The VP8 fragment of VP4 is the rhesus rotavirus hemagglutinin.

The amino-terminal trypsin cleavage fragment of VP4, called VP8, was expressed from a recombinant baculovirus in Sf-9 cells. The baculovirus-expressed VP8 protein is antigenically conserved as demonstrated by its recognition by a library of neutralizing monoclonal antibodies. In Sf-9 cell sonicates, the expressed VP8 protein is capable of agglutinating human type O erythrocytes, indicating that the functionally intact rhesus rotavirus viral hemagglutinin is contained in the 247-amino acid VP8 trypsin cleavage fragment. Amino acid similarities between VP8 and the amino-terminal 282 amino acids of the reovirus sigma 1 protein suggests that the sigma 1 hemagglutination function resides within these amino-terminal amino acids as well. When the expressed VP8 protein was used to immunize mice, a broadly cross-reactive neutralizing antibody response was obtained. Antibodies elicited to the expressed VP8 protein neutralized viruses of serotypes 1-4 and 6 but not porcine strains OSU (st5) or Gottfried (st4). The neutralizing antibody response to VP8 appeared to be more cross-reactive than the immune response to expressed VP4 or to whole RRV virion. This suggests that subunit protein immunizations may broaden the neutralizing antibody immune responses to rotaviruses and enhance protective immunity to serotypically distinct strains.     

Humoral immune responses to VP4 and its cleavage products VP5* and VP8* in infants vaccinated with rhesus rotavirus.

The humoral immune response to rhesus rotavirus (RRV) VP4 and its cleavage products VP5* and VP8* was determined in paired serum samples from 44 infants vaccinated with RRV or human rotavirus-RRV reassortants and 5 placebo recipients. Our aim was to try to measure the response to those regions of VP4 most closely related to protection. An enzyme-linked immunosorbent assay (ELISA) was used to measure the immunoglobulin G immune response to baculovirus-expressed full-length RRV VP4, full-length VP8*, and the amino-terminal polypeptide of VP5* called VP5*(1) (amino acids 248 to 474). The two antigenic regions of VP4 selected for study, VP5*(1) and VP8*, have previously been shown to contain most of the cross-reactive and strain-specific neutralization epitopes, respectively, while the remaining carboxy-terminal half of VP5* (amino acids 475 to 776) has not been clearly associated with neutralization. All three recombinant proteins were antigenically conserved, since they reacted with a library of neutralizing monoclonal antibodies directed at VP4. There was a high percentage of seroresponders to VP4 (61%) or to VP8* (52%), but fewer infants seroresponded to VP5*(1) (11%). In addition, infants responding to VP5*(1) had considerably lower titers than to VP4 or VP8*. Immune response to VP4 correlated strongly with the responses detected by the plaque reduction neutralization assay but did not correlate with the responses detected by the ELISA to whole RRV. These data imply that the VP5*(1) region is less immunogenic than the VP8* region of VP4 in infants immunized with RRV or RRV reassortants. The low immunogenicity of VP5* might adversely affect the efficacy of RRV vaccine candidates.     

Molecular analysis of the VP7, VP4, VP6, NSP4, and NSP5/6 genes of a buffalo rotavirus strain: identification of the rare P[3] rhesus rotavirus-like VP4 gene allele

We report the detection and molecular characterization of a rotavirus strain, 10733, isolated from the feces of a buffalo calf affected with diarrhea in Italy. Strain 10733 was classified as a P[3] rotavirus, as the VP8* trypsin cleavage product of the VP4 protein revealed a high amino acid identity (96.2%) with that of rhesus rotavirus strain RRV (P5B[3]), used as the recipient virus in the human-simian reassortant vaccine. Analysis of the VP7 gene product revealed that strain 10733 possessed G6 serotype specificity, a type common in ruminants, with an amino acid identity to G6 rotavirus strains ranging from 88 to 98%, to Venezuelan bovine strain BRV033, and Hungarian human strain Hun4. Phylogenetic analysis based on the VP7 gene of G6 rotaviruses identified at least four lineages and an apparent linkage between each lineage and the VP4 specificity, suggesting the occurrence of repeated interspecies transmissions and genetic reassortment events between ruminant and human rotaviruses. Moreover, strain 10733 displayed a bovine-like NSP4 and NSP5/6 and a subgroup I VP6 specificity, as well as a long electropherotype pattern. The detection of the rare P[3] genotype in ruminants provides additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.     

Amino acid sequence analysis of bovine rotavirus B223 reveals a unique outer capsid protein VP4 and confirms a third bovine VP4 type.

The nucleotide and deduced amino acid sequence of the gene 4 of bovine rotavirus strain B223 is described. The open reading frame is predicted to encode a VP4 of 772 amino acids, shorter than described for any other rotavirus strain sequenced to date. B223 VP4 shows 70 to 73% similarity to other rotavirus VP4 proteins, demonstrating the presence of a unique VP4 type, and confirming a third VP4 allele in the bovine rotavirus population. Multiple sequence alignment with several other rotavirus strains created gaps in the sequence to account for a shorter VP4. The alignment shows a two contiguous amino acid deletions within the trypsin cleavage region of B223 VP4. Comparisons of two regions flanking the trypsin cleavage site, (aa 224 to 235, and aa 257 to 271) which show high homologies between strains, demonstrate that the region 5' to the trypsin cut site has a low homology (66%) to other rotavirus strains, although the region 3' to the trypsin cleavage site shows high homologies (86 to 93%) with other rotavirus strains. The lack of a conserved proline residue within the 5' flanking region suggests a possible altered local conformation of this site in B223 VP4. A second gap inserted into the VP4 of B223 on multiple sequence alignment is a three contiguous amino acid deletion at position 613-615 in the VP5* subunit. Previously defined biologic properties of this strain in relation to the determination of the amino acid composition of VP4 are discussed.     

Sequence Analysis of the VP4, VP6, VP7, and NSP4 Gene Products ofthe Bovine Rotavirus WC3

The bovine rotavirus (BRV) WC3 serves as the background strain in the development of a multivalent reassortant vaccine against rotavirus gastroenteritis in infants. The genes encoding the outer capsid spike protein VP4, the inner capsid protein VP6, the outer capsid glycoprotein VP7, and the viral enterotoxin NSP4 of BRV WC3 were sequenced. Comparative analysis of the deduced amino acids of the sequenced genes indicated that the BRV WC3 strain shares a high degree of amino acid identity with serotype P7[5] VP4 (93–96%), serotype G6 VP7 (91–97%), subgroup (SG) I VP6 (96–99%), and NSP4 genogroup A (96–98%) BRV strains. Our results confirm and extend previous studies which suggested that the VP4 of BRV WC3 was closely related to that of the P7[5] prototype, BRV UK. In addition, the VP6 and VP7 of BRV WC3 were very similar to the VP6 and VP7 of both SG I and G6 BRV NCDV and UK strains. However, the NSP4 of BRV WC3 was more closely related to that BRV NCDV, the P6[1] prototype, than to that of BRV UK.     

Determination of human rotavirus VP4 using serotype-specific cDNA probes

Summary The VP4 genetic groups of 151 field strains of human rotaviruses obtained from infants and young children with diarrhea from four locations in Malaysia were analyzed. The strains were adapted to growth in tissue culture and studied further by molecular hybridization of northern blotted RNA to PCR-generated cDNA probes representing amino acids 84–180 of the KU strain VP4, 83–181 of the DS-1 strain VP4, and 83–180 of either the 1076 or K8 strain VP4, representing VP4 genetic groups 1–4 (P1A, P1B, P2, and P3), respectively. The majority (79% of the field strains hybridized with the KU VP4 genetic group 1 probe and were associated with G1, G3, G4, untypable, or mixed G serotypes. VP4 genetic group 1 (P1A) strains were the most common in all locations in Malaysia between 1978–1988. Three strains which exhibited G3 and subgroup I specificity hybridized with the K8 VP4 genetic group 4 probe. These three VP4 genetic group 4 (P3) strains were detected in two different years and locations, extending the initial detection of this VP4 genetic group (the K8 strain) in Japan to a larger geographical area of Asia.     

Homotypic protection against rotavirus-induced diarrhea in infant mice breast-fed by dams immunized with the recombinant VP8* subunit of the VP4 capsid protein

The outer capsid proteins VP4 and VP7 induce neutralizing antibody against rotavirus. We have investigated in a mouse model the protection mediated by immunization with VP8*, the amino-terminal tryptic fragment of VP4. BALB/c female mice immunized with simian rotavirus SA11 VP6 and VP8* proteins expressed in Escherichia coli were mated with seronegative males. Litters were orally challenged with the SA11 strain (P5B[2], G3) or with the murine rotavirus strain EDIM (P10[16], G3) to verify the degree of protection against diarrhea induced in the newborns. Only those pups born to dams immunized with VP8* did not develop diarrhea after having been orally challenged with the SA11 strain. Pups born to naive dams but foster nursed by VP8*-immunized dams did not develop diarrhea after having been orally infected with the SA11 strain, but they suffered diarrhea when challenged with the EDIM strain. These results support the concepts that (1) VP8* is a highly immunogenic polypeptide that induces effective homotypic protection against disease in pups born to dams immunized with this antigen and (2) in newborn mice the protection against disease is mediated by neutralizing secretory antibodies present in the milk rather than by serum antibodies transferred through the placenta to the offspring.     

VP7 and VP4 genotypes of rotaviruses cocirculating in Iran, 2015 to 2017: Comparison with cogent sequences of Rotarix and RotaTeq vaccine strains before their use for universal mass vaccination

The present study was conducted to analyze the genotypic diversity of circulating species A rotavirus (RVA) strains in Iran and also to investigate comparative analysis between the genotypes of VP4 and VP7 of cocirculating RVA and vaccine strains before the vaccine is introduced in the national immunization program. The G3-lineage I was found in this study as the most common G genotype which was followed by G9-lineage III, G1-lineages I, II, G12-lineage III, G2-lineage IV, and G4-lineage I. Also, P[8]-lineages III, IV was found as the predominant P genotype which was followed by P[4]-lineage V, and P[6]-lineage I. Overally, G3P[8] was determined as the most common combination. Moreover, the analysis of the VP7 antigenic epitopes showed that several amino acid differences existed between circulating Iranian and the vaccine strains. The comparison of genotype G1 of Iranian and vaccine strains (RotaTeq and Rotarix), and genotypes G2, G3, and G4 of Iranian and RotaTeq vaccine strains revealed three to five amino acids differences on the VP7 antigenic epitopes. Furthermore, analyzing of the VP8* epitopes of Iranian P[8] strains indicated that they contained up to 11 and 14 amino acid differences with Rotarix and RotaTeq, respectively. Based on different patterns of amino acid substitutions in circulating and vaccine strains, the emergence of antibody escaping mutants and potentially the decrease of immune protection might ensue in vaccinated children. However, considering the broad cross-protective activity of RVA vaccines, their efficacy should be monitored after the introduction in Iran.     

Isolation of a human rotavirus containing a bovine rotavirus VP4 gene that suppresses replication of other rotaviruses in coinfected cells

Summary Bovine-human reassortant strains containing ten human rotavirus gene segments and segment 4, encoding VP4, of a bovine rotavirus were isolated from the stool of an infected Bangladeshi infant during cell culture adaptation. Two plaque purified variants of this reassortant, one making very large (429-L4) and the other tiny (429-S4) plaques, were further analyzed. The electropherotypes of these variants were identical except for slight mobility differences in segment 4. The predicted sequence of amino acids (aa) 16–280 in VP4 proteins revealed four differences between variants even in this limited region, so no single difference could be linked to plaque size. The small plaque variant S4 was phenotypically unstable and mutated to a large plaque-former within a single cell culture passage. The predicted sequence of aa 16–280 of a large plaque variant derived from S4 revealed six changes, only one of which was common to that of the L4 strain, thus suggesting that multiple amino acid changes in VP4 may affect plaque size. Although the large plaque variant L4 grew faster and was released from cells more rapidly than S4, its replication and that of other rotaviruses tested (i.e. RRV, NCDV and Wa) was suppressed by S4 in coinfected cells. Using an RRV×S4 reassortant containing only RRV segment 4, it was established that suppression was linked to the S4 VP4 protein. This suppression could not be associated with inhibition of viral adsorption and, therefore, appeared to occur following internalization. Thus, a new property of the rotavirus VP4 protein has been identified in a bovine-human rotavirus reas-sortant.     

Evidence of conserved epitopes in variable region of VP8* subunit of VP4 protein of rotaviruses of P[8]-1 and P[8]-3 lineages

Although antibody responses to the human rotavirus VP4 protein have been reported, few studies have analyzed the specificity of these responses to the VP8* subunit. This study investigated antibody responses generated against the variable region of the VP4 protein (VP8* subunit) in children infected with rotavirus genotype P[8]. Recombinant VP8* subunit (rVP8*) and truncations corresponding aa 1-102
(peptide A) and 84-180 (peptide B) of rotavirus strains P[8]-1 and P[8]-3 lineages were expressed in Escherichia coli and examined for antibody reactivity using ELISA and Western blot assays. Sera from infected children had IgG antibodies that reacted with full-length rVP8*, peptide A and B of both lineages, with stronger reactivity observed against peptide B. In addition, anti-strain Wa (P[8]-1) and anti-rVP8* (P[8]-3) rabbit polyclonal antiserum reacted against peptide B sequences of both lineages. These data indicate that the VP8* variable region of rotavirus belonging to P[8]-1 and P[8]-3 lineages have conserved epitopes recognized by antibodies elicited during natural infections.     

Development of competitive ELISA for serodiagnosis on African horsesickness virus using baculovirus expressed VP7 and monoclonal antibody

VP7, the sero-group common antigen, of African horsesickness virus (AHSV-4) was expressed in insect cells by recombinant baculovirus. To develop a specific diagnostic method, monoclonal antibody (Mab) against VP7 was prepared and investigated as diagnostic reagent with the baculovirus expressed VP7. However, the Mab against VP7 of AHSV cross-reacted with Chuzan virus by the indirect immunofluorescence assay (IFA), confirming the presence of conserved domain of VP7 among Orbiviruses. This study describes two types of ELISA; Mab linked indirect (I-ELISA) and competitive-ELISA (C-ELISA) using baculovirus expressed VP7 as an antigen. These ELISAs were compared for serodiagnosis of AHSV showing that C-ELISA was more specific than I-ELISA. The results indicated that C-ELISA is applicable to serodiagnosis of AHSV regardless of serotypes.     

An Anti-apoptosis Gene of the Bcl-2 Family from Marine Birnavirus Inhibiting Apoptosis of Insect Cells Infected with Baculovirus

VP5 is a 15-kDa nonstructural protein encoded by a small open reading frame in 5′-terminal of segment A of the Marine Birnavirus (MABV) (strainY-6) genome. Comparisons of the amino acid sequence of the VP5 with other Bcl-2 family member proteins indicated that the VP5 protein contains Bcl-2 homology (BH) domains BH1, BH2, BH3, and BH4, but without the transmembrane region. The VP5 gene from MABV was fused to enhancing green fluorescence protein (eGFP) gene and inserted into the baculovirus genome under the control of polyhedrin gene promoter, and then was highly expressed in insect cells. The expressed VP5 was capable of enhancing insect cell viability, prevented membrane blebbing and delayed DNA internucleosomal cleavage when cells were infected with the recombinant virus. The results suggested that the VP5 of MABV is a novel anti-apoptosis gene, which could regulate the cell apoptosis-off system.     

Nucleotide sequence and expression inE. coli of the complete P4 type VP4 from a G2 serotype human rotavirus

Summary The complete sequence of a P4 type VP4 gene from a G2 serotype human rotavirus, IS2, isolated in India has been determined. Although the IS2 VP4 is highly homologous to the other P4 type alleles, it contained acidic amino acid substitutions at several positions that make it acidic among the P4 type alleles that are basic. Moreover, comparative sequence analysis revealed unusual polymorphism in members of the P4 type at amino acid position 393 which is highly conserved in members of other VP4 types. To date, expression of complete VP4 inE. coli has not been achieved. In this study we present successful expression inE. coli of the complete VP4 as well as VP8* and VP5* cleavage subunits in soluble form as fusion proteins of the maltose-binding protein (MBP) and their purification by single-step affinity chromatography. The hemagglutinating activity exhibited by the recombinant protein was specifically inhibited by the antiserum raised against it. Availability of pure VP4 proteins should facilitate development of polyclonal and monoclonal antibodies (MAbs) for P serotyping of rotaviruses.     

Sequence comparison of the VP7 gene encoding the outer capsid glycoprotein among animal and human group C rotaviruses

Summary The nucleotide sequences of the outer capsid glycoprotein (VP7) genes from the Shintoku bovine and the HF and WH porcine group C rotaviruses were determined and compared with those of the published corresponding genes from the Cowden porcine and Ehime human group C rotaviruses. The VP7 genes of all 5 strains were 1063 nucleotides in length and possess one open reading frame encoding a polypeptide of 332 amino acids. Comparative analysis of the deduced amino acid sequences indicated that 85.2–97.0% identity was observed for the VP7 of the serotypically related strains of group C rotaviruses (Cowden, WH and Ehime) whereas 69.9–74.7% identity was observed among the serotypically distinct strains (Shintoku; Cowden, WH and Ehime; and HF). At least 8 variable regions in the VP7 were recognized among serotypically distinct strains, and these locations were similar to those of the variable regions in the VP7 of group A rotaviruses.     

Mapping of epitopes of VP2 protein of chicken anemia virus using monoclonal antibodies

To map the epitopes of VP2 protein of chicken anemia virus (CAV), VP2 was expressed as a fusion protein in Escherichia coli BL21 (DE3). The Western blot demonstrated that recombinant VP2 protein could be recognized by sera of chickens infected with CAV. Female BALB/c mice were immunized with purified recombinant VP2 produced in E. coli BL21 (DE3) and seven VP2-specific monoclonal antibodies (MAbs) were developed. The results of Western blot showed that all the seven MAbs recognized the recombinant VP2 protein expressed in the baculovirus and reacted with MDCC-MSB1 cells infected with CAV by indirect immunofluorescence assay. The VP2 protein was dissected into 21 overlapping fragments, expressed as fusion peptides in E. coli and used for epitope mapping by pepscan analysis. ELISA and Western blot assays indicated that most of MAbs reacted with the 12th and 13th fragments (amino acids 111-136) and one of them reacted with the 3rd fragment (amino acids 21-36). The linear immunodominant epitope of VP2 was located mainly in amino acid residues 111-126 and 121-136.     

Recombinant proteins VP1 and VP3 of hepatitis A virus prime for neutralizing response

Six overlapping genomic regions of capsid proteins VP1 and VP3 of hepatitis A virus (HAV) inserted into the expression vectors pBD or pUR respectively expressed beta-galactosidase-HAV fusion proteins. The recombinant proteins were poorly soluble so they were difficult to detect by human anti-HAV sera in radioimmunoassay, but the fusion proteins dissolved in sodium dodecyl sulfate reacted with human and rabbit anti-HAV-positive sera in immunoblots. Antisera against VP1 and VP3 recombinant proteins reacted with the respective structural proteins of HAV in immunoblots. Two recombinant proteins, one including the first 120 amino acids of the N-terminus of VP1 and the other containing all of VP1 except for the first 60 N-terminal amino acids, induced a transient neutralizing antibody response in rabbits. Antisera directed against other regions of VP1 and VP3 neither neutralized viral infectivity nor recognized native virus in a competitive radioimmunoassay. However, when immunized animals were challenged with a sub-immunogenic dose of HAV, all animals responded with stable virus-neutralizing antibodies.     

VP1 pseudocapsids,but not a glutathione-S-transferase VP1 fusion protein,prevent polyomavirus infection in a T-cell immune deficient experimental mouse model

The ability to vaccinate against polyomavirus infection in a T-cell deficient as well as a normal immune context was studied using polyomavirus major capsid protein (VP1) pseudocapsids (VP1-ps) or a glutathione-S-transferase-VP1 (GST-VP1) fusion protein. VP1-ps (1 or 10 microg) were administered subcutaneously, alone or together with Freund's complete and incomplete adjuvant, to CD4(-/-)8(-/-) T-cell deficient or normal C57Bl/6 mice on four occasions. Alternatively, CD4(-/-)8(-/-) and normal mice were inoculated with either GST-VP1 or Py-VP1-ps (5 microg). Following immunisation, antibody titres were tested by ELISA to VP1-ps or GST-VP1 or by haemagglutination inhibition (HAI). Mice were then infected with polyomavirus. Three weeks post-infection, the mice were killed and examined for the presence of polyomavirus DNA by PCR. Viral DNA was not detected in CD4(-/-)8(-/-) mice immunised with either VP1-ps alone or in combination with Freund's complete and incomplete adjuvant, or in any of the normal mice immunised with VP1-ps or GST-VP1. However, viral DNA was detected in 2/5 of the CD4(-/-)8(-/-) mice immunised with GST-VP1 and in non-immunised controls. Greater antibody titres were observed to VP1-ps than to GST-VP1 in CD4(-/-)8(-/-) mice after VP1-ps compared to GST-VP1 immunisation and antibody responses were better in normal than in immune-deficient mice. Only immunisation with VP1-ps resulted in haemagglutination inhibition. Complete protection against polyomavirus infection in the T-cell deficient context was obtained with VP1-ps, but not with GST-VP1, immunisation using the present vaccination protocol.     

Identification of a novel VP4 genotype carried by a serotype G5 porcine rotavirus strain

Rotavirus genome segment 4, encoding the spike outer capsid VP4 protein, of a porcine rotavirus (PoRV) strain, 134/04-15, identified in Italy was sequenced, and the predicted amino acid (aa) sequence was compared to those of all known VP4 (P) genotypes. The aa sequence of the full-length VP4 protein of the PoRV strain 134/04-15 showed aa identity values ranging from 59.7% (bovine strain KK3, P8[11]) to 86.09% (porcine strain A46, P[13]) with those of the remaining 25 P genotypes. Moreover, aa sequence analysis of the corresponding VP8* trypsin cleavage fragment revealed that the PoRV strain 134/04-15 shared low identity, ranging from 37.52% (bovine strain 993/83, P[17]) to 73.6% (porcine strain MDR-13, P[13]), with those of the remaining 25 P genotypes. Phylogenetic relationships showed that the VP4 of the PoRV strain 134/04-15 shares a common evolutionary origin with porcine P[13] and lapine P[22] rotavirus strains. Additional sequence analyses of the VP7, VP6, and NSP4 genes of the PoRV strain 134/04-15 revealed the highest VP7 aa identity (95.9%) to G5 porcine strains, a porcine-like VP6 within VP6 genogroup I, and a Wa-like (genotype B) NSP4, respectively. Altogether, these results indicate that the PoRV strain 134/04-15 should be considered as prototype of a new VP4 genotype, P[26], and provide further evidence for the vast genetic and antigenic diversity of group A rotaviruses.     

The VP1 protein of avian encephalomyelitis virus is a major host-protective immunogen that serves as diagnostic potential

Avian encephalomyelitis virus (AEV) is an important pathogen of poultry and is classified as a member of Picornaviridae. To investigate the protective immunity induced by AEV structural proteins, recombinant VP1, VP0, and VP3 proteins were expressed in a baculovirus system. The result of in vivo protection assays shows that the VP1 protein is a major host-protective immunogen against AEV challenge and demonstrates further that the antibody raised against VP1 protein could neutralize more effectively AEV infection than antibody against VP3 or VP0 protein in a virus neutralization test. These purified recombinant proteins were subsequently evaluated as enzyme-linked immunosorbent assay (ELISA) antigens for detection of AEV infection. A total number of 50 positive sera and 30 negative sera were tested for ELISA validation. Results obtained by testing 193 sera from chickens suspected of being infected AEV further showed that the diagnostic sensitivities of the VP1, VP3, and VP0 protein-based ELISAs were 98.1, 80.6, and 51.9%, and their specificities were 100, 87.9, and 81.8%, respectively. Both sensitivity and specificity of the VP1 protein-based ELISA were comparable with a commercially available test, indicating that the VP1 protein has a highly promising and reliable diagnostic potential, and thus is a suitable antigen for ELISA detection of AEV antibodies in chickens.