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Journal of Virology, July 2008, p. 6753-6757, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00601-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Generation of Recombinant Rotavirus with an Antigenic Mosaic of Cross-Reactive Neutralization Epitopes on VP4

Satoshi Komoto, Masanori Kugita, Jun Sasaki, and Koki Taniguchi^*

Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan

Received 18 March 2008/ Accepted 14 April 2008

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Recombinant rotavirus (RV) with cDNA-derived chimeric VP4 was generated using recently developed reverse genetics for RV. The rescued virus, KU//rVP4(SA11)-II(DS-1), contains SA11 (simian RV strain, G3P[2])-based VP4, in which a cross-reactive neutralization epitope (amino acids 381 to 401) on VP5* is replaced by the corresponding sequence of a different P-type DS-1 (human RV strain, G2P[4]). Serological analyses with a panel of anti-VP4- and -VP7-neutralizing monoclonal antibodies revealed that the rescued virus carries a novel antigenic mosaic of cross-reactive neutralization epitopes on its VP4 surface. This is the first report of the generation of a recombinant RV with artificial amino acid substitutions.

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Rotavirus (RV), a member of the family Reoviridae, is the leading etiological agent of severe gastroenteritis in infants and young children worldwide (15, 16). RV particles possess three concentric capsid layers that enclose an 11-segmented genome of double-stranded RNA (dsRNA) (6). The outer capsid VP4 is a spike protein essential for viral attachment and entry. It is also the major antigenic component on the surface of this virus. VP4 defines P serotypes, and at least 14 P serotypes have been assigned; however, since it is difficult to differentiate P serotypes serologically, P genotypes based on the VP4 sequence have been proposed and 26 P genotypes (P[1] to P[26]) have been reported (6).

The expression of foreign epitopes on viral surfaces has been reported for several positive- and negative-stranded RNA viruses (reviewed in references 1, 7, 14, 21, and 28). In contrast, Reoviridae members such as RV have been refractory to direct genetic manipulation, except for mammalian orthoreovirus (10, 17-19), and therefore could not be used as vectors for the expression of foreign epitopes. Only recently has reverse genetics for RV, which enables one to generate infectious RVs containing a cDNA-derived gene segment, been successful (11). This RV system is based on helper virus-driven reverse genetics, which was originally developed for influenza viruses by Enami and colleagues (4, 5, 12).

In the present study, we used this system to introduce site-specific mutations into one of the three cross-reactive neutralization epitopes of RV VP4, which resulted in the preparation of previously undescribed recombinant RV expressing chimeric VP4 on its surface. The use of this approach to construct chimeric RVs may lead to a new generation of effective vaccine vectors against RV disease, as well as research on the molecular biology of RV.

Generation of recombinant RV carrying chimeric VP4. A previous study revealed cross-reactive neutralization epitopes I, II, and III on VP5* of RV (26). We chose epitope II for expression in the chimeric VP4 molecules because its sequence is highly hydrophobic and it is thought to be especially immunodominant (13, 26). To replace the epitope II sequence in VP4 of the SA11 virus (simian RV strain, G3P[2]), genetic manipulation was carried out in a pX8dT-based (20) T7 RNA polymerase-driven plasmid, pT7/VP4(SA11), encoding the full-length VP4 gene of SA11 (Fig. 1) (11). In the mutated plasmid, pT7/VP4(SA11)-II(DS-1) (Fig. 1), the amino acid sequence (amino acids 381 to 401) of epitope II to be expressed was replaced by the corresponding one from a different P-type virus, DS-1 (human RV strain, G2P[4]), by using a QuikChange II site-directed mutagenesis kit (Stratagene) with primers (+) 5'-AGaCTACCAGTTGGAaAATGGCCTaTTaTgAaTGGGGGAG-3' and (–) 5'-CAtTcAtAAtAGGCCATTtTCCAACTGGTAGtCTAAAATT-3'. The nucleotides shown in lowercase are mutated to modify the amino acid sequence of epitope II from that of SA11. As shown in Fig. 1, the sequence differences between the SA11 and DS-1 viruses in epitope II are found at five amino acids. To generate recombinant RVs carrying chimeric VP4, reverse genetics for RV (11) was performed. Briefly, a monolayer of COS-7 cells, which had been infected beforehand with a recombinant vaccinia virus expressing T7 RNA polymerase (rDIs-T7pol.) (9), was transfected with the constructed pT7/VP4(SA11)-II(DS-1) plasmid and then infected with the KU helper virus (human RV strain, G1P[8]). When cultures of transfected cells were passaged in fresh MA104 cells in the presence of two neutralizing monoclonal antibodies (N-MAbs), YO-2C2 (22) and ST-1F2 (23), that specifically neutralize the KU helper virus, an RV-induced cytopathic effect was detected. The rescued virus, named KU//rVP4(SA11)-II(DS-1), was plaque purified three times.

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FIG. 1. Schematic representation of SA11 virus-based transcription plasmids encoding the full-length VP4 gene. Plasmid pT7/VP4(SA11) contains the authentic full-length VP4 gene cDNA of SA11, flanked by the T7 RNA polymerase promoter and hepatitis delta virus (HDV) ribozyme, followed by the T7 RNA polymerase terminator. Manipulation of the VP4 gene by means of amino acid mutations (positions are indicated by bold letters and asterisks below the sequences) was carried out in pT7/VP4(SA11); the mutant plasmid pT7/VP4(SA11)-II(DS-1) contains five amino acid mutations within the epitope II sequence. Numbers indicate the amino acid positions in the SA11 VP4 sequence. PT7, Rib, and TT7 denote the T7 RNA polymerase promoter, the HDV ribozyme, and the T7 RNA polymerase terminator, respectively.

RNA analysis of the rescued virus. Virion dsRNAs from the rescued KU//rVP4(SA11)-II(DS-1) virus were extracted and then analyzed by polyacrylamide gel electrophoresis (Fig. 2A) as described previously (11). As expected, the VP4 dsRNA of the rescued KU//rVP4(SA11)-II(DS-1) virus (Fig. 2A, lane 3) migrated to almost the same position as the corresponding segments of the SA11 virus (lane 4) and the recombinant KU//rVP4(SA11) virus possessing a cDNA-derived authentic SA11 VP4 genome with a KU backbone (lane 2) (11), the mobility being slower than that of the VP4 segment of the KU helper virus (lanes 1 and 5). Direct sequencing of the rescued virus also indicated that the designed six nucleotides mutations in the epitope II sequence were stably introduced into the VP4 dsRNA segment of the infectious RV (Fig. 2B). These results confirmed that the rescued KU//rVP4(SA11)-II(DS-1) virus is a KU-based recombinant virus carrying a chimeric VP4 gene and proved that substitutions at this particular site are compatible with the preparation of a fully functional virus.

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FIG. 2. Rescue of recombinant viruses containing a cDNA-derived chimeric VP4 genome. (A) Polyacrylamide gel electrophoresis of dsRNAs extracted from the rescued VP4 gene transfectants. Lanes 1 and 5, dsRNAs from the KU helper virus; lanes 2 and 3, dsRNAs from the recombinant KU//rVP4(SA11) virus containing the cDNA-derived authentic VP4 gene (lane 2) and the rescued KU//rVP4(SA11)-II(DS-1) virus (lane 3); lane 4, dsRNAs of SA11 used for VP4 gene cloning. The numbers on the left indicate the order of the genomic dsRNA segments of the KU helper virus. (B) Site-specific mutations introduced into the dsRNA genome of the rescued VP4 gene transfectant. The full-length VP4 genes of the recombinant KU//rVP4(SA11) and KU//rVP4(SA11)-II(DS-1) viruses were amplified by reverse transcription-PCR to yield a 2,386-bp product. The 2,386-bp fragments were directly sequenced, which demonstrated the site-specific mutations introduced within the VP4 genome of the rescued KU//rVP4(SA11)-II(DS-1) virus (positions are indicated by asterisks below the sequence).

Serological characterization of the rescued virus. The antigenic properties of the rescued KU//rVP4(SA11)-II(DS-1) virus were investigated by an enzyme-linked immunosorbent assay (ELISA) and a plaque neutralization assay, using a panel of anti-VP4 and -VP7 N-MAbs (22-26). Different anti-VP4 N-MAbs are found to recognize each of the three cross-reactive neutralization epitopes (26): epitope I (defined by YO-2C2), epitope II (defined by KU-6B11, ST-1F2, and S2-2F2), and epitope III (defined by KU-4D7). Notably, KU-6B11 and ST-1F2 recognize the epitope II sequences of at least the P[2] and P[8] types, and S2-2F2 is directed to that of the P[4] type. First, antigen capture ELISA was performed using these anti-VP4 (YO-2C2, KU-6B11, ST-1F2, S2-2F2, and KU-4D7) and anti-VP7 (KU-4, YO-1E2, and S2-2G10) N-MAbs, as described previously (11) (Fig. 3A). The SA11 (G3P[2]), recombinant KU//rVP4(SA11) (G1P[2]), and KU (G1P[8]) viruses reacted with KU-6B11 (directed to epitope II), which is found to react commonly with the P[2] and P[8] types, but did not react with S2-2F2 (epitope II), which recognizes the P[4] type, such as the DS-1 virus (G2P[4]). The KU helper virus also showed reactivity with ST-1F2 (epitope II), which selectively reacts with the P[8] type. On the other hand, the rescued KU//rVP4(SA11)-II(DS-1) virus lost reactivity with KU-6B11 and ST-1F2 but showed reactivity with S2-2F2, whereas the DS-1 virus reacted with KU-6B11 moderately. In addition, this rescued KU//rVP4(SA11)-II(DS-1) virus showed no reactivity with YO-2C2 (epitope I) or KU-4D7 (epitope III), as found for SA11 and KU//rVP4(SA11) viruses containing authentic SA11 VP4. Further, the KU//rVP4(SA11)-II(DS-1), KU//rVP4(SA11), and KU helper viruses reacted with anti-VP7 KU-4, specific for the G1 type, but not with YO-1E2 or S2-2G10, specific for G3 or G2, respectively. These results demonstrated that the rescued KU//rVP4(SA11)-II(DS-1) virus is a KU-based recombinant virus expressing both the DS-1- and SA11-derived epitopes on its VP4 surface, that is, this chimeric virus carries the designed antigenic mosaic of cross-reactive neutralization epitopes on its VP4 surface.

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FIG. 3. Serological analyses of rescued viruses. (A) Reactivity patterns of the rescued viruses with a panel of anti-VP4 (YO-2C2, KU-6B11, ST-1F2, S2-2F2, and KU-4D7) and anti-VP7 (KU-4, YO-1E2, and S2-2G10) N-MAbs by antigen capture ELISA: phosphate-buffered saline (PBS) alone (no virus) (virus 1), SA11 (virus 2), KU//rVP4(SA11) (virus 3), KU//rVP4(SA11)-II(DS-1) (virus 4), KU (virus 5), and DS-1 (virus 6). Values are expressed as relative absorbances, with values for KU (in cases of YO-2C2, KU-6B11, ST-1F2, KU-4D7, and KU-4), DS-1 (S2-2F2 and S2-2G10), and SA11 (YO-1E2) normalized to 1.0. The experiment was repeated three times with similar results, and representative results are shown. (B) Neutralization of the rescued viruses by anti-VP4 N-MAbs (KU-6B11 and S2-2F2). For the plaque neutralization assay, CV-1 cells per well of a six-well plate were infected with approximately 100 PFU of virus which had been preincubated with different N-MAb dilutions at 37°C for 1 h. After 1 h of adsorption, the virus-antibody mixtures were removed prior to the addition of the primary overlay medium. At 2 days postinfection, the cell monolayers were stained with secondary overlay medium containing 0.7% agarose and 0.005% neutral red. Values are expressed as relative virus titers, with the value for no N-MAb normalized to 100%. The data shown are the mean viral titers and standard deviations for three independent cell cultures.

Next, a plaque neutralization test involving two representative anti-VP4 N-MAbs (KU-6B11 and S2-2F2) was performed. As shown in Fig. 3B, the KU//rVP4(SA11) virus (G1P[2]) was neutralized by KU-6B11, which recognizes the P[2] type, but not by S2-2F2, which recognizes the P[4] type. Conversely, the rescued KU//rVP4(SA11)-II(DS-1) virus was neutralized by S2-2F2 but not by KU-6B11, although S2-2F2 neutralized this chimeric virus less efficiently than it neutralized DS-1 (G2P[4]). The less efficient neutralization of this chimeric virus by S2-2F2 suggests that although the replacement of the epitope II sequence did not affect the property of VP4 as far as binding with this N-MAb is concerned, it may have caused a slight conformational change around the domain that resulted in less-efficient neutralization by this N-MAb. These results may reflect the finding that epitope II appears to be conformational (26). Also, the observation that DS-1 but not chimeric KU//rVP4(SA11)-II(DS-1) was moderately recognized (Fig. 3A) and neutralized (Fig. 3B) by KU-6B11 is possibly due to a slight conformational change around epitope II in the chimeric virus with the genetic manipulations. In any case, these results revealed that the antigenic properties of the rescued KU//rVP4(SA11)-II(DS-1) virus changed from those of the parental KU//rVP4(SA11) virus due to substitutions within the epitope II sequence, leading to the expression of the replaced DS-1-derived epitope II on its VP4 surface.

Infectivity of the rescued virus. The VP4 spike protein possesses a single internal hydrophobic domain (amino acids 384 to 401) that exhibits sequence similarity with the internal fusogenic domain of the E1 glycoprotein of some alphaviruses (13) and that is involved in the permeabilization of the recombinant VP5* model and cellular membranes (2, 3, 8). Interestingly, this putative fusion sequence significantly overlaps with the epitope II domain (amino acids 381 to 401) studied above. As this potent fusogenic domain is likely to be critical for viral infection, the chimeric KU//rVP4(SA11)-II(DS-1) virus may have altered biological characteristics (infectivity) compared with the parental KU//rVP4(SA11) virus, owing to the five amino acids mutations in the epitope II domain. To assess the infectivity of the recombinant virus, SA11, KU//rVP4(SA11), KU//rVP4(SA11)-II(DS-1), KU, and DS-1 were used for the infection of MA104 cells (Fig. 4A). As observed in a previous study with the parental KU//rVP4(SA11) virus carrying authentic SA11 VP4 (11), titers similar to those of SA11 were attained. In contrast, the rescued KU//rVP4(SA11)-II(DS-1) virus, which expresses chimeric VP4 possessing the DS-1-derived epitope II sequence (hydrophobic domain), displayed impaired growth (<10-fold-lower titer) compared to KU//rVP4(SA11). Human RVs showed significantly lower growth than the others carrying the SA11-based VP4. We then examined the plaque sizes in CV-1 cells for these viruses by measuring the mean diameters of each of 20 plaques from two independent assays (Fig. 4B). The virus growth titers correlated well with the sizes of the plaques formed. That is, the viruses with high infectivity [SA11 and KU//rVP4(SA11)] formed large plaques (diameters, 4.99 ± 0.50 and 5.44 ± 0.60 mm, respectively), as has been demonstrated by SA11 VP4-carrying viruses (27), while the human viruses with low infectivity (KU and DS-1) formed small plaques (diameters, 2.46 ± 0.48 and 0.96 ± 0.23 mm, respectively). On the other hand, the chimeric KU//rVP4(SA11)-II(DS-1) virus formed smaller-sized plaques than the SA11 and KU//rVP4(SA11) viruses but larger ones than the KU and DS-1 viruses (diameter, 3.80 ± 0.36 mm). Therefore, these results indicate that the amino acid substitutions in the epitope II (hydrophobic domain) sequence and/or a slight conformational change in and around the domain replaced with that of the slow-growing human DS-1 virus may significantly affect the infectivity of the fast-growing parental KU//rVP4(SA11) virus, leading to attenuation. Further mutational studies involving reverse genetics will provide information on which amino acid residues within this domain are involved in the control of the virus growth rate.

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FIG. 4. Infectivity of rescued viruses containing a cDNA-derived VP4 genome. (A) Single-step growth curves of the rescued viruses. A monolayer of MA104 cells was infected with the SA11, KU//rVP4(SA11), KU//rVP4(SA11)-II(DS-1), KU, and DS-1 viruses at a multiplicity of infection of 5, incubated for the intervals shown, and then lysed by freeze-thaw cycles. Virus titers were determined by a plaque assay with CV-1 cells. The data shown are the mean viral titers and standard deviations for three independent cell cultures. (B) Plaques of the rescued viruses. The viruses were directly plated on CV-1 cells for plaque formation. After 2 days, infected cells were stained with secondary overlay medium containing 0.7% agarose and 0.005% neutral red. The experiment was repeated three times with similar results, and representative results are shown.

In summary, a recombinant RV with an antigenic mosaic of cross-reactive neutralization epitopes on its VP4 surface could be engineered by means of reverse genetics, which indicates that RVs may be used as expression vectors for foreign epitopes. It is expected that this approach will be valuable for the development of novel RV vaccines and vaccine vectors, as well as for an understanding of the molecular basis of RV pathogenesis, although the efficiency of this system is not optimal yet.

 
We thank Akiko Yui for technical assistance.

This study was supported in part by Grants-in-Aids for Scientific Research and Young Scientists (B), the 21st Century Center of Excellence Program of Fujita Health University, and the Open Research Center Program of Fujita Health University from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

 
* Corresponding author. Mailing address: Department of Virology and Parasitology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan. Phone: 81-562-93-2467. Fax: 81-562-93-4008. E-mail: kokitani{at}fujita-hu.ac.jp

Published ahead of print on 23 April 2008.

Top ABSTRACT TEXT REFERENCES

 

1
1. Conzelmann, K. K. 2004. Reverse genetics of Mononegavirales. Curr. Top. Microbiol. Immunol. 283:1-41.[Medline]
2
2. Denisova, E. R., W. Dowling, R. LaMonica, R. Shaw, S. Scarlata, F. Ruggeri, and E. R. Mackow. 1999. Rotavirus capsid protein VP5* permeabilizes membranes. J. Virol. 73:3147-3153.[Abstract/Free Full Text]
3
3. Dowling, W., E. Denisova, R. LaMonica, and E. R. Mackow. 2000. Selective membrane permeabilization by the rotavirus VP5* protein is abrogated by mutations in an internal hydrophobic domain. J. Virol. 74:6368-6376.[Abstract/Free Full Text]
4
4. Enami, M., W. Luytjes, M. Krystal, and P. Palese. 1990. Introduction of site-specific mutations into the genome of influenza virus. Proc. Natl. Acad. Sci. USA 87:3802-3805.[Abstract/Free Full Text]
5
5. Enami, M., G. Sharma, C. Benhan, and P. Palese. 1991. An influenza virus containing nine different RNA segments. Virology 185:291-298.[CrossRef][Medline]
6
6. Estes, M. K., and A. Z. Kapikian. 2006. Rotaviruses, p. 1917-1974. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 5th ed. Lippincott Williams & Wilkins, Philadelphia, PA.
7
7. Evans, D. J. 1999. Reverse genetics of Picornaviruses. Adv. Virus Res. 53:209-228.[Medline]
8
8. Golantsova, N. E., E. E. Gorbunova, and E. R. Mackow. 2004. Discrete domains within the rotavirus VP5* direct peripheral membrane association and membrane permeability. J. Virol. 78:2037-2044.[Abstract/Free Full Text]
9
9. Ishii, K., Y. Ueda, K. Matsuo, T. Kitamura, K. Kato, Y. Izumi, K. Someya, T. Ohsu, M. Honda, and T. Miyamura. 2002. Structural analysis of vaccinia virus DIs strain: application as a new replication-deficient viral vector. Virology 302:433-444.[CrossRef][Medline]
10
10. Kobayashi, T., A. A. R. Antar, K. W. Boehme, P. Danthi, E. A. Eby, K. M. Guglielmi, G. H. Holm, E. M. Johnson, M. S. Maginnis, S. Naik, W. B. Skelton, J. D. Wetzel, G. J. Wilson, J. D. Chappell, and T. S. Dermody. 2007. A plasmid-based reverse genetics system for animal double-stranded RNA viruses. Cell Host Microbe 1:147-157.[CrossRef][Medline]
11
11. Komoto, S., J. Sasaki, and K. Taniguchi. 2006. Reverse genetics system for introduction of site-specific mutations into the double-stranded RNA genome of infectious rotavirus. Proc. Natl. Acad. Sci. USA 103:4646-4651.[Abstract/Free Full Text]
12
12. Luytjes, W., M. Krystal, M. Enami, J. D. Parvin, and P. Palese. 1989. Amplification, expression, and packaging of a foreign gene by influenza virus. Cell 59:1107-1113.[CrossRef][Medline]
13
13. Mackow, E. R., R. D. Shaw, S. M. Matsui, P. T. Vo, M. N. Dang, and H. B. Greenberg. 1988. The rhesus rotavirus gene encoding VP3: location of amino acids involved in homologous and heterologous rotavirus neutralization and identification of a putative fusion region. Proc. Natl. Acad. Sci. USA 85:645-649.[Abstract/Free Full Text]
14
14. Neumann, G., and Y. Kawaoka. 2004. Reverse genetics systems for the generation of segmented negative-sense RNA viruses entirely from cloned cDNA. Curr. Top. Microbiol. Immunol. 283:43-60.[Medline]
15
15. Parashar, U. D., E. G. Hummelman, J. S. Bresee, M. A. Miller, and R. I. Glass. 2003. Global illness and deaths caused by rotavirus disease in children. Emerg. Infect. Dis. 9:565-572.[Medline]
16
16. Parashar, U. D., C. J. Gibson, J. S. Bresee, and R. I. Glass. 2006. Rotavirus and severe childhood diarrhea. Emerg. Infect. Dis. 12:304-306.[Medline]
17
17. Roner, M. R., and W. K. Joklik. 2001. Reovirus reverse genetics: incorporation of the CAT gene into the reovirus genome. Proc. Natl. Acad. Sci. USA 98:8036-8041.[Abstract/Free Full Text]
18
18. Roner, M. R., K. Bassett, and J. Roehr. 2004. Identification of the 5' sequences required for incorporation of an engineered ssRNA into the reovirus genome. Virology 329:348-360.[Medline]
19
19. Roner, M. R., and J. Roehr. 2006. The 3' sequences required for incorporation of an engineered ssRNA into the reovirus genome. Virol. J. 3:1.[CrossRef][Medline]
20
20. Schnell, M. J., T. Mebatsion, and K. K. Conzelmann. 1994. Infectious rabies viruses from cloned cDNA. EMBO J. 13:4195-4203.[Medline]
21
21. Shen, Y., and L. Post. 2006. Virus vectors and their applications, p. 539-564. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 5th ed. Lippincott Williams & Wilkins, Philadelphia, PA.
22
22. Taniguchi, K., S. Urasawa, and T. Urasawa. 1985. Preparation and characterization of neutralizing monoclonal antibodies with different reactivity patterns to human rotaviruses. J. Gen. Virol. 66:1045-1053.[Abstract/Free Full Text]
23
23. Taniguchi, K., Y. Morita, T. Urasawa, and S. Urasawa. 1987. Cross-reactive neutralization epitopes on VP3 of human rotavirus: analysis with monoclonal antibodies and antigenic variants. J. Virol. 61:1726-1730.[Abstract/Free Full Text]
24
24. Taniguchi, K., T. Urasawa, Y. Morita, H. B. Greenberg, and S. Urasawa. 1987. Direct serotyping of human rotavirus in stools by an enzyme-linked immunosorbent assay using serotype 1-, 2-, 3-, and 4-specific monoclonal antibodies to VP7. J. Infect. Dis. 155:1159-1166.[Medline]
25
25. Taniguchi, K., Y. Hoshino, K. Nishikawa, K. Y. Green, W. L. Maloy, Y. Morita, S. Urasawa, A. Z. Kapikian, R. M. Chanock, and M. Gorziglia. 1988. Cross-reactive and serotype-specific neutralization epitopes on VP7 of human rotavirus: nucleotide sequence analysis of antigenic mutants selected with monoclonal antibodies. J. Virol. 62:1870-1874.[Abstract/Free Full Text]
26
26. Taniguchi, K., W. L. Maloy, K. Nishikawa, K. Y. Green, Y. Hoshino, S. Urasawa, A. Z. Kapikian, R. M. Chanock, and M. Gorziglia. 1988. Identification of cross-reactive and serotype 2-specific neutralization epitopes on VP3 of human rotavirus. J. Virol. 62:2421-2426.[Abstract/Free Full Text]
27
27. Taniguchi, K., K. Nishikawa, N. Kobayashi, T. Urasawa, H. Wu, M. Gorziglia, and S. Urasawa. 1994. Differences in plaque size and VP4 sequence found in SA11 virus clones having simian authentic VP4. Virology 198:325-330.[CrossRef][Medline]
28
28. Walpita, P., and R. Flick. 2005. Reverse genetics of negative-stranded RNA viruses: a global perspective. FEMS Microbiol. Lett. 244:9-18.[CrossRef][Medline]

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Journal of Virology, July 2008, p. 6753-6757, Vol. 82, No. 13
0022-538X/08/$08.00+0     doi:10.1128/JVI.00601-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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