VP7 Serotype Research Articles

Specificity and affinity of neuraminic acid exhibited by canine rotavirus strain K9 carbohydrate-binding domain (VP8*).

The outer capsid spike protein VP4 of rotaviruses is a major determinant of infectivity and serotype specificity. Proteolytic cleavage of VP4 into 2 domains, VP8* and VP5*, enhances rotaviral infectivity. Interactions between the VP4 carbohydrate-binding domain (VP8*) and cell surface glycoconjugates facilitate initial virus-cell attachment and subsequent cell entry. Our saturation transfer difference nuclear magnetic resonance (STD NMR) and isothermal titration calorimetry (ITC) studies demonstrated that VP8*64-224 of canine rotavirus strain K9 interacts with N-acetylneuraminic and N-glycolylneuraminic acid derivatives, exhibiting comparable binding epitopes to VP8* from other neuraminidase-sensitive animal rotaviruses from pigs (CRW-8), cattle (bovine Nebraska calf diarrhoea virus, NCDV), and Rhesus monkeys (Simian rhesus rotavirus, RRV). Importantly, evidence was obtained for a preference by K9 rotavirus for the N-glycolyl- over the N-acetylneuraminic acid derivative. This indicates that a VP4 serotype 5A rotavirus (such as K9) can exhibit a neuraminic acid receptor preference that differs from that of a serotype 5B rotavirus (such as RRV) and the receptor preference of rotaviruses can vary within a particular VP4 genotype.

Relative Roles of GM1 Ganglioside, N -Acylneuraminic Acids, and α2β1 Integrin in Mediating Rotavirus Infection

N-acetyl- and N-glycolylneuraminic acids (Sia) and α2β1 integrin are frequently used by rotaviruses as cellular receptors through recognition by virion spike protein VP4. The VP4 subunit VP8*, derived from Wa rotavirus, binds the internal N-acetylneuraminic acid on ganglioside GM1. Wa infection is increased by enhanced internal Sia access following terminal Sia removal from main glycan chains with sialidase. The GM1 ligand cholera toxin B (CTB) reduces Wa infectivity. Here, we found sialidase treatment increased cellular GM1 availability and the infectivity of several other human (including RV-3) and animal rotaviruses, typically rendering them susceptible to methyl α-d-N-acetylneuraminide treatment, but did not alter α2β1 usage. CTB reduced the infectivity of these viruses. Aceramido-GM1 inhibited Wa and RV-3 infectivity in untreated and sialidase-treated cells, and GM1 supplementation increased their infectivity, demonstrating the importance of GM1 for infection. Wa recognition of α2β1 and internal Sia were at least partially independent. Rotavirus usage of GM1 was mapped to VP4 using virus reassortants, and RV-3 VP8* bound aceramido-GM1 by saturation transfer difference nuclear magnetic resonance (STD NMR). Most rotaviruses recognizing terminal Sia did not use GM1, including RRV. RRV VP8* interacted minimally with aceramido-GM1 by STD NMR. Unusually, TFR-41 rotavirus infectivity depended upon terminal Sia and GM1. Competition of CTB, Sia, and/or aceramido-GM1 with cell binding by VP8* from representative rotaviruses showed that rotavirus Sia and GM1 preferences resulted from VP8*-cell binding. Our major finding is that infection by human rotaviruses of commonly occurring VP4 serotypes involves VP8* binding to cell surface GM1 glycan, typically including the internal N-acetylneuraminic acid. Rotaviruses, the major cause of severe infantile gastroenteritis, recognize cell surface receptors through virus spike protein VP4. Several animal rotaviruses are known to bind sialic acids at the termini of main carbohydrate chains. Conversely, only a single human rotavirus is known to bind sialic acid. Interestingly, VP4 of this rotavirus bound to sialic acid that forms a branch on the main carbohydrate chain of the GM1 ganglioside. Here, we use several techniques to demonstrate that other human rotaviruses exhibit similar GM1 usage properties. Furthermore, binding by VP4 to cell surface GM1, involving branched sialic acid recognition, is shown to facilitate infection. In contrast, most animal rotaviruses that bind terminal sialic acids did not utilize GM1 for VP4 cell binding or infection. These studies support a significant role for GM1 in mediating host cell invasion by human rotaviruses.

Whole genomic analyses of asymptomatic human G1P[6], G2P[6] and G3P[6] rotavirus strains reveal intergenogroup reassortment events and genome segments of artiodactyl origin

Although P[6] group A rotaviruses (RVA) cause diarrhoea in humans, they have been also associated with endemics of predominantly asymptomatic neonatal infections. Interestingly, strains representing the endemic and asymptomatic P[6] RVAs were found to possess one of the four common human VP7 serotypes (G1–G4), and exhibited little antigenic/genetic differences with the VP4 proteins/VP4 encoding genome segments of P[6] RVAs recovered from diarrhoeic children, raising interest on their complete genetic constellations. In the present study, we report the overall genetic makeup and possible origin of three such asymptomatic human P[6] RVA strains, RVA/Human-tc/VEN/M37/1982/G1P2A[6], RVA/Human-tc/SWE/1076/1983/G2P2A[6] and RVA/Human-tc/AUS/McN13/1980/G3P2A[6]. G1P[6] strain M37 exhibited an unusual genotype constellation (G1-P[6]-R1-C1-M1-A1-N1-T2-E1-H1), not reported previously, and was found to originate from possible intergenogroup reassortment events involving acquisition of a DS-1-like NSP3 encoding genome segment by a human Wa-like RVA strain. On the other hand, G2P[6] strain 1076 exhibited a DS-1-like genotype constellation, and was found to possess several genome segments (those encoding VP1, VP3, VP6 and NSP4) of possible artiodactyl (ruminants) origin on a human RVA genetic backbone. The whole genome of G3P[6] strain McN13 was closely related to that of asymptomatic human Wa-like G3P[6] strain RV3, and both strains shared unique amino acid changes, which might have contributed to their attenuation. Taken together, the present study provided insights into the origin and complex genetic diversity of P[6] RVAs possessing the common human VP7 genotypes. This is the first report on the whole genomic analysis of a G1P[6] RVA strain.

Antigenic and Molecular Characterization of Unusual Rotavirus Strains in Burkina Faso in 1999

Thirty-six of 37 rotavirus strains recovered from the diarrheal stools of 166 children <3 years of age in Burkina Faso were characterized at both the antigenic and molecular levels. The rotavirus strains were confirmed by polyacrylamide gel electrophoresis; 30 displayed predominantly short electropherotype patterns, and 6 had a long RNA pattern. The strains were subgrouped by monoclonal antibody enzyme immunoassay for VP6 and were typed as subgroup I (29 of 30 short rotavirus strains) and subgroup II (5 of 6 long strains). The VP7 serotyping and genotyping showed that all 6 viruses with long electropherotype patterns were G1. The short strains were determined to be VP7 serotype G2 by reverse-transcription polymerase chain reaction (PCR) in 27 strains and nucleic acid sequencing of selected strains, although only 1 reacted with the G2-specific monoclonal antibodies. Finally, the short patterns were shown by the PCR genotyping method to be VP4 genotype P[6], and the long patterns were shown to be P[8]. The predominant strain found in Burkina Faso in this small study was an unusual G2P[6] strain that showed a short RNA electropherotype and VP6 subgroup I specificity and failed to react with a panel of G2-specific monoclonal antibodies.

Unexpectedly high burden of rotavirus gastroenteritis in very young infants.

BackgroundThe highest incidence of rotavirus gastroenteritis has generally been reported in children 6-24 months of age. Young infants are thought to be partially protected by maternal antibodies acquired transplacentally or via breast milk. The purpose of our study was to assess the age distribution of children with confirmed community-acquired rotavirus gastroenteritis presenting to an urban referral hospital.MethodsChildren presenting to The Children's Hospital of Philadelphia with acute gastroenteritis have been monitored for the presence of rotavirus antigen in the stool by ELISA (followed by genotyping if ELISA-positive) since the 1994-95 epidemic season.ResultsOver the last 12 rotavirus seasons prior to the introduction of the pentavalent rotavirus vaccine in 2006, stool specimens from 1646 patients tested positive for community-acquired rotavirus infection. Gender or age was not recorded in 6 and 5 cases, respectively. Overall, 58% of the cases occurred in boys. G1 was the predominant VP7 serotype, accounting for 72% of cases. The median (IQR) age was 11 (5-21) months. A total of 790 (48%) cases occurred in children outside the commonly quoted peak age range, with 27% in infants <6 months of age and 21% in children >24 months of age. A total of 220 (13%) cases occurred during the first 3 months of life, and the highest number of episodes per month of age [97 (6%)] was observed during the second month of life.ConclusionsThe incidence of community-acquired rotavirus gastroenteritis monitored over 12 seasons in the prevaccine era at a major university hospital was nearly constant for each month of age during the first year of life, revealing an unexpectedly high incidence of symptomatic rotavirus disease in infants <3 months old. A sizeable fraction of cases occurred in children too young to have been vaccinated according to current recommendations.

A longitudinal cohort study in calves evaluated for rotavirus infections from 1 to 12 months of age by sequential serological assays

Using an immunocytochemical staining assay involving six different recombinant baculoviruses with each expressing one of the major bovine rotavirus VP7 (G6, G8 and G10) and VP4 (P6[1], P7[5] and P8[11]) serotypes, we analyzed IgG antibody responses to individual proteins in archival serum samples collected from 31 calves monthly from 1 to 12 months of age during 1974-1975 in Higley, Arizona. Seroresponses to VP7 and VP4, as determined by a fourfold or greater antibody response, were not always elicited concurrently following infection: in some calves, (1) seroresponses to VP7 were detected earlier than to VP4 or vice versa; and (2) a subsequent second seroresponse was detected for VP7 or VP4 only. In addition, a second infection was more likely to be caused by different G and/or P types. Analyses of serum samples showed that the most frequent G-P combination was G8P6[1], followed by G8P7[5], G8P8[11] and G6P6[1].

Group A Human Rotavirus Genomics: Evidence that Gene Constellations Are Influenced by Viral Protein Interactions

Group A human rotaviruses (HRVs) are the major cause of severe viral gastroenteritis in infants and young children. To gain insight into the level of genetic variation among HRVs, we determined the genome sequences for 10 strains belonging to different VP7 serotypes (G types). The HRVs chosen for this study, D, DS-1, P, ST3, IAL28, Se584, 69M, WI61, A64, and L26, were isolated from infected persons and adapted to cell culture to use as serotype references. Our sequencing results revealed that most of the individual proteins from each HRV belong to one of three genotypes (1, 2, or 3) based on their similarities to proteins of genogroup strains (Wa, DS-1, or AU-1, respectively). Strains D, P, ST3, IAL28, and WI61 encode genotype 1 (Wa-like) proteins, whereas strains DS-1 and 69M encode genotype 2 (DS-1-like) proteins. Of the 10 HRVs sequenced, 3 of them (Se584, A64, and L26) encode proteins belonging to more than one genotype, indicating that they are intergenogroup reassortants. We used amino acid sequence alignments to identify residues that distinguish proteins belonging to HRV genotype 1, 2, or 3. These genotype-specific changes cluster in definitive regions within each viral protein, many of which are sites of known protein-protein interactions. For the intermediate viral capsid protein (VP6), the changes map onto the atomic structure at the VP2-VP6, VP4-VP6, and VP7-VP6 interfaces. The results of this study provide evidence that group A HRV gene constellations exist and may be influenced by interactions among viral proteins during replication.

Changes in macromolecular transport appear early in Caco-2 cells infected with a human rotavirus

Objective. Rotavirus is a major cause of viral gastroenteritis, but its interaction with intestinal mucosa is poorly understood. The aim of this study was to examine the effect of Wa rotavirus (VP7 serotype 1) on barrier function in confluent Caco-2 cell monolayers. Wa is the most common serotype causing severe diarrhoea in humans. Material and methods. We examined light and electron microscopic morphology, macromolecular transport, paracellular permeability, electrical parameters, disaccharidases and cytoskeletal structure in Wa- and in control sham-infected cells using a homologous human virus-cell system resembling human infection. Results. During the first 48 h following Wa infection, there was no evidence of loss of integrity or of cytopathic effect in the monolayer. A significant cytopathic effect was noticed after 48 h. Further studies examined the initial 24-h period during which there was no evidence of significant injury. Apical-to-basolateral transcytosis of the macromolecule horseradish peroxidase (HRP) was selectively inhibited at 4 and 24 h post-infection with Wa. There were no significant changes in basolateral-to-apical transcytosis, endocytosis or in apical-to-apical recycling of HRP after Wa infection. G- and F-actin levels were significantly reduced within an area corresponding to the viroplasm in Wa-infected cells but not elsewhere in the monolayer. Conclusions. The early stages of rotavirus infection, before gross epithelial injury, are associated with a selective reduction in the apical uptake and transcytosis of macromolecules. We speculate that this is an epithelial defence mechanism.

Group A rotavirus strains circulating in the eastern center of Tunisia during a ten‐year period (1995–2004)

An epidemiological survey investigating rotavirus infections in children was undertaken in the Eastern Center of Tunisia between January 1995 and December 2004. A total of 982 faecal specimens collected from children less than 5 years in age were screened by enzyme-linked immunosorbent assay (ELISA) or latex agglutination assay for the presence of group A rotavirus antigen. Rotavirus-positive samples were used for G and P typing by multiplex semi-nested reverse transcription-PCR. Rotaviruses were detected in 22% (n = 220) of stools. Of these, 164 were typed for VP7: G genotypes found were G1 (59%), G2 (2%), G3 (9%), G4 (10%), G8 (1%), and G9 (1%). Sixteen specimens (9%) showed mixed G profiles. A total of 119 specimens were typed for VP4. P genotypes detected were P[8] (32%), P[6] (15%), and P[4] (13%). Mixed P profiles were also detected (6%). Although the distribution of the detected genotypes appeared to change annually, G1P[8] rotavirus strains always predominated during the 10-year period of study. This is the first report of rotaviruses in Tunisia with unconventional VP7 serotypes such as G8 and G9, highlighting the need for continual surveillance of emerging strains in Northern Africa. Indeed, the new commercial vaccines only contain the VP7 genes that dictate G1 or G1 to G4 specificities. These vaccines may protect less well against unusual strains circulating in countries planning to implement a rotavirus vaccine strategy.

An outbreak of rotavirus infection among adults in an institution for rehabilitation: long-term residence in a closed community as a risk factor for rotavirus illness

An outbreak of group A rotavirus infection resulted in gastroenteritis among disabled adults in an isolated rehabilitation institution in Kobe, Japan. Of the 95 residents, 16 were diagnosed with rotavirus illness. The causative agent was a single strain of typical human group A rotavirus belonging to VP7 serotype G2, VP4 genotype P[4], and NSP4 genotype A. Mean duration of stay was significantly longer for residents with rotavirus illness (22.1+/-11.8 years) than for residents without the disease (13.5+/-10.6 years; P=0.01). Age, sex, disability and location of resident rooms displayed no significant relationships with illness. These observations suggest that long-term residence in a closed community, which might be related to absence of immuno-stimulation, represents a risk factor for rotavirus illness.

A Hexavalent Human Rotavirus–Bovine Rotavirus (UK) Reassortant Vaccine Designed for Use in Developing Countries and Delivered in a Schedule with the Potential to Eliminate the Risk of Intussusception

There is an urgent need for a rotavirus vaccine, because up to 592,000 infants and young children <5 years old die each year from rotavirus diarrhea, predominantly in the developing countries. We have developed a tetravalent human-bovine rotavirus (UK) reassortant vaccine with VP7 (G) specificity for serotypes 1, 2, 3, and 4, which has been shown to be safe, immunogenic, and effective in preventing severe rotavirus diarrhea. However, because of the emergence of VP7 (G) serotype 9 as an epidemiologically important serotype and the importance of VP7 (G) serotype 8 in focal areas, we are planning to add human-bovine (UK) reassortants with G8 and G9 specificity to the tetravalent vaccine, thereby formulating a "designed" hexavalent vaccine for universal use. In addition, we propose that the vaccine be administered orally in a 2-dose schedule, with the first dose given at 0-4 weeks of age and the second dose given at 4-8 weeks of age, when infants are relatively refractory to developing intussusception, thereby avoiding the age period when naturally occurring intussusception is most prevalent (i.e., ages 3-4 months through age 9 months). In this way, there may be the potential to eliminate or at least significantly decrease the risk of intussusception associated with rotavirus vaccination.

A porcine G9 rotavirus strain shares neutralization and VP7 phylogenetic sequence lineage 3 characteristics with contemporary human G9 rotavirus strains

Of five globally important VP7 (G) serotypes (G1–4 and 9) of group A rotaviruses (the single most important etiologic agents of infantile diarrhea worldwide), G9 continues to attract considerable attention because of its unique natural history. Serotype G9 rotavirus was isolated from a child with diarrhea first in the United States in 1983 and subsequently in Japan in 1985. Curiously, soon after their detection, G9 rotaviruses were not detected for about a decade in both countries and then reemerged in both countries in the mid-1990s. Unexpectedly, however, such reemerged G9 strains were distinct genetically and molecularly from those isolated in the 1980s. Thus, the origin of the reemerged G9 viruses remains an enigma. Sequence analysis has demonstrated that the G9 rotavirus VP7 gene belongs to one of at least three phylogenetic lineages: lineage 1 (strains isolated in the 1980s in the United States and Japan), lineage 2 (strains first isolated in 1986 and exclusively in India thus far), and lineage 3 (strains that emerged/reemerged in the mid-1990s). Currently, lineage 3 G9 viruses are the most frequently detected G9 strains globally. We characterized a porcine rotavirus (A2 strain) isolated in the United States that was known to belong to the P[7] genotype but had not been serotyped by neutralization. The A2 strain was found to bear serotype G9 and P9 specificities as well as NSP4 [B] and subgroup I characteristics. By VP7-specific neutralization, the porcine G9 strain was more closely related to lineage 3 viruses than to lineage 1 or 2 viruses. Furthermore, by sequence analysis, the A2 VP7 was shown to belong to lineage 3 G9. These findings raise intriguing questions regarding possible explanations for the emergence of variations among the G9 strains.

Rotavirus serotype G9 strains belonging to VP7 gene phylogenetic sequence lineage 1 may be more suitable for serotype G9 vaccine candidates than those belonging to lineage 2 or 3.

A safe and effective group A rotavirus vaccine that could prevent severe diarrhea or ameliorate its symptoms in infants and young children is urgently needed in both developing and developed countries. Rotavirus VP7 serotypes G1, G2, G3, and G4 have been well established to be of epidemiologic importance worldwide. Recently, serotype G9 has emerged as the fifth globally common type of rotavirus of clinical importance. Sequence analysis of the VP7 gene of various G9 isolates has demonstrated the existence of at least three phylogenetic lineages. The goal of our study was to determine the relationship of the phylogenetic lineages to the neutralization specificity of various G9 strains. We generated eight single VP7 gene substitution reassortants, each of which bore a single VP7 gene encoding G9 specificity of one of the eight G9 strains (two lineage 1, one lineage 2 and five lineage 3 strains) and the remaining 10 genes of bovine rotavirus strain UK, and two hyperimmune guinea pig antisera to each reassortant, and we then analyzed VP7 neutralization characteristics of the eight G9 strains as well as an additional G9 strain belonging to lineage 1; the nine strains were isolated in five countries. Antisera to lineage 1 viruses neutralized lineage 2 and 3 strains to at least within eightfold of the homotypic lineage viruses. Antisera to lineage 2 virus neutralized lineage 3 viruses to at least twofold of the homotypic lineage 2 virus; however, neutralization of lineage 1 viruses was fourfold (F45 and AU32) to 16- to 64-fold (WI61) less efficient. Antisera to lineage 3 viruses neutralized the lineage 2 strain 16- to 64-fold less efficiently, the lineage 1 strains F45 and AU32 8- to 128-fold less efficiently, and WI61 (prototype G9 strain) 128- to 1024-fold less efficiently than the homotypic lineage 3 viruses. These findings may have important implications for the development of G9 rotavirus vaccine candidates, as the strain with the broadest reactivity (i.e., a prime strain) would certainly be the ideal strain for inclusion in a vaccine.

Human rotavirus strains bearing VP4 gene P[6] allele recovered from asymptomatic or symptomatic infections share similar, if not identical, VP4 neutralization specificities

A rotavirus VP4 gene P[6] allele has been documented in a number of countries to be characteristically associated with an endemic predominantly asymptomatic infection in neonates in maternity hospital nurseries. The mechanisms underlying the endemicity and asymptomatic nature of such neonatal infections remain unknown. Rotavirus strains sharing this same P genotype, however, have more recently been recovered from an increasing number of symptomatic diarrheal episodes in infants and young children in various parts of the world. Previously, we have shown that an asymptomatic P[6] rotavirus neonatal infection is not associated with a unique VP7 (G) serotype but may occur in conjunction with various G types. Although amino acid sequence comparisons of the VP4 gene between selected “asymptomatic” and “symptomatic” P[6] rotavirus strains have been reported and yielded information concerning their VP4 genotypes, serotypic comparisons of the outer capsid spike protein VP4 of such viruses have not been studied systematically by two-way cross-neutralizations. We determined the VP4 neutralization specificities of four asymptomatic and four symptomatic P[6] strains: two each of asymptomatic and symptomatic strains by two-way tests, and two each of additional asymptomatic and symptomatic strains by one-way tests. Both asymptomatic and symptomatic P[6] strains were shown to bear similar, if not identical, VP4 neutralization specificities. Thus, P[6] rotavirus strains causing asymptomatic or symptomatic infections did not appear to belong to unique P (VP4) serotypes. In addition, a close VP4 serotypic relationship between human P[6] rotavirus strains and the porcine P[6] rotavirus Gottfried strain was confirmed.

Integrin-using rotaviruses bind alpha2beta1 integrin alpha2 I domain via VP4 DGE sequence and recognize alphaXbeta2 and alphaVbeta3 by using VP7 during cell entry.

Integrins alpha2beta1, alphaXbeta2, and alphaVbeta3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the alpha2beta1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the alphaXbeta2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of alpha2beta1, alphaXbeta2, and alphaVbeta3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound alpha2beta1, and VP7 interacted with alphaXbeta2 and alphaVbeta3 at a postbinding stage. DGEA inhibited rotavirus binding to alpha2beta1 and infectivity, whereas GPRP binding to alphaXbeta2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed alpha2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the alpha2 I domain. In a novel process, integrin-using viruses bind the alpha2 I domain of alpha2beta1 via DGE in VP4 and interact with alphaXbeta2 (via GPR) and alphaVbeta3 by using VP7 to facilitate cell entry and infection.

Rotavirus diarrhea severity is related to the VP4 type in Mexican children.

This report is of a community-based case control study to assess whether the severity of acute diarrhea by rotavirus (RV) in young children is associated with a particular VP7 (G) or VP4 (P) RV serotype. Five hundred twenty children younger than 2 years of age with diarrhea lasting less than 3 days were age and gender matched with 520 children with no diarrhea. The G and P serotypes were determined with specific monoclonal antibodies, and the VP4 serotype specificity in a subgroup was confirmed by genotyping. Infection with a G3 serotype led to a higher risk of diarrhea than infection with a G1 serotype. Infection with a G3-nontypeable-P serotype was associated with more severe gastroenteritis than infection with a G3 (or G1) P1A[8] serotype. A child with diarrhea-associated dehydration was almost five times more likely to be infected with a G3-nontypeable-P serotype than a child without dehydration (P < 0.001). Moreover, the two predominant monotypes within serotype P1A[8] had significantly different clinical manifestations. In this study, the severity of RV-associated diarrhea was related to different P serotypes rather than to G serotypes. The relationship between serotype and clinical outcomes seems to be complex and to vary among different geographic areas.

Construction and characterization of rhesus monkey rotavirus (MMU18006)- or bovine rotavirus (UK)-based serotype G5, G8, G9 or G10 single VP7 gene substitution reassortant candidate vaccines

Group A rotaviruses are the single most important etiologic agents of severe diarrhea of infants and young children worldwide and have been estimated to be responsible for approximately 650,000–800,000 deaths annually in children <5-year-old in the developing countries. Thus, the development of a safe and effective rotavirus vaccine has been a global public health goal. Epidemiologic surveillance of rotavirus VP7 (G) serotypes–genotypes conducted in various populations throughout the world has repeatedly shown that approximately 90% of the typeable rotavirus isolates belong to G1–G4. For these reasons, we have developed a rhesus rotavirus (RRV)-based or bovine rotavirus (UK)-based quadrivalent vaccine which is designed to provide antigenic coverage for G1–G4. More recently, G serotypes–genotypes other than G1–G4, including G5, G8–G10, have been detected in various parts of the world. Although the occurrence of such uncommon G types, except for G9, has been focal, still, in order to “be ready and prepared”, we have constructed and characterized eight additional reassortant rotavirus vaccines, each of which bears a single human or bovine VP7 gene encoding G serotype 5, 8, 9 or 10 specificity and the remaining 10 genes of RRV strain MMU18006 or bovine rotavirus strain UK. These candidate vaccines could be evaluated singly in special populations or in combination with a RRV- or an UK-based quadrivalent vaccine to broaden its G serotype specificity.

Molecular Epidemiology of Rotaviruses in Nigeria: Detection of Unusual Strains with G2P[6] and G8P[1] Specificities

We noted with interest the recent report by Adah and colleagues on the detection of G2P[6] rotavirus strains in Nigeria (1). We have also been conducting epidemiological surveillance of rotavirus strains recovered from young children with diarrheal disease in Lagos, Nigeria (2), and identified similar strains during 2001. Adah and colleagues identified 12 strains that had the unusual profile of VP7 serotype G2 and VP4 genotype P[6]. Most G2 rotavirus strains bear a specific VP4 genotype of P[4] (4), which is well recognized as belonging to the DS-1 genogroup (7). However, Adah did not extend the characterization of these strains to analyze the potential reassortment of various genes. The DS-1 genogroup can be phenotypically measured on at least three structural genes and one nonstructural gene. These include the VP6 subgroup, VP7 serotype, and VP4 genotype, as well as the migration of the NSP5 gene (7). DS-1 genogroup rotaviruses typically share subgroup I, VP7 serotype G2, and VP4 genotype P[4], and the strains carry a short RNA electropherotype. Analysis of these four markers would provide valuable epidemiological data on the potential reassortment of these unusual rotavirus strains. In our studies, we have continued the surveillance and characterization of rotavirus strains from young children admitted with acute infantile diarrhea requiring hospitalization (2). In the present report, we identified 21 cases of rotavirus among 150 children (14%). Fecal samples were collected from children less than 5 years of age with acute diarrhea attending the Gbaja Health Center, Massey Street Children's Hospital, and Lagos University Teaching Hospital (all within Lagos State, Nigeria) between January and December 2001. Ten percent suspensions of the samples in phosphate-buffered saline were tested for the presence of group A rotavirus antigen by using a commercial enzyme immunoassay (Rotavirus IDEIA; Dako, Cambridge, United Kingdom). All 21 strains were examined for the diversity of the RNA electropherotypes by polyacrylamide gel electrophoresis. Standard RNA extraction was performed by phenol-chloroform treatment and ethanol precipitation as described in detail elsewhere (8). Silver staining was used to identify the double-stranded RNA segments as described previously (8). In addition, the VP6 subgroup specificity of the rotavirus strains was determined by using the VP6 monoclonal antibodies developed by Greenberg et al. (6). These monoclonal antibodies, specific for subgroup I (255/60) and subgroup II rotaviruses (631/9) rotaviruses, have been extensively used in studies worldwide. The methods for their use have been described in detail elsewhere (6). The VP4 types were identified as described by Gentsch et al. (2, 3). The viral RNA was extracted by treatment with Genetron and purified by RNAid (Bio 101, Inc., La Jolla, Calif.) before analysis by the reverse transcription-PCR (RT-PCR) method with primers con2 and con3. The PCR products were then typed with a cocktail of primers for the different human VP4 genotypes (3). The VP7 genotypes were examined by the RT-PCR typing method of Gouvea et al. (2, 5). The purified RNA was reverse transcribed, and primers directed to the terminal sequences were used to amplify the entire gene (5). These techniques have been described in detail elsewhere, and similar conditions were used here (2). In this study, the unusual G2P[6] strains constituted 8 of 21 rotavirus-positive specimens (38%). Analysis of the two other epidemiological markers showed that all eight strains carried a VP6 subgroup I specificity and were characterized by a short RNA electropherotype, although two variations were seen (Fig. ​(Fig.11). FIG. 1. Polyacrylamide gel electrophoresis of the unusual G2P[6] rotavirus strains recovered in Lagos. The two short strains that carried this unusual genotypic configuration (lanes 2 and 3) are illustrated with a long G8P[1] strain (lane 1) for comparison. Taken together, these observations indicate that the P[6] VP4 gene may have reassorted onto the DS-1 genogroup background of these strains. However, further analysis is required to identify whether other genes also reassorted during the generation of these unusual rotaviruses. Furthermore, this report shows the importance of including all epidemiological markers when characterizing unusual rotavirus strains.

A群ロタウイルスが関与した成牛下痢症

Bacterial and parasitic pathogens were negative in fecal samples taken from three adult diarrheal cows on a dairy farm in Akita Prefecture in January 2001, Electron microcopy revealed only rotavirus particles in the samples. RNA-polyacrylamide gel electrophoresis detected group-A rotavirus (GAR) RNA segments. GAR was isolated from two samples with MA104 cells, the VP7 serotype of which was G6 and the VP4 genotype of which was P [1] Significant GAR antibody response was observed in two affected and one non-affected cow. Results indicate that GAR infection caused diarrhea in the adult cows in this study.

Rotavirus strains circulating in Africa during 1996–1999: emergence of G9 strains and P[6] strains

Rotavirus infection is associated with 150,000–200,000 deaths annually in Africa. Although the withdrawal of the RotaShield ® vaccine has been a major setback in rotavirus vaccine development, new vaccine candidates are under development and approaching phase II and III trials. Before these trials could be conducted in Africa, a comprehensive survey of the circulating VP7 serotypes and VP4 genotypes is required. During the past 3 years, over 3000 rotavirus-positive specimens from several African countries have been analysed. RT-PCR techniques for the VP7 and VP4 genotypes and by monoclonal antibodies to the VP6 subgroup and VP7 serotype have been performed. Almost 75% of the strains were typed by the VP7 monoclonal antibodies or RT-PCR. VP4 genotyping was done in approximately half of these strains. The predominant strains circulating across Africa during 1996–1999 were P[6]G1 and P[6]G3 strains. Geographic differences were noted and West Africa displayed the most diverse strains with G3/8 and G1/3 “mosaic” viruses occurring commonly. G9 strains were identified in several countries indicating that the strain is emerging in Africa too. G9 was the predominant strain in certain countries during 1999. The circulating types observed will have implications for the new rotavirus vaccine candidates.