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PubMed2002

Molecular detection and sequence analysis of human caliciviruses from acute gastroenteritis outbreaks in Hungary.

Three viral gastroenteritis (VGE) outbreaks that occurred in 1998-1999, in Hungary were investigated for the presence of human caliciviruses (HuCVs). HuCVs in stool specimens were detected by reverse transcription-polymerase chain reaction (RT-PCR) using primer pair 289/290, which was designed based on the RNA-dependent RNA polymerase (RdRp) sequence. RT-PCR results were confirmed by sequencing showing that all three outbreak strains belonged to genogroup II of "Norwalk-like viruses" (NLVs). Two strains had high sequence identity with strains in known genetic clusters (Hawaii and Lordsdale clusters). The third strain (MOH) had distinct RdRp sequence, sharing 77/86% (nt/aa) identity with Snow Mountain virus (SMV), the closest genogroup II virus. To characterize MOH further, we cloned, sequenced, and expressed in baculovirus its capsid gene. It had 75/79% (nt/aa) identity with SMV, but 97/98% (nt/aa) identity with NLV/Hillingdon/90/UK, a recently identified genetic cluster of HuCVs. The recombinant MOH (rMOH) capsid protein self-assembled into virus-like particles (VLPs), which is antigenically distinct from other recombinant HuCV capsid antigens available in our laboratory. Further study of this VLP will have important applications in antigenic characterization and diagnosis of HuCVs.

Acute Disease↗
PubMed2001

Nucleotide sequence analysis and development of consensus primers of RT-PCR for detection of Norwalk-like viruses prevailing in Japan.

A total of 177 different nucleotide sequences of the RNA polymerase region of Norwalk-like viruses (NLVs) genomes, collected via a nation-wide survey project in Japan between 1989 and 1998, were examined by reverse transcription-polymerase chain reaction (RT-PCR) employing various primer pairs. The nucleotide sequences of different strains showed great diversity, with a range of 57 to 100% identities among strains. The strains could be classified into five clusters: Norwalk (NV), Snow Mountain agent/Bristol virus (SMA/BV), Toronto virus/Mexico virus (TV/MX), and Japan specific cluster 1 and 2 (JP-1 and JP-2). Within each cluster there is greater than 85% identity of amino acid sequence (more than 75% identity of nucleotide sequences), based on sequence homology analysis. We believe that two of the five clusters, JP-1and JP-2, define new specific clusters found in Japan according to phylogenetic and pair-wise comparison studies. An RT-PCR procedure was designed using new consensus primer pairs, P1/P2, P1/P3, and Y1/Y2 based on multiple alignment of collected nucleotide sequences, that are expected to detect nearly all NLVs prevailing in Japan. The usefulness of the primers was tested by ten different laboratories in Japan using a panel of ten fecal samples containing different virus strains. The identification of these primer pairs will facilitate routine diagnosis of NLV infection by RT-PCR and offers the potential for their direct detection in food and environmental samples.

Caliciviridae Infections↗
PubMed1990

Antigenic relatedness among the Norwalk-like agents by serum antibody rises.

The Norwalk, Snow Mountain (SMA), and Hawaii agents are etiologically associated with separate outbreaks of acute viral gastroenteritis. Previous cross-challenge of volunteers, immune electron microscopy, and/or enzyme-immunoassay analysis suggested that these agents are antigenically distinct. We examined paired sera from human volunteers challenged with these agents for the presence of homologous and heterologous serum antibody titer rises to the agents. Two-way cross-reactions occurred between Hawaii agent and SMA. A one-way cross-reaction occurred between Norwalk agent and SMA, as volunteers challenged with Norwalk agent had heterologous serum antibody titer rises to SMA, but the reverse did not occur. The Norwalk and Hawaii agents had minimal cross-reaction, with only one volunteer challenged with Hawaii agents having a heterologous rise to Norwalk agent. These observations indicate varying degrees of antigenic relatedness among these agents.

Antibodies, Viral↗
PubMed1993

Efficacy of a recombinant Norwalk virus protein enzyme immunoassay for the diagnosis of infections with Norwalk virus and other human "candidate" caliciviruses.

A recombinant Norwalk virus antigen enzyme immunoassay (rNV EIA) for the detection fo Norwalk virus (NV) antibodies was evaluated using sera from volunteers and patients infected with Norwalk, Hawaii, Snow Mountain Agent, small round structured viruses (SRSV), and human calicivirus (HuCV) strains. The results for NV antibodies correlated well with previous results obtained using a NV radioimmunoassay. The test rNV EIA was found to be highly specific for NV. The results of tests on sera obtained from patients involved in confirmed outbreaks of SRSV in the UK showed that only 1/12 was due to Norwalk virus. Further evidence is presented that SRSV strain UK2 is antigenically related to NV.

Adult↗
PubMed1994

Sequence similarity of human caliciviruses and small round structured viruses.

The application of reverse transcription-polymerase chain reaction (RT-PCR) using primers directed to the RNA dependent RNA polymerase region within ORF1 of Norwalk virus (NV) showed that 31 percent of morphologically typical human caliciviruses (HuCV) and 57% of small round structured viruses (SRSVs) produced a product of 470 bp similar to the NV control, NV 8FIIa/68/US. Alignment of the amino acid sequences of morphologically typical HuCVs with previously published sequences for SRSVs, NV, and Snow Mountain agent (SMA) showed a high degree of homology (90-92%) with SMA and a lesser extent of homology with NV (60-61%). The amino acid sequence of two strains of HuCV, HuCV/3C/92/UK, and HuCV/5C/92/UK differed by only one or two amino acids respectively in the RNA dependent RNA polymerase region from that of two strains of SRSV obtained from children in the United Kingdom, SRSV/4S/90/UK and Japan, SRSV/OTH-25/89/J which were found to have identical amino acid sequences. The use of an EIA for detection of NV antigen employing antisera raised to recombinant NV protein indicated that HuCVs and SRSVs obtained from children and adults in the United Kingdom were antigenically distinct from the prototype Norwalk virus, NV/8fIIa/68/US.

Adult↗
PubMed1995

Enzyme immunoassay using baculovirus-expressed human calicivirus (Mexico) for the measurement of IgG responses and determining its seroprevalence in London, UK.

The use of an enzyme immunoassay (EIA) employing a baculovirus-expressed recombinant human calicivirus (Mexico virus, MxV) for the detection of IgG-specific antibodies is described. MxV appeared to be related antigenically to a strain of small round structured virus, SRSV/UK4/Leeds/91, which had previously been shown by solid phase immune electron microscopy (SPIEM) to be related to Snow Mountain agent (SMA). One other outbreak which occurred in San Anita, USA in 1980 and was due to consumption of contaminated water was caused by a virus antigenically related to MxV. Volunteers and patients who developed significant IgG responses to rMxV showed anamnestic IgG responses (2 to 4-fold) in the recombinant Norwalk virus (rNV) IgG assay. Patients and volunteers who were known to have been infected with several other strains of calicivirus/small round spherical viruses (SRSV) including NV and SRSV UK3 showed no significant antibody response to rMxV in the EIA. A seroepidemiological survey of sera from 338 children in London showed that infection with MxV occurred earlier in life than NV. Primary infections with MxV were common after the age of 6 months. Over 70% of children had evidence of infection by the ages of 2 years, whereas only 12% of these children had been infected with NV. High concentrations of maternal antibody were present during the first month of life which was detected in 96% of the neonates. The results suggest that the high sensitivity of the EIA may be detecting maternal antibody throughout the first 8 months of life.

Adolescent↗
PubMed1995

Study of Norwalk-related viruses in Mexican children.

Two-hundred Mexican children monitored from birth to 2 years of age in a cohort study of diarrhea were tested for Norwalk virus (NV) and Norwalk-related virus infection. Blood was collected quarterly and tested by an enzyme immunoassay (EIA) using the recombinant NV (rNV) particles as antigen. Stool was collected weekly and tested by an EIA using hyperimmune anti-sera from animals immunized with rNV and a reverse transcription-polymerase chain reaction (RT-PCR) with primers in the RNA polymerase region of NV. A high prevalence of serum antibody to NV (85% at age 2 years) was found by the antibody EIA. In 54 stool specimens selected from children who developed a high titer of serum antibody to rNV, none was positive for NV by the antigen EIA, but 6 yielded products by the RT-PCR. One stool specimen (MX virus) yielded a 3.3 kb RT-PCR product from the 3' end of the viral genome. The MX virus cDNA has a genomic organization like other caliciviruses. Sequence comparison showed that MX virus shares 80% nucleic acid and 91% amino acid sequence identity with Snow Mountain agent (SMA), but only 62% and 60% identity, respectively, with NV in the RNA polymerase region, suggesting that MX virus is a SMA-like virus.

Amino Acid Sequence↗
PubMed2005

Evidence of the etiological predominance of norovirus in gastroenteritis outbreaks--emerging new-variant and recombinant noroviruses in Hungary.

Between January 2001 and December 2003, stool specimens from 262 (45%) of 581 reported outbreaks of gastroenteritis were investigated for noroviruses in Hungary. Specimens collected from outbreaks of non-bacterial gastroenteritis were examined by reverse-transcription polymerase chain reaction and enzyme immunoassay. In 253 (97%) of 262 outbreaks, norovirus was detected and confirmed by sequencing in 211 (81%). Hospitals (35%), day care centers (30%), and elderly homes (27%) were the most common settings. Diversity and frequency of the genotypes changed over time but with predominance (95%) of genogroup (GG) II strains. Strains grouped into 11 genotypes including an epidemic spread of new-variant GGII4 (Lordsdale virus) and a recently emerged group of natural recombinant strains (GGIIb/Hilversum polymerase) with four capsid types (Hawaii, Mexico, Snow Mountain, and Lordsdale). Clusters of epidemics including food-borne outbreaks were detected. According to this study, noroviruses are the predominant etiological agents causing gastroenteritis outbreaks in Hungary.

Caliciviridae Infections↗
PubMed1989

Prediction of the conformation of ice-nucleation protein by conformational energy calculation.

The active conformation of an ice-nucleation protein, whose major portion consists of a long polypeptide segment of nearly repetitive octapeptides, is predicted by the analyses of conformational energy and the mechanism of crystal growth. The protein ideally has an exact octapeptide repetition and is assumed to have a helical conformation. The present study searched for low-energy helical conformations and each of the obtained low-energy conformations examined as to whether it has a surface structure that can promote crystal formation. Two conformations obtained were good candidates for an ice nucleus. Both were found to have on their surfaces an arrangement of hydrogen-bonding sites, which fits well with those of hydrogen bonds in hexagonal ice crystal. Further, one of the two conformations had a hexagonal conformational symmetry consistent with the hexagonal ice crystal structure. The other conformation had a pentagonal conformational symmetry that could enable the growth of an ice crystal--dendritic polycrystalline snow crystal--which grows on metastable cubic ice.

Amino Acid Sequence↗
PubMed1996

Heparin causes the accumulation of heparan sulfate in cultures of arterial smooth muscle cells.

Heparin infusion in an experimental animal model of arterial injury causes a significant increase in the proteoglycan (PG) component of the extracellular matrix in the injured arteries (Snow, A. D., Bolender, R. P., Wight, T. N., and Clowes, A. W. (1990) Am. J. Pathol. 137, 313-330). The mechanisms responsible for this heparin-induced increase in arterial PGs are not understood. To address this question, we have examined the effect of heparin on PG synthesis and accumulation by aortic smooth muscle cells in culture. Heparin causes a dose-dependent increase in the accumulation of PGs while the greatest percentage change among the different types of PGs is a doubling of the amount of heparan sulfate (HS) accumulated in the cell layer. There is also a selective enrichment of HS in the trypsin-resistant component of the cell layer indicating a specific modification in intracellular PGs. This change is due to the accumulation of large HS glycosaminoglycans (GAGs) which elute at K(av) approximately 0.3 on Sepharose CL-6B (Mr approximately 50,000) under dissociative conditions and which are absent from the controls. These GAGs are located inside the cell as is indicated by their retention after heparitinase or trypsin treatment of the cell layer. Furthermore, the increased accumulation of labeled HS in steady-state heparin-treated cells above controls does not appear for several hours after the addition of [35S]sulfate, indicating that HS accumulation is due to decreased degradation of the HS and not new synthesis. This possibility is further supported by the fact that degradation of the intracellular large HS in trypsinized cells is delayed for 2 h by incubation with heparin. These results suggest that heparin may reduce the intracellular degradation of HS through competitive inhibition of endogenous heparitinase.

Animals↗
PubMed1998

Language development in children with simple-partial left-hemisphere epilepsy.

The nature of cerebral involvement in the acquisition of language was addressed in this longitudinal study of children with an early diagnosis of epilepsy with simple-partial seizures (SPE) and with epileptogenic foci localized in the left frontal (LF) lobe. Yearly evaluations of six SPE-LF children on tests of linguistic comprehension (pointing, understanding of narrative, and understanding of prepositions) and production (repetition, lexical diversity, and grammatical production) were carried out between the ages of 3 and 8 years and compared to those of large samples of control children on the same tasks and at each age level. Linguistic production of all children were transcribed, coded, and analyzed using the Child Language Data Exchange System (MacWhinney & Snow, 1991). Individual evolution trajectories revealed that SPE-LF children showed a clear dissociation in linguistic performance between comprehension and production. Linguistic comprehension gradually improved to reach normal performance levels by age 7 while linguistic production, even at later stages, remained quite poor. This dissociation in the development of linguistic performance in SPE-LF children suggests a complex interplay between brain maturation dynamics and dysfunction modulating the succession of stages in language development. The observed persistent deficits in specific aspects of linguistic performance argue for an early involvement of the anterior areas of the left cerebral hemisphere in the production of language.

Age Factors↗
PubMed1997

River water with high iron concentration and low pH causes mortality of lamprey roe and newly hatched larvae.

The effects of prevailing water quality of Perhonjoki River in Western Finland on the hatchability of lamprey roe and survival of newly hatched larvae were investigated. The river flows through sulfide-rich soils, and is characterized by high average total iron (1.5-3 mg liter-1) and aluminum (0.45-0.6 mg liter-1) concentrations and relatively low pH commonly 5.5 to 6.0, with acidification peaks below pH 5. The hatchability of lamprey roe was markedly reduced under these conditions as compared with ground water with low iron (0.002 mg liter-1) and aluminum (0.11 mg liter-1) concentrations but at the same pH. Furthermore, the hatchability of roe and survival of newly hatched larvae was especially affected by an increase in total iron concentration to values that may be observed after heavy rains and during the snow-melt period (4-6 mg liter-1). The results suggest that increases in total iron concentration may play a role in the reproductive failures of lampreys observed in Finnish rivers.

Aluminum↗
PubMed1997

Biology of A beta amyloid in Alzheimer's disease.

The genetic associations with the pathological features of AD are diverse: A rapidly growing number of mutations in presenilin 1 and 2 on chromosomes 14 and 1, respectively, are found in many early-onset FAD patients (Lendon et al., 1997). In addition, beta PP mutations are found in a small percentage of early-onset FAD kindreds. The apoE4 allele on chromosome 19 is associated with the presence of the most common form of AD, sporadic AD (Wisniewski & Frangione, 1992; Namba et al., 1991). However, it is clear that other proteins are also involved in the pathogenesis of AD, since some early-onset FAD kindreds do not have linkage to PS1, PS2, apoE, or beta PP, while at least 50% of late-onset AD is unrelated to apoE. Other proteins which have been implicated in the formation of senile plaques, but so far are not known to have any genetic linkage to AD, include proteoglycans (Snow et al., 1987), apoA1 (Wisniewski et al., 1995a), alpha 1-antichymotrypsin (Abraham et al., 1988), HB-GAM (Wisniewski et al., 1996a), complement components (McGeer & Rogers, 1992), acetylcholinesterase (Friede, 1965), and NAC (Ueda et al., 1993). Which of these proteins will be the most important for the etiology of the most common form of AD, late-onset sporadic AD, remains an open question. Three of the genes which are now known to be linked to AD, including PS1, beta PP, and apoE, have been established immunohistochemically and biochemically to be components of senile plaques (see Fig. 1). This raises at least two possibilities: either each of these proteins is part of one pathway with A beta-related amyloid formation as a final causative pathogenic event or amyloid deposition in AD is a reactive process related to dysfunction of a number of different CNS proteins. Whether or not amyloid formation is directly causative in the pathogenesis of AD, current data suggest that new therapeutic approaches which may inhibit the aggregation and/or the conformational change of sA beta to A beta fibrils (Soto et al., 1996) have the greatest likelihood to make a significant impact on controlling amyloid accumulation in AD.

Alzheimer Disease↗
PubMed1997

The role of human caliciviruses in epidemic gastroenteritis.

Members of the Caliciviridae family of small, positive-sense RNA viruses exhibit a broad host range. The Norwalk and Norwalk-like caliciviruses in this family are major etiologic agents of epidemic gastroenteritis in humans. This illness characteristically lasts 24-48 h and often occurs in group settings such as families, schools, institutions, or communities. The spread of the human caliciviruses is considered to be predominantly by person-to-person contact via the fecal-oral-route. However, the ingestion of calicivirus-contaminated food or water can result in large-scale common-source outbreaks. Many basic features concerning the biology and replication of the human caliciviruses are not known because they have not yet been grown in cell culture and the virus does not appear to replicate in animal models other than the chimpanzee. Sequence analysis of RT-PCR-generated DNA fragments derived from serotypically distinct reference strains (such as the Norwalk, Hawaii, and Snow Mountain viruses) and other circulating strains associated with gastroenteritis has provided evidence for marked genetic diversity among these viruses. Moreover, analysis of the antigenic relationships among these viruses using paired sera from individuals infected with well-characterized reference strains or from animals immunized with recombinant "virus-like particles" (VLPs) suggests that several serotypes of these viruses are circulating worldwide. The availability of molecular techniques for the detection of these fastidious viruses has enabled epidemiologic studies that have strengthened the association of human caliciviruses with acute gastroenteritis and has demonstrated a potential role for antigenic diversity in the natural history of these pathogens.

Animals↗
PubMed1997

Haemorrhagic fevers and ecological perturbations.

Hemorrhagic fever is a clinical and imprecise definition for several different diseases. Their main common point is to be zoonoses. These diseases are due to several viruses which belong to different families. The Flaviviridae have been known for the longest time. They include the Amaril virus that causes yellow fever and is transported by mosquitoes. Viruses that have come to light more recently belong to three other families: Arenaviridae, Bunyaviridae, and Filoviridae. They are transmitted by rodents (hantaviruses and arenaviruses) or from unknown reservoirs (Ebola Marburg). The primary cause of most outbreaks of hemorrhagic fever viruses is ecological disruption resulting from human activities. The expansion of the world population perturbs ecosystems that were stable a few decades ago and facilitates contacts with animals carrying viruses pathogenic to humans. Another dangerous human activity is the development of hospitals with poor medical hygiene. Lassa, Crimean-Congo or Ebola outbreaks are mainly nosocomial. There are also natural environmental changes: the emergence of Sin Nombre in the U.S. resulted from heavier than usual rain and snow during spring 1993 in the Four Corners. Biological industries also present risks. In 1967, collection of organs from monkeys allowed the discovery in Marburg of a new family of viruses, the Filoviridae. Hemorrhagic fever viruses are cause for worry, and the avenues to reduce their toll are still limited.

Animals↗
PubMed1996

Genetic and antigenic diversity of human caliciviruses (HuCVs) using RT-PCR and new EIAs.

RT-PCR using primers from conserved regions of calicivirus genomes, followed by sequencing, permits characterization of genetic variation within the family. EIAs based on baculovirus-expressed viral capsid proteins and hyperimmune antisera against the capsid proteins were developed to detect HuCV antigens and antibodies. Serologic surveys using recombinant Norwalk virus (rNV) and recombinant Mexico virus (rMX, a SMA-like virus) EIAs showed that infections by HuCVs are common and that children acquire antibodies to HuCVs at an early age in both developed and developing countries. Three HuCV genogroups have been described that are represented by Norwalk virus (NV), Snow Mountain agent (SMA), and Sapporo virus, although recently accumulated sequences of HuCV strains indicate these genogroups can be further divided. These genogroups also correspond to distinct antigenic groups based on the results of the recombinant EIAs. The three genogroups co-circulate and have a worldwide distribution, although the SMA genogroup seems to be predominant currently. Application of these new assays for further characterization of the genetic and antigenic properties of HuCVs remains an important task for HuCV research.

Antibodies, Viral↗
PubMed1996

The epidemiology of human calicivirus/Sapporo/82/Japan.

Based on genome analysis of the RNA-dependent RNA polymerase region, it has been proposed that human caliciviruses (HuCV) can be classified into at least three genogroups: genogroup I is represented by Norwalk virus (NV), genogroup II by Snow Mountain agent (SMA) and genogroup III by HuCV/Sapporo/82/Japan (HuCV/Sa/82/J) virus. HuCV/Sa/82/J strain is genetically unique and more closely related to animal caliciviruses than are other known HuCVs, such as NV and SMA. HuCV/Sa/82/J strain was detected in four outbreaks of HuCV gastroenteritis occurring between 1977 and 1982 in an infant home in Sapporo. The HuCVs detected from these four outbreaks all showed a typical "Star of David" configuration by electron microscopy (EM), and they were identical antigenically and genetically. This strain has also been detected in other prefectures in Japan, as well as in the USA, UK, Saudi Arabia and Kenya. Seroepidemiological studies have shown a worldwide distribution of this virus, including Japan, USA, UK, Southeast Asia, Canada, China and Kenya. This virus has been circulating in Sapporo for at least 19 years (1977-1995). HuCV/Sa/82/J strain is thought to be one of the common causes of viral gastroenteritis worldwide. The HuCV/Sa/82/J strain has been detected mainly in infants. Age-related prevalence of antibody to this strain also shows that infections commonly occur in children less than 5 years old, although viruses in the NV and SMA genogroups commonly infect adults. The pattern of acquisition of antibodies to strain HuCV/Sa/82/J is similar to that of other common viral infections. HuCV/Sa/82/J strain is unique virologically and clinically among caliciviruses.

Age Factors↗
PubMed1996

Reverse transcription-polymerase chain reaction detection and sequence analysis of small round-structured viruses in Japan.

Between 1985 and 1995, mass outbreaks of acute gastroenteritis caused by small round-structured virus (SRSV), occurred in eight prefectures in Japan. Fecal samples from 59 patients ill during these outbreaks were recently examined in our laboratory by electron microscopy (EM) and by reverse transcription-polymerase chain reaction (RT-PCR). For RT-PCR, we prepared two sets of primers, a set corresponding to the polymerase region of open reading frame 1 (ORF-1) and a set corresponding to the capsid region of ORF-2 of Norwalk virus (NV). The SRSV nucleic acid detection rate with these primers was more than double that achieved with EM. Most samples found by EM to contain virus particles were also positive by PCR. When the two sets of primers were used separately, the virus detection rate differed depending on the primer used, suggesting that the viral strains examined were not genetically not homogeneous. We then selected nine strains of the virus, cloned their PCR products and analyzed their base sequences. The base sequences of these strains were compared with those of reference strains including prototype NV and Snow Mountain agent (SMA). This comparison yielded the following findings: (1) SRSVs that cause mass outbreaks of gastroenteritis in Japan are genetically variable; (2) SRSV strains that are genetically similar to SMA and SRSV-OTH 25/89/J(OTH25) are dominant in Japan, but strains similar to NV are also present in this country; and (3) a strain (MI1/94) which is genetically identical to Southampton virus (SHV) was detected. Detection of SRSV using sensitive RT-PCR and analysis of the sequences of the amplification products seems to provide a useful means of studying the molecular epidemiology of SRSV.

Adult↗