Showing papers by "Harry B. Greenberg published in 1988"

The rhesus rotavirus gene encoding protein VP3: location of amino acids involved in homologous and heterologous rotavirus neutralization and identification of a putative fusion region.

Abstract: The complete gene 4 nucleotide sequence was determined for rhesus rotavirus and each of 11 viral variants selected by neutralizing monoclonal antibodies. Gene 4 is 2362 bases in length and encodes a protein, VP3, of 776 amino acids with a calculated Mr of 86,500. A conserved trypsin cleavage site, located at amino acid 247, divides VP3 into VP8 and VP5. Neutralizing monoclonal antibodies directed at VP3 were used to select variants that escaped neutralization. Each variant contains a single gene 4 mutation that permits viral growth in the presence of the antibody. Variant mutations were identified in six distinct neutralization regions in VP8 and VP5. Five of the six neutralization regions were found in VP8. The VP8 regions were primarily associated with strain-specific or limited heterotypic rotavirus neutralization. One region was identified in VP5 by three monoclonal antibodies that neutralize a broad range of rotavirus serotypes. The VP5 neutralization region is largely hydrophobic and is similar to putative fusion sequences of Sindbis and Semliki Forest viruses.

Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis.

Abstract: Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 [VP3]; 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. Cleavage of VP3 does not alter viral binding to cell monolayers. In previous electron microscopic studies of infected cell cultures, it has been demonstrated that rotavirus particles enter cells by both endocytosis and direct cell membrane penetration. To determine whether trypsin treatment affected rotavirus internalization, we studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Endocytosis inhibitors (sodium azide, dinitrophenol) and lysosomotropic agents (ammonium chloride, chloroquine) had a limited effect on the entry of infectious virus into cells. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 medicated 51Cr, [14C]choline, and [3H]inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.

Relative concentrations of serum neutralizing antibody to VP3 and VP7 proteins in adults infected with a human rotavirus.

Abstract: Two outer capsid rotavirus proteins, VP3 and VP7, have been found to elicit neutralizing-antibody production, but the immunogenicity of these proteins during human rotavirus infection has not been determined. The relative amounts of serum neutralizing antibody against the VP3 and VP7 proteins of the CJN strain of human rotavirus were, therefore, determined in adult subjects before and after infection with this virus. Reassortant strains of rotavirus that contained the CJN gene segment for only one of these two neutralization proteins were isolated and used for this study. The geometric mean titer of serum neutralizing antibody to a reassortant virus (CJN-M) that contained VP7 of CJN and VP3 of another human rotavirus was 12.7 times less than that of antibody to CJN before infection and 20.3 times less after infection. This indicated that most neutralizing antibody was against the VP3 rather than the VP7 protein of CJN. This result was confirmed with other reassortants between CJN and animal rotavirus strains (EDIM and rhesus rotavirus). These findings suggest that VP3 is the primary immunogen that stimulates neutralizing antibody during at least some rotavirus infections of humans. Images

Functional and topographical analyses of epitopes on the hemagglutinin (VP4) of the simian rotavirus SA11.

Abstract: An immunochemical analysis of the hemagglutinin (VP4) of the simian rotavirus SA11 was performed to better understand the structure and function of this molecule. Following immunization of mice with double-shelled virus particles and VP4-enriched fractions from CsCl gradients, a battery of anti-SA11 hybridomas was generated. A total of 13 clones secreting high levels of anti-VP4 monoclonal antibody (MAb) was characterized and compared with two cross-reactive anti-VP4 MAbs generated against heterologous rhesus (RRV) and porcine (OSU) rotavirus strains. These cross-reactive MAbs effectively neutralized SA11 infectivity in vitro. The epitopes recognized by these 15 MAbs were grouped into six antigenic sites on the SA11 hemagglutinin. These sites were identified following analysis of the MAbs by using a simple competitive binding enzyme-linked immunosorbent assay (ELISA) and biological assays. Three of the antigenic sites were involved in neutralization of virus infectivity in vitro. All the MAbs with neutralization activity and two nonneutralizing MAbs were able to inhibit viral hemagglutination of human erythrocytes. Competitive binding ELISA data showed a positive cooperative binding effect with some pairs of the anti-VP4 MAbs, apparently due to a conformational change induced by the binding of the first MAb. Some of the MAbs also bound better to trypsin-treated virus than to non-trypsin-treated virus. A topographic map for VP4 is proposed on the basis of the observed properties of each antigenic site.

Isolation of a bovine rotavirus with a "super-short" RNA electrophoretic pattern from a calf with diarrhea.

Abstract: A rotavirus with a "super-short" RNA electropherotype was isolated from a calf with diarrhea and was designated VMRI strain. Segments 10 and 11 of this rotavirus migrated more slowly than did those of bovine rotavirus strains NCDV, B641, and B223. The electrophoretic pattern of the VMRI strain was similar to that reported for rotaviruses with super-short RNA electropherotypes from humans and rabbits. Northern (RNA) blot hybridization indicated that gene 11 of the VMRI strain was altered and migrated between gene segments 9 and 10. The subgroup of the VMRI strain was shown to be subgroup I. The VMRI strain of bovine rotavirus was neutralized by antisera containing polyclonal antibodies to rotavirus serotype 6 (bovine rotavirus serotype I) strains NCDV and B641 and by ascitic fluid containing monoclonal antibodies directed to VP7 of serotype 6 rotavirus. The VMRI strain was not neutralized by either polyclonal or monoclonal antibodies to strain B223 (bovine rotavirus serotype II). Collective data on the neutralization of the VMRI strain with monoclonal antibodies and polyclonal antibodies suggest that this virus is a member of the NCDV group (serotype 6) of rotaviruses (bovine rotavirus serotype I). Images

Reactogenicity and antigenicity of rhesus rotavirus vaccine (MMU-18006) in newborn infants in Venezuela.

Abstract: The reactogenicity and antigenicity of the rhesus rotavirus vaccine, strain MMU18006, developed at the Laboratory of Infectious Diseases (National Institute of Allergy and Infectious Diseases, National Institutes of Health) were examined in a double blind, placebo-controlled study of 40 newborn infants in Caracas, Venezuela. The children were observed for the first few days after birth in the hospital nursery and by home visits for 10 days after vaccination to detect any adverse reactions. No reactions could be attributed to the vaccine. Serologic responses to the vaccine were evaluated in paired sera obtained at birth (cord blood) and 4 weeks after vaccination. Serologic responses to the vaccine were not observed by complement fixation, neutralization or a rhesus rotavirus VP7 epitope-specific competition assay. However, such responses were found in 9 of 14 tested infants by an immunoglobulin A-specific enzyme-linked immunosorbent assay. Seventeen of the 20 vaccinees also shed rhesus rotavirus vaccine in stool during the postvaccination period.

Serotypic analysis of VP3 and VP7 neutralization escape mutants of rhesus rotavirus.

Abstract: Neutralization escape mutants of simian rotaviruses (rhesus rotavirus and SA11) were tested in hemagglutination inhibition and neutralization assays against hyperimmune and infection sera to determine if mutation in an immunodominant epitope could enable neutralization escape. An SA11 mutant with a new glycosylation site at amino acid 211 of VP7 was shown to escape neutralization by hyperimmune but not infection sera.

Phenotypic mixing during coinfection of cells with two strains of human rotavirus.

Abstract: Coinfection of MA-104 cells with serotype 1 (Wa strain) and serotype 3 (P strain) human rotaviruses resulted in progeny viruses that were phenotypically mixed in their major outer-shell structural-protein VP7 and in the genome segment that encodes this protein (segment 9). Segments from the Wa virus predominated in these progeny whether they were of parental or reassortant genotype. Neutralization with monoclonal antibodies specific for VP7 proteins of the coinfecting viruses caused an alteration in genomic distribution favoring the strain heterologous to the neutralizing monoclonal antibody. Because the percentage of change in distribution of segment 9 was similar to that of the other combined segments, there appeared to be no greater association between VP7 and segment 9 than with other segments of the homologous virus during encapsulation. From these results, it was calculated that the progeny of coinfection with P segment 9 were 77.4% mosaic structures and 14.8% pseudotypes; progeny of coinfection with Wa segment 9 were 40.2% mosaic structures and 1.3% pseudotypes. Similar determinations were made for the reassortant progeny alone.

Phenotypic Mixing during Coinfection ofCells withTwoStrains of HumanRotavirus

Abstract: Coinfection of MA-104 cells with serotype 1 (Wa strain) and serotype 3 (P strain) human rotaviruses resulted in progeny viruses that were phenotypically mixed in their major outer-shell structural-protein VP7 and in the genome segment that encodes this protein (segment 9). Segments from the Wa virus predominated in these progeny whether they were of parental or reassortant genotype. Neutralization with monoclonal antibodies specific for VP7 proteins of the coinfecting viruses caused an alteration in genomic distribution favoring the strain heterologous to the neutralizing monoclonal antibody. Because the percentage of change in distribution of segment 9 was similar to that of the other combined segments, there appeared to be no greater association between VP7 and segment 9 than with other segments of the homologous virus during encapsulation. From these results, it was calculated that the progeny of coinfection with P segment 9 were 77.4% mosaic structures and 14.8% pseudotypes; progeny of coinfection with Wa segment 9 were 40.2% mosaic structures and 1.3% pseudotypes. Similar determinations were made for the reassortant progeny alone.