Humoral immune response to rotavirus NSP4 enterotoxin in Spanish children

The rotavirus non-structural protein 4 (NSP4) has been shown to play a crucial role in rotavirus-induced diarrhea, acting as a viral enterotoxin. It has also been demonstrated that antibody to NSP4 can reduce the severity of rotavirus-induced diarrhea in newborn mice. Two recombinant baculoviruses, expressing the NSP4 protein from the SA11 and Wa rotavirus strains, genotypes A and B, respectively, were used to produce and purify these glycoproteins, which were applied as antigen in an enzyme-linked immunosorbent assay (ELISA) to test the specific antibody response to NSP4 in human sera. Serum samples from 30 children convalescing from a rotavirus infection, from 54 healthy children under 5-years-old, and from 49 adults were tested to determine the presence of antibodies to the viral enterotoxin and to rotavirus structural proteins. Seventy percent of the sera from rotavirus-infected children showed an IgG antibody response to either one or both NSP4 proteins used in this study, although the response was weak. However, IgG antibodies towards either one or both NSP4 proteins were only detected in 26% of the non-convalescent healthy children and in only 18% of the adults. No serum IgA antibodies towards NSP4 were found in this study. IgG antibody recognition of the NSP4 protein from the SA11 and Wa rotavirus strains was not always heterotypic.     

Evaluation of serum antibody responses against the rotavirus nonstructural protein NSP4 in children after rhesus rotavirus tetravalent vaccination or natural infection

The immune response elicited by the rotavirus nonstructural protein NSP4 and its potential role in protection against rotavirus disease are not well understood. We investigated the serological response to NSP4 and its correlation with disease protection in sera from 110 children suffering acute diarrhea, associated or not with rotavirus, and from 26 children who were recipients of the rhesus rotavirus tetravalent (RRV-TV) vaccine. We used, as antigens in an enzyme-linked immunosorbent assay (ELISA), affinity-purified recombinant NSP4 (residues 85 to 175) from strains SA11, Wa, and RRV (genotypes A, B, and C, respectively) fused to glutathione S-transferase. Seroconversion to NSP4 was observed in 54% (42/78) of the children who suffered from natural rotavirus infection and in 8% (2/26) of the RRV-TV vaccine recipients. Our findings indicate that NSP4 evokes significantly (P < 0.05) higher seroconversion rates after natural infection than after RRV-TV vaccination. The serum antibody levels to NSP4 were modest (titers of < or = 200) in most of the infected and vaccinated children. A heterotypic NSP4 response was detected in 48% of the naturally rotavirus-infected children with a detectable response to NSP4. Following natural infection or RRV-TV vaccination, NSP4 was significantly less immunogenic than the VP6 protein when these responses were independently measured by ELISA. A significant (P < 0.05) proportion of children who did not develop diarrhea associated with rotavirus had antibodies to NSP4 in acute-phase serum, suggesting that serum antibodies against NSP4 might correlate with protection from rotavirus diarrhea. In addition, previous exposures to rotavirus did not affect the NSP4 seroconversion rate.     

Evaluation of Serum Antibody Responses against the Rotavirus Nonstructural Protein NSP4 in Children after Rhesus Rotavirus Tetravalent Vaccination or Natural Infection

The immune response elicited by the rotavirus nonstructural protein NSP4 and its potential role in protection against rotavirus disease are not well understood. We investigated the serological response to NSP4 and its correlation with disease protection in sera from 110 children suffering acute diarrhea, associated or not with rotavirus, and from 26 children who were recipients of the rhesus rotavirus tetravalent (RRV-TV) vaccine. We used, as antigens in an enzyme-linked immunosorbent assay (ELISA), affinity-purified recombinant NSP4 (residues 85 to 175) from strains SA11, Wa, and RRV (genotypes A, B, and C, respectively) fused to glutathione S-transferase. Seroconversion to NSP4 was observed in 54% (42/78) of the children who suffered from natural rotavirus infection and in 8% (2/26) of the RRV-TV vaccine recipients. Our findings indicate that NSP4 evokes significantly (P < 0.05) higher seroconversion rates after natural infection than after RRV-TV vaccination. The serum antibody levels to NSP4 were modest (titers of ≤200) in most of the infected and vaccinated children. A heterotypic NSP4 response was detected in 48% of the naturally rotavirus-infected children with a detectable response to NSP4. Following natural infection or RRV-TV vaccination, NSP4 was significantly less immunogenic than the VP6 protein when these responses were independently measured by ELISA. A significant (P < 0.05) proportion of children who did not develop diarrhea associated with rotavirus had antibodies to NSP4 in acute-phase serum, suggesting that serum antibodies against NSP4 might correlate with protection from rotavirus diarrhea. In addition, previous exposures to rotavirus did not affect the NSP4 seroconversion rate.     

人轮状病毒NSP4蛋白131和133位氨基酸变异对毒力影响的研究

目的研究两株A组人轮状病毒NSP4蛋白131位和133位氨基酸位点的变异对毒力的影响。方法从昆明地区2002年和2005年秋冬季婴幼儿腹泻流行期不同程度腹泻患儿中分离得到两株轮状病毒流行株02k38和05k44,RT-PCR扩增NSP4全长基因,进行cDNA序列测序。通过pGEX-5X-1载体,转化E.coliBL21以谷胱苷肽S-转移酶融合蛋白的形式表达两株病毒的NSP4蛋白C-端86～170位氨基酸（GST-NSP486-170）;并酶切融合头纯化得到两种NSP486-170蛋白,在ICR乳鼠中比较这两种蛋白致小鼠腹泻的差异。结果两种NSP486-170蛋白毒性无差异。结论两株轮状病毒131位和133位氨基酸位点的变异不影响毒力的改变,其致腹泻活性没有明显差异（P〉0.05）。     

Genomic characterization of a novel group A lamb rotavirus isolated in Zaragoza, Spain

An ovine rotavirus (OVR) strain, 762, was isolated from a 30-day-old lamb affected with severe gastroenteritis, in Zaragoza, Spain, and the VP4, VP7, VP6, NSP4, and NSP5/NSP6 genes were subsequently characterized molecularly. Strain OVR762 was classified as a P[14] rotavirus, as the VP4 and VP8* trypsin-cleavage product of the VP4 protein revealed the highest amino acid (aa) identity (94% and 97%, respectively) with that of the P11[14] human rotavirus (HRV) strain PA169, isolated in Italy. Analysis of the VP7 gene product revealed that OVR762 possessed G8 serotype specificity, a type common in ruminants, with the highest degree of aa identity (95-98%) shared with serotype G8 HRV, bovine rotavirus, and guanaco (Lama guanicoe) rotavirus strains. Moreover, strain OVR762 displayed a bovine-like NSP4 (genotype E2) and NSP5/NSP6 (genotype H3), and a VP6 genotype I2, as well as a long electropherotype pattern. This is the first report of a lamb rotavirus with P[14] and G8 specificities, providing additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.     

Species-specific but not genotype-specific primary and secondary isotype-specific NSP4 antibody responses in gnotobiotic calves and piglets infected with homologous host bovine (NSP4[A]) or porcine (NSP4[B]) rotavirus

Using recombinant baculoviruses expressing rotavirus NSP4 [A], [B], [C], and [D] genotypes of bovine, porcine, human, simian, or murine origin, we analyzed serum antibody responses to NSP4s in gnotobiotic calves and piglets infected by the oral/alimentary or intraamniotic route with bovine (NSP4[A]) (Wyatt, R.G., Mebus, C.A., Yolken, R.H., Kalica, A.R., James, H.D., Jr., Kapikian, A.Z., Chanock, R.M., 1979. Rotaviral immunity in gnotobiotic calves: heterologous resistance to human virus induced by bovine virus. Science 203(4380), 548-550) or porcine (NSP4[B]) (Hoshino, Y., Saif, L.J., Sereno, M.M., Chanock, R.M., Kapikian, A.Z., 1988. Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen. J. Virol. 62(3), 744-748) rotaviruses. Following primary infection and challenge with virulent rotaviruses, the animals developed higher or significantly higher antibody titers to homologous host homotypic NSP4s than to heterologous host homotypic or heterologous host heterotypic NSP4s, indicating that antibody responses were species specific rather than genotype specific. Antibody responses to NSP4s corresponded closely with the phylogenetic relationships of NSP4s within a species-specific region of amino acids (aa) 131-141. In contrast, NSP4 genotypes determined by amino acid full-length sequence identity predicted poorly their "serotypes". In piglets, antibodies to NSP4 induced by previous oral infection failed to confer protection against challenge from a porcine rotavirus bearing serotypically different VP4 and VP7 but essentially identical NSP4 to the porcine rotavirus in primary infection. Thus, in an approach to immunization with a live oral rotavirus vaccine, the NSP4 protein does not appear to play an important role in protection against rotavirus disease and infection.     

Systemic and intestinal antibody responses to NSP4 enterotoxin of Wa human rotavirus in a gnotobiotic pig model of human rotavirus disease

Antibody responses to the Wa human rotavirus (HRV) nonstructural protein NSP4, a viral enterotoxin, were evaluated in neonatal gnotobiotic (Gn) pigs. Gn pigs were inoculated orally with one dose of 10(5) fluorescent focus units (FFU) of virulent Wa HRV (HRV-V), to mimic natural infection, or with three doses of 5 x 10(7) FFU attenuated Wa HRV (HRV-A) at 10-day intervals, to mimic oral attenuated rotavirus vaccines, or they were mock inoculated (mock). Subsets of pigs were challenged with 10(6) FFU of virulent Wa HRV at post-inoculation day 28 (PID 28). Post-challenge, the HRV-V pigs were completely protected against diarrhea and virus shedding, whereas the HRV-A pigs had a 50% protection rate against diarrhea and a 67% protection rate against virus shedding. All mock-inoculated pigs shed virus and had diarrhea post-challenge. Isotype antibody titers to NSP4 were compared in serum and intestinal contents, at post-inoculation day (PID) 28 and at post-challenge day 7 (PCD 7/PID 35) by indirect ELISA, using purified recombinant NH2-6xHis-tagged NSP4 of virulent Wa HRV. Pre-challenge, both the HRV-V and HRV-A-inoculated pigs had similar moderate titers of serum IgG antibodies to NSP4. However, only the HRV-V-inoculated pigs developed detectable serum and intestinal IgA antibody titers to NSP4 pre-challenge, compared with the HRV-A-inoculated pigs. The mock-inoculated pigs had no IgM, IgA, or IgG antibodies to NSP4 pre-challenge. All Wa HRV-inoculated pigs developed low to moderate titers of serum IgM, IgG, and IgA antibodies to NSP4 post-challenge, but the mock-inoculated pigs had only IgM antibodies post-challenge. Both Wa HRV-inoculated groups developed low titers of IgA antibody to NSP4 in the small intestinal contents post-challenge, but titers were 5.8-fold higher in the HRV-V pigs. Our results concur with findings that both rotavirus vaccinated and naturally infected children seroconvert with modest IgG antibodies to NSP4 [Johansen et al. (1999) J Med Virol 59:369-367]. These data suggest that Gn pigs could be a useful model to evaluate serum and intestinal IgA antibodies to NSP4 and their role in protection against HRV infection. Further experiments may clarify whether (1) the NSP4 antibodies detected pre-challenge in the HRV-V pigs contribute to the higher protection rates observed, or (2) the reduced or delayed NSP4 antibody responses of the HRV-A pigs are associated with the lower protection rates in these pigs.     

Rotavirus cross-species pathogenicity: molecular characterization of a bovine rotavirus pathogenic for pigs

Rotaviruses which cause disease in heterologous animal species have been reported but the molecular basis of cross-species infectivity and disease is not established. We report the molecular characterization of a cloned rotavirus, PP-1, which was originally obtained from cattle and which had been biologically characterized in vivo in two target animal species, gnotobiotic pigs and calves. In pigs, PP-1 caused severe clinical disease but in experimental calves it replicated subclinically. PP-1 was characterized as a G3 reassortant with a porcine VP4 and NSP4 but a bovine NSP1. The PP-1 VP4 had 96 to 97% deduced amino acid identity to P[7] porcine rotaviruses and P[7] specificity was confirmed with VP4-specific monoclonal antibodies. Sequence analysis of the PP-1 NSP1 showed 94 to 99.6% deduced amino acid identity to bovine rotaviruses but the NSP4 protein had 94 to 98% identity to the NSP4 genotype B porcine rotaviruses. G-typing PCR initially classified PP-1 as a G10 rotavirus but sequence analysis revealed 92 to 96% identity of the PP-1 VP7 with porcine, simian, and human G3 rotaviruses. These results, combined with the in vivo properties of PP-1 in the two target species, supported the concept that species-specific VP4 and NSP4, but not NSP1, are required to induce rotavirus disease, at least in calves and pigs. The results illustrate experimentally that rotaviruses circulating in one animal species can pose a risk to another by the emergence of a pathogenic reassortant rotavirus under appropriate conditions.     

中国轮状病毒非结构蛋白NSP4基因变异特征的分析

目的　研究我国轮状病毒流行株NSP4基因的变异特点。方法　对近年来从我国不同地区获得的 2 7份人轮状病毒流行株的NSP4基因用RT PCR进行扩增 ,克隆后进行全长cDNA序列分析 ,并利用Clustal× 1.8,TreeView3 2及DNAStar软件与参比株Wa、KUN、AU 1、EW及来自GenBank的OSU、SA11、Hochi、US2 44、Bristol株的NSP4序列进行分析比较。采用PCR分型方法对VP7血清型进行鉴定 ,确定轮状病毒G型与NSP4基因型的关系。结果　氨基酸同源性比较表明 ,我国轮状病毒不同流行株NSP4之间同源性为 81.7%～ 99.4% ,据此可将 2 7株RVNSP4分为 2组 ,分别以Wa株和KUN株为代表 ,其中以Wa组为主。组内同源性分别为 92 .0 %～ 99.4%和 92 .0 %～ 98.9% ,组内变异率分别为 0～ 8 5 %及 1 2 %～ 8 5 %。两组间变异率达 16 6%～ 2 1 0 %。氨基酸进化树提示在Wa组内包括 3个亚组。轮状病毒G血清型与NSP4基因型之间的联系不确定。结论　我国流行株NSP4基因主要可分为Wa组和KUN组 ,在Wa组内可形成三个亚组 ,并且在高变区有特征性的氨基酸位点。NSP4的变异与年份有关而与地域关系不密切     

Conserved structural features of nonstructural glycoprotein NSP4 between group A and group C rotaviruses

Summary.  The nonstructural glycoprotein NSP4 of group C human rotavirus strain Ehime 9301 was determined to be 150 amino acids in length and 96% identical with the NSP4 of another group C human rotavirus strain Bristol. Both NSP4 sequences were virtually unrelated to group A rotavirus NSP4s. However, the structural features of group A and group C rotavirus NSP4s were similar with hydrophobic domains being in the amino terminus and a coiled coil domain after the membrane-spanning domain, although group C rotavirus NSP4 lacked one amino-terminal hydrophobic domain. Received January 10, 1997 Accepted April 24, 1997     

Characterization of human symptomatic rotavirus isolates MP409 and MP480 having ‘long’ RNA electropherotype and subgroup I specificity, highly related to the P6[1],G8 type bovine rotavirus A5, from Mysore, India

Summary.  In an epidemiological study of symptomatic human rotaviruses in Mysore, India during 1993 and 1994, isolates MP409 and MP480 were isolated from two children suffering from severe, acute dehydrating diarrhea. Both isolates exhibited ‘long’ RNA pattern and subgroup I specificity suggesting the likelihood of their animal origin. Both isolates did not react with monoclonal antibodies (MAbs) specific for serotypes G1 to G6 as well as G10. To determine the genetic origin of these isolates, complete nucleotide sequences of genes encoding the outer capsid proteins VP4 and VP7, nonstructural proteins NSP1 and NSP3 and viral enterotoxin protein NSP4 from MP409 and partial sequences of genes from MP480 were determined. Comparison of the 5′ and 3′ terminal sequences of 250 nucleotides revealed complete identity of the gene sequences in both strains suggesting that MP409 and MP480 are two different isolates of a single strain. Comparison of the nucleotide and deduced amino acid sequences of VP4, VP7, NSP1 and NSP3 of MP409 with published sequences of strains belonging to different serotypes revealed that both outer capsid proteins VP4 and VP7 and NSP1 are highly related to the respective proteins from the P6[1], G8 type bovine rotavirus A5 isolated from a calf with diarrhoea in Thailand and that the NSP3 is highly homologous to that of bovine rotaviruses. The NSP4 protein showed greatest sequence identity with NSP4s belonging to the KUN genetic group to which NSP4s from human G2 type strains and bovine rotaviruses belong. MP409 and MP480 likely signify interspecies transmission of P6[1], G8 type strains from cattle to humans and represent the first P6[1] type rotaviruses isolated in humans. These and our previous studies on the asymptomatic neonatal strain I321 are of evolutionary and epidemiological significance in the context of close association of majority of the Indian population with cattle. Received September 29, 1999 Accepted February 4     

Mutations selected in rotavirus enterotoxin NSP4 depend on the context of its expression

The rotavirus NSP4 protein is cytotoxic when transiently expressed in cells and is capable of inducing secretory diarrhea in neonatal mice. NSP4 consists of 175 amino acids, and sequences important for its toxic effects have been mapped to the carboxy-terminal half of the protein. In this report, we compared NSP4-encoding nucleotide sequences recovered from cell lines engineered to express NSP4 from human rotavirus strain Wa with NSP4 sequences recovered from cells persistently infected with either Wa or simian rotavirus strain SA11. In cells stably transfected with Wa NSP4, we found that proline(138) was changed to either serine or threonine. However, in cells persistently infected with SA11, we found that phenylalanine(33) was changed to leucine, and in cells persistently infected with Wa, no changes were observed in NSP4. Expression of Wa NSP4 in Caco-2 cells resulted in increased cell-doubling times and decreased cell viability in comparison to cells expressing NSP4-serine(138) or NSP4-threonine(138). This result suggests that sequence polymorphism at residue 138 in Wa NSP4 influences the cytotoxicity of the protein. Therefore, mutations in the carboxy-terminal half of NSP4 are selected when NSP4 is expressed in cells in the absence of other viral proteins, but not in the context of viral replication. These findings suggest that cytotoxic functions of NSP4 are not operant during natural rotavirus infection.     

Viral proteins VP2, VP6, and NSP2 are strongly precipitated by serum and fecal antibodies from children with rotavirus symptomatic infection

Rotavirus-specific IgA has been correlated with immune protection against rotavirus reinfection and symptomatic disease. Systemic and mucosal antibody responses were determined by an enzyme-linked immunosorbent assay in 11 infants with severe rotavirus gastroenteritis. Geometric mean titers of antirotavirus serum IgG and IgA antibodies were significantly higher during the convalescence of the disease (P < 0.001 vs. acute-phase titers). Rotavirus-specific fecal sIgA antibodies increased 4 times during the convalescence in 9 (81.8%) children (P < 0.001). The serum IgG and IgA antibody and fecal sIgA antibody responses to individual rotavirus polypeptides were characterized by radioimmunoprecipitation assay (RIPA) using Staphylococcus aureus protein A and the lectin jacalin to precipitate IgG- and IgA-immune complexes, respectively. The main IgG response was directed toward the structural viral proteins VP2, VP4, and VP6 and toward the nonstructural protein NSP2. Serum IgA reactivity was detected by RIPA in all serum samples, with major responses to VP2, VP6, and NSP2. Interestingly, fecal sIgA in convalescent samples reacted strongly toward NSP2 and VP6. These data reinforce the antigenic importance of rotaviral proteins other than VP4 and VP7, such as VP2, VP6, and NSP2, as main targets in the immune response to rotavirus. J. Med. Virol. 56:58–65, 1998. © 1998 Wiley-Liss, Inc.     

Full genomic analysis of a porcine–bovine reassortant G4P[6] rotavirus strain R479 isolated from an infant in China

During the 2004 surveillance of rotaviruses in Wuhan, China, a G4P[6] rotavirus strain R479 was isolated from a stool specimen collected from a 2‐year‐old child with diarrhea. The strain R479 had an uncommon subgroup specificity I + II, and analysis of the VP6 gene suggested that it was related to porcine rotaviruses. In the present study, full‐length nucleotide sequences of all the RNA segments of R479 were determined and analyzed phylogenetically to identify the origin of individual RNA segments. According to the rotavirus genotyping system based on 11 RNA segments, the genotype of R479 was expressed as G4‐P[6]‐I5‐R1‐C1‐M1‐A1‐N1‐T7‐E1‐H1. This genotype includes the porcine‐like VP6 genotype (I5) and bovine‐like NSP3 genotype (T7). Phylogenetic analysis revealed that R479 genes encoding VP1, VP2, VP3, VP6, VP7, VP8*, NSP1, NSP4, and NSP5 were more closely related to those of porcine rotaviruses than human or other animal rotaviruses. In contrast, it was remarkable that the NSP3 gene of R479 was genetically closely related to only a bovine rotavirus strain UK. The NSP2 gene of R479 was also unique and clustered with only the G5P[8] human strain IAL28 and G3P[24] simian strain TUCH. These results suggested that R479 may be a reassortant virus having the NSP3 gene from a bovine rotavirus in the genetic background of a porcine rotavirus, with an NSP2 gene related to the porcine‐human reassortant strain IAL28. To our knowledge, R479 is the first porcine–bovine reassortant rotavirus isolated from a human. J. Med. Virol. 82:1094–1102, 2010. © 2010 Wiley‐Liss, Inc.     

Group C rotavirus NSP4 induces diarrhea in neonatal mice

Summary.  Nonstructural glycoprotein NSP4 of group A rotavirus induces diarrhea in neonatal mice by functioning as an enterotoxin. Previously, our laboratory reported that the structural features of group A and group C rotavirus NSP4 proteins are well conserved despite a lack of sequence homology between group A and group C rotavirus NSP4 proteins [Horie Y, et al., Arch Virol (1997) 142: 1865–1872]. To test whether group C rotavirus NSP4 has an enterotoxigenic activity, we expressed in Escherichia coli the carboxy two-thirds (corresponding to amino acid residues 55–150) of the NSP4 protein derived from group C rotavirus strain Ehime 9301. This truncated NSP4 protein was able to induce diarrhea in 5-day-old CD-1 mice when administered intraperitoneally. Thus, group C rotavirus NSP4 acts as an enterotoxin like group A rotavirus NSP4. Received September 22, 2000 Accepted November 18, 2000     

Detection of a porcine rotavirus strain with VP4, VP7 and NSP4 genes of different animal origins

A new rotavirus strain, sh0902, was detected in diarrheic piglets on a farm in Shanghai, China, and its genotype was characterized as G1P[7]. Analysis of the VP4, VP7 and NSP4 genes demonstrated VP4 homology to bovine and swine rotavirus strains; the nucleotide (nt) and amino acid (aa) identities were 99.7% and 99.5%, respectively. The VP7 gene was highly homologous to that of a giant panda rotavirus strain, with 98.5% similarity at the nt level and 99% similarity at the aa level. The nucleotide sequence of the NSP4 gene displayed high homology to human rotavirus strain R479, with 99.7% identity at the nt level and 99.3% identity at the aa level. This is the first report of an unusual porcine rotavirus strain with VP4, VP7 and NSP4 genes that are highly homologous to bovine, swine, giant panda and human strains isolated at geographically distant sites (South Korea, China and India). Our data indicate that rotaviruses have circulated among humans and animals and undergone genome reassortment.     

Molecular characterization of a rare G3P[3] human rotavirus reassortant strain reveals evidence for multiple human-animal interspecies transmissions

An unusual strain of human rotavirus G3P[3] (CMH222), bearing simian-like VP7 and caprine-like VP4 genes, was isolated from a 2-year-old child patient during the epidemiological survey of rotavirus in Chiang Mai, Thailand in 2000-2001. The rotavirus strain was characterized by molecular analysis of its VP4, VP6, VP7, and NSP4 gene segments. The VP4 sequence of CMH222 shared the greatest homology with those of caprine P[3] (GRV strain) at 90.6% nucleotide and 96.4% amino acid sequence identities. Interestingly, the VP7 sequence revealed highest identity with those of simian G3 rotavirus (RRV strain) at 88% nucleotide and 98.1% amino acid sequence identities. In contrast, percent sequence identities of both the VP4 and VP7 genes were lower when compared with those of human rotavirus G3P[3] reference strains (Ro1845 and HCR3). Analyses of VP6 and NSP4 sequences showed a close relationship with simian VP6 SG I and caprine NSP4 genotype C, respectively. Phylogenetic analysis of VP4, VP6, VP7, and NSP4 genes of CMH222 revealed a common evolutionary lineage with simian and caprine rotavirus strains. These findings strongly suggest multiple interspecies transmission events of rotavirus strains among caprine, simian, and human in nature and provide convincing evidence that evolution of human rotaviruses is tightly intermingled with the evolution of animal rotaviruses.     

Species specificity and interspecies relatedness of NSP4 genetic groups by comparative NSP4 sequence analyses of animal rotaviruses

Summary.  Previous sequence analyses of the rotavirus nonstructural NSP4 from human and some animal rotavirus strains revealed the presence of three distinct NSP4 alleles or genetic groups. To examine the species of origin relatedness and diversity of NSP4, the nucleotide and deduced amino acid sequences of the gene encoding the NSP4 from 15 animal rotavirus strains of porcine, equine, bovine, lapine and canine origin were determined and compared to human and other animal strains sequenced previously. Lapine and equine strains were shown to belong to the NSP4 genotype A. Murine NSP4 sequences formed a previously unrecognized fourth distinct NSP4 genotype (genotype D) that was more divergent compared to NSP4 genotype A, B, and C than the latter three are among each other. Within NSP4 genotypes, strains isolated from rabbits, horses, cows (genotype A) and pigs (genotype B) clustered according to species of origin, suggesting a conserved pattern of evolution within species. NSP4 sequence comparison among one wildtype and two tissue culture-adapted lapine strains, known to cause disease in neonatal rabbits, failed to identify amino acid changes within the variable region spanning amino acids 130 to 141, suggesting that disease in rabbits is the result of the lapine virus infection and replication, including production of the NSP4 enterotoxin. Accepted September 3, 1999/Received July 19, 1999     

Ricin toxin B subunit enhancement of rotavirus NSP4 immunogenicity in mice

A 90-amino acid peptide from the simian rotavirus SA-11 nonstructural protein, NSP4 was linked to the N-terminus of the Ricinus communis A-B toxin B subunit protein (RTB) and synthesized in Escherichia coli. Recombinant RTB and the NSP4(90)::RTB fusion protein bound artificial receptor glycoprotein asialofetuin in an in vitro enzyme-linked immunosorbent assay (ELISA), demonstrating biological activity of the recombinant protein. Mice co-inoculated with purified recombinant RTB plus NSP4(90) peptide proteins or heat denatured NSP4(90)::RTB fusion protein generated higher titers of serum anti-NSP4(90) IgG antibodies than mice immunized with NSP4(90) peptide alone, indicating the presence of adjuvant functions for N-terminal linked RTB. Serum anti-NSP4(90) IgG titers were highest in mice immunized with native recombinant NSP4(90)::RTB fusion protein, confirming the immunostimulatory function of RTB. Results of experiments described here demonstrate the feasibility of using RTB mediated adjuvant functions for stimulation of the antigenicity of a rotavirus nonstructural protein. The ability of recombinant NSP4(90)::RTB fusion protein synthesized in E. coli to bind glycoprotein receptor molecules effectively indicates that protein linkage to the RTB N-terminus and synthesis of the recombinant NSP4(90)::RTB fusion protein in bacteria do not interfere with the immunostimulatory properties of the RTB subunit.     

Serologic and genomic characterization of a G12 human rotavirus in Thailand

The G and P type specificity of the human rotavirus strain T-152 (G12P[9]) isolated in Thailand was serologically confirmed with G12-specific monoclonal antibodies prepared in this study by using a reference G12 strain, L26, as an immunizing antigen and a P[9]-specific monoclonal antibody, respectively. The genomic relationship of strain T-152 with representative human rotavirus strains was examined by means of Northern blot analysis. The results showed that T152 is closely related to strain AU-1 (G3P[9]). Gene 5 (NSP1 gene) of T152, which did not hybridize with those of any other strains examined, was characterized by sequence determination. The T152 NSP1 gene is 1,652 nucleotides in length, encodes 493 amino acids, and exhibits low identity to those of representative human and animal rotaviruses.