ABSTRACT Noroviruses (NVs) are important human pathogens that cause acute gastroenteritis. Genetically related animal enteric NVs have also been described, but there is no evidence of interspecies transmission of NVs. In this study we characterized antibody prevalence among domestic pigs by using recombinant capsid antigens of two human NVs (Norwalk and Hawaii) and one swine NV (SW918) that is genetically related to GII human NVs. Recombinant SW918 capsid protein expressed in baculovirus self-assembled into virus-like particles (VLPs) that were detected by antibodies against GII (Hawaii and Mexico), but not GI (Norwalk and VA115), human NVs. NVs recognize human histo-blood group antigens as receptors, but SW918 VLPs did not bind to human saliva samples with major histo-blood group types. Seventy-eight of 110 (71%) pig serum samples from the United States and 95 of 266 (36%) pig serum samples from Japan possessed antibodies against SW918. Serum samples from pigs in the United States were also tested for antibodies against human NVs; 63% were positive for Norwalk virus (GI) and 52% for Hawaii virus (GII). These results indicate that NV infections are common among domestic pigs; the finding of antigenic relationships between SW918 and human NVs and the detection of antibodies against both GI and GII human NVs in domestic animals highlights the importance of further studies on NV gastroenteritis as a possible zoonotic disease.

Noroviruses, an important cause of acute gastroenteritis, have been found to recognize human histo-blood group antigens (HBGAs) as receptors. Four strain-specific binding patterns to HBGAs have been described in our previous report. In this study, we have extended the binding patterns to seven based on 14 noroviruses examined. The oligosaccharide-based assays revealed additional epitopes that were not detected by the saliva-based assays. The seven patterns have been classified into two groups according to their interactions with three major epitopes (A/B, H, and Lewis) of human HBGAs: the A/B-binding group and the Lewis-binding group. Strains in the A/B binding group recognize the A and/or B and H antigens, but not the Lewis antigens, while strains in the Lewis-binding group react only to the Lewis and/or H antigens. This classification also resulted in a model of the norovirus/HBGA interaction. Phylogenetic analyses showed that strains with identical or closely related binding patterns tend to be clustered, but strains in both binding group can be found in both genogroups I and II. Our results suggest that noroviruses have a wide spectrum of host range and that human HBGAs play an important role in norovirus evolution. The high polymorphism of the human HBGA system, the involvement of multiple epitopes, and the typical protein/carbohydrate interaction between norovirus VLPs and HBGAs provide an explanation for the virus-ligand binding diversities.

Norovirus and Sapovirus are two genera of the family Caliciviridae that contain viruses that can cause acute gastroenteritis in humans. Noroviruses (NOR) are genetically highly diverse but limited studies of the genetic diversity of sapoviruses (SAP) have been reported. In this study we characterized twenty-five SAP detected in our laboratory from outbreaks or sporadic cases of acute gastroenteritis in children from different geographical locations and in adults involved in a cruise ship outbreak investigation and a nursing home outbreak. Based on significant differences of partial RNA polymerase sequences (278-286 nt), the 25 strains were grouped into 12 genetic clusters, including 9 potential new clusters. Extended sequence analysis of the capsid gene of selected strains representing five potential new clusters supported this grouping. Four strains (Hou7-1181/90, Mex340/90, Cruise ship/00 and Argentina39) had <84% amino acid (aa) identity to each other and to the published sequences in the GenBank. Mex14917/00 was almost identical to Stockholm/97/SE whose RNA polymerase sequence was unknown. Phylogenetic and distance analyses of the capsid region of the four new strains showed that Hou7-1181/90 and Argentina39 represent two new genogroups and Mex340/90 and Cruise ship/00 belong to two new clusters within the London/92 genogroup. Thus, based on the capsid sequences we propose to classify the currently known SAP into nine genetic clusters within five genogroups, including one genogroup that is represented by an animal calicivirus, the porcine enteric calicivirus (PEC).

The baculovirus expression system has been widely used to produce the capsid proteins of Norovirus (NV) and the proteins form virus-like particles (VLPs) that are useful in many studies, such as immunology, diagnosis, and host-receptor interaction. We report here the application of the E. coli expression system in the production of recombinant NV capsid proteins. In a direct comparison of a previous well-characterized NV strain (VA387), we have demonstrated that the E. coli-expressed capsid proteins maintain the same antigenicity and receptor binding specificity as that of the baculovirus-expressed capsid, although the E. coli-expressed VA387 proteins did not form VLPs. Using the E. coli-expression system, we characterized the receptor-binding patterns of three additional NV strains (OIF1998, Parris Island and VA115), in which OIF1998 binds to HBGA of nonsecretors but did not bind or binds weakly to the HBGA of secretors, as seen in strain VA207. Parris Island binds to HBGA of types A and B but not type O secretors and nonsecretors. VA115 did not show specific binding to any A, B, O secretor nor nonsecretor, which is also observed when the capsid protein of this strain was expressed in baculovirus. Our data indicate that VLP formation is not required for receptor binding, and that the bacteria expression system offers a simple alternative for large production of NV capsid protein for various research purposes, particularly for strains generating low yields in the insect cells.

ABSTRACT Noroviruses (NORs) are an important cause of acute gastroenteritis. Recent studies of NOR receptors showed that different NORs bind to different histo-blood group antigens (HBGAs), and at least four distinct binding patterns were observed. To determine the structure-function relationship for NORs and their receptors, two strains representing two of the four binding patterns were studied. Strain VA387 binds to HBGAs of A, B, and O secretors, whereas strain MOH binds to HBGAs of A and B secretors only. Using multiple sequence alignments, homology modeling, and structural analysis of NOR capsids, we identified a plausible “pocket” in the P2 domain that may be responsible for binding to HBGA receptors. This pocket consists of a conserved RGD/K motif surrounded by three strain-specific hot spots (N 302 , T 337 , and Q 375 for VA387 and N 302 , N 338 , and E 378 for MOH). Subsequent mutagenesis experiments demonstrated that all four sites played important roles in binding. A single amino acid mutation at T 337 (to A) in VA387 or a double amino acid mutation at RN 338 (to TT) in MOH abolished binding completely. Change of the entire RGD motif to SAS abolished binding in case of VA387, whereas single amino acid mutations in that motif did not have an apparent effect on binding to A and B antigens but decreased binding to H antigen. Multiple mutations at the RGK motif of MOH (SIRGK to TFRGD) completely knocked out the binding. Mutation of N 302 or Q 375 in VA387 affected binding to type O HBGA only, while switch mutants with three amino acid changes at either site from MOH to VA387 resulted in a weak binding to type O HBGAs. A further switch mutant with three amino acid changes at E 378 from MOH to VA387 diminished the binding to type A HBGA only. Taken together, our data indicate that the binding pocket likely exists on NOR capsids. Direct evidence of this hypothesis requires crystallography studies.

We characterized the binding of 8 Noroviruses (NORs) to histo-blood group antigens (HBGAs) in human saliva using recombinant NOR (rNOR) capsid proteins. Among the 8 rNORs tested, 6 formed viruslike particles (VLPs) when the capsid proteins were expressed in insect cells, all of which revealed variable binding activities with saliva; the remaining 2 rNORs did not form VLPs, and the proteins did not bind, or bound weakly, to saliva. Four distinct binding patterns were associated with different histo-blood types, defined by Lewis, secretor, and ABO types. Three patterns (VA387, NV, and MOH) recognized secretors, and 1 pattern (VA207) recognized Lewis-positive nonsecretors. The 3 secretor-recognizing patterns were defined as A/B (MOH), A/O (NV), and A/B/O (VA387) binders. Oligosaccharides containing the Lewis and ABH antigenic epitopes were involved in binding. Our findings suggest that different strains of NORs may recognize different human HBGAs on intestinal epithelial cells as receptors for infection.

人类杯状病毒(human calicivirus,HuCV)是引起儿童和成人非菌性胃肠炎的主要病原之一.为了掌握HuCV在我国的流行情况,1998年7月至2001年6月,从长春市儿童医院2343例5岁以下腹泻患儿中共收集粪便标本1264份,其中1056份来自2135例住院患儿.对轮状病毒检测为阴性的588份标本,经多价酶免疫试验(EIA)和两组引物反转录-聚合酶链反应(RT-PCR)检测HuCV,202份为阳性,其中住院患儿标本178份,HuCV检出率为16.9%.HuCV腹泻以2岁以下儿童为主(占96%),流行高峰季节为11月至次年3月.选择17株HuCV进行分子鉴定,15株属GⅡ-4群,1株属GⅡ-3群,另1株属GⅠ-2群,表明GⅡ-4群HuCV是我国流行的优势株.根据HuCV住院患儿的监测资料初步估计,HuCV腹泻住院率约为0.5‰～2.4‰.讨论了长春地区HuCV的流行趋势和疾病负担.以上结果为我国HuCV腹泻的预防和控制提供了科学依据.

Human caliciviruses were detected by EIA and/or RT‐PCR in stool specimens from children with diarrhea treated at out‐ or in‐patient facilities between 1995 and 1998 in Mendoza, Argentina. Mexico virus–like strains detected by primers NV36/51 were transiently prevalent in 1995/1996. Significantly more human caliciviruses were detected when primers were designed from contemporaneously circulating strains. Nucleotide sequences of a highly conserved region in the RNA polymerase gene of 10 selected human caliciviruses were determined. Eight strains were Norwalk‐like viruses and two strains were Sapporo‐like viruses. Seven of the eight Norwalk‐like viruses also were positive by the recombinant Mexico virus antigen EIA. The seven Mexico virus EIA‐positive strains revealed two patterns in the RNA polymerase sequences: two strains were closest to Mexico virus and the other five strains were closest to Lordsdale virus. One of the five “Lordsdale” viruses was found to be a naturally occurring recombinant between the Mexico virus and Lordsdale human calicivirus genetic clusters [Jiang et al., ( 1999b ) Archives of Virology 144:2377–2387]. The Mexico virus EIA‐negative strain had 73–77% nucleotide identity with the closest related Norwalk‐like viruses, indicating it might belong to a new genetic cluster of the Norwalk‐like virus genus. The two Sapporo‐like viruses were distinct genetically; one belonged to the Houston/90 or Parkville cluster and the other to a new cluster. Some strains appeared to have short periods of prevalence and locally adapted primer pairs significantly increased detection rates. The finding of high diversity of circulating strains, including recombinant strains and strains with previously unrecognized genetic identities, highlights a need for studies of human caliciviruses in these children and other populations. J. Med. Virol. 67:289–298, 2002. © 2002 Wiley‐Liss, Inc.

The interfacial reactions between Al2O3 particles and 5083 Al–Mg alloys during fabrication by compocasting and subsequent remelting at 800 °C for 0.5 and 4 h have been studied using analytical transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is found that the reaction product is changed with increasing remelting time. MgO is the main reaction product after casting and remelting for 0.5 h. The MgO crystals are then very small (about 5–20 nm in size) and the reaction zone is about 50–80 nm thick for the as-cast material and about 100–150 nm after remelting for 0.5 h. However, after remelting for 4 h, MgO is transformed into MgAl2O4 and the interfacial region is then constituted of large MgAl2O4 crystals of about 1 μm in size. This transformation indicates that the nature of the reaction products is controlled by the current Mg content in the matrix.

Human caliciviruses (HuCVs) are antigenically diverse. The antigenic relationships among different HuCVs have been difficult to study because HuCVs cannot be passaged in the laboratory. In this study, we describe cloning, sequencing and expression of the viral capsid proteins of three HuCVs that were identified in outbreaks of acute gastroenteritis in Virginia in 1997-1998. Yields of the capsid proteins similar to previously expressed recombinant Norwalk virus were obtained using the baculovirus expression system. Recombinant VA97207 capsid protein (rVA97207) and rVA98387, but not rVA98115, formed virus-like particles (VLPs). All three recombinant capsid antigens detected seroresponses in patients involved in outbreaks of acute gastroenteritis associated with genetically homologous or related HuCVs. The antigenic relationships of the three strains were further characterized using hyperimmune antisera against the three capsid antigens as well as four previously characterized recombinant capsid antigens of Norwalk (rNV), Mexico (rMxV), Hawaii (rHV), and Grimsby viruses (rGrV). VA98387 shared 98% aa identity with GrV; rVA98387 was detected by antisera to GrV. VA98115 shared 87% aa identity with Desert Storm virus and 65% aa identity with prototype Norwalk virus (NV); rVA98115 reacted weakly with NV antisera. VA97207 shared 80% aa identity with Amsterdam and 75% aa identity with Leeds strains and rVA97207 was not detected by any of the heterologous antibodies. In conclusion, VA97207 and VA98115 may belong to CV antigenic types not previously expressed, while VA98387 is a GrV-like virus. Low levels of cross-reactive antibodies were detected between types. Further studies to characterize these antigens and to develop enzyme immune assays (EIAs) for these strains are in progress.

Human caliciviruses (HuCVs) contain two genera: "Norwalk-like viruses" (NLVs) and "Sapporo-like viruses" (SLVs). The importance of the two genera as a cause of acute gastroenteritis of infants and children remains unknown. Beginning in 1989, a birth cohort of children in Mexico was enrolled and monitored for acute gastroenteritis. A subset of 115 diarrhea stool specimens from 76 children and 66 non-diarrhea stool specimens from 64 children was examined for HuCVs by RT-PCR by using a primer pair (p289/290) that detects both NLVs and SLVs. Twenty-two (19%) of the 115 diarrhea stool specimens and 5 (7%) of 66 non-diarrhea stool specimens produced RT-PCR products of expected size (319 bp for NLVs and 331 bp for SLVs). Twenty of the twenty-seven strains were cloned and sequenced. Pairwise sequence analysis showed that 9 (60%) and 6 (40%) of the 15 strains from the diarrhea stools were NLVs and SLVs, respectively. The same proportions of NLVs (60%) and SLVs (40%) were observed in the non-diarrhea stools. Strains in the NLV genus could be further divided into four clusters: Lordsdale, MxV, and HV and one potentially new cluster. Strains in the SLV genus could be divided into three clusters: Sapporo/82, Lon/92, and a potentially new cluster. Strains from the Lordsdale cluster were the most common among these children. The findings of both genera and multiple clusters of HuCVs co-circulating and the identification of new strains of HuCVs in the population justify the need for future studies of HuCVs in infants and children.

The application of molecular technologies, such as the expression of viral proteins in baculovirus, has provided a powerful approach to the diagnosis of human calicivirus (HuCV) infections. The baculovirus-expressed HuCV capsid protein self-assembles into virus-like particles, providing excellent reagents for immunologic assays, such as enzyme immunoassays (EIAs). Following the expression of the capsid protein of Norwalk virus, the capsid proteins of 8 other HuCV strains have been expressed in baculovirus. The unlimited supply of baculovirus-produced reagents for HuCVs allows these EIAs to be applied in large-scale clinical and epidemiological studies. Both the antigen and antibody-detection EIAs are highly sensitive. The antigen-detection EIAs are highly specific, but the antibody-detection EIAs are more broadly reactive. This article reviews baculovirus expression techniques used to produce HuCV capsid antigens, development of EIAs using these antigens, and application of these EIAs in studies of HuCV infection and illness.

Sapporo-like caliciviruses reveal typical calicivirus morphology and cause acute gastroenteritis. This study describes the expression in baculovirus of capsid proteins of two Sapporo-like calicivirus strains (Hou/86 and Hou/90). Eight different constructs of the capsid genes were compared for production of the proteins. Constructs containing short (9 or 19 nt) upstream sequences failed to produce capsid proteins but extension of the upstream sequence to 73 nt resulted in production of capsid proteins. Expressed capsid protein with the MEG tri-peptide as the N-terminus self-formed virus-like particles (VLPs). Expressed protein with an upstream AUG failed to form VLPs. Addition of His-tag to the N-terminus of capsid protein also blocked VLP formation. Of three Norwalk-Hou/90 chimeric capsid gene constructs, one resulted in production of chimeric capsid and the protein did not form VLPs. Recombinant capsid proteins for each of Hou/86 and Hou/90 were further characterized. The expressed capsid antigens of the two strains were antigenically distinct but shared a common epitope(s). Further study of these proteins should allow development of immunologic assays for diagnosis and should help to clarify the epidemiology of Sapporo-like caliciviruses in humans.

We identified a Norwalk-like calicivirus (CV) whose genome likely was derived from naturally occurring recombination. This strain (Arg320) was detected by the EIA developed against recombinant Mexico virus (rMxV) capsids, but the viral RNA polymerase sequence was closer to Lordsdale virus, in a separate genetic cluster of Norwalk-like viruses. A 3.3 kb cDNA from the RNA polymerase region to the 3' end of the genome of Arg320 was cloned and sequenced. The sequence demonstrated that the capsid region of Arg320 shared 95% amino acid identity with MxV, but 68% identity with Lordsdale virus, while the RNA polymerase region shared 95% identity with Lordsdale virus, but 87% identity with MxV. Pair-wise sequence comparisons identified a potential recombination site at the polymerase/capsid junction. This is the first example of a naturally occurring recombinant in the CV family. Further studies to search for and characterize other strains may be necessary for understanding the genetic diversity of the family.

A primer pair (p289/290) based on the RNA polymerase sequence of 25 prototype and currently circulating strains of human caliciviruses (HuCVs) was designed for the detection of both Norwalk-like caliciviruses (NLVs) and Sapporo-like caliciviruses (SLVs) by reverse transcription-polymerase chain reaction (RT-PCR). This primer pair produces RT-PCR products of 319 bp for NLVs and 331 bp for SLVs. The usefulness of this primer pair was shown by its detection of prototype NLVs (Norwalk, Snow Mountain, Hawaii and Mexico viruses) and SLVs (Sapporo/82, Hou/86, Hou/90 and Lon/92) and currently circulating strains of NLVs and SLVs in children and adults. This primer pair also detected more viruses in either NLV or SLV genera than previously designed primers. This primer pair is useful for broad detection of HuCVs for clinical and epidemiologic studies as well as for environmental monitoring.

The primate calicivirus, Pan-1, was originally isolated from several primate species. It displayed typical calicivirus morphology by electron microscopy. We determined the genomic sequence of Pan-1 by cDNA cloning and direct RNA sequencing. Pan-1 shares a similar genomic organization and a high degree of sequence identity with feline caliciviruses. The Pan-1 genome contains 8,304 nucleotides, plus a poly-A tail, and is longer than any other calicivirus strains with a completely known sequence. The extra sequences of Pan-1 include a unique 424-nucleotide sequence at the 5' end of ORF1, additional amino acids at the N-terminus of the capsid, and a longer 3' UTR.

Three types of composites, reinforced by as-received SiC, artificially oxidized SiC or Al2O3 particles, were fabricated by a compocasting method and extruded at 480 °C with an extrusion ratio of 16:1. Extrusion of these composites causes large scale dynamic recrystallization resulting in a fine matrix microstructure. The extrusion increases not only the ultimate tensile strength, but also the elongation to fracture of the composites. In the first part of our series of three articles, the microstructure of as-cast composites, extruded composites and extruded monolithic 5083 aluminum alloy is studied. The effects of reinforcing particles and other parameters on the dynamic recrystallization behaviour of the composites are discussed.

Caliciviruses (CVs) include at least 42 distinct serotypes. Seventeen CV serotypes have been isolated from marine sources and are called San Miguel sea lion caliciviruses (SMSVs). CVs also have been isolated from reptiles, primates, and other terrestrial animals. Nucleotide sequences from portions of genome of prototype strains for six SMSV serotypes, the reptile CV, Cro-1, the cetacean CV, Tur-1, and the primate CV, Pan-1, are presented. cDNA products of the polymerase (all strains characterized) and capsid (SMSV-17) regions were produced by reverse transcription-polymerase chain reaction using Pan-1 primers. Comparisons of nucleotide and amino acid identity among these and published CV sequences indicated that the nine characterized CVs fall into a phylogenetic group that includes SMSV-1 and SMSV-4 and that is more closely related to other characterized animal CVs than to most human CVs. The phylogenetic analysis also indicated that distinct genera exist among the Caliciviridae. SMSV-17 and SMSV-4 are predicted to be closer to each other than other caliciviruses of known serotype; 574 (82%) of the 704 amino acids in the SMSV-17 and SMSV-4 capsid genes were identical.