Investigation of Norovirus genogroups (GI, GII and GIV) in stool of pet dogs with diarrhea / Investigação de genogrupos de norovírus (GI, GII e GIV) nas fezes de cães de estimação com diarréia

In this study, we searched the existence of human norovirus (NoV) GI, GII and GIV in the stool of 128 pet dogs with diarrhea, of different sex, age and breed, in Burdur, Turkey, using Real-Time PCR method. Human NoV GII was found in only 5 of the 128 dog stool samples (3.91%). It was discovered that human NoV existed most in crossbreed, female and aged 24 months or over dogs. These dogs found with human NoV GII were either bought from pet shops, stray dogs or taken as puppy of another pet dog. The sheltering conditions of these dogs were moderate and they were fed with home food residue and dry food. It was also found that most of them were vaccinated and had certain walking sites. The owners of the animals detected with infection generally did not have the habit of washing their hands or changing their clothes before or after caring their pets. We strongly advice that dog owners' personal hygiene, the necessity of changing their clothes during their contact with animals, the environment provided for the dog, the sensitivity in caring, use of strong and effective disinfectant, keeping the dogs away from toilets and sewerage systems, as well as not feeding them with food residues are crucial issues in dogs' care. Owners of the dogs with NoV GII were middle aged or elderly people, male, and there were no children in their houses. As these dogs are treated like the owner's child, it is assumed that they could be transmitted with NoV GII as a result of close interaction with their owner.(AU)

Neste estudo pesquisamos a existência de norovírus humano (NoV) GI, GII e GIV nas fezes de 128 cães com diarréia, de diferentes sexos, idades e raças, em Burdur, Turquia, utilizando o método de PCR em tempo real. NoV GII humano foi encontrado em apenas 5 das 128 amostras de fezes de cães (3,91%). Foi descoberta NoV humana, principalmente em cruzamentos, fêmeas e cães com idade igual ou superior a 24 meses. Os cães encontrados com NoV GII humano foram comprados de lojas de animais, eram vira-latas ou foram tomados como filhotes de outro cão de estimação. As condições de abrigo desses cães eram moderadas. Os cães foram alimentados com restos de comida caseira e comida seca. Verificou-se também que a maioria dos animais foi vacinada e tinham locais adequados para caminhada. Os donos dos animais detectados com infecção geralmente não tinham o hábito de lavar as mãos ou trocar de roupa antes ou depois de cuidar de seus animais de estimação. Aconselhamos que a higiene pessoal dos donos, a necessidade de trocar de roupa durante o contato com animais, o ambiente fornecido para o cão, a sensibilidade no cuidado, o uso de desinfetantes eficazes, manter os cães longe de banheiros e esgotos, assim como evitar alimentá-los com resíduos alimentares, são questões cruciais no cuidado dos cães. Os proprietários dos cães com NoV GII são de meia-idade ou idosos, a maioria do sexo masculino, e não havia crianças em suas casas. Como esses cães são tratados como um filho, presume-se que eles foram infectados com o NoV GII como resultado de uma interação próxima com o proprietário.(AU)

Genomic sequences analysis of human Norovirus GII.4 variant JN010 in Jinan / 中华实验和临床病毒学杂志

Objective@#To analyse the genomic characteristics of Novirus(NoV) GII.4 genotype JN010 strain isolated form Jinan in 2017.@*Methods@#Seven pairs of primers were designed and used to amplify the JN010 genome. Sequence analyses, alignment and phylogenetic trees of ORF1 and ORF2 genes were performed using the software Lasergene7.1 and MEGA5.2. At the same time the major protein VP1 amino acid mutations were analyzed.@*Results@#The 7 516 bp complete genome sequence of JN010 strain was obtained, the most mutation sites were located in P2 subdomain of VP1. Two substitutions I293N and H373N of VP1 were locate neighboring epitope A, and R297H mutation happened within epitope A and the site A that binding with histo-blood group antigens(HBGAs). The JN010 strain was GII.Pe/GII.4 genotype and genetically closest to the strains found in Osaka of Japan(GenBank accession number LC066046) and the strains in Zhongshan city of Guangdong (GenBank accession number KY407064) respectively according to ORF1 and ORF2 gene homologous and phylogenetic analysis.@*Conclusions@#The NoV GII.4 variant strain JN010 has occurred mutations in the key site of the epitope A and site A that bind with HBGAs, and maybe affect its antigenicity and interaction with HBGAs.

Molecular screening of blue mussels indicated high mid-summer prevalence of human genogroup II Noroviruses, including the pandemic "GII. 4 2012" variants in UK coastal waters during 2013

Abstract This molecular study is the first report, to the best of our knowledge, on identification of norovirus, NoV GII.4 Sydney 2012 variants, from blue mussels collected from UK coastal waters. Blue mussels (three pooled samples from twelve mussels) collected during the 2013 summer months from UK coastal sites were screened by RT-PCR assays. PCR products of RdRP gene for noroviruses were purified, sequenced and subjected to phylogenetic analysis. All the samples tested positive for NoVs. Sequencing revealed that the NoV partial RdRP gene sequences from two pooled samples clustered with the pandemic "GII.4 Sydney variants" whilst the other pooled sample clustered with the NoV GII.2 variants. This molecular study indicated mussel contamination with pathogenic NoVs even during mid-summer in UK coastal waters which posed potential risk of NoV outbreaks irrespective of season. As the detection of Sydney 2012 NoV from our preliminary study of natural coastal mussels interestingly corroborated with NoV outbreaks in nearby areas during the same period, it emphasizes the importance of environmental surveillance work for forecast of high risk zones of NoV outbreaks.

The molecularly etiological study on the GII.17 norovirus causing an acute gastroenteritis outbreak in Shenyang / 中华实验和临床病毒学杂志

Objective@#To define the etiology and genetics of the pathogen causing an acute gastroenteritis outbreak in a college of Shenyang.@*Methods@#A total of 15 anal swab samples were collected from students or kitchen staffs of the college where outbroke the epidemic of acute gastroenteritis in December 2015 in Shenyang, Liaoning province. Real-time PCR was performed to identify the infection of Norovirus(NoV). The NoV genes were amplified and sequenced for those positive samples, followed to perform phylogenetic analysis.@*Results@#Of the 15 specimens, 12 were NoV positive by Real-time PCR, phylogenetic analysis showed that the infected virus of the outbreak was belonged to NoV genotype GII.17. The homology between the present virus and new mutated GII.17 strain of KR020503 identified in 2014—2015 epidemic outbreak in China was over 99%.@*Conclusions@#The new mutated NoV GII.17 caused the acute gastroenteritis outbreak in a college of Shenyang in 2015, NoV GII.17 was detected firstly in Northeast China.

The Genetic Diversity of Norovirus from Children with Diarrhea in Gwangju Metropolitan City, Korea: 2008-2012

In order to study the characteristics of norovirus in Gwangju metropolitan city, We examined norovirus in 13,931 fecal specimens collected through five years (2008-2012) from children admitted with a chief complain of acute diarrhea. Among a total of norovirus (NoV) was most frequently detected (3,025 cases, 21.7%). Concerning the frequency of virus detected by month, NoV tended to break out frequently from October to March in the following year. NoV was detected most highly in 0~3 year infants. Through examinations on NoV genotypes, among 3,025 cases that turned out to be positive, the genotypes of 2,652 cases were determined with various results including 13 types of GI and 17 types of GII. The results of analysis on GI genotypes were as follows: GI-4 (21.9%), GI-2 (15.2%), and GI-6 (10.5%). GII genotypes were as follows: GII-4 (63.9%), GII-3 (18.9%), GII-8 (4.2%), GII-2 (3.9%), GII-6 (3.3%), and GII-1 (1.9%). Eight types of variants for GII-4 genotype (427 cases) were identified. The majority of the GII-4 variants was GII-4_Farmington (181 cases, 42.4%), which peaked in 2012, while GII-4_2008b (173 cases, 40.5%) showed a high prevalence in 2011. Concerning the circulation of variants, as many as eight types of GII-4 variants were identified in 2012, showing more varieties than in other years. Therefore, this study can be used as fundamental data for the development of vaccine candidate for the prevention of viral diarrheal diseases with high-incidence.

Human Norovirus Genogroups Detected from Acute Gastroenteritis Patients in Seoul from May 2013 to April 2015

Norovirus is an important cause of acute nonbacterial gastroenteritis in communities worldwide. It was evaluated the prevalence of norovirus infections in patients with acute gastroenteritis occurring in Seoul from May 2013 to April 2015, with regular surveillance. 7.3% (252/3,485) of the fecal specimens were determined to be positive for noroviruses by reverse transcription-polymerase chain reaction (RT-PCR). Norovirus genogroup distribution was 19.1% (48/252) genogroup GI, 71.4% (180/252) genogroup GII, and 9.5% (24/252) genogroup G1+GII respectively. It was most norovirus detection rates from November 2013 to March 2015. And it was rotavirus 0.2% (7/3,485), astrovirus 0.03% (1/3,485), sapovirus 0.03% (1/3,485) and, it was non-detective on adenovirus. Norovirus genotypes identified were nine kinds of genogroup GI (GI-1, GI-2, GI-3, GI-4, GI-6, GI-7, GI-8, GI-12, GI-14) and eight kinds of genogroup GII (GII-2, GII-3, GII-4, GII-5, GII-6, GII-7, GII-14, GII-16, GII-17). The genetic characteristics of norovirus and the epidemiological patterns of a viral pathogen from acute gastroenteritis patients may give potentially effective data for epidemiological studies in Seoul, Korea.

Expression of human norovirus VP1 gene and VP1-specific monoclonal antibodies / 동물의과학연구지

Norovirus (NoV) is an etiologic agent of human and animal acute gastroenteritis and is a member of the family Caliciviridae. NoV is classified based on nucleotide sequences of the VP1 gene into at least six genogroups (GI-GVI), among which GI, GII, and GIV are known to infect humans and GII is the most prevalent genogroup. In this study, VP1, the full gene of GII human NoV, was cloned from a human fecal sample and expressed using a baculovirus expression system. Human NoV VP1-specific monoclonal antibodies (MAbs) were produced using expressed recombinant VP1. Expressed VP1 in the recombinant virus was confirmed by polymerase chain reaction (PCR), indirect fluorescence antibody (IFA) test, and Western blot analysis. Eight hybridomas secreting VP1-specific MAbs against human GII NoV were generated and characterized. All of the MAbs produced in this study reacted with human GII NoV VP1-recombinant baculoviruses but not with other non-human calicivirus recombinant baculoviruses. These MAbs reacted specifically with human NoV GII.4-2009 virus-like particles (VLPs), and some MAbs showed cross-reactivity with other GII.4 variant VLPs. Expressed human GII NoV VP1-recombinant protein and MAbs specific to this protein can be used as useful reagents for detecting and characterizing human NoV.

Molecular Characteristics of Noroviruses Genogroup I and Genogroup II Detected in Patients With Acute Gastroenteritis

Noroviruses are the leading cause of epidemic gastroenteritis, including foodborne outbreak, in Korea. The prevalence of human noroviruses was studied in diarrheal stool samples of patients with acute gastroenteritis by conventional duplex reverse transcription (RT)-PCR. Diarrheal stool samples were collected from 1,685 patients from the local hospitals in Seoul. The prevalence of the noroviruses was 22.8% (222/972 patients) in 2012 and 11.2% (80/713 patients) in 2013, with a total of 17.9% (302/1,685 patients). Genotyping was performed on 302 norovirus-positive stool samples to reveal 5.6% prevalence of genogroup I (GI) (17/302) and 94.4% prevalence of genogroup II (GII) (285/302). The patients with norovirus-associated acute gastroenteritis mostly showed prevalence of GII norovirus, especially GII.4 (64.6%; 195/302).

Genetic Characterization of Norovirus GII.3 Circulating in Korea

Noroviruses (NoV) are the major viral pathogen that causes epidemic acute gastroenteritis and outbreaks of food-borne illness. The major genotypes responsible for the epidemics of NoV are GII.4 and GII.3. However, most studies of NoV have been associated with GII.4 genotype and only few studies have been done with GII.3 genotype. Here, we selected 18 GII.3 strains, which recently circulated in Korea, and determined the partial sequences of the capsid gene. Phylogenetic analysis comparing these sequences with 29 reference strains from the GenBank database was performed using the MEGA program. All NoV GII.3 strains formed 2 distinct genetic lineages and variant groups. Lineage A showed 94.1~97.6%, 90.2~94.6% nucleotide identities from lineage B and variant group, respectively. Lineage B showed 90.2~94.6% nucleotide identities from variant group. These different genetic lineages based on the phylogenetic analysis of capsid sequences imply that the circulating Korean NoV GII.3 strains have potential antigenic variation.

Mechanism of anti-diabetic action, efficacy and safety profile of GII purified from fenugreek (Trigonella foenum-graceum Linn.) seeds in diabetic animals.

Mechanism of action of GII (100 mg/kg body weight, po for 15 days) purified from fenugreek (T. foenum-graecum) seeds was studied in the sub-diabetic and moderately diabetic rabbits. In the sub-diabetic rabbits it did not change much the content of total lipids, glycogen and proteins in the liver, muscle and heart (glycogen was not studied in the heart). However, in the moderately diabetic rabbits same treatment decreased total lipids more in the liver (21%) than those in the heart and muscle. Total protein content increased (14%) in the liver but negligible change (5-7%) was observed in heart and muscle. Glycogen increased (17%) in the liver but not in the muscle of the moderately diabetic rabbits (glycogen was not estimated in the heart). Among the enzymes of glycolysis, activity of glucokinase was not affected in the liver of both the sub-diabetic and moderately diabetic rabbits. Phosphofructokinase and pyruvate kinase activity in both sub-diabetic and moderately diabetic rabbits increased (13-50%) indicating stimulation of glycolysis. The activity of gluconeogenic enzymes glucose-6-phosphatase and fructose-1,6-diphosphatase of the sub-diabetic rabbits decreased in the liver (15-20%) but not in the kidneys. In the moderately diabetic rabbits after treatment with GII, glucokinase in the liver was not affected much (-9%) but increased well in the muscle (40%). Phosphofructokinase and pyruvate kinase were moderately increased both in the liver and the muscle (18-23%). The gluconeogenic enzyme glucose-6-phosphatase decreased reasonably well in the liver and kidneys (22, 32%). Fructose-1,6-diphosphatase decreased only slightly (10, 9%) in the moderately diabetic rabbits. Thus GII seems to decrease lipid content of liver and stimulate the enzymes of glycolysis (except glucokinase) and inhibit enzymes of gluconeogenesis in the liver of the diabetic especially moderately diabetic rabbits.

Anti-hyperglycemic compound (GII) from fenugreek (Trigonella foenum-graecum Linn.) seeds, its purification and effect in diabetes mellitus.

An anti-hyperglycemic compound named GII was purified from the water extract of the seeds of fenugreek (T. foenum-graecum) and shown to be different from trigonelline and nicotinic acid isolated earlier from the same plant. GII (50 mg/kg body weight, po) reduced blood glucose in glucose tolerance test (GTT) in the sub-diabetic and moderately diabetic rabbits and significantly reduced the area under the curve (AUC) of GTT. Treatment for 7 days of the sub-diabetic rabbits with GII (50 mg/kg body weight, po) improved glucose tolerance without reducing fasting blood glucose (FBG) which was nearly normal. The results suggest that there is no risk of hypoglycemia in near normal animals (may be humans also) with abnormal GTT. Treatment of the moderately diabetic rabbits with GII (100 mg/kg body weight for 3 weeks) reduced FBG to nearly normal value and improved GTT. GII was more effective than the standard drug tolbutamide. Intermittent therapy given on days 1–5, 11–15, 26–30 and 56–60 to moderately diabetic rabbits leaving in between days without treatment brought down FBG to normal and AUC during GTT was normal. After 15 days treatment with GII (100 mg/kg body weight for 3 weeks) glycosylated hemoglobin came down and insulin increased to normal values in the sub-diabetic, moderately diabetic and severely diabetic rabbits. GII treatment (100 mg/kg body weight for 15 days) brought down all the altered serum lipids (TC, HDLC, TAG, PLs and FFAs) to normal levels. The results suggest that intermittent therapy, instead of daily therapy is possible and GII has good potential as an oral anti-diabetic drug with intermittent therapy.

Identification and molecular characterization of norovirus in São Paulo State, Brazil / Identificação e caracterização molecular de norovírus em surtos de gastroenterites no Estado de São Paulo

Norovirus (NoV), previously called Norwalk-like virus, represents an important group of human pathogens associated with outbreaks of nonbacterial gastroenteritis. Epidemiological surveys of outbreaks have shown that the most important routes of transmission are person-to-person contacts and contaminated food and water, with the virus affecting adults and children. NoV is classified into genogroups, being genogroups GI, GII and GIV found in humans. In view of the high genetic diversity of NoV and the lack of information about this virus in Brazil, the aim of the present study was the molecular characterization of NoV isolated from diarrheic stool samples of patients from São Paulo State. In this study, 204 stool specimens collected during diarrhea outbreaks were analyzed by RT-PCR, and 12 were sequenced for genogroup confirmation. One-step PCR was performed in order to amplify the B region of ORF 1 using the MON 431, 432, 433 and 434 primer pool. From total, 32 (15.7 percent) stool specimens were positive for NoV genogroup GII. Comparison of the sequences of the PCR products to GenBank sequences showed 88.8 percent to 98.8 percent identity, suggesting the presence of genogroup GII in gastroenteritis outbreaks in São Paulo State.

Norovírus (NoV), anteriormente chamado Norwalk-like vírus, constituem um importante grupo de patógeno humano associado a surtos de gastroenterites não bacterianas. Levantamentos epidemiológicos de surtos demonstram que as mais importantes vias de transmissão são contatos pessoa-pessoa, água e alimentos contaminados, afetando adultos e crianças. Os NoV são classificados em genogrupos, sendo os genogrupos GI, GII e GIV encontrados em humanos. A grande diversidade genética dos NoV e a falta de informação sobre esses vírus no Brasil determinaram o tema deste estudo que teve como objetivo a identificação e caracterização molecular de NoV em amostras fecais de pacientes com gastroenterites, provenientes de surtos de diarréia no estado de São Paulo. Neste estudo, 204 amostras de fezes coletadas durante surto de diarréia foram analisadas por RT-PCR, e 12 foram seqüenciadas para confirmar o genogrupo. O método one step PCR foi empregado para a amplificação da região B da ORF1 com o pool de primers MON 431, 432, 433 e 434. Do total, 32 (15,7 por cento) amostras foram positivas para norovírus genogrupo GII. A comparação das seqüências dos produtos de PCR às seqüências do GenBank demonstrou 88,8 por cento a 98,8 por cento de identidade, sugerindo a presença de norovírus genogrupo GII em surtos de gastroenterites no Estado de São Paulo.