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Neuro-Oncology  2014;16(Suppl 2):ii95.
OBJECTIVE: Data from mouse models suggest that high-grade gliomas (HG-gliomas) origin from somatic mutant neural stem and precursor cells (NPCs). We found that endogenous NPCs also defend the brain against HG-gliomas. Here, we describe the signal transduction mechanism of NPC-mediated tumor suppression and present data on the tropism of endogenous NPCs to human HG-gliomas. METHODS: For our experiments, we used transgenic mouse models, human HG-glioma biopsies, molecular biology techniques (microarrays, real-time PCR, mass-spectrometry, array-CGH, fluorescence in situ hybridization), cell-culture (primary NPC- and glioma cultures of human and murine origin), immunohistochemistry and survival studies in orthotopic mouse tumour models. RESULTS: NPCs migrate from the subventricular zone to HG-gliomas, reduce glioma expansion and prolong survival by releasing a group of fatty acid ethanolamides that have agonistic activity on the vanilloid receptor (transient receptor potential vanilloid subfamily member-1; TRPV1). TRPV1 expression is much higher in HG-gliomas than in tumor-free brain and TRPV1 stimulation triggers tumor cell death. NPC-mediated tumor suppression can be mimicked in the adult brain by systemic administration of the synthetic vanilloids suggesting that TRPV1 agonists hold potential as new HGastrocytoma therapeutics. Furthermore, we analyzed the tumor-parenchyma interface of neurosurgical resections for the presence of NPCs and distinguished these physiological cells from the tumor mass. We observed that PSA-NCAM-positive NPCs, which are genetically distinct from the the tumor mass, accumulate at the border of high-grade gliomas and display a marker profile consistent with immature migratory neuronal progenitor cells. CONCLUSION: Overall, our data reveal a mechanism for NPC attraction CNS tumors and suggest that NPCs accumulate in human high-grade astrocytomas. In mouse models NPCs suppress HG-gliomas and the endogenous anti-tumorigenic factors can be used as pre-clinical HG-glioma therapeutics.
PMCID: PMC4185733
Neuro-Oncology  2014;16(Suppl 2):ii108.
OBJECTIVE: Data from transgenic mouse models show that neuronal progenitor cells (NPCs) can migrate towards experimental brain tumors and modulate the course of pathology. However, the pathways attracting NPCs to CNS neoplasms were unknown and it was unexplored if NPCs migrate towards metastatic brain tumors in humans. METHODS: We analyzed the tumor-parenchyma interface of neurosurgical resections for the presence of (NPCs) and distinguished these physiological cells from the tumor mass ( by fluorescence in situ hybridization, FISH). Also, we performed experiments with transgenic mouse models, cell-culture (primary NPC- and glioma cultures of human and murine origin), cell migration assays and immunohistochemistry. RESULTS: We observed that PSA-NCAM-positive NPCs accumulate at CNS metastases. These metastatic tumors are distinguished from neural cells by defined sets of markers. Transplanting murine glioma cells embedded in a cell impermeable hollow fiber capsule into the brains of nestin-gfp reporter mice shows that diffusible factors are sufficient to induce a neurogenic reaction. In vitro, vascular endothelial growth factor (VEGF) secreted from glioma cells increases the migratory and proliferative behavior of adult human brain derived neural stem and progenitor cells via stimulation of VEGF receptor-2 (VEGFR-2). In vivo, inhibiting VEGFR-2 signaling with a function-blocking antibody leads to a reduction in NPC migration in response to tumors. CONCLUSION: Overall, our data reveal a mechanism for NPC attraction to CNS tumors and suggest that NPCs accumulate in human high-grade astrocytomas.
PMCID: PMC4185790
3.  Emergence of G12 Rotavirus Strains in Delhi, India, in 2000 to 2007▿  
Journal of Clinical Microbiology  2008;46(4):1343-1348.
The prospect that rotavirus diarrhea in children may soon be prevented by vaccines has placed a new priority on understanding the diversity of rotavirus strains and the mechanism by which these strains evolve over time. We have characterized a total of 465 rotavirus strains collected in North India from 2000 to 2007 for G and P types by reverse transcription-PCR and sequencing. The novel G12 rotavirus strains recently detected in other countries were first detected in India in 2001 and have emerged as the predominant strains in Delhi, India, during 2005 to 2007. While the VP7 sequence was highly homologous among G12 strains isolated in Delhi, suggesting recent emergence from a common ancestor, the strains had a diverse constellation of other gene segments, demonstrating substantial reassortment. For the entire period, the common rotavirus G types G1 (26%), G2 (25%), and G9 (14%) comprised 65% of the strains, and common P types, P[4] (19%), P[6] (22%), and P[8] (35%), comprised 76% of the total P types. Of note, we detected a high percentage of unusual (17%) strains and fecal specimens with mixed (12% G and 15% P) rotavirus infections having a variety of genomic constellations. For the first time, we identified two novel rotavirus strains with unusual G/P combinations, G2P[11] and G3P[11], in patients with diarrhea. The study highlights the great diversity among rotaviruses isolated from Indian children, the opportunity for genetic reassortment between strains, and the emergence of a novel G12 strain in our country. Due to the demonstrated effect of antigenic diversity on rotavirus vaccines, it will be important to continue careful monitoring of these strains as rotavirus vaccine programs are implemented in India.
PMCID: PMC2292948  PMID: 18272705
4.  First Detection of G12 Rotaviruses in Newborns with Neonatal Rotavirus Infection at All India Institute of Medical Sciences, New Delhi, India▿  
Journal of Clinical Microbiology  2007;45(11):3824-3827.
Rotavirus genotype G12 strains were detected for the first time among newborns with asymptomatic rotavirus infection (74% of 39 rotavirus strains isolated from the infected infants were genotype G12) in the nursery of the All India Institute of Medical Sciences during a period from 2005 to 2006. Sequence analysis of the VP7 genes from these neonatal strains indicated a high level of homology to other G12 strains reported worldwide, suggesting the recent emergence of these strains in humans. Such nosocomial infections of newborns represent a potential source of introduction of novel rotavirus serotypes into the community.
PMCID: PMC2168534  PMID: 17728476
5.  Molecular Epidemiology of Group A Rotavirus Diarrhea among Children in Buenos Aires, Argentina, from 1999 to 2003 and Emergence of the Infrequent Genotype G12 
Journal of Clinical Microbiology  2006;44(6):2046-2050.
To examine the epidemiology of rotaviruses in Buenos Aires, Argentina, we screened 1,212 stool samples from children with diarrhea in the southern district of Buenos Aires from 1999 to 2003. We identified 187 samples (15.4%) that were positive for group A rotavirus by use of antigen enzyme-linked immunosorbent assay. Among these specimens, 112 were available for typing: 93 (83.0%) were single-type infections, 9 (8.0%) were mixed-type infections with more than one G or P type, and 10 (8.9%) were G and/or P nontypeable. In contrast to the findings in our last study, from 1996 to 1998, genotype P[4], G2 strains were almost completely absent and P[8], G1 and P[8], G4 strains were dominant, representing more than 80% of the G and P types found. Genotypes G2 and G9 were detected in few samples, and type G3 was completely absent. We identified several uncommon genotype G12 strains, representing the first detections outside of Asia and the United States, by sequencing. Using a genotype G12-specific reverse transcription-PCR, we identified eight (6.7%) positive samples for the 1999 to 2003 period. The high degree of sequence identity between recent G12 isolates from Argentina, the United States, and Asian countries suggests a relatively recent introduction(s) of these strains into humans from a common progenitor. The Argentinean G12 strains belonged to genotype P[9], similar to most of the recently described Asian G12 strains. The finding of G12 strains in several other regions of the world raises the possibility that G12 may be emerging globally and suggests that surveillance for this strain should be conducted routinely.
PMCID: PMC1489448  PMID: 16757596
6.  Serum Antibody Responses in Children with Rotavirus Diarrhea Can Serve as Proxy for Protection 
We examined sera from 42 patients 1 to 30 months of age for rotavirus immunoglobulin M (IgM), IgA, IgG, and IgG subclasses and sought to determine if serum antibody could serve as a reliable marker for prediction of disease severity. Infants in the first few months of life usually had high maternal IgG titers and, when they were infected with rotavirus, had low IgM titers or no IgM in acute-phase sera and poor seroconversions 3 weeks later, suggesting that maternal antibodies had inhibited viral replication and antibody responses. All patients ≥6 months of age had IgM in acute-phase sera, indicating that IgM is a good marker for acute rotavirus infection. IgG was the best overall predictor of an infection, as the convalescent-phase sera of 81% of the patients had a fourfold rise in the IgG titer. IgA titers in convalescent-phase sera and conversion rates were higher among patients ≥12 months of age than among children younger than 12 months. IgG1 was the predominant subclass detected in the acute-phase sera of some children and in all 28 convalescent-phase serum samples examined. Patients with preexisting acute-phase IgG titers of ≥100 or ≥200 had diarrhea that was less severe or of a shorter duration. These results indicate that serum IgG is the most reliable marker for seroconversion and is a consistent proxy for protection against severe disease.
PMCID: PMC549315  PMID: 15699422
7.  Duration of symptoms and spread of colorectal cancer: a short history does not mean early disease. 
The 5-year survival rates for colorectal cancer are generally lower in the UK than other European countries. In an attempt to improve prognosis, central government has stipulated that patients with suspicious symptoms ought to be seen within 2 weeks of referral from a primary care physician. In order to evaluate whether symptom duration affects stage at presentation of colorectal cancer, a retrospective analysis of all patients presenting over a 2-year period to a large district general hospital was performed. There was no significant difference (P = 0.885) in Dukes' staging in patients with symptoms lasting less or more than 6 months. Though seeing patients with symptoms suspicious of colorectal cancer in specialist out-patient clinics within 2 weeks of presentation to the primary care physician would probably reduce the number of patients presenting as an emergency, it is unlikely to improve prognosis. Thus funds diverted towards the 2-week wait are probably best utilised for other procedures such as colonoscopy and for improving care once the diagnosis of cancer has been made. Diagnosis of colorectal cancer at an earlier stage is best achieved by screening of the population.
PMCID: PMC2504185  PMID: 12484575
8.  Cytokines as Mediators for or Effectors against Rotavirus Disease in Children 
Rotavirus is the most common cause of severe gastroenteritis in young children, but the pathogenesis and immunity of this disease are not completely understood. To examine the host response to acute infection, we collected paired serum specimens from 30 children with rotavirus diarrhea and measured the levels of nine cytokines (interleukin-1β [IL-1β], IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, gamma interferon [IFN-γ], and tumor necrosis factor alpha [TNF-α]) using a microsphere-based Luminex Flowmetrix system. Patients with acute rotavirus infection had elevated median levels of seven cytokines in serum, and of these, the levels of three (IL-6, IL-10, and IFN-γ) were significantly (P < 0.05) higher than those in serum from control children without diarrhea. Patients with fever had significantly (P < 0.05) higher levels of IL-6 in serum than control children, and those with fever and more episodes of diarrhea had significantly (P < 0.05) higher levels of TNF-α than those without fever and with fewer episodes of diarrhea. We further demonstrated a negative association (P < 0.05) between the levels of IL-2 and the number of stools on the day on which the first blood sample was collected. Finally, patients with vomiting had significantly (P < 0.05) lower levels of IFN-γ than those without vomiting. Our pilot study provides evidence that the types and magnitudes of cytokine responses to rotavirus infection in children influence or reflect the clinical outcome of disease. These findings suggest that certain cytokines may play an important role in the pathogenesis of and the protection against rotavirus disease in children and, consequently, may provide directions and insights that could prove critical to the prevention or treatment of this important disease.
PMCID: PMC262432  PMID: 14607858
9.  Unexpected Detection of Animal VP7 Genes among Common Rotavirus Strains Isolated from Children in Mexico 
Journal of Clinical Microbiology  2003;41(9):4400-4403.
In the course of characterizing 103 rotaviruses from children in Mexico, we found that the majority of strains were globally common types (55.4% of total), while uncommon types represented 5.7%, mixed infections with common types represented 14.8%, and partially or fully nontypeable isolates represented about 24%. Serotype G9 was detected for the first time in Mexico. We sequenced a subset of strains that were G nontypeable by reverse transcriptase PCR and found surprisingly that two strains having common human rotavirus P genotypes (8 and 6) had serotype G3 and G4 VP7 gene sequences that shared closer homology with canine and porcine strains, respectively, than with human strains, suggesting that these isolates represented reassortants between human and animal rotaviruses.
PMCID: PMC193830  PMID: 12958276
10.  Characterization of Serotype G9 Rotavirus Strains Isolated in the United States and India from 1993 to 2001 
Journal of Clinical Microbiology  2003;41(7):3100-3111.
The emergence of rotavirus serotype G9 as a possible fifth globally common serotype in the last decade, together with its increasing detection in association with various genome constellations, raises questions about the origins and epidemiological importance of recent G9 isolates. We examined a collection of 40 G9 strains isolated in the United States from 1996 to 2001 and in India since 1993 to determine their VP7 gene sequences, P types, E types, subgroup specificities, and RNA-RNA hybridization profiles. With the exception of two U.S. strains, all of the study strains shared high VP7 gene sequence homology (<2.5% sequence divergence on both the nucleotide and amino acid levels) and were more closely related to other recent isolates than to the first G9 strains isolated in the 1980s. The VP7 gene sequence and RNA-RNA hybridization profiles of the long-E-type strains showed greater variation than the short-E-type strains, suggesting that the latter strains are the result of a relatively recent reassortment event of the G9 VP7 gene into a short-E-type lineage. No evidence for reassortment of genes other than VP4 and VP7 between major human rotavirus genogroups was observed. Except for Om46 and Om67, which formed a distinct clade, phylogenetic analysis showed that most of the study strains grouped together, with some subgroups forming according to genetic constellation, geographic location, and date of isolation. The high potential of G9 strains to generate different P and G serotype combinations through reassortment suggests that it will be important to determine if current vaccines provide heterotypic protection against these strains and underscores the need for continued surveillance for G9 and other unusual or emerging rotavirus strains.
PMCID: PMC165321  PMID: 12843049
11.  What is the lag time between income inequality and health status? 
PMCID: PMC1731662  PMID: 10827916
12.  Where children sit in motor vehicles: a comparison of selected European and American cities 
Injury Prevention  1998;4(2):98-102.
Objectives—To ascertain whether there are differences in child seating location between selected cities in the US and continental Europe, and if differences exist, to ascertain what factors predict them.
Setting—Boston and New Orleans, which have no laws regarding child seating location, and Paris, Frankfurt, and Brussels, which for approximately 20 years have had laws requiring children under the ages of 10 or 12 to sit in the rear.
Methods—Observations were made in the first quarter of 1997 at several locations in or near each city. The vehicle seating capacity, total number of occupants, the seating location of adults and children, and driver shoulder belt use were recorded for each vehicle with at least one child. The predictors of a vehicle having a child in the front were estimated using logistic regression.
Results—Data on 5501 children riding in 3778 vehicles were collected. Adjusting for differences in vehicle seating capacity, occupant mix, and driver shoulder belt use, vehicles in the European cities are significantly less likely to have a child in the front seat than vehicles in the American cities.
Conclusions—Cities with no history of laws prohibiting children from sitting in the front, vehicles with low seating capacity, vehicles with no adult (other than the driver) or many child passengers, and unbelted drivers were associated with a higher likelihood of children riding in the front seat. It is feasible for a society to insist, through custom and/or law, that children sit in the back seat.
PMCID: PMC1730365  PMID: 9666361
13.  Surveillance of Rotavirus Strains in the United States: Identification of Unusual Strains 
Journal of Clinical Microbiology  2000;38(7):2784-2787.
Rotavirus strains from 964 fecal specimens collected from children at 11 U.S. hospital laboratories from November 1997 to March 1998 and from samples collected at 12 laboratories from November 1998 to March 1999 were typed for G and P proteins. Serotype G1 was the predominant serotype in 1997–1998 (88%), followed by G2 (6.2%), G9 (3.3%), and G3 (1.5%). This pattern was similar to that seen in 1998–1999: G1 (79%), G2 (15%), G9 (3.0%), G4 (1.6%), and G3 (0.3%). Novel P[9] strains were identified in both seasons, and analysis of a 364-nucleotide fragment from gene segment 4 of one of the strains demonstrated 97.3% nucleotide identity with the prototype P3[9],G3 strain, AU1, isolated in Japan. This is the first report of a human AU1-like strain in the United States. These results reinforce our initial findings that serotype G9 persists in the United States but has not become a predominant strain and that the common serotypes G1 to G4 account for almost 90% of strains in circulation. Other uncommon strains exist in the United States but may have been overlooked before because of their low prevalence and the use of inadequate diagnostic tools.
PMCID: PMC87033  PMID: 10878089
15.  Detection and Characterization of Novel Rotavirus Strains in the United States 
Journal of Clinical Microbiology  1998;36(11):3223-3229.
We recently established a rotavirus strain surveillance system in the United States to monitor the prevalent G serotypes before and after the anticipated implementation of a vaccination program against rotavirus and to identify the emergence of uncommon strains. In this study, we examined 348 rotavirus strains obtained in 1996 to 1997 from children with diarrhea in 10 U.S. cities. Strains were characterized for P and G types, subgroups, and electropherotypes by using a combination of monoclonal antibody immunoassay, reverse transcription-PCR, and hybridization. The four strains most commonly found worldwide comprised 83% of the isolates (P[8]G1, 66.4%; P[4]G2, 8.3%; P[8]G3, 6.9%; P[8]G4, 1.4%), but 9.2% were unusual strains (P[6]G9, 5.5%; P[8]G9, 1.7%; P[6]G1, 1.4%; and P[4]G1 and P[8]G2, 0.3% each). Strains not typeable for P or G type accounted for 5.5% of the total, while 2.3% of the strains had more than one G type (mixed infections). All P[6]G9 strains tested had short electropherotypes and subgroup I specificity and were detected in 4 of 10 cities, while P[8]G9 strains had long electropherotypes and subgroup II VP6 antigens. Both sequence analysis of the VP7 open reading frame (about 94 to 95% amino acid identity with the VP7 gene of G9 prototype strain WI61) and binding to a G9-specific monoclonal antibody strongly suggest that U.S. G9 strains belong to serotype G9. The high detection rates of unusual rotaviruses with G9 (7.2%) or P[6] (6.9%) specificity in multiple U.S. cities suggest the emergence of new strains or inadequate diagnosis in the past. The epidemiologic importance of these strains remains to be determined.
PMCID: PMC105305  PMID: 9774569
16.  Rotavirus. 
Emerging Infectious Diseases  1998;4(4):561-570.
Rotavirus, the most common diarrheal pathogen in children worldwide, causes approximately one third of diarrhea-associated hospitalizations and 800,000 deaths per year. Because natural infection reduces the incidence and severity of subsequent episodes, rotavirus diarrhea might be controlled through vaccination. Serotypespecific immunity may play a role in protection from disease. Tetravalent rhesus-human reassortant rotavirus vaccine (RRV-TV) (which contains a rhesus rotavirus with serotype G3 specificity and reassortant rhesus-human rotaviruses with G1, G2, and G4 specificity) provides coverage against the four common serotypes of human rotavirus. In clinical trials in industrialized countries, RRV-TV conferred 49% to 68% protection against any rotavirus diarrhea and 61% to 100% protection against severe disease. This vaccine was licensed by the U.S. Food and Drug Administration on August 31, 1998, and should be cost-effective in reducing diarrheal diseases in industrialized countries. The vaccine's efficacy and cost-effectiveness in developing countries should be evaluated.
PMCID: PMC2640254  PMID: 9866732
17.  Serodiagnosis of human and animal pythiosis using an enzyme-linked immunosorbent assay. 
Conventional serodiagnosis of Pythium insidiosum infections involves the use of the immunodiffusion (ID) test. This test specifically diagnoses human and animal pythiosis. The test, however, has limited sensitivity and does not detect some culturally proven cases of the disease. Because of the increased recognition of pythiosis among humans and animals, we developed and evaluated an enzyme-linked immunosorbent assay (ELISA) using a soluble antigen from broken hyphae of P. insidiosum. Studies were carried out with sera from five humans and eight animals with culturally and/or histologically proven pythiosis. Some of these sera were negative in the ID test for pythiosis. Heterologous case sera from thirteen humans and two horses, plus 5 sera from healthy humans and 17 from healthy animals, were tested. Of the pythiosis case sera tested, the ID test detected only 8 of 13 (61.5%), whereas the ELISA detected all of them (100%). The ID and ELISA tests were entirely specific and gave negative results or low titers respectively, with sera from humans and animals with heterologous fungal infections or with no apparent illness. No correlation was found between the height of the ELISA titers and negative or positive sera in the ID test. Our results indicate that the ELISA is a reliable serodiagnostic test for pythiosis. It is as specific as the ID test but more sensitive.
PMCID: PMC170646  PMID: 9384295
18.  A one-tube method of reverse transcription-PCR to efficiently amplify a 3-kilobase region from the RNA polymerase gene to the poly(A) tail of small round-structured viruses (Norwalk-like viruses). 
Journal of Clinical Microbiology  1997;35(3):570-577.
Amplification of a 3-kb genome region from the RNA polymerase gene to the 3' poly(A) tail of small round-structured virus (SRSV) by reverse transcription-PCR (RT-PCR) has been difficult to achieve because of a stable secondary structure in a region between the RNA polymerase gene and the 5' end of the second open reading frame. We have developed a one-tube RT-PCR method to efficiently amplify this region. The method comprises three procedures: purification of poly(A)+ RNA from a starting RNA solution by oligo(dT)30 covalently linked to latex particles, buffer exchange, and continuous RT and PCR in a single tube containing all reaction components. The key elements of this method are (i) first-strand cDNA synthesis with the Superscript II version of RNase H- Moloney murine leukemia virus reverse transcriptase at 50 degrees C for 10 min by using the RNA-oligo(dT)30 hybrid on the latex particles as the template and primer, and (ii) PCR by Taq and Pwo DNA polymerases mixed together with a mixture of 12 phased oligo(dT)25 antisense primers. The detection threshold of the one-tube RT-PCR method was as little as 0.2 ng of the crude RNA used as the source of the template. Using this method, we obtained 3-kb products from 24 SRSV strains previously characterized into four genetic groups. These included 5 P1-A, 4 P1-B, 5 P2-A, and 10 P2-B strains. Because SRSVs have not yet been cultivated in vitro, this novel method should facilitate molecular characterization of SRSVs to provide a firm scientific foundation for improvements and refinements of SRSV diagnostics.
PMCID: PMC229629  PMID: 9041391
19.  Unusual diversity of human rotavirus G and P genotypes in India. 
Journal of Clinical Microbiology  1996;34(2):436-439.
Between April and December 1993, we determined P and G genotypes of group A rotavirus strains obtained from children admitted to diarrhea treatment centers in five Indian cities. From a total of 63 rotavirus-positive specimens, we identified 10 different strains with five different G genotypes and four distinct P types by using reverse transcription-PCR. The common worldwide strains G1P8, G2P4, G3P8, and G4P8 were underrepresented among Indian children (33%), whereas strains of P type 6 (G1P6, G2P6, G3P6, G4P6, and G9P6), which primarily infect asymptomatic newborns but are rare in children with diarrhea were common in India (43%). Of these, G9P6, a strain not previously reported to be found in children with diarrhea, was the most prevalent (22%). Eleven percent of the strains were nontypeable, and another 11% of the specimens had mixed infections. Using digoxigenin-labeled, genotype-specific hybridization probes, we confirmed all G9 strains and mixed infections tested and identified three nontypeable strains (one G9 and two P8). The epidemiological significance of G9 rotavirus strains, if confirmed in other settings, may have important implications for vaccine development.
PMCID: PMC228815  PMID: 8789033
20.  Typing of human astroviruses from clinical isolates by enzyme immunoassay and nucleotide sequencing. 
Journal of Clinical Microbiology  1995;33(4):797-801.
A typing enzyme immunoassay (TYPE-EIA) was used to determine the antigenic types of 64 astrovirus-positive specimens from nine collections from seven countries. Six of the seven known astrovirus types were detected in the collections, with HAstV-1 predominating in all collections for one from the United Kingdom. Selected specimens were analyzed further by reverse transcriptase PCR and nucleotide sequencing of 348 bp within the capsid protein precursor region of the genome. The phylogenetic groupings (genotypes) determined from the sequences were entirely consistent with the antigenic groupings (serotypes) of isolates obtained by using the TYPE-EIA. The genetic variation within genotypes was small compared with the variation between genotypes, allowing unambiguous categorization of all specimens. Although some strains from widely separated geographic areas had identical sequences, in general, within a region most strains of the same type were identical. The TYPE-EIA may help further our understanding of the epidemiology of astrovirus and the possible role of serotype-specific immunity, while further knowledge of sequences could facilitate the development of simpler molecular methods of typing astrovirus strains.
PMCID: PMC228043  PMID: 7790440
21.  Use of solid-phase immune electron microscopy for classification of Norwalk-like viruses into six antigenic groups from 10 outbreaks of gastroenteritis in the United States. 
Journal of Clinical Microbiology  1995;33(2):501-504.
Norwalk-like viruses observed in fecal specimens from 10 outbreaks of gastroenteritis investigated in the United States between 1987 and 1992 were analyzed by solid-phase immune electron microscopy. Outbreak virus strains were classified into six antigenic groups: the four types (UK1 to UK4) previously defined in the United Kingdom, Norwalk virus, and the Oklahoma agent that was newly defined in this study. The diversity of antigenic types demonstrated in these outbreaks was greater than previously recognized and will serve as a basis for characterization of these strains at the molecular level.
PMCID: PMC227978  PMID: 7714218
22.  Detection and differentiation of antigenically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-PCR and southern hybridization. 
Application of reverse transcription (RT)-PCR to detect small round-structured viruses (SRSVs) from fecal specimens of patients with gastroenteritis has been insensitive because of the tremendous sequence heterogeneity between strains. We have designed two RT-PCR primer sets (G-1 and G-2) based on the nucleotide sequence diversity in the RNA polymerase gene of SRSVs belonging to two distinct genogroups represented by Norwalk virus (primers G-1) and Snow Mountain agent (primers G-2). All 22 SRSV strains examined that had been classified previously by solid-phase immune electron microscopy into four antigenic types (UK1, UK2, UK3, and UK4) could be detected by RT-PCR with these two primer sets. The G-1 primer set detected 6 UK2 strains, and the G-2 primers detected 16 strains, including 7 UK1, 5 UK3, and 4 UK4 strains. On the basis of nucleotide sequences of 81-bp fragments of the RT-PCR products from 13 strains determined in this study, together with those previously reported for 17 SRSV strains, we designed four sets of internal oligonucleotide probes (P1-A, P1-B, P2-A, and P2-B) for Southern hybridization, using chemiluminescent detection. The P1-A probe hybridized with PCR products from the UK2 strains; the P1-B probe, with products from two of the seven UK1 strains; the P2-A probe, with four of the remaining five UK1 strains; and the P2-B probe, with products from both UK3 and UK4 strains, as well as with one strain originally typed as UK1 which showed cross-reactivity with UK4 upon retesting by solid-phase immune electron microscopy. RT-PCR with both the G-1 and the G-2 primer sets can increase the detection rate of the many antigenically distinct SRSVs and, when combined with Southern hybridization, may predict the antigenic type of the SRSV associated with infection.
PMCID: PMC227881  PMID: 7699068
23.  Characterization of rotavirus strains from newborns in New Delhi, India. 
Journal of Clinical Microbiology  1994;32(7):1820-1822.
Between 1986 and 1993, 72% of rotavirus strains isolated from newborns at five hospitals in New Delhi, India, had long electropherotypes, subgroup II VP6 antigens, and G and P genotypes (G9P11) identical to those of prototype strain 116E. A novel strain with a G9P6 genotype, representing 13% of the isolates, was identified. These results demonstrate that G9P11 and G9P6 rotavirus strains are common in nurseries in New Delhi.
PMCID: PMC263808  PMID: 7929782
24.  Serum antibody response to recombinant major inner capsid protein following human infection with group B rotavirus. 
Journal of Clinical Microbiology  1994;32(6):1599-1603.
Recombinant major inner capsid protein (VP6) of the IDIR strain of group B rotavirus (GBR) was incorporated in a solid-phase immunoassay to access antibody response to infection in humans. Expression of VP6 in insect cells permitted design of a highly sensitive assay that avoided the contaminants present in GBR antigens obtained from fecal specimens. Among patients infected with the ADRV strain of GBR in China, increased reactivity with recombinant VP6 was observed in convalescent-phase sera in comparison with sera obtained shortly after infection (P = 0.0084). Anti-VP6 antibodies were detectable as soon as 7 days after onset of gastrointestinal symptoms, and serum reactivity persisted in specimens drawn more than 1 year after infection. Solid-phase immunoassay with recombinant VP6 was next employed in order to assess anti-GBR antibody in 513 serum specimens obtained from 423 Maryland residents (ages, 7 months to 96 years; median age, 42 years). Four individuals (< 1%) exhibited serum antibodies directed against the recombinant VP6 (ages, 54 to 95 years; mean age, 77 years). Examination of 129 additional serum specimens including some from other geographic regions of the United States failed to reveal the presence of anti-GBR antibody. Anti-GBR antibody was also not detected in any of 131 serum specimens from 60 staff and residents of a nursing home in Switzerland. While infection of humans with GBR has been uncommon in these locations outside of China, the detection of serum antibodies in older individuals in the United States either indicated an unknown, age-related risk factor or may have indicated infection in the more distant past. The availability of these reagents should allow surveys for GBR infection among additional populations that have not previously been investigated.
PMCID: PMC264049  PMID: 8077413
25.  Characterization of a variant strain of Norwalk virus from a food-borne outbreak of gastroenteritis on a cruise ship in Hawaii. 
Journal of Clinical Microbiology  1994;32(4):861-866.
A gastroenteritis outbreak affecting at least 217 (41%) of 527 passengers on a cruise ship was caused by a variant strain of Norwalk virus (NV) that is related to but distinct from the prototype NV strain. Consumption of fresh-cut fruit served at two buffets was significantly associated with illness (P < or = 0.01), and a significant dose-response relationship was evident between illness and the number of various fresh-cut fruit items eaten. Seven (58%) of 12 paired serum specimens from ill persons demonstrated at least fourfold rises in antibody response to recombinant NV capsid antigen. A 32-nm small round-structured virus was visualized by electron microscopy in 4 (29%) of 14 fecal specimens, but none of the 8 specimens that were examined by an enzyme immunoassay for NV antigen demonstrated antigen. Four (40%) of 10 fecal specimens were positive by reverse transcriptase-PCR by using primer pairs selected from the polymerase region of NV. In a 145-bp region, the PCR product shared only 72% nucleotide sequence identity with the reference NV strain and 77% nucleotide sequence identity with Southampton virus but shared 95% nucleotide sequence identity with UK2 virus, a United Kingdom reference virus strain. In addition, the outbreak virus was serotyped as UK2 virus by solid-phase immune electron microscopy. The genetic and antigenic divergence of the outbreak strain from the reference NV strain highlights the need for more broadly reactive diagnostic assays and for improved understanding of the relatedness of the NV group of agents.
PMCID: PMC263153  PMID: 8027335

Results 1-25 (57)