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H F Clark

Publications and source records attributed to H F Clark.

At least 37 records · Page 2Linked to original sources
PubMed1993

Nucleotide sequence of the VP4-encoding gene of an unusual human rotavirus (HCR3).

Human rotavirus strain HCR3 was isolated from the stool of a clinically normal infant and identified as a serotype G3 rotavirus; however, it could not be grouped into any known human VP4 genetic groups by a polymerase chain reaction assay. The fourth gene of strain HCR3, which encodes the outer capsid protein VP4, was sequenced. This gene is 2362 nucleotides in length and contains one open reading frame capable of encoding a protein of 776 amino acids. The VP4 protein of strain HCR3 shared 67.5-73.5% amino acid identity with those of strains KU, RV-5, 1076, and K8, representing four human genetic groups, and relatively high homology (84.7%) with a fifth genetic group represented by strain 69M, whose VP4 shows more similarity to animal than to human strains. Strain HCR3 shared higher VP4 amino acid homology with various animal rotaviruses, ranging from 74.5 to 89.4%. These observations suggest that the VP4 outer capsid protein of strain HCR3 represents a new VP4 genetic group that is more closely related to animal rotaviruses than to human rotaviruses.

Amino Acid Sequence↗
PubMed1993

Restriction endonuclease analysis of the vp7 genes of human and animal rotaviruses.

The vp7 genes of 194 strains of group A rotaviruses representing all known G types were analyzed with three restriction enzymes by direct digestion of amplified cDNA copies or by deduction of the restriction patterns from known sequences. Mammalian rotavirus strains were classified into 28 restriction patterns consisting of combinations of the 6 profiles (s1 to s6) obtained by digestion with Sau96I endonuclease, 9 profiles (h1 to h9) obtained with HaeIII, and 15 profiles (b1 to b15) obtained with BstYI. Digestion with Sau96I and HaeIII identified restriction sites common to all, or almost all, rotavirus strains studied, whereas BstYI was the most discriminating among rotavirus strains. A clear correlation between some restriction patterns or individual profiles and G type and/or host species of origin was found. Several discriminatory restriction sites consisted of type-specific nucleic acid sequences that encoded conserved amino acid residues. Although not directly involved in antigenic diversity, these sites appear to indicate the G type of the isolate. The technique permits rapid comparison of a large number of virus isolates directly from fecal specimens and provides useful markers for investigating the evolution of rotavirus vp7 genes and tracing vaccine virus and interspecies transmission.

Animals↗
PubMed1993

Murine rotavirus genes encoding outer capsid proteins VP4 and VP7 are not major determinants of host range restriction and virulence.

Simian rotavirus (RRV) and murine rotavirus (EDIM-RW) differ dramatically in the oral inoculum required to cause diarrheal disease in neonatal mouse pups and in their ability to spread and cause disease in uninoculated littermates. A genetic approach was used to explore the molecular basis of these differences. Reassortant viruses were produced in vivo by coinfecting infant mice with RRV and EDIM-RW. Reassortant viruses were isolated by plaque purification of progeny virus obtained from mouse pup intestines on MA104 cells. The plaque-purified reassortants were evaluated for 50% diarrhea dose (DD50) and for the ability to spread and cause diarrhea in uninoculated littermates. The parental RRV strain had a DD50 of 10(5) PFU per animal, while the EDIM-RW parental strain had a DD50 of less than 1 PFU per animal. RRV never spreads from inoculated to uninoculated littermates and causes disease. Twenty-three reassortants were tested. Of great interest were the reassortants D1/5 and C3/2, which derived genes 4 and 7 (encoding VP4 and VP7) from RRV. These viruses had a DD50 similar or identical to that of EDIM-RW and spread efficiently from inoculated mouse pups to uninoculated pups. We conclude that the major outer capsid proteins VP4 and VP7 are not primarily responsible for virulence or host range restriction in the mouse model using a homologous murine rotavirus.

Animals↗
PubMed1993

Group A rotaviruses produce extrahepatic biliary obstruction in orally inoculated newborn mice.

Extrahepatic biliary atresia is a devastating disease occurring in 1 in 10,000 to 14,000 infants annually in the United States. We have recently described preliminary data suggesting an association of group C rotavirus with biliary atresia in two infants. However, a group C rotavirus animal model of biliary atresia is not presently available. On the other hand, some strains of the better-characterized and much more common group A rotaviruses produce hepatobiliary disease in infant mice. This disease shares many characteristics of the human infection. The present report describes extrahepatic biliary obstruction in immunocompetent BALB/c infant mice infected with a human or animal strain of group A rotavirus. Two-d-old BALB/c mice orally inoculated with hepatobiliary tropic rotavirus were shown to have active virus replication in the biliary tract and liver as early as 48 h postinoculation. At approximately 7 d postinoculation, between one fourth and one half of infant mice, depending on the virus strain, showed signs of inflammation and swelling in the bile ducts. The obstruction was complete in about one half of symptomatic animals. Although there was no obvious atresia as described in human infants, the obstruction was irreversible about 50% of the time, and the resulting fibrosis and bile ductular proliferation in the liver were strikingly similar to those seen in the liver of the human infant with biliary atresia.

Administration, Oral↗
PubMed1991

Prevalence of serum neutralizing antibody to serotype 9 rotavirus WI61 in children from South America and central Europe.

Neutralizing serum antibody to serotype 9 rotavirus WI61 was detected in 41% of 870 Ecuadorian children and 26% of 140 German children. In both areas an age-related prevalence increase was observed. We identified 11 serum samples from Ecuadorian children which neutralized exclusively serotype 9 rotavirus. Thirteen of 71 (18%) German children hospitalized with serologically defined primary rotavirus gastroenteritis showed a seroconversion to serotype 9 rotavirus; however, in 10 of these 13 patients, the infecting serotype could be identified as serotype 1, 3, or 4. Furthermore, all 13 patients showed fourfold increases in titer to at least one further serotype.

Antibodies, Viral↗
PubMed1990

Serotype 1 reassortant of bovine rotavirus WC3, strain WI79-9, induces a polytypic antibody response in infants.

A reassortant rotavirus, strain W179-9, was constructed bearing gene 9 of serotype 1 rotavirus strain WI79 and all other genes derived from bovine (serotype 6) rotavirus strain WC3. The antigenic phenotype of WI79-9 is bivalent: serotype 1 and serotype 6. WI79-9 administered orally at a dose of 10(7.5) p.f.u. induced no adverse effects in 48 infants of age 2-11 months. Serotype 1- and serotype 6-specific serum neutralizing antibody titres were induced with approximately equal frequency in these infants. Serotype 1-specific antibody responses were inhibited in infants previously seropositive to type 1. The immune response rate was enhanced by administration of a second, 'booster dose'.

Adult↗
PubMed1990

Immune protection of infants against rotavirus gastroenteritis by a serotype 1 reassortant of bovine rotavirus WC3.

The safety and protective efficacy of a serotype 1 reassortant of bovine rotavirus WC3, disignated strain WI79-9, was evaluated in a double-blind placebo-controlled trial. Rotavirus reassortant WI79-9 contains a gene segment 9 coding for the surface structural protein vp7 of a human serotype 1 rotavirus, with all other gene segments derived from WC3 rotavirus, which had previously been shown to be safe and immunogenic in infants. Infants 2-11 months of age were given two doses of vaccine (10(7.3) plaque-forming units/dose) or of placebo 28 days apart. Adverse reactions to the vaccine were not detected. The incidence of serum plaque reduction neutralization antibody responses to two doses of vaccine was serotype 6, 97%; serotype 3, 68%; and serotype 1, 22%. Active surveillance during the subsequent rotavirus season revealed 8 cases of rotavirus gastroenteritis in 39 placebo control infants and no cases in 38 WI79-9 vaccine recipients (protection = 100%, P = .003). Six cases of rotavirus gastroenteritis were caused by type 1 and two by type 3 virus. Although vaccination with WI79-9 affected only the incidence of rotavirus gastroenteritis, the vaccinated infants exhibited a significantly reduced incidence of total days of diarrhea, fever, and illness associated with gastroenteritis in general.

Antibodies, Viral↗
PubMed1990

Serotypes and electropherotypes of human rotavirus in the USA: 1987-1989.

The epidemiology of rotavirus gastroenteritis was investigated for two consecutive seasons (1987-1988 and 1988-1989) in seven locales in the continental USA. The 281 representative fecal samples obtained from children with diarrhea were electropherotyped and serotyped by an enzyme immunoassay with serotype-specific monoclonal antibodies and a new amplification typing technique (polymerase chain reaction typing). Serotype 1 was predominant in both years, particularly in the North and East; serotype 3 was second in frequency and found most often in the South; serotype 2 was detected only occasionally; serotypes 4, 8, and 9 were never found. Rotavirus strains were grouped into five major electropherotypes, each corresponded to a single serotype, and the relative migration of the gene segments 7-9 could be used to distinguish serotype 1 from serotype 3. The amplification typing technique proved to be of great value in typing the 17% of rotavirus-positive specimens untypable by the serologic technique.

Antibodies, Monoclonal↗
PubMed1990

Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens.

The rotavirus gene segment coding for the major outer capsid glycoprotein vp7 was amplified directly from stool specimens by the polymerase chain reaction (PCR). Double-stranded RNA extracted from stool samples was used as the template for reverse transcription, which was followed immediately and in the same reaction mix with amplification, using the Taq polymerase. Various conditions were examined to optimize the yield of the amplified gene. The concentrations of MgCl2, dimethyl sulfoxide, and template RNA were critical. The choice of primer pairs allowed amplification of the entire segment or specific portions. By using type-specific primers derived from distinct regions on the gene, we devised a PCR typing method in which each human serotype virus produced a characteristic segment size, readily identifiable in agarose gels. The PCR typing method was applied to 10 rotavirus reference strains, including all 6 known human serotypes (serotypes 1, 2, 3, 4, 8, and 9), and to 34 stool specimens previously serotyped by an enzyme immunoassay with monoclonal antibodies. An absolute correlation was found between the molecular and serologic methods. In addition, 14 stool specimens nonserotypable by an enzyme immunoassay with monoclonal antibodies could be typed by the PCR method. Besides the application for rotavirus detection and typing directly from stools, the PCR method provides a rapid and efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.

Base Sequence↗
PubMed1989

Discrepant rotavirus results in two laboratories using the same enzyme immunoassay.

Rotavirus testing was performed on 66 stool samples in two separate laboratories using the same enzyme immunoassay. Sixteen of 46 of the positive results reported by the reference laboratory were negative in the hospital laboratory. In addition, RNA gel electrophoresis had negative results in the 16 discrepant samples. This indicates the need to repeat or confirm positive results.

Child↗
PubMed1988

Protective effect of WC3 vaccine against rotavirus diarrhea in infants during a predominantly serotype 1 rotavirus season.

We used a double-blind, placebo-controlled trial to study the efficacy of WC3 rotavirus vaccine administered to 104 infants (ages, three to 12 months) before the rotavirus season. Forty-nine infants received vaccine; 55 received placebo. Rotavirus disease during this season was predominantly caused by a serotype 1 strain. In placebo recipients there were 14 cases of rotavirus diarrhea (attack rate, 25%); 11 were moderate to severe (attack rate, 20%). Vaccinees experienced only three cases of rotavirus disease (attack rate, 6.1%), all mild. When all cases (whether associated with rotavirus or not) of clinically significant diarrhea (CSD) were evaluated, WC3 vaccine provided statistically significant (P less than .01) protection against the total number of episodes of CSD and reduced the number of days of CSD-associated diarrhea, vomiting, fever, or illness. Seventy-one percent of the WC3-vaccinated infants had serum antibody responses to the vaccine. The 14 placebo recipients who experienced natural disease predominantly had antibody responses to serotype 1. Sera taken after the rotavirus season revealed a nearly identical rate (40%) of natural rotavirus infection in the vaccinated and placebo groups.

Antibodies, Viral↗
PubMed1988

Rotavirus serotype-specific neutralizing activity in human milk.

A plaque reduction neutralization assay was used to determine rotavirus serotype-specific neutralizing activity in human breast milk from 25 mothers of upper socioeconomic background and 20 mothers of a lower socioeconomic status. Levels of neutralizing activity, as well as those of rotavirus-specific antibodies detected by enzyme-linked immunosorbent assay (ELISA), were comparable for each socioeconomic group. Overall, neutralizing activity in human milk was detected in the majority of samples and can be increased many months postpartum. The prevalence of neutralization antibodies (titers, greater than or equal to 1:10) was 77% against Wa (serotype 1), 86% against SA-11 (serotype 3), and 75% against NCDV (bovine) rotavirus. Rotavirus-specific IgA and IgG antibodies detected by ELISA (titers, greater than or equal to 1:10) were present in 35% and 55% of breast milks, respectively. Sequential analysis of repeated breast milk samples from five individual mothers revealed that rotavirus neutralizing activity fluctuated over time, with high activity observed in one mother's milk at 18 months postpartum. Mothers who breast-fed for six months or more tended to have higher milk neutralizing titers against rotavirus.

Adolescent↗
PubMed1987

Rotavirus isolate WI61 representing a presumptive new human serotype.

A virus (strain WI61) representing a presumptive new human serotype was isolated from an 18-month-old child with gastroenteritis admitted to Children's Hospital of Philadelphia in February 1983. The WI61 virus was clearly distinguished by cross-neutralization tests from human rotaviruses of serotypes 1, 2, 3, and 4, human 69M, and representative bovine (NCDV), porcine (OSU), and chicken (Ch2) rotaviruses. Antisera generated in guinea pigs hyperimmunized to WI61 virus displayed a partial cross-reactivity with rotaviruses of human serotypes 1, 2, 3, and 4. By means of studies with reassortant rotaviruses, it was presumptively determined that the WI61 virus cross-reactive antigenic determinants are localized on the vp3 surface polypeptide coded by gene segment 4. The characteristic RNA genome electropherotype of WI61 virus was observed in 5 of 59 cases of infant gastroenteritis detected in 1983 and 1984 but has not been observed in a subsequently at Children's Hospital. Serotype WI61-specific neutralizing antibodies were observed in a majority of sera of normal adults and infants of less than 4 or greater than 12 months of age collected in the Philadelphia area. Median antibody titers to WI61 equaled or exceeded those to rotaviruses of serotype 1 or 3. Each of seven samples of commercial cow's milk exhibited neutralizing antibodies to WI61 virus at a titer greater than or equal to that to serotype 1 or 3 or bovine (strain NCDV) rotavirus. However, WI61 rotavirus did not induce disease or a specific serum-neutralizing antibody response when fed to a caesarean-derived colostrum-deprived newborn calf. WI61 rotavirus caused diarrhea in newborn mice with a 50% diarrhea-inducing dose of 10(7.0) PFU.

Animals↗
PubMed1987

Gastroenteritis caused by human rotaviruses (serotype three) in a suckling mouse model.

The pathogenic potential of human rotaviruses of serotypes 1 through 4 was evaluated in suckling mice. Oral inoculation of three different human rotaviruses of serotype 3 into 5-6 day old CD-1 mice caused disease characterized by diarrhea and dehydration. The mean 50% diarrhea inducing dose (DD50) was 5 X 10(5) pfu. Histopathological examination of small intestines revealed villus epithelial cell vacuolization localized to the distal one-third of the villus. Only Serotype 3 rotaviruses exhibited a rapid phase of viral growth in the intestine between 7 and 12 hours post-inoculation. Larger inocula of rotavirus serotypes 1, 2, and 4 did not cause disease or typical histopathologic changes. However, immunoperoxidase staining for rotavirus antigen was positive in all serotypes tested indicating that infection can occur without apparent disease and is not serotype specific. This convenient in-vivo model can be used to evaluate attenuation of human origin vaccine candidates of serotype 3.

Animals↗
PubMed1986

Immune response of infants and children to low-passage bovine rotavirus (strain WC3).

A bovine rotavirus (strain WC3) was isolated from a calf in Pennsylvania and adapted to growth in continuous Cercopithecus cell line CV1. A pool for human vaccine trials was produced at the 12th cell culture passage level. After preliminary testing in adults and older children, a dose of 3 X 10(7) plaque-forming units was given by mouth to 52 infants and children aged 5 months to 6 years. No clinical sequelae were detected, and shedding in feces was detected in only 30% of tested infants. A serum-neutralizing antibody response was induced in 95% of 21 infants aged 5 to 11 months; response rates were slightly reduced in older infants. The antibody response was primarily directed toward bovine rotavirus, but a response to human serotype 3 rotavirus was also observed in approximately 50% of vaccinees. After vaccination with WC3, infants with preexisting antibody to rotaviruses of human serotype 1 or 3 frequently exhibited a booster response to those serotypes. WC3 is a candidate rotaviral vaccine deserving larger trials in children.

Adult↗
PubMed1986

Response of adult human volunteers to oral administration of bovine and bovine/human reassortant rotaviruses.

Small groups of adult volunteers, in sequence, were inoculated orally with inactivated purified bovine rotavirus of strain NCDV, with live NCDV purified or unpurified and with two different NCDV X human rotavirus reassortant viruses. One of five volunteers given 200 micrograms of ultravioletinactivated NCDV developed a virus-neutralizing (VN) and a binding antibody response detected by enzyme-linked immunosorbent assay (ELISA). Four of 10 volunteers given from 1 X 10(6) to 1 X 10(8) p.f.u. of live NCDV developed VN antibody, but nine of 10 responded when ELISA, HAI and radioimmuno-precipitation tests for serum antibody were also considered. Two different NCDV X human serotype 1 Wa strain virus reassortants, each containing Wa gene segment 9 and the serotype 1 neutralization phenotype, were administered orally in doses up to 10(6) p.f.u. The reassortants were relatively ineffective in eliciting a serum antibody response at the dosage level employed.

Administration, Oral↗