Multiplex PCR allows simultaneous detection of pathogens in ships' ballast water.

There is enormous potential for global transfer of microorganisms, including pathogens, in ships' ballast water. We contend that a major advancement in the study of ballast-water microorganisms in particular, and of aquatic pathogens in general, will be expedited sample analysis, such as provided by the elegant technology of DNA microarrays. In order to use DNA microarrays, however, one must establish the appropriate conditions to bind target sequences in samples to multiple probes on the microarrays. We conducted proof-of-concept experiments to optimize simultaneous detection of multiple microorganisms using polymerase chain reaction (PCR) and Southern hybridization. We chose three target organisms, all potentially found in ballast water: a calicivirus, the bacterium Vibrio cholerae, and the photosynthetic protist Aureococcus anophagefferens. Here, we show simultaneous detection of multiple pathogens is possible, a result supporting the promising future use of microarrays for simultaneous detection of pathogens in ballast water.

Isolation of reptilian calicivirus Crotalus type 1 from feral pinnipeds.

Ten virus isolates were obtained from three species of marine mammals sampled on San Miguel Island (California, USA) and 1,200 km north on Rogue Reef (Oregon, USA) during tagging operations in 1986-87. Seven of these 10 were derived from 30 sampled Steller sea lion (Eumetopias jubatus pups, while two of 10 were isolated from one of 19 sampled California sea lion (Zalophus californianus californianus pups, and the remaining isolate was derived from 30 sampled northern fur seal (Callorhinus ursinus) pups. All 10 isolates were identified as belonging to a single serotype, reptilian calicivirus Crotalus type 1 (RCV Cro-1), previously isolated from both healthy and diseased snakes and frogs in a California zoologic collection. The marine samples also showed that nine of 30 Steller sea lion pups, one of 19 California sea lion pups and zero of 30 fur seal pups were producing type specific neutralizing antibodies to RCV Cro-1. This represents the first reported instance of the isolation from marine sources of calicivirus originally isolated from a terrestrial species.

Motional characteristics of K+ and Na+ in intact and sucrose-permeabilized rat lymphocytes.

Most, if not all, cells maintain an unequal distribution of Na+ and K+ against their environment. These two monovalent ions are in constant exchange between the cell and the extracellular space since both ions have proved to be permeable through the cell membrane. The distribution of Na+ and K+ in intact and "sucrose-permeabilized" rat lymphocytes were studied ("sucrose-permeabilization" means homogenization in isotonic sucrose solution). Both the intact and the permeabilized lymphocytes were incubated in Hanks' solution and then transferred into K+, Na(+)-free isotonic sucrose solution. Alternatively, the cells were incubated only in the sucrose solution or in Hanks' solution. The Na+ and K+ content of the cells were determined at the conclusion of each period of incubation in the same or different medium. We found that K+ did not equilibrate under any conditions in intact lymphocytes but Na+ responded to changes of the incubation media. In the permeabilized cells Na+ freely equilibrated with the extracellular medium while K+ did not, although its concentration decreased compared to that of intact cells.

Reclassification of the Caliciviridae into distinct genera and exclusion of hepatitis E virus from the family on the basis of comparative phylogenetic analysis.

Caliciviridae and Picornaviridae belong to the same subphylum and genera within Picornaviridae are well characterized. Until 1998, Caliciviridae included one genus Calicivirus, containing strains with distinct structural and genomic features. Phylogenetic analyses of capsid genes revealed five clusters within Caliciviridae corresponding to differences in genome organization. In order to determine to what taxonomic level these clusters correspond, genomic sequences of caliciviruses, picornavirus prototypes, and two togavirus strains were analyzed. Distance and maximum likelihood methods were used to estimate the phylogenetic relationships among strains. Analysis of the capsid gene revealed separation of five main clusters (Norwalk-like, and Sapporo-like human caliciviruses, hepatitis E virus, vesicular exanthem of swine-like, and lapine caliciviruses) and distances corresponding to those observed among picornavirus genera. Utilizing more conserved (presumed helicase and polymerase) regions for the analyses, only major groups of caliciviruses were separated with confidence, with distances also comparable to those separating picornavirus genera. Analysis in these regions that included togavirus sequences moved HEV strains out of the calicivirus cluster. Our findings support the reclassification of caliciviruses into four genera. The phylogenetic position of hepatitis E virus, by analysis of non-structural genes, is outside of the caliciviruses, in an uncertain taxonomic position.

Quantitative trait locus analysis of fatty acid concentrations in maize.

A study was conducted to determine the number and chromosomal location of quantitative trait loci (QTL) influencing the concentration of five fatty acids in 200 F2S1 lines derived from an Illinois High Oil (IHO) by Illinois Low Oil (Early Maturity) (ILO(EM)) cross. Restriction fragment length polymorphism (RFLP) analysis was performed on the 200 S1 lines and concentrations of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids were determined in self-pollinated kernels harvested from plants grown in replicated field trials during 1992 and 1993. A series of 74 cDNA and genomic clones were used and these revealed 80 polymorphic loci spaced, on average, 24 cM apart throughout the maize genome. Analysis of variance detected significant (p < 0.05) associations between several RFLP loci and the concentration of each fatty acid. A total of 15 RFLP loci clustered in 12 chromosomal regions were associated with the concentration of 16:0, 17 loci clustered in 10 regions were associated with the concentration of 18:0, 12 loci clustered in eight regions were associated with the concentration of 18:1 and 18:2, and 17 loci clustered in eight regions were associated with the concentration of 18:3. Multiple linear regression models consisting of four RFLP loci explained 24 and 62% of the total phenotypic and genotypic variation (R2) among the 200 F2S1 lines for 16:0, five loci explained 51 and 71% of the variation for 18:0, three loci explained 67 and 79% of the variation for 18:1, two loci explained 67 and 81% of the variation for 18:2, and seven loci explained 52 and 78% of the variation for 18:3 in these 200 F2S1 lines. The ratio of 18:1 to 18:2 was tightly interrelated as the same QTL were associated with the concentrations of 18:1 and 18:2. A quantitative trait locus that explained 63% of the phenotypic variation in the ratio of 18:1 to 18:2 is tightly linked to umc65 on chromosome 6 in the region of the linoleic acid1 locus.

Molecular epidemiology of 'Norwalk-like viruses' associated with gastroenteritis outbreaks in New Zealand.

Outbreaks of gastroenteritis are a major public health problem in New Zealand. The introduction of molecular detection methods has now shown that the 'Norwalk-like viruses' (NLVs) are the major cause of food and waterborne nonbacterial gastroenteritis. Reverse transcription and polymerase chain reaction (RT-PCR) were used to determine the presence of NLVs in faecal specimens from 83 nonbacterial gastroenteritis outbreaks occurring in New Zealand between August 1995 and July 1999. Further characterisation of the NLVs for epidemiological purposes was carried out by dot blot DNA hybridisation and DNA sequencing of representative outbreak strains. The majority of NLV strains occurring in New Zealand since August 1995 are similar to those occurring overseas. The predominant New Zealand strain is genetically similar to the Bristol/Lordsdale virus group. Several New Zealand outbreaks were attributed to Auckland virus, a Mexico-like NLV strain identified as the most likely cause of gastroenteritis after consumption of contaminated oysters in 1994. A new strain, designated Napier virus, has been identified in six outbreaks since 1996. A number of strains closely resembling internationally recognised strains, including Southampton virus, Saratoga virus; Desert Shield virus and Melksham virus have been associated with gastroenteritis outbreaks across New Zealand. Application of these typing methods has provided information on disease transmission for epidemiological investigations of public health significance.

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.

Genomic mapping of a calicivirus VPg.

We identified a primate calicivirus (Pan-1) VPg in Pan-1-infected cells. The Pan-1 VPg was associated with both genomic and subgenomic RNAs. RNase digestion of Pan-1 RNA yielded a residual protein of 16 kDa. The N-terminal sequence of Pan-1 VPg was determined by direct amino acid sequencing and mapped to a region of the genome equivalent to picornavirus VPgs. Alignment of this protein sequence with similar regions of other calicivirus genomes allowed identification of conserved amino acid motifs and potential boundaries of the calicivirus VPg genes. Proteinase K treatment abolished the infectivity of Pan-1 RNA, suggesting that Pan-1 VPg is required for RNA infectivity.

Characterisation of a South African human astrovirus as type 8 by antigenic and genetic analyses.

Human astroviruses (HAstV) can, on the basis of immunoassays using type-specific rabbit antisera, be classified into eight serotypes that correlate with genotypes. Very few isolates of HAstV type 8 have been described and there is a paucity of data available with regard to the antigenic and genetic relationships between HAstV type 8 (HAstV-8) and HAstV types 1 (HAstV-1) to 7 (HAstV-7). A wild-type HAstV from a South African paediatric patient with diarrhoea was analysed antigenically, by immune electron microscopy and enzyme immunoassay, and genetically in selected regions of the ORF1a, ORF1b and ORF2 and characterised as a HAstV-8. This HAstV-8 strain exhibited greatest homology with HAstV-4 in the 5' end of the capsid gene and ORF1a and 1b, and greatest homology with HAstV-5 in the 3' end of the capsid region. This study confirms, by both antigenic and genetic analyses, that HAstV-8 represents a distinct antigenic and genotype and is the first report of a HAstV-8 from a hospitalised paediatric patient with diarrhoea in southern Africa.

Phylogenetic analysis of the Caliciviruses.

A phylogenetic portrait of the genus Calicivirus in the family Caliciviridae was developed based upon published sequences and newly characterized calicivirus (CV) strains, including additional Sapporo-like HuCV strains in pediatric diarrhea stool specimens from South Africa, the United Kingdom, and the United States. Distance and parsimony methods were applied to nucleotide and amino acid sequences of human and animal calicivirus 3D RNA-dependent RNA polymerase (approximately 470nt) and capsid hypervariable regions (approximately 1,200nt) to generate phylogenetic trees. Pairwise amino acid identity in the 3D region among the Sapporo-like strains ranged from 61% to 100%. Human and animal caliciviruses (HuCVs and AnCVs) separated into five genogroups: small round-structured viruses (SRSV), Sapporo-like, and hepatitis E virus (HEV)-like HuCVs and rabbit-, and vesicular exanthema of swine virus (VESV)-like AnCVs, each with a distinct genome organization. Each genogroup, including the Sapporo-like HuCVs, subdivided further into subgenogroups. The capsid region trees had higher levels of confidence than the 3D region trees and limited conclusions about genogroups could be drawn from the 3D region analyses. This analysis suggested that CVs include five potential virus subfamilies.

Molecular epidemiology of human astrovirus diarrhea among children from a periurban community of Mexico City.

Human astroviruses (HAstVs) were detected in 23 stool samples from 365 diarrhea episodes among 214 children (<18 months old) prospectively monitored for diarrhea in Mexico City. Stool samples were tested by EIA and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. EIA was less sensitive (74%) and equally specific, compared with RT-PCR analysis using type-common primers for HAstV detection. Of 31 HAstV isolates, EIA typed 18 (69%) of 26 EIA-positive samples, and RT-PCR analysis typed 26 (84%) of 31 RT-PCR-positive samples. Phylogenetic analysis of the 3' end of the capsid region (363 nucleotides) confirmed the type assignment by EIA and RT-PCR analysis and determined the type for 5 previously untyped samples. Six HAstV antigenic types cocirculated in the community: HAstV-2 (42%), HAstV-4 (23%), HAstV-3 (13%), HAstV-1 (10%), HAstV-5 (6%), and HAstV-7 (6%). RT-PCR and sequence analysis provided more detailed epidemiology of HAstV in the community than did antigenic detection methods.

Molecular characterization of a novel recombinant strain of human astrovirus associated with gastroenteritis in children.

We report a naturally occurring human astrovirus (HAstV) strain detected in two different geographic locations. We identified two isolates of this strain in a diarrhea outbreak at a child care center in Houston, Texas; and two isolates in diarrhea stool samples from two children in Mexico City. All four isolates were detected in stool samples by enzyme immunoassay (EIA). One of the Mexican isolates was typed by EIA and all four isolates were HAstV-5 by typing RT-PCR. The four isolates were >97% nucleotide-identical in two different genomic regions: ORF1a (246 nt), and the 3' end of the genome (471 nt). One isolate from each geographic location was further sequenced in the transition region from ORF1b to ORF2 (1255 nt) and this region of the two isolates showed > or = 99% nt identity. Phylogenetic analyses of sequences of eight HAstV antigenic types and the novel strain in the transition region demonstrated the new strain being closely related to HAstV-3 in ORF1b, but closest to HAstV-5 in ORF2. These results and high sequence identity among all HAstV antigenic types in the transition region and RNA structural predictions supported a potential recombination site at the ORF1b/ORF2 junction. This is the first evidence that recombination occurs among human astroviruses.

Prevalence and genetic diversity of human caliciviruses (HuCVs) in Mexican children.

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.

Partial characterization of the genome of nine animal caliciviruses.

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.

Sapporo-like human caliciviruses are genetically and antigenically diverse.

The Sapporo-like human caliciviruses (HuCVs) comprise one of three genogroups of HuCVs associated with acute gastroenteritis. Phylogenetic analysis has shown that Sapporo-like HuCVs are related more closely to animal caliciviruses than to other known HuCVs. We produced 3.2 kb cDNA fragments from the 3' end to three Sapporo-like HuCVs that were associated with acute gastroenteritis in children (Houston/86, Houston/90, and London/92). Sequence analysis of the 3.2 kb cDNAs showed that two of the three viruses had a genomic organization similar to that of other Sapporo-like strains and the third strain (London/92) lacked an open reading frame overlapping the 5' end of the capsid gene. Alignment of the capsid sequences of these three strains showed 44-78% amino acid identity among the three strains. Phylogenetic analysis of the aligned sequences indicated the three strains are related but each belongs to a distinct genetic cluster. The genetic differences are associated with antigenic differences in that an enzyme immune assay (EIA) specific for the prototype Sapporo/82 strain detected the Houston/86 strain, but not the Houston/90 and London/92 strains. In vitro transcription and translation of viral cDNA containing the predicted capsid gene of Houston/90 resulted in a protein of 63 K, which is immunoprecipitated by sera from children infected with the strain. Genetically and antigenically distinct strains in the Sapporo-like HuCVs have not been described previously and the occurrence of such diverse strains in the same community likely increases the importance of these strains as a cause of illness in children.

In vitro isolation and characterization of a calicivirus causing a vesicular disease of the hands and feet.

We report that a calicivirus of oceanic origin, San Miguel sea lion virus serotype 5 (SMSV-5), is a human pathogen. This biotype was isolated originally from blisters on the flippers of northern fur seals (Callorhinus ursinus) and replicates readily in primate and human cell lines. It infects a phylogenetically diverse array of hosts (poikilotherms to primates) and induces type-specific neutralizing antibodies in exposed humans. Group antibody against a pooled antigen of SMSV-5 and two other serotypes was also observed in 18% of 300 blood donors from a population in the northwestern United States. The human calicivirus isolate designated SMSV-5 Homosapien-1 (SMSV-5 Hom-1) was recovered from a laboratory worker with systemic illness, including vesicular lesions on all four extremities. We believe this newly described human disease represents a paradigmatic shift in calicivirus disease recognition.

Molecular epidemiology of childhood astrovirus infection in child care centers.

This study assessed the role of human astrovirus (HAstV) in outbreaks and sporadic cases of diarrhea among children attending child care centers (CCCs) and determined the infecting astrovirus antigenic types by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis. Eight astrovirus outbreaks occurred in 6 CCCs. Of 179 children with diarrhea, 36 (20%) had astrovirus-associated diarrhea. Diarrhea stools obtained during diarrhea outbreaks were more likely to contain astrovirus (40/476) than were samples not associated with a diarrhea outbreak (14/452) (P<.001). Type-specific RT-PCR and DNA sequencing identified 5 outbreaks associated with HAstV-1 and 3 outbreaks with HAstV-2. Sequential outbreaks in 2 CCCs occurred with a different type in the same year. Phylogenetic analysis identified 6 clades of HAstV-1 and 2 clades of HAstV-2 during this 1-year surveillance. Astrovirus was a significant cause of diarrhea outbreaks, and 2 antigenic types were present in the community during 1 diarrhea season.

Taxonomy of the caliciviruses.

The International Committee on Taxonomy of Viruses (ICTV) has recently approved several proposals submitted by the present Caliciviridae Study Group. These proposals include the division of the family into 4 new genera designated Lagovirus, Vesivirus, "Norwalk-like viruses (NLVs), and "Sapporo-like viruses (SLVs); the latter 2 genera were assigned temporary names until acceptable names can be determined by the scientific community. The genera have been further divided into the following species: Feline calicivirus and Vesicular exanthema of swine virus (genus Vesivirus), Rabbit hemorrhagic disease virus and European brown hare syndrome virus (genus Lagovirus), Norwalk virus (genus NLV), and Sapporo virus (genus SLV). In addition, the ICTV approved a proposal to remove the hepatitis E virus from the Caliciviridae into an "unassigned classification status.