We have constructed various baculovirus recombinants expressing selected rotavirus proteins including VP4 (P1A, P1B, P2, P3, P4, P5B, P6 or P7), VP7 (G1, G2, G3, G4, G6 or G9) or NSP4 (genotype A, B, C or D). Previously, by using immunocytochemistry assay involving such recombinants, we analyzed sera obtained from gnotobiotic calves or piglets infected orally with homologous bovine (NSP4A) or porcine (NSP4B) virus. We reported that following primary infection and subsequent challenge with virulent rotaviruses, naive calves and piglets developed either higher or significantly higher IgA andor IgG antibody titers to the homologous-host homotypic NSP4A or B proteins than to the heterologous-host homotypic or heterologous host heterotypic rotavirus NSP4s, indicating that primary and secondary antibody responses were species-specific rather than genotype-specific. Unexpectedly, such isotype antibody responses were shown to be correlated closely with the molecular phylogenetic relationships of NSP4 proteins within a species-specific region of amino acids 131-141, reaffirmng that NSP4-specific antibody responses were primarily species-specific. In piglets, antibodies to NSP4 induced by previous oral infection failed to confer protection against oral challenge with a porcine rotavirus bearing serotypically different VP4 and VP7 but essentially identical NSP4 to the porcine rotavirus used in primary infection. Thus, as a potential novel approach to immunization with a live oral rotavirus vaccine, the NSP4 protein did not appear to be important in protection against rotavirus disease and infection. ? Previously, a total of 18 adult volunteers was given orally 1 ml of a 0.2% stool filtrate containing the virulent human rotavirus strain D (G1P1A8, NSP4B) (Kapikian, et al., 1983. J. Infec. Dis. 147:95). Four of 5 who shed rotavirus after challenge developed diarrhea. In an attempt to identify correlates of resistance to rotaviral diarrheal disease andor infection, we analyzed serum IgA and IgG antibody titers in 16 of the 18 volunteers. We used an immunocytochemistry assay involving a total of 18 different recombinant baculoviruses expressing each of the following major serotypegenotype of rotavirus proteins for the serologic assays: VP4 (P1A8; P1B4; P2A6; P3.9; P4.10); VP7 (G1-4, G9); NSP4 (A-D); as well as VP6 and NSP2. The prechallenge IgG antibody titers to types G1 and G3 VP7, types P1A8 and P2A6 VP4 and type A NSP4 in the non-infected group were significantly higher than those in the asymptomatically infected and symptomatically infected groups. Logistic regression analysis showed that the higher probability of resistance to asymptomatic and symptomatic rotavirus infection correlated with higher prechallenge IgG antibody titers to the homotypic VP7 (G1) (p is equal to 0.0077 for asymptomatic infection; p is equal to 0.0004 for symptomatic infection) and the homotypic VP4 (P1A8) (p is equal to 0.0091 for asymptomatic infection; p is equal to 0.0011 for symptomatic infection), suggesting that protection against rotavirus infection and disease may be VP7- andor VP4-homotypic although they may not be the sole determinants of protective immunity.? We analyzed archival serum samples collected from 31 calves monthly from birth to 12 months of age during 1974-1975 season in Arizona. The specimens had been sent to NIH for collaborative studies in the mid 1970s by the late Dr. Francis R. Abinanti of the University of Arizona College of Agriculture, Tucson, Arizona. By determining G and P type-specific IgG antibody titers in such samples by an immunocytochemistry assay using recombinant baculoviruses expressing rotavirus VP7 with G6, G8 or G9 specificity and VP4 with P6 or P7 specificity, we studied the frequency of rotavirus infection in calves as well as the distribution of the G and P types of infecting rotaviruses. All calves tested experienced at least one rotavirus infection as determined by a 4-fold or greater antibody response during the12 month period: 15 experienced a single rotavirus infection and 16 developed multiple rotavirus infections. Analyses of serum samples showed that G8 (35.5%) serotype infected calves most frequently during the study period followed by G6 (22.6%) and G10 (16.1%); mixed G types were detected in 25.1% of samples. Similarly, an IgG response to P6 was detected in 41.9% of the samples followed by P7 (22.6%), whereas 35.5% of samples demonstrated responses to multiple P types. The most frequent G-P combination of infecting rotavirus deduced from analyses of antibody responses in calves was G8P7 followed by G6P6 and G8P6 or 7. Of interest was the finding that serum IgG antibody responses to outer capsid proteins VP7 and VP4 of an infecting rotavirus strain were not always induced simultaneously; in some calves an antibody response to VP7 was detected earlier than that to VP4 whereas in others antibody response to VP4 preceded that to VP7. This may be due to the presence or absence in calves of preexisting maternally derived G andor P type-specific antibodies at the time of rotavirus infection.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Intramural Research (Z01)
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