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.

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.

Baculovirus expression and antigenic characterization of the capsid proteins of three Norwalk-like viruses.

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.

Surveillance for rotavirus in Argentina.

Group A rotaviruses are the major cause of severe gastroenteritis in young children worldwide. Because rotavirus vaccination appeared imminent, a nationwide surveillance program was organized between October 1996 and October 1998 in the largest Argentine cities. Surveillance for disease burden, rotavirus detection, and rotavirus typing was undertaken at nine locations. Results showed rotavirus to be associated with 42% of diarrhea admissions. Although the prevalent G types changed from year to year, common G types were found in 96% of the cases and were usually associated with common P types. Uncommon G types, G9 and G5, were found at low prevalence and uncommon G/P combinations occurred at almost every study site. These data suggest that a rotavirus vaccine could substantially decrease the rotavirus disease burden in Argentina, but that introduction of a vaccine should be accompanied by a concurrent surveillance system.

Rotavirus-associated medical visits and hospitalizations in South America: a prospective study at three large sentinel hospitals.

BACKGROUND: Knowledge of the impact of rotavirus-associated disease on the health care systems of South America can aid in defining strategies for diagnosis, management and prevention. Up to date information on the impact of rotavirus disease in South America is scarce. AIM: To determine prospectively the impact of rotavirus disease as a cause of medical visits and hospitalizations at three large sentinel pediatric hospitals in Argentina, Chile and Venezuela. METHODS: A 2-year prospective surveillance for rotavirus-associated medical visits and hospitalizations was conducted during 1997 through 1998 at three large sentinel public hospitals, one each in Argentina, Chile and Venezuela. A common surveillance protocol was implemented at the three sites, and a representative number of nonbloody diarrhea stool samples from children <36 months of age were tested for rotavirus by enzyme-linked immunosorbent assay. RESULTS: For our target age group, acute diarrhea-associated medical visits/hospitalizations represented 41%/2%, 5%/6% and 9%/13% of all medical visits/all hospitalizations at the Argentinean, Chilean and Venezuelan sites, respectively (P < 0.001 for difference among the three sites). Rotavirus detection rates among a total of 5,801/1,256 medical visit/hospitalization diarrhea stool samples tested were 39%/71% in Argentina, 34%/47% in Chile and 29%/38% in Venezuela (P < 0.01 by chi square for difference among the three sites). Rotavirus was associated with a mean of 1.5, 1.8 and 3% of total medical visits and 1.6, 2.8 and 5% of hospitalizations among children <36 months of age at the Argentinean, Chilean and Venezuelan sites, respectively. Seasonality was evident for medical visits at all three sites (although less striking in Chile) with peak activity occurring between November and May. Rotavirus-associated hospitalizations had a marked peak in Venezuela, represented largely by short stays, but not in Argentina and Chile. CONCLUSIONS: Rotavirus was a significant cause of medical visits at all three sentinel sites. Rotavirus caused less hospitalizations than previously reported in Argentina and Chile. On the basis of our findings we estimate that approximately 106,000/ 21,000, 48,000/8,000 and 98,000/31,000 rotavirus-associated medical visits/hospitalizations occur yearly in Argentina, Chile and Venezuela, respectively.

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.

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.

Serum antibody as a marker of protection against natural rotavirus infection and disease.

To determine whether naturally acquired serum IgA and IgG antibodies were associated with protection against rotavirus infection and illness, a cohort of 200 Mexican infants was monitored weekly for rotavirus excretion and diarrhea from birth to age 2 years. Serum samples collected during the first week after birth and every 4 months were tested for anti-rotavirus IgA and IgG. Children with an IgA titer >1:800 had a lower risk of rotavirus infection (adjusted relative risk [aRR], 0.21; P<.001) and diarrhea (aRR, 0. 16; P=.01) and were protected completely against moderate-to-severe diarrhea. However, children with an IgG titer >1:6400 were protected against rotavirus infection (aRR, 0.51; P<.001) but not against rotavirus diarrhea. Protective antibody titers were achieved after 2 consecutive symptomatic or asymptomatic rotavirus infections. These findings indicate that serum anti-rotavirus antibody, especially IgA, was a marker of protection against rotavirus infection and moderate-to-severe diarrhea.

Human caliciviruses are a significant pathogen of acute sporadic diarrhea in children of Santiago, Chile.

Human caliciviruses (HuCVs) are increasingly recognized as common pathogens that cause acute sporadic diarrhea in children; however, regional antigenic and genetic diversity complicate detection techniques. Stool samples from children seeking medical attention in 2 outpatient clinics, a large emergency department, and 2 hospital wards were evaluated for HuCVs by reverse transcription-polymerase chain reaction, using primers based on a conserved sequence of the polymerase region of a previously sequenced Chilean strain. HuCVs were detected in 53 (8%) of 684 children 1 month to 5 years of age (mean, 13 months). Detection occurred year-round without a clear seasonal peak, and detection frequency declined from 16% in 1997 to 2% in 1999. The decline may have been due to a change in virus genotype. HuCVs are a significant pathogen of acute sporadic diarrhea in Chilean children, and continuous characterization of genetic diversity will be crucial for appropriate detection.

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.

Concentration and detection of caliciviruses in water samples by reverse transcription-PCR.

Human caliciviruses (HuCVs) cause waterborne outbreaks of gastroenteritis. Standard indicators of a safe water supply do not adequately predict contamination of water by viruses, including HuCVs. We developed a method to concentrate and detect HuCVs in water samples by using a cultivable primate calicivirus (Pan-1) as a model. Viable Pan-1 was seeded in different types of water and then filtered with a 1MDS filter, eluted with beef extract (BE), and reconcentrated by polyethylene glycol (PEG) precipitation. The viruses in the final samples were tested by plaque assay or by reverse transcription (RT)-PCR following extraction of the RNA with Trizol. Pan-1 was more sensitive to high-pH treatment than poliovirus was; a pH 9.0 BE solution was found to recover 35% more viable Pan-1 than a pH 9.5 BE solution recovered. Pan-1 was recovered from small volumes of deionized, finished, ground, and surface waters at efficiencies of 94, 73, 67, and 64%, respectively, when samples were assayed after elution without further concentration. When larger volumes of water (up to 40 liters) were tested after elution and concentration with PEG, 38, 19, and 14% of the seeded Pan-1 were recovered from finished, ground, and surface waters, respectively. The limit of detection of Pan-1 by RT-PCR was estimated to be 0.75 to 1.5 PFU in 40 liters of finished water. This method may be adapted for monitoring HuCVs in drinking water and other types of water for public health safety.

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.

Genomic and antigenic variation among rotavirus strains circulating in a large city of Argentina.

Knowledge of the antigenic diversity of rotaviruses circulating in a region should be acquired before introducing a rotavirus vaccine. In a collection of 151 rotavirus-positive samples from Mendoza, Argentina, strain diversity was evaluated utilizing G-typing monoclonal antibodies (MAbs), reverse-transcriptase-polymerase chain reaction (RT-PCR) G and P typing, and electropherotyping (PAGE). The G type of 137 (91%) specimens was determined. Typing MAb reactivity with the homologous type ranged from 25-94%. For the seven G1 MAbs utilized, 28 patterns of reactivity among 68 G1 strains occurred. For the 48 G2 strains, six patterns of reactivity occurred utilizing three G2-specific MAbs. Of the 92 samples G- and P-typed by reverse-transcriptase-polymerase chain reaction, 89% had single G/P combinations: eight G1[P4], one G1[P6], twelve G1[P8], 58 G2 [P4], and two G2 [P6]. Nine samples had more than one G type with a single P type, one sample had two P types associated with one G type, and one sample contained multiple G and P types. Twenty-nine PAGE patterns occurred for all G types, but differences of antigenic reaction did not predict differences in migration of gene segments 7, 8, and 9. For three specimens showing discordant results between G type by enzyme-linked immunosorbent assay (EIA) and RT-PCR, we observed unexpected electropherotypes. Complementary evaluation by RT-PCR and MAb-based EIA with multiple typing MAbs revealed genetic and antigenic diversity of circulating rotaviruses, including extensive intratypic variation of the G1 and G2 neutralization antigens, in Mendoza during a single season of rotavirus activity.

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.

Diagnosis of human caliciviruses by use of enzyme immunoassays.

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.

Design and evaluation of a primer pair that detects both Norwalk- and Sapporo-like caliciviruses by RT-PCR.

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.

[Detection of Norwalk and Mexico viruses, two human Caliciviruses in stools of Chilean children]. / Detección de virus Norwalk y México, dos calicivirus humanos en deposiciones de niños chilenos.

BACKGROUND: Human calciviruses (HuCVs) cause diarrhea outbreaks associated with consumption of contaminated food and water. Seroepidemiological studies in developing countries, suggest that HuCVs can cause acute gastroenteritis in children. AIM: To study the presence of Norwalk (NV) and Mexico (MX) virus, two HuCVs, in stools of Chilean children from different settings. SUBJECTS AND METHODS: ELISA tests for NV and MX were performed in 677 stool samples for children aged 0 to 132 years old, with acute diarrhea occurring in day care centers or consulting in outpatient clinics or emergency rooms. We also studied eight samples from children involved in a diarrhea outbreak that occurred in a rural community in 1992. A subset of samples was tested with polymerase chain reactions using different primers. RESULTS: Only one sample from a child with acute diarrhea occurring in a day care center was positive for HuCV by polymerase chain reaction. Three samples from the outbreak were positive by the latter method and by ELISA. The HuCV obtained from the day care center was genetically different from other known HuCV. CONCLUSIONS: Despite the high seroprevalence, NV and MX viruses were detected in a very low proportion of Chilean children stools.