains (VP2, ≤ 96%; NSP2 and NSP5, ≤ 97%, NSP3, ≤ 94%), or, to porcine strains (VP1, ≤ 89%). Other genes had moderate sequence similarities (VP4, ≤ 87%; VP6, ≤ 81%; VP7, ≤ 82%) with reference strains from ruminants. The NSP4 gene shared limited sequence identity (≤ 71%) with other mammalian and avian rotavirus NSP4 types, and was designated a novel genotype, E15. This study demonstrates genetic diversity in the outer capsid and some backbone genes of an old-world camelid rotavirus strain and uncovers its common evolutionary roots with strains from other ruminants.

Surveillance of human rotaviruses in 2007-2011, Hungary: exploring the genetic relatedness between vaccine and field strains.

BACKGROUND: The availability of rotavirus vaccines has resulted in an intensification of post vaccine strain surveillance efforts worldwide to gain information on the impact of vaccines on prevalence of circulating rotavirus strains. OBJECTIVES: In this study, the distribution of human rotavirus G and P types in Hungary is reported. In addition, the VP4 and VP7 genes of G1P[8] strains were sequenced to monitor if vaccine-derived strains were introduced and/or some strains/lineages were selected against. STUDY DESIGN: The study was conducted in 8 geographic areas of Hungary between 2007 and 2011. Rotavirus positive stool samples were collected from diarrheic patients mostly <5 years of age. Viral RNA was amplified by multiplex genotyping RT-PCR assay, targeting the medically most important G and P types. When needed, sequencing of the VP7 and VP4 genes was performed. RESULTS: In total, 2380 strains were genotyped. During the 5-year surveillance we observed the dominating prevalence of genotype G1P[8] (44.87%) strains, followed by G4P[8] (23.4%), G2P[4] (14.75%) and G9P[8] (6.81%) genotypes. Uncommon strains were identified in a low percentage of samples (4.12%). Phylogenetic analysis of 318 G1P[8] strains identified 55 strains similar to the Rotarix strain (nt sequence identities; VP7, up to 97.9%; VP4, up to 98.5%) although their vaccine origin was unlikely. CONCLUSIONS: Current vaccines would have protected against the majority of identified rotavirus genotypes. A better understanding of the potential long-term effect of vaccine use on epidemiology and evolutionary dynamics of co-circulating wild type strains requires continuous strain surveillance.

Detection of hepatitis E virus in samples of animal origin collected in Hungary.

Hepatitis E virus (HEV) is an enterically transmitted human pathogen. HEV infections are mainly associated with acute, self-limited, icteric hepatitis with an average mortality rate of 1%. Animal reservoirs are considered to play an important role in the maintenance of the virus and in the spread of HEV to humans. HEV-induced seroconversion was described in several species, however clinical hepatitis in animals has not been observed to date. HEV strains from animals are genetically closely related to human HEV isolates, which supports the opinions on the zoonotic transmission of the virus. In this expansive study the occurrence of HEV was investigated in Hungarian wild and domesticated animal samples. HEV RNA was detected by reverse transcription-polymerase chain reaction in liver samples of wild boars, roe deer, and deer. The investigations of domestic swine samples detected HEV in 39% of the investigated Hungarian pig farms. Simultaneous investigation revealed no definite difference between liver and faeces samples of domestic pigs in the frequency of HEV positivity. The highest (36%) incidence of HEV infection was found among the 11-16-week-old pigs. Samples from domestic cattle and rodents collected in pig farms, forests and meadows were tested negative for HEV RNA. Phylogenetic analysis of partial sequences amplified within the ORF1 and ORF2 regions of selected strains revealed that the detected viruses belong to three subgroups of the third genogroup of HEV, and are closely related to human and swine HEV strains detected in different countries. The investigations revealed widespread distribution of HEV in Hungarian wild ungulate and domesticated swine populations, with considerable genetic diversity among the strains.

Trends in the epidemiology of human G1P[8] rotaviruses: a hungarian study.

Epidemiological trends of the globally most common rotavirus genotype, G1P[8], were investigated in Hungary during a 16-year period by sequencing and phylogenetic analysis of the surface antigens. Antigen shift among epidemiologically major G1P[8] strains was observed in 6 seasons, as indicated by changes in the sublineages of the G1 VP7 and the P[8] VP4 genes. The temporal clustering of some rotavirus VP4 and VP7 gene sublineages and the periodic emergence and/or resurgence of previously unrecognized rotavirus sublineages in the study population suggest a dynamic nature for these common strains. Recently established international strain surveillance networks may help to identify and track the spread of epidemiologically important rotavirus strains across countries and continents.

[Post vaccination rotavirus surveillance in Hungary, in 2007]. / Posztvakcinációs rotavírus-surveillance Magyarországon, 2007.

Vaccination is the main strategy to control severe dehydrating gastroenteritis caused by rotaviruses in early childhood. The availability of new generation rotavirus vaccines has led to an intensification of strain surveillance worldwide, in part, to gauge the impact of the possible vaccine-driven immune selection of wild-type rotavirus strains. In the present study, authors describe the strain prevalence data obtained in 2007, with the involvement of different regions of Hungary. Genomic RNA was extracted from rotavirus-positive stool samples collected mainly from children and then subjected to genotyping using multiplex RT-PCR assay. Type-specific primers targeted G1 to G4, G6, G8 to G10, and G12 VP7 specificities, and P[4], P[6], and P[8] to P[11] VP4 specificities were used. Out of 489 rotavirus-positive specimens, collected from 482 patients, 466 and 474 were successfully G and P typed, respectively, and both G and P type specificities could be assigned for 457 strains. Prevalence data showed the predominance of G4P[8] (31.5%) strains, followed by G1P[8] (28.3%), G2P[4] (19.3%), and G9P[8] (10.2%). Minority strains were G1P[4] (0.4%), G2P[8] (1.3%), G3P[9] (0.2%), G4P[6] (0.7%), G6P[9] (0.4%), G8P[8] (0.2%), G9P[4] (0.2%), G9P[6] (0.8%), and G12P[8] (0.4%). Mixed infections were found in 1.2% of the samples, while 4.9% remained partially or fully non-typified. Our data indicate that the antigen specificities of medically important rotavirus strains identified in this 1-year study are well represented in the vaccines available in the pharmaceutical private market in Hungary. Depending on the vaccination coverage achievable in the forthcoming years, the post-vaccination rotavirus strain surveillance may allow us to gain comprehensive information on the impact of rotavirus vaccines on the prevalence of circulating rotavirus strains.

Frequent rearrangement may explain the structural heterogeneity in the 11th genome segment of lapine rotaviruses - short communication.

In rotaviruses, intragenic recombination or gene rearrangement occurs almost exclusively in the genome segments encoding for non-structural proteins. Rearranged RNA originates by mechanisms of partial sequence duplications and deletions or insertions of non-templated nucleotides. Of interest, epidemiological investigations have pointed out an unusual bias to rearrangements in genome segment 11, notably in rotavirus strains of lapine origin, as evidenced by the detection of numerous lapine strains with super-short genomic electropherotype. The sequence of the full-length genome segment 11 of two lapine strains with super-short electropherotype, LRV-4 and 3489/3, was determined and compared with rearranged and normal cognate genome segments of lapine rotaviruses. The rearranged genome segments contained head-to-tail partial duplications at the 3' end of the main ORF encoding NSP5. Unlike the strains Alabama and B4106, intermingled stretches of non-templated sequences were not present in the accessory RNA of LRV-4 and 3489/3, while multiple deletions were mapped, suggesting the lack of functional constraints. Altogether, these findings suggest that independent rearrangement events have given origin to the various lapine strains that have super-short genome pattern.

Searching for HAdV-52, the putative gastroenteritis-associated human adenovirus serotype in Southern Hungary.

Human adenovirus (HAdV) serotype 52 has recently been discovered in the United States in samples from human patients with gastroenteritis of unknown etiology and is suspected to be a new human enteric pathogen. The aim of the present pilot study was to investigate whether this virus is circulating in the population of Southern Hungary by screening stool specimens collected from gastroenteritis cases and communal sewage samples in the area of Baranya County. A total of 209 diarrheic stool (124 from children and 85 from adults) and 45 influent sewage samples were screened for HAdV-52 by PCR using a primer pair specific to the gene of 12.5K protein in the E3 genomic region. The novel human adenovirus was not detected in any of the tested samples, suggesting that HAdV-52 was not circulating in the target population and the area during the study period. Since temporal and geographical fluctuations may markedly affect the epidemiology of human enteric pathogens, additional investigations are required to gain more in-depth insights into the ecology of this novel adenovirus.

First detection of P[6],G9 rotaviruses in Hungary--an imported strain from India?

EuroRotaNet was launched to monitor rotavirus strain prevalence during and after introduction of rotavirus vaccines in Europe. In early 2007, we detected P[6],G9 rotaviruses to appear in Hungary, representing the first documented occurrence of this strain in our surveillance area. Epidemiologic data suggested that this strain was introduced from India.

Diarrhoeagenic Escherichia coli are not a significant cause of diarrhoea in hospitalised children in Kuwait.

BACKGROUND: The importance of diarrhoeagenic Escherichia coli (DEC) infections in the Arabian Gulf including Kuwait is not known. The prevalence of DEC (enterotoxigenic [ETEC], enteropathogenic [EPEC], enteroinvasive [EIEC], enterohemorrhagic [EHEC] and enteroaggregative [EAEC]) was studied in 537 children < or = 5 years old hospitalised with acute diarrhoea and 113 matched controls from two hospitals during 2005-07 by PCR assays using E. coli colony pools. RESULTS: The prevalence of DEC varied from 0.75% for EHEC to 8.4% for EPEC (mostly atypical variety) in diarrhoeal children with no significant differences compared to that in control children (P values 0.15 to 1.00). Twenty-seven EPEC isolates studied mostly belonged to non-traditional serotypes and possessed beta and theta intimin subtypes. A total of 54 DEC isolates from diarrhoeal children and 4 from controls studied for antimicrobial susceptibility showed resistance for older antimicrobials, ampicillin (0 to 100%), tetracycline (33 to 100%) and trimethoprim (22.2 to 100%); 43.1% of the isolates were multidrug-resistant (resistant to 3 or more agents). Six (10.4%) DEC isolates produced extended spectrum beta-lactamases and possessed genetic elements (blaCTX-M, blaTEM and ISEcp1) associated with them. CONCLUSION: We speculate that the lack of significant association of DEC with diarrhoea in children in Kuwait compared to countries surrounding the Arabian Gulf Region may be attributable to high environmental and food hygiene due to high disposable income in Kuwait.

[Molecular epidemiology of hepatitis E virus in Hungary: endemic, food-borne zoonosis]. / A hepatitis E-vírus molekuláris epidemiológiája hazánkban: endémiás, élelmiszer közvetítette zoonosis.

UNLABELLED: Hepatitis E virus (HEV) is one of a common cause of acute, fecally transmitted hepatitis in developing countries. Identification of HEV in indigenous human infection and in domestic pig raises the possibility that HEV infection is also a zoonosis. AIM/METHODS: Molecular detection and epidemiology of HEV in humans with acute hepatitis and in domestic (pig, cattle) and wild (boar and roe-deer) animals by ELISA and RT-PCR in Hungary. RESULTS: Between 2001 and 2006, a total of 116 (9.6%) human sera were positive by HEV IgM ELISA and 13 (24.5%) of 53 samples were also confirmed by RT-PCR and sequencing. Forty-two, 11 and 9 samples were RT-PCR-positive from swine (feces: 22.7%; liver: 30.8%), roe-deer (liver: 34.4%) and wild boar (liver: 12.2%), respectively. Except for an imported infection caused by genotype 1, 19 sequences (human: 12, swine: 4, roe-deer: 1, wild boar: 2) belong to genotype 3 HEV. Genetically identical strains were detected in human and roe-deer and in 2 other human clusters. CONCLUSIONS: HEV is an endemic agent in Hungary. Consumption of raw or undercooked meat-products is one of the possible sources of the indigenous HEV infections. Cross-species infection with genotype 3 HEV involves a food-borne transmission route in Hungary.

Characterization and zoonotic potential of endemic hepatitis E virus (HEV) strains in humans and animals in Hungary.

BACKGROUND: Hepatitis E virus (HEV) is a common cause of acute, fecally transmitted hepatitis in developing countries. Identification of HEV in indigenous human infection and in domestic pig raising the possibility that HEV infection is also a zoonosis. OBJECTIVES/STUDY DESIGN: Molecular detection and epidemiology of HEV in humans (South-East Hungary) with acute hepatitis and in domestic (pig, cattle) and wild (boar and roe-deer) animals (countrywide) by ELISA and RT-PCR. RESULTS: Between 2001 and 2006, a total of 116 (9.6%) of 1203 human sera were positive by HEV IgM ELISA and 13 (24.5%) of 53 samples were also confirmed by RT-PCR and sequencing. Forty-two (27.3%) of 154, 11 (34.4%) of 32 and 9 (12.2%) of 74 samples were RT-PCR-positive from swine (feces: 22.7%; liver: 30.8%), roe-deer (liver) and wild boar (liver), respectively. Except for an imported infection caused by genotype 1, 19 sequences (human: 12, swine: 4, roe-deer: 1, wild boar: 2) belong to genotype 3 HEV. Genetically identical strains were detected in human and roe-deer and in 2 other human clusters. CONCLUSIONS: HEV is an endemic agent in Hungary. Consumption of raw or undercooked meat-products is one of the possible sources of the indigenous HEV infections. Cross-species infection with genotype 3 HEV potentially involves a food-borne transmission route in Hungary.

Assignment of the group A rotavirus NSP4 gene into genotypes using a hemi-nested multiplex PCR assay: a rapid and reproducible assay for strain surveillance studies.

The rotavirus non-structural protein NSP4 has been implicated in a number of biological functions during the rotavirus cellular cycle and pathogenesis, and has been addressed as a target for vaccine development. The NSP4 gene has been classified into six genotypes (A-F). A semi-nested triplex PCR was developed for genotyping the major human NSP4 genotypes (A-C), which are common in human rotavirus strains but are also shared among most mammalian rotavirus strains. A total of 192 previously characterized human strains representing numerous G and P type specificities (such as G1P[8], G1P[4], G2P[4], G3P[3], G3P[8], G3P[9], G4P[6], G4P[8], G6P[4], G6P[9], G6P[14], G8P[10], G8P[14], G9P[8], G9P[11], G10P[11], G12P[6] and G12P[8]) were tested for NSP4 specificity by the collaborating laboratories. An additional 35 animal strains, including the reference laboratory strains SA11 (simian, G3P[2]), NCDV (bovine, G6P[1]), K9 and CU-1 (canine, G3P[3]), together with 31 field isolates (canine, G3P[3]; feline, G3P[9]; porcine, G2P[23], G3P[6], G4P[6], G5P[6], G5P[7], G5P[26], G5P[27], G9P[6] and G9P[7]) were also successfully NSP4-typed. Four human G3P[9] strains and one feline G3P[9] strain were found to possess an NSP4 A genotype, instead of NSP4 C, suggesting a reassortment event between heterologous strains. Routine NSP4 genotyping may help to determine the genomic constellation of rotaviruses of man and livestock, and identify interspecies transmission of heterologous strains.

Genetic diversity and zoonotic potential of human rotavirus strains, 2003-2006, Hungary.

Rotavirus strain surveillance is being conducted in many countries before and after introduction of newly licensed vaccines to assess the impact of the vaccines on rotavirus strains. Here we describe a strain surveillance study in the Budapest area of Hungary (2003-2006) based on RNA profile analysis, genotyping by multiplex PCR and nucleotide sequencing. Among 1,983 G-typed rotaviruses we identified G1 (22%), G2 (4.8%), G3 (3.5%), G4 (18.5%), G6 (1.1%), G8 (<0.1%, n = 1), G9 (42%), and G12 (3.4%) specificities. Information on P genotype incidence was determined for a subset of samples (n = 814). In addition to the globally important strains, a variety of uncommon antigen combinations were also found, for example, P[9],G3; P[14],G6; or P[14],G8. Sequence and phylogenetic analysis of the VP7, VP4, VP6, and NSP4 genes of selected strains with uncommon antigen combinations demonstrated high similarity with certain bovine, porcine, feline, equine, and lapine rotaviruses, respectively. Continued surveillance is needed to assess the role of animal rotaviruses in human diseases.

First detection of Tula hantaviruses in Microtus arvalis voles in Hungary.

Tula hantavirus (TULV) is a member of the genus Hantavirus, family Bunyaviridae and is mainly carried by the European common vole (Microtus arvalis). In order to detect TULV, we tested Microtus arvalis (MAR) and Microtus subterraneus (MSU) voles captured in two different locations of the Southern Transdanubian region of Hungary. The viral genome was detectable in 37% of the tested MAR voles but, interestingly, was absent in all MSU. Phylogenetic analysis performed with a partial coding sequence of the capsid gene showed that Hungarian TULV strains clustered with viruses detected in western Slovakia and in the Czech Republic. To the best of our knowledge, this is the first report on the identification of TULV detected in MAR voles in the Transdanubian region of Hungary.

Detection and quantification of group C rotaviruses in communal sewage.

Group C rotaviruses have been recognized as a cause of acute gastroenteritis in humans, cattle, and swine, although the true epidemiologic and clinical importance of this virus in these hosts has not yet been fully established. A real-time PCR assay based on a broadly reactive primer pair was developed and used to quantitatively determine the viral load of group C rotaviruses in environmental samples. A total of 35 raw and 35 treated sewage samples collected at the same sampling time in four Hungarian sewage treatment plants during a survey in 2005 were tested for the presence of group C rotaviruses. The overall detection rates were 91% (32 of 35) for the influent and 57% (20 of 35) for the effluent samples. Molecular characterization of the amplified partial VP6 gene revealed the cocirculation of human and animal (i.e., bovine and porcine) strains that were easily distinguishable by melting curve analysis. Human strains yielded relatively high viral loads (mean, 1.2 x 10(7); median, 6.9 x 10(5) genome equivalents per liter influent sewage) and appeared to display seasonal activity over the study period, whereas animal strains appeared to circulate throughout the year at much lower average titers (bovine strains mean, 9.9 x 10(4); median, 3.0 x 10(4); porcine strains mean, 3.9 x 10(4); median, 3.1 x 10(4) genome equivalents per liter influent sewage). Our findings suggest that monitoring of communal sewage may provide a good surrogate for investigating the epidemiology and ecology of group C rotaviruses in humans and animals.

Genetic drift of norovirus genotype GII-4 in seven consecutive epidemic seasons in Hungary.

BACKGROUND: Noroviruses are common pathogens in human gastroenteritis outbreaks worldwide. They belong to a genetically diverse group of RNA viruses with multiple genogroups (G) and genotypes. Genotype GII-4 norovirus (Lordsdale) is the predominant agent in epidemics. OBJECTIVES: To investigate the genetic variation in GII-4 strains isolated during seven epidemic seasons in Hungary from November 2000 to June 2007. STUDY DESIGN: Using the prospective epidemiological surveillance of norovirus outbreaks in Hungary, GII-4 strains were selected for further genetic analysis. After phylogenetic analysis, RNA-polymerase (open reading frame 1; ORF1), capsid (ORF2) and the ORF1/ORF2 junction were analysed by RT-PCR and sequencing. RESULTS: Three hundred and seventy-seven (76.8%) of 491 confirmed norovirus outbreaks were caused by genotype GII-4. GII-4 was the predominant genotype in six of the seven epidemic seasons. Four main GII-4 variants--epidemic point mutants--(GII-4-2000, GII-4-2002, GII-4-2004 and GII-4-2006b) were detected, with each variant predominating in two consecutive epidemic seasons. CONCLUSIONS: Genotype GII-4 was confirmed as the predominant genetic type in epidemic norovirus seasons in Hungary. Genetic drift successfully promotes the re-emergence of GII-4 variants in the population. The elevated number of norovirus outbreaks in the population predicts the emergence of new GII-4 genetic variants as part of an international epidemic.

[EuroRotaNet--European rotavirus strain surveillance network established with Hungarian participation]. / Hazai részvétellel megalakult az Európai Rotavírus-törzs-figyelô Hálózat (EuroRotaNet).

Group A rotaviruses are the most common cause of severe gastroenteritis worldwide. The incidence and distribution of group A rotavirus sero/genotypes varies between geographical areas during a rotavirus season, and from one season to the next. In addition, cocirculation of genetically diverse multitypic rotaviruses and of intratypic variants in any one place and time is common. Assuming widespread use of rotavirus vaccine in the near future, comprehensive surveillance of natural rotavirus infections is vital. EuroRotaNet has been established in order to gather comprehensive information on the rotavirus types co-circulating throughout Europe. The main objectives of the network are to (i) develop methods and algorithms for effective rotavirus strain typing and characterisation, (ii) describe in detail the molecular epidemiology of rotavirus infections in Europe, (iii) monitor the effectiveness of current genotyping methods and respond to changes associated with genetic drift and shift, and (iv) monitor the emergence and spread of novel rotavirus strains within Europe. This infrastructure may serve as a platform for future surveillance activities and nested studies for evaluating the effectiveness of a rotavirus vaccine in the general population. Studies to monitor the reduction in disease associated with common rotavirus types, the possible vaccine-induced emergence of antibody escape mutants of genotypes other than those included in the vaccine and of reassortment between vaccine and naturally circulating wildtype strains are required.

First detection of Dobrava hantavirus from a patient with severe haemorrhagic fever with renal syndrome by SYBR Green-based real time RT-PCR.

Dobrava hantavirus (DOBV) infection was diagnosed in a previously healthy 46-y-old hunter suffering from severe haemorrhagic fever with renal syndrome (HFRS). Specific IgM antibodies against DOBV were identified by an immunofluorescence assay, while viral nucleic acid was detected by the molecular method, confirming the diagnosis. Our results reveal an existing risk of DOBV transmission to humans in Hungary.

[Molecular detection of adenovirus type-8 epidemic keratoconjunctivitis in Hungary]. / Adenovírus 8-as típusa okozta járványos keratoconjunctivitis molekuláris diagnosztikája.

INTRODUCTION: Both infectious and non-infectious forms of acute conjunctivitis are known. Viruses, especially different types of adenoviruses are the etiological agents of infectious epidemic conjunctivitis (conjunctivitis epidemica). AIMS: The author's aims were to describe an outbreak of keratoconjunctivitis and to detect the viral agent by molecular methods in Hungary. MATERIALS AND METHODS: Classical epidemiological methods were used for investigation. Polymerase chain reaction (PCR) followed by sequencing were used for the detection of adenoviral hexon region from freshly collected conjunctival swabs. RESULTS: Between 9 October and 18 December 2006, a total of 60 patients became ill with keratoconjunctivitis in 7 settlements in Southwest Hungary. Mean age was 51,2 years. Conjunctivitis (100%), lacrimation (94%), foreign body sensation (83%), and dim vision (76%) were the main clinical symptoms. Both eyes were affected in half of the cases. Direct contact was the main transmission route including nosocomial spread associated with ophthalmology practices. Five (62.5%) of 8 conjunctival swabs were PCR-positive for adenovirus type 8 (HAdV8/Baranya/2006/HUN; EF210714) which was genetically identical to adenovirus strain detected in Austria in 2004 (DQ149614). CONCLUSIONS: The outbreak of keratoconjunctivitis was partially associated with nosocomial infection caused by type 8 adenovirus. Both the recognition of the clinical illness, laboratory diagnosis and public health measures are necessary for the prevention of keratoconjunctivitis infection and epidemic.