Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains

Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.     

Rotavirus vaccines: recent developments and future considerations

Two new vaccines have recently been shown to be safe and effective in protecting young children against severe rotavirus gastroenteritis. Although both vaccines are now marketed worldwide, it is likely that improvements to these vaccines and/or the development of future generations of rotavirus vaccines will be desirable. This Review addresses recent advances in our knowledge of rotavirus, the host immune response to rotavirus infection and the efficacy and safety of the new vaccines that will be helpful for improving the existing rotavirus vaccines, or developing new rotavirus vaccines in the future.     

Evolutionary history and global spread of the emerging g12 human rotaviruses

G12 rotaviruses were first detected in diarrheic children in the Philippines in 1987, but no further cases were reported until 1998. However, G12 rotaviruses have been detected all over the world in recent years. Here, we report the worldwide variations of G12 rotaviruses to investigate the evolutionary mechanisms by which they managed to spread globally in a short period of time. We sequenced the complete genomes (11 segments) of nine G12 rotaviruses isolated in Bangladesh, Belgium, Thailand, and the Philippines and compared them with the genomes of other rotavirus strains. Our genetic analyses revealed that after introduction of the VP7 gene of the rare G12 genotype into more common local strains through reassortment, a vast genetic diversity was generated and several new variants with distinct gene constellations emerged. These reassortment events most likely took place in Southeast Asian countries and spread to other parts of the world. The acquirement of gene segments from human-adapted rotaviruses might allow G12 to better propagate in humans and hence to develop into an important emerging human pathogen.     

Genetic engineering of rotaviruses by reverse genetics

The rotavirus genome is composed of 11 gene segments of dsRNA. A recent breakthrough in the field of rotaviruses is the development of a reverse genetics system for generating recombinant rotaviruses possessing a gene segment derived from cloned cDNA. Although this approach is a helper virus‐driven system that is technically limited and gives low levels of recombinant viruses, it allows alteration of the rotavirus genome, thus contributing to our understanding of these medically important viruses. So far, this approach has successfully been applied to three of the 11 viral segments in our laboratory and others, and the efficiency of recovery of recombinant viruses has been improved. However, we are still waiting for the development of a helper virus‐free reverse genetics system for generating an infectious rotavirus entirely from cDNAs, as has been achieved for other members of the Reoviridae family.     

Reassortant rotaviruses containing structural proteins vp3 and vp7 from different parents induce antibodies protective against each parental serotype.

Genetic studies of reassortant rotaviruses have demonstrated that gene segments 4 and 9 each segregate with the serotype-specific neutralization phenotype in vitro. Reassortant rotaviruses derived by coinfection of MA-104 cells with the simian strain SA11 and the antigenically distinct bovine strain NCDV were used to determine which viral genes coded for proteins which induced a protective immune response in vivo. In addition, reassortant rotaviruses containing only the gene segment 4 or 9 protein products (vp3 and vp7, respectively) from SA11 or NCDV were used to determine the serotypic specificities of both vp3 and vp7 in several mammalian rotavirus strains. vp3 and vp7 from the murine strain Eb were shown to be indistinguishable from the corresponding proteins from strain SA11. Adult mice orally inoculated with strain Eb developed neutralizing antibodies to both vp3 and vp7. The two naturally occurring bovine rotavirus strains NCDV and UK were shown to contain antigenically similar vp7 but distinct vp3 proteins. Mouse dams orally immunized with a reassortant virus containing only gene 9 from NCDV passively protected their progeny against UK challenge, whereas mouse dams orally immunized with a reassortant virus containing only gene 4 from NCDV did not. Finally, we constructed reassortant viruses that immunized against rotaviruses of two distinct serotypes. SA11 X NCDV reassortants that contained vp3 and vp7 from different parents induced a protective immune response against both parental serotypes. vp3 and vp7 were independently capable of inducing a protective immune response after oral immunization. An understanding of the serotypic specificities of both vp3 and vp7 of human rotavirus isolates will be necessary for the development of successful strategies to protect infants against severe rotavirus infections.     

In silico study of rotavirus VP7 surface accessible conserved regions for antiviral drug/vaccine design

Background

Rotaviral diarrhoea kills about half a million children annually in developing countries and accounts for one third of diarrhea related hospitalizations. Drugs and vaccines against the rotavirus are handicapped, as in all viral diseases, by the rapid mutational changes that take place in the DNA and protein sequences rendering most of these ineffective. As of now only two vaccines are licensed and approved by the WHO (World Health Organization), but display reduced efficiencies in the underdeveloped countries where the disease is more prevalent. We approached this issue by trying to identify regions of surface exposed conserved segments on the surface glycoproteins of the virion, which may then be targeted by specific peptide vaccines. We had developed a bioinformatics protocol for these kinds of problems with reference to the influenza neuraminidase protein, which we have refined and expanded to analyze the rotavirus issue.

Results

Our analysis of 433 VP7 (Viral Protein 7 from rotavirus) surface protein sequences across 17 subtypes encompassing mammalian hosts using a 20D Graphical Representation and Numerical Characterization method, identified four possible highly conserved peptide segments. Solvent accessibility prediction servers were used to identify that these are predominantly surface situated. These regions analyzed through selected epitope prediction servers for their epitopic properties towards possible T-cell and B-cell activation showed good results as epitopic candidates (only dry lab confirmation).

Conclusions

The main reasons for the development of alternative vaccine strategies for the rotavirus are the failure of current vaccines and high production costs that inhibit their application in developing countries. We expect that it would be possible to use the protein surface exposed regions identified in our study as targets for peptide vaccines and drug designs for stable immunity against divergent strains of the rotavirus. Though this study is fully dependent on computational prediction algorithms, it provides a platform for wet lab experiments.     

A组人轮状病毒全基因组克隆和基因型分析

运用基因克隆和重组技术,从一急性胃肠炎患儿样品中克隆到一株轮状病毒（RV）全基因组cDNA。核苷酸序列测定结果表明,该株RV基因组11条RNA共含有18613个核苷酸（3302bp—751bp）,编码5791个氨基酸。全基因组研究结果表明,该株RV（TB-Chen）为A组,II亚组,基因型为G2P[4]/NSP4[A]。这是首个A组人RV全基因组研究结果的报道,对研究和开发RV疫苗具有积极的意义。     

Serial observations of chronic rotavirus infection in an immunodeficient child.

Chronic rotavirus infection of an infant with severe combined immunodeficiency (SCID) was studied by virological examinations in association with long-term observation of his symptoms and immune status. During eleven months of hospitalization, the patient was suffering from incurable severe diarrhea with persisting excretion of rotaviruses detected by electron microscopy and the reversed-passive hemagglutination (R-PHA) test and had transient hepatitis symptom despite multiple administrations of human gammaglobulin and high calorie fluids. The detected viruses were morphologically recognized as rotavirus with double capsid structure. Polyacrylamide gel electrophoretic (PAGE) analysis of their genomic RNAs showed the long electropherotype of group A virus with abnormal migration profiles changing considerably from the early to the late phase of illness: (1) The 11th segment became undetectable; (2) the molecular weight of the 6th segment slightly increased; (3) seven to fourteen extra segments appeared; and (4) PAGE patterns of viral genomic RNAs changed every three or four months. These findings suggest that chronic infection with rotavirus accompanied the generation of extra viral genomic segments and their unusual assortments in an immunodeficient host.     

Full genomic analysis of human rotavirus strain B4106 and lapine rotavirus strain 30/96 provides evidence for interspecies transmission

The Belgian rotavirus strain B4106, isolated from a child with gastroenteritis, was previously found to have VP7 (G3), VP4 (P[14]), and NSP4 (A genotype) genes closely related to those of lapine rotaviruses, suggesting a possible lapine origin or natural reassortment of strain B4106. To investigate the origin of this unusual strain, the gene sequences encoding VP1, VP2, VP3, VP6, NSP1, NSP2, NSP3, and NSP5/6 were also determined. To allow comparison to a lapine strain, the 11 double-stranded RNA segments of a European G3P[14] rabbit rotavirus strain 30/96 were also determined. The complete genome similarity between strains B4106 and 30/96 was 93.4% at the nucleotide level and 96.9% at the amino acid level. All 11 genome segments of strain B4106 were closely related to those of lapine rotaviruses and clustered with the lapine strains in phylogenetic analyses. In addition, sequence analyses of the NSP5 gene of strain B4106 revealed that the altered electrophoretic mobility of NSP5, resulting in a super-short pattern, was due to a gene rearrangement (head-to-tail partial duplication, combined with two short insertions and a deletion). Altogether, these findings confirm that a rotavirus strain with an entirely lapine genome complement was able to infect and cause severe disease in a human child.     

Ganglioside GM(1a) on the cell surface is involved in the infection by human rotavirus KUN and MO strains.

Rotavirus is the most common cause of severe gastroenteritis in infants and children worldwide. The cell attachment of most animal rotaviruses, which belong to the neuraminidase-sensitive strains, requires sialic acid residues on the host cell membranes. On the other hand, most human rotaviruses are classified as neuraminidase-insensitive strains. The involvement of gangliosides on the host cell surface in human rotavirus infection was investigated by immunostaining analysis of target cells, and by assaying the neutralization of infection by rotavirus and the blocking of target cellular receptors. In host cells (MA104 cells) pretreated with Arthrobacter ureafaciens neuraminidase, which were still infected by human rotaviruses (KUN and MO strains), GM(3) was hydrolyzed markedly by the neuraminidase, while GM(1a) was not hydrolyzed at all. Infection by the rotaviruses was strongly inhibited by exogenous ganglioside GM(1a), but not GA(1). Infection was also inhibited by pretreatment of the MA104 cells with cholera toxin B-subunit, which specifically blocked ganglioside GM(1a) on the plasma membrane. The treatment of MA104 cells with the endoglycoceramidase attenuated human rotavirus infection. From these findings, we concluded that GM(1a) on the plasma membrane of the host cells was involved in the infection by human rotavirus KUN and MO strains.     

Recombinant porcine rotavirus VP4 and VP4-LTB expressed in Lactobacillus casei induced mucosal and systemic antibody responses in mice

Background  

Porcine rotavirus infection is a significant cause of morbidity and mortality in the swine industry necessitating the development of effective vaccines for the prevention of infection. Immune responses associated with protection are primarily mucosal in nature and induction of mucosal immunity is important for preventing porcine rotavirus infection.     

Comparison of the genomes of simian, bovine, and human rotaviruses by gel electrophoresis and detection of genomic variation among bovine isolates.

By co-electrophoresis in polyacrylamide gels, the segmented double-standed RNA genome of the simian rotavirus, SA 11, was compared with those of human and bovine rotaviruses. A comparison between SA 11 virus and the Northern Ireland cell culture adapted bovine virus showed that the electrophoretic mobilities of each of the 11 corresponding segments differed. In other comparisons, four to seven segment variations were more common. When the genomes of various bovine rotaviruses were compared, eight different electropherotypes were detected. Four of these electropherotypes were obtained from one property during a single outbreak of disease. In view of such genetic diversity, a scheme for the systematic designation of different rotavirus samples is proposed. The significance of the variations in relation to the molecular epidemiology of bovine rotavirus infections is discussed.     

Understanding reduced rotavirus vaccine efficacy in low socio-economic settings

Introduction

Rotavirus vaccine efficacy ranges from >90% in high socio-economic settings (SES) to 50% in low SES. With the imminent introduction of rotavirus vaccine in low SES countries, understanding reasons for reduced efficacy in these settings could identify strategies to improve vaccine performance.

Methods

We developed a mathematical model to predict rotavirus vaccine efficacy in high, middle and low SES based on data specific for each setting on incidence, protection conferred by natural infection and immune response to vaccination. We then examined factors affecting efficacy.

Results

Vaccination was predicted to prevent 93%, 86% and 51% of severe rotavirus gastroenteritis in high, middle and low SES, respectively. Also predicted was that vaccines are most effective against severe disease and efficacy declines with age in low but not high SES. Reduced immunogenicity of vaccination and reduced protection conferred by natural infection are the main factors that compromise efficacy in low SES.

Discussion

The continued risk of severe disease in non-primary natural infections in low SES is a key factor underpinning reduced efficacy of rotavirus vaccines. Predicted efficacy was remarkably consistent with observed clinical trial results from different SES, validating the model. The phenomenon of reduced vaccine efficacy can be predicted by intrinsic immunological and epidemiological factors of low SES populations. Modifying aspects of the vaccine (e.g. improving immunogenicity in low SES) and vaccination program (e.g. additional doses) may bring improvements.     

Use of PCR and nucleotide sequence analysis of the VP7 gene for detection of bovine rotaviruses and identification of their G serotypes

A Vp7 gene fragment PCR protocol was developed to detect the bovine rotaviruses and to identify their G serotypes. The most widespread bovine rotaviruses of G serotypes (G6, G8 and G10) can be distinguished on the basis of the PCR fragment size, while other G serotypes can be differentiated through a comparative analysis of the VP7 gene fragment nuclcotide sequence. Twenty-four bovine rotavirus field isolates were detected, and their G stereotypes were determined by using the method in question. Fourteen isolates were shown to be of G6 serotype; four of them were of G8, five--of G10, and one isolate was of G11 serotype. A possibility of detecting more than one isolate by this method was shown. Finally, a feasibility of using the method for searching for rotaviruses of new bovine rotavirus G serotypes and for rotaviruses, which do not belong to a so far described G serotypes, is discussed.     

Heterogeneity and temporal dynamics of evolution of G1 human rotaviruses in a settled population

A rotavirus sample collection from 19 consecutive years was used to investigate the heterogeneity and the dynamics of evolution of G1 rotavirus strains in a geographically defined population. Phylogenetic analysis of the VP7 gene sequences of G1P[8] human rotavirus strains showed the circulation of a heterogeneous population comprising three lineages and seven sublineages. Increases in the circulation of G1 rotaviruses were apparently associated with the introduction of novel G1 strains that exhibited multiple amino acid changes in antigenic regions involved in rotavirus neutralization compared to the strains circulating in the previous years. The emergence and/or introduction of G1 antigenic variants might be responsible for the continuous circulation of G1 rotaviruses in the local population, with the various lineages and sublineages appearing, disappearing, or cocirculating in an alternate fashion under the influence of immune-pressure mechanisms. Sequence analysis of VP4-encoding genes of the G1 strains revealed that the older strains were associated with a unique VP4 lineage, while a novel VP4 lineage emerged after 1995. The introduction of human rotavirus vaccines might alter the forces and balances that drive rotavirus evolution and determine the spread of novel strains that are antigenically different from those included in the vaccine formulations. The continuous emergence of VP7-VP4 gene combinations in human rotavirus strains should be taken into consideration when devising vaccination strategies.     

Sequence analysis of gene 11 equivalents from "short" and "super short" strains of rotavirus

The molecular basis for the aberrant migration pattern of the gene 11 equivalent in rotaviruses with "short" (human DS-1) and "super short" (human 69M and bovine VMRI) electropherotypes was investigated. The mRNAs of these viruses were synthesized in vitro, and the entire gene 11 equivalent of each of these viruses was sequenced with specific synthetic oligonucleotide primers. These sequences were compared with previously published sequences of "long" pattern rotavirus gene 11 segments. The increased lengths of the gene 11 equivalents of DS-1, 69M, and VMRI are due to a prolonged, 3' untranslated region in this gene segment. The 3' untranslated region of the VMRI gene 11 equivalent contains a clear duplication of a portion of its coding sequence. A stretch of 18 consecutive nucleotides within the 330-nucleotide, 3' untranslated region of 69M is identical to a section of UK coding sequence. The DS-1 and the remainder of the 69M 3'-end additional sequences are similar to each other, but neither is similar to any other currently available rotavirus gene sequence. This finding suggests that a process other than homologous duplication is involved in the evolution of these sequences. The widespread occurrence of human and animal rotaviruses with short and super short electropherotypes provides evidence that intragenic and possibly intergenic recombinational events associated with an error-prone viral RNA polymerase may play a role in increasing the genetic repertoire of rotaviruses.