Isolation And Serotypic Characterization Of Human And Animal Rotaviruses

National Institute Of Allergy And Infectious Diseases

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Abstract

In the past year, we have advanced the objectives of this project as follows: [unreadable] (1) Serotype G8 virus, which can be found in cows at relatively high frequency, was first isolated in a study performed between 1979 and 1981, from stool specimens collected from children with diarrhea in Jakarta and Medan, Indonesia in association with P10 (prototype strain 69M). Since then only a few reports of such a G serotype have been published, except for many countries in Africa where it has been detected in an increasing frequency since 1997-1998. Interestingly, the majority of these G8 human strains were not associated with the P10 type, but present a great diversity of P-G combinations such as G8:P8, G8:P4, G8:P6, G8:P1, G8:P2 and G8:P14. Sequence and phylogenetic analyses of the VP7 gene of various G8 virus strains have demonstrated the presence of at least four phylogenetic lineages. The study of the relationship of the phylogenetic lineages and the neutralization specificity of selected G8 strains belonging to lineage 1, 2, 3 or 4 is important because of the increasing epidemiologic importance of G8 viruses and also to include the G8 component in certain rotavirus vaccines (e.g., UK-based reassortant vaccine being developed in India). We generated (i) single VP7 gene substitution reassortants each of which bore 10 genes of bovine rotavirus UK strain and only the VP7 gene of G8 rotavirus strain belonging to one of four VP7 gene lineages, and (ii) hyperimmune guinea pig antiserum to each reassortant. Preliminary results obtained by two-way cross-neutralization assays show that, unlike G9 rotavirus VP7s which display lineage-specific neutralization profiles, no significant difference in neutralization specificities is detected among the four G8 VP7 lineages tested. [unreadable] (2) Rotavirus genotype G1:P8 is the most commonly detected G-P combination globally. Increasing evidence suggests that genetic drift observed among community G1 strains is generating antigenic variants, an observation which may be important for the development of vaccination strategies. The human G1 rotavirus VP7 gene, which encodes the major protective and neutralization antigen, has been reported to belong to one of at least four phylogenetic lineages. By analyzing the four G1 phylogenetic lineages detected in infants and young children hospitalized with diarrhea Childrens Hospital National Medical Center in Washington, DC, during 1974-1991 as well as selected G1 samples collected in other regions of the world, we studied the epidemiological and evolutionary dynamics of the G1 VP7 genes. The VP7 G1 lineage distribution showed a marked temporal variation over time. For example, the G1-III lineage was present from 1974 to 1980 and became undetectable after 1980; lineage G1-I was detectable from 1977-1981 and then in 1991; and lineage G1-II was detected for the first time in 1988 as a predominant strain and remained predominant in 1989 and 1991; and lineage G1-IV was detectable throughout the study period except for 1975 and 1981. In order to assess the importance of specific amino-acid(s) identified for VP7 gene lineage differentiation, a site-specific selection analysis was performed using the SLAC method, and the positions of representative changes were mapped on an unrooted tree generated for the Washington DC G1 VP7 gene sequences. Interestingly, not only non-synonymous but also synonymous changes were shown to be associated with lineage differentiation. Analysis of the estimates of the divergence time of the more recent ancestor indicated that the four G1 lineages have been present for the last 60 years in the DC area. This estimate is similar to the HA and NA subtypes of avian influenza virus and the measles virus H gene. The nucleotide substitution rate of the DC G1 rotavirus VP7 genes was estimated at 8.91 x 10 to the minus 4 power/site/year, which falls close to the substitution rates observed for other RNA viruses. The deduced VP7 amino acid sequence analyses of the DC G1 viruses revealed the presence of mutations inside as well as outside of the previously recognized antigenic regions A, C, and F, some of which were lineage-specific. In order to detect amino acid position(s) under selection pressure, we conducted a site-by-site Maximum Likelihood analysis of the VP7 gene sequences of G1 rotaviruses collected globally, and found that 6 amino acid positions were shown to be under positive-selection pressure. Interestingly, positions 23, 37, 41, 49 and 281 were not previously recognized by immunological methods. These findings appear to suggest that the genetic heterogeneity of human G1 rotavirus VP7 genes is driven by both antigenic and genetic drift. [unreadable] (3) Group A rotavirus diarrhea is a potentially life-threatening disease that affects millions of infants and young children annually worldwide. A total of 5621 stool specimens were collected from hospitalized infants and young children (n = 4203) with diarrhea between 1974 and 1991 at the Childrens Hospital National Medical Center in Washington, DC. Although these archival specimens had been evaluated previously for rotaviruses by EM, IEM and/or ELISA, they were reexamined by current PCR-ELISA techniques to dissect VP7/VP4 specificities. Rotavirus infections were detected primarily between December and May throughout the study period. Nine hundred and twenty-six samples collected from 769 infants and young children were positive for rotavirus. Of the 769 patients who were rotavirus positive, 481 (62.5%) shed G1 strains, 177 (23%) shed G3 strains, 38 (5%) shed G2 strains, 35 (4.6%) shed G4 strains and 4 (0.5%) shed G9 strains; 34 samples (4.4%) were G non-typeable. P genotype was determined for 684 of the 769 (88.9%) samples. Among the 769 strains, P8 was the predominant genotype (83.9%) followed by P4 (3.6%) and P6 (1.4%), and 85 samples (11.1%) were P non-typeable. No G or P type mixed infections were detected. Of the 769 strains, G1P8 represented the most common combination (55.7%) followed by G3P8 (19.5%) and G4P8 (4.2%) whereas for 119 (15.5%) strains the G or P genotype could not be determined. Unexpectedly, three samples collected in 1980 and one sample collected in 1988 were genotyped as G9P8; these three strains represented the earliest known G9 strains ever detected.[unreadable] (4) The genome of group A rotavirus (the most important etiologic agent of severe diarrhea in infants and young children worldwide) consists of 11 segments of double-stranded RNA. Since polyacrylamide gel electrophoresis (PAGE) is simple and cost-effective to perform, it has been used extensively in rotavirus research such as molecular epidemiology and gene-protein coding assignments of selected genome segments. In a standard 7.5% or 12% PAGE gel, the rotavirus gene encoding the outer capsid glycoprotein VP7, which can induce protective neutralizing antibodies, has been assigned to genome segment 7, 8 or 9 depending upon the rotavirus strain. In this project, we demonstrated that (i) the relative position of the VP7 gene of serotype G2 rotaviruses (strains DS-1, 1076 and S2) varied depending upon the concentration of acrylamide in a PAGE gel; (ii) an altered migration pattern of the G2 virus VP7 gene in a PAGE gel that influenced the VP7 gene coding assignment occurred not only in a homologous G2 virus gene background but also in a heterologous G3 virus rhesus monkey rotavirus gene background; and (iii) the VP7 gene bearing G1, G3, G4 or G9 specificity did not display this altered migration pattern when the PAGE running conditions were varied.