Characterization of Neutralization Specificities of Outer Capsid Spike Protein VP4 of Selected Murine, Lapine, and Human Rotavirus Strains

Virology 299, 64–71 (2002) doi:10.1006/viro.2002.1474

Characterization of Neutralization Specificities of Outer Capsid Spike Protein VP4 of Selected Murine, Lapine, and Human Rotavirus Strains Yasutaka Hoshino, 1 Ronald W. Jones, and Albert Z. Kapikian Epidemiology Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-8026 Received November 26, 2001; returned for revision January 4, 2002; accepted February 22, 2002 Neutralization specificities of outer capsid spike protein VP4 of murine rotavirus strains EW (P?[16],G3) and EHP (P?[20],G3) and lapine rotavirus strains Ala (P?[14],G3), C11 (P?[14],G3), and R2 (P?[14],G3) as well as human rotavirus strains PA169 (P?[14],G6) and HAL1166 (P?[14],G8) were determined by two-way cross-neutralization. This was done by generating and characterizing (i) three murine ⫻ human, three lapine ⫻ human, and two human ⫻ human single gene substitution reassortant rotaviruses, each of which bore identical human rotavirus DS-1 strain VP7 (G2), and (ii) guinea pig hyperimmune antiserum raised against each reassortant. Reference rotavirus strains employed in the study represented 10 established VP4 (P) serotypes, including 1A[8], 1B[4], 2A[6], 3[9], 4[10], 5A[2], 5B[2], 5B[3], 6[1], 7[5], 8[11], 9[7], and 10[16] as well as a P serotype unknown P[18]. Murine rotavirus strains EW and EB were demonstrated to share the same P serotype (P10[16]) distinct from (i) 9 established P serotypes, (ii) lapine and human rotavirus strains bearing the P[14] genotype, and (iii) an equine rotavirus strain bearing the P[18] genotype. Both lapine (Ala, C11, and R2) and human (PA169 and HAL1166) rotaviruses were shown to belong to the same VP4 serotype, which represented a distinct new P serotype (P11[14]). P serotype 13[20] was assigned to murine rotavirus EHP strain VP4, which was shown to be distinct from all the P serotypes/genotypes examined in the present study.

proteins has been established based on a criterion of ⬎20-fold antibody differences between the homologous and heterologous reciprocal neutralizing antibody titers (Estes, 1996; Hoshino and Kapikian, 1996). Establishing the VP4 (P) or VP7 (G) serotype specificity of a rotavirus requires type-specific high titered polyclonal hyperimmune antisera. In order to circumvent the lack of readily available reagents for serotyping and to enable the classification of a large number of rotavirus field isolates, a G or P genotyping assay was developed in which “genotype-specific” primers were employed in RT–PCR. The numbers assigned for G serotypes (types determined by neutralization assay) and G genotypes (types determined by a nonserological assay) are identical, and thus a single number is used (e.g., G1). However, the numbers assigned for P serotypes and P genotypes are different; therefore, a P serotype is designated with a P followed by the assigned number, whereas the P genotype is designated by a P followed by the assigned number in brackets (e.g., P1A[8]). If the genotype but not the serotype is known, then the P is followed by the bracketed number only. The current status of VP4 (P) serotype classification has been summarized (Table 4). It is still unclear whether neutralizing antibodies are of major importance in protecting infants and young children against rotavirus diarrhea. However, there is increasing evidence from rotavirus vaccine field trials indicating that serotype-specific immunity is important (re-

INTRODUCTION The group A rotaviruses are the single most important etiologic agents of acute severe diarrhea in infants and young children as well as in the young of many animal species worldwide (Bern and Glass, 1994; Kapikian et al., 2001). Rotavirus diarrhea is estimated to be responsible for approximately 600,000–800,000 deaths each year in children aged less than 5 years in developing countries and more than 500,000 physician visits and 50,000 hospitalizations annually in this same age group in the United States alone (Kapikian et al., 2001; Glass et al., 1994). Thus, the development of a safe and effective vaccine has been a global public health goal and vigorously pursued by various groups around the world (Kapikian et al., 2001; Bresee et al., 1999). A complete rotavirus particle carries two outer capsid proteins, VP4 and VP7, which have been demonstrated to induce protective neutralizing antibodies independently (Greenberg et al., 1983; Hoshino et al., 1985, 1988; Offit and Blavat, 1986; Offit et al., 1986a,b; Matsui et al., 1989; Andrew et al., 1992; Both et al., 1993; Coste et al., 2000; Gil et al., 2000). Thus, a dual system of rotavirus classification to designate the neutralization specificity of both VP7 (G serotype) and VP4 (P serotype) outer capsid 1

To whom reprint requests should be addressed at ES/LID/NIAID/NIH, Building 50, Room 6308, 50 South Drive MSC 8026, Bethesda, MD 208928026. Tel: 301-594-1851. Fax: 301-480-1387. E-mail: [email protected] 0042-6822/02

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NEUTRALIZATION SPECIFICITIES OF VP4

viewed by Hoshino and Kapikian, 2000). In addition, various practical animal models, such as mice and rabbits, have been used to investigate mechanisms involved in rotavirus disease, infection, and immunity (Coelho et al., 1981; Thouless et al., 1988; Conner et al., 1988; Ijaz et al., 1989; Ward et al., 1990; Conner and Ramig, 1996; Rose et al., 1998). A murine rotavirus was the first group A rotavirus to be described as a causative agent of diarrhea in mouse pups and has been used extensively in a mouse model to study rotavirus infection and immunity. Serotypic characterization of the VP7 protein and complete nucleotide and deduced amino acid sequences of the VP4 gene of selected murine and lapine rotavirus strains have been determined (Sato et al., 1982b; Thouless et al., 1986; Greenberg et al., 1986; Tanaka et al., 1988; Nishikawa et al., 1989; Dunn et al., 1994; Ciarlet et al., 1997). However, characterization of the VP4 neutralization specificities of such strains has never been performed except for the murine rotavirus EB, which was shown by one-way neutralization to be distinct from 5 of 9 known P serotypes (1A[8], 1B[4], 2A[6], 2B[6], 3[9], 4[10], and 9[7]) and thus assigned to P serotype 10[16] (Sereno and Gorziglia, 1994). Of interest is the finding that lapine rotavirus strains and certain human rotavirus strains share the same VP4 genotype specificity (P[14]) (Ciarlet et al., 1997). The purpose of this study was to characterize the neutralization specificity of the VP4 protein of selected murine, lapine, and human rotavirus strains by two-way cross-neutralization based on a criterion of a ⬎20-fold antibody difference. RESULTS AND DISCUSSION Generation and characterization of murine ⫻ human G2 rotavirus reassortants We generated (i) three single gene substitution murine ⫻ human rotavirus reassortants, each of which had 10 genes from human rotavirus DS-1 strain (P1B[4],G2) and only the VP4 gene from murine rotavirus strains EB, EW, or EHP (Fig. 1) and (ii) hyperimmune guinea pig antiserum to each of the three murine ⫻ human rotavirus reassortants. Each reassortant was confirmed by VP7specific monoclonal antibody (mAb)-based serotyping enzyme-linked immunosorbent assay (ELISA) to carry G2 specificity (data not shown). When hyperimmune antiserum raised to EB ⫻ DS1(P10[16],G2) or EW ⫻ DS-1 (P[16],G2) was tested in plaque reduction neutralization (PRN) assays against various rotavirus strains representing a battery of P serotypes and genotypes, each serum neutralized only the VP7-homotypic DS-1 strain, the VP4-homotypic EB (P10[16],G3) and EW (P[16],G3) strains, as well as the homotypic reassortants, suggesting that strains EB and EW belonged to the same P serotype (Table 1). Since the VP4-specific neutralizing antibody titers were considerably lower than those to the G2 parent and the reassor-

65

FIG. 1. Electrophoretic migration patterns of genomic RNAs of murine rotavirus EB strain (lane 1), reassortant EB ⫻ DS-1 (lane 2), human rotavirus DS-1 strain (lane 3), reassortant EW ⫻ DS-1 (lane 4), murine rotavirus EW strain (lane 5), murine rotavirus EHP strain (lane 6), reassortant EHP ⫻ DS-1 (lane 7), and human rotavirus DS-1 strain (lane 8) in a 10% polyacrylamide gel. Genomic RNAs were electrophoresed at 8 mA for 15 h and the resulting migration patterns were visualized by staining of the gel with silver nitrate.

tant, we evaluated a high titered hyperimmune antiserum to the EB strain by neutralization against selected rotavirus strains representing the 10 established P serotypes and 2 genotypes. This confirmed that the VP4 neutralization specificities of the EB and EW strains were essentially similar, if not identical. The VP7-homotypic DS-1 strain and the VP4-homotypic EHP strain (P[20]) were the only viruses to be neutralized significantly by anti-EHP ⫻ DS-1 (P[20],G2) guinea pig hyperimmune antiserum, indicating that murine rotavirus EHP strain represented a P serotype distinct from the battery of P serotypes established thus far (Table 1). When the reassortants EB ⫻ DS-1 and EW ⫻ DS-1 were examined by PRN against guinea pig hyperimmune antisera raised to rotavirus strains belonging to a battery of P serotypes as well as three strains of unknown serotype (P[14], P[18], or P[20]), they were neutralized only by VP7-homotypic anti-DS-1 antiserum and VP4homotypic anti-EB antiserum (Table 2), which indicated again that strains EB and EW shared the same VP4 serotype specificity, which was distinct from all the P serotypes and strains of unknown serotype tested in this study. Similarly, VP7-homotypic antiserum to DS-1 and VP4-homotypic antiserum to EHP were the only hyperimmune antisera that neutralized EHP ⫻ DS-1 reassortant, confirming that strain EHP carried a P serotype

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HOSHINO, JONES, AND KAPIKIAN TABLE 1 Characterization by Neutralization of VP4 of Selected Murine Rotavirus Strains Reciprocal of 60% PRN antibody titer of guinea pig hyperimmune antiserum to indicated rotavirus Rotavirus strain

Species of origin

P type [genotype]

G type

EW ⫻ DS-1 (P[16],G2)

EB ⫻ DS-1 (P10[16],G2)

EB (P10[16],G3)

EHP ⫻ DS-1 (P[20],G2)

Wa DS-1 DS-1 ⫻ UK ST3 K8 69M Ro1845 Ro1845 ⫻ DS-1 SA11 SA11 ⫻ DS-1 MMU18006 MMU18006 ⫻ DS-1 NCDV UK B223 OSU EW EB PA169 Ala L338 EHP EW ⫻ DS-1 EB ⫻ DS-1 EHP ⫻ DS-1

Human Human RT* Human Human Human Human RT Vervet monkey RT Rhesus monkey RT Bovine Bovine Bovine Porcine Murine Murine Human Lapine Equine Murine RT RT RT

1A[8] 1B[4] 1B[4] 2A[6] 3[9] 4[10] 5A[3] 5A[3] 5B[2] 5B[2] 5B[3] 5B[3] 6[1] 7[5] 8[11] 9[7] [16] 10[16] [14] [14] [18] [20] [16] 10[16] [20]

1 2 6 4 1 8 3 2 3 2 3 2 6 6 10 5 3 3 6 3 13 3 2 2 2

⬍80 10,240*** ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 NT** ⬍80 NT ⬍80 NT ⬍80 ⬍80 ⬍80 ⬍80 640**** 320 ⬍80 ⬍80 ⬍80 ⬍80 20,480***** NT NT

⬍80 10,240 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 NT ⬍80 NT ⬍80 NT ⬍80 ⬍80 ⬍80 ⬍80 320 320 ⬍80 ⬍80 ⬍80 ⬍80 NT 40,960 NT

⬍80 ⬍80 NT 80 80 80 NT ⬍80 NT 80 NT 80 ⬍80 ⬍80 ⬍80 ⬍80 5,120 10,240 ⬍80 NT ⬍80 NT 1,280 2,560 80

⬍80 10,240 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 NT 80 NT ⬍80 NT 80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 80 ⬍80 ⬍80 2,560 NT NT 40,960

* RT, reassortant. ** NT, not tested. *** VP7 homologous values in bold. **** VP4 homologous values underlined. ***** VP7 and VP4 homologous values in bold and underlined.

distinct from 10 P serotypes established thus far and 2 P genotypes (P[14] and P[18]) tested in this study. Generation and characterization of single gene substitution lapine ⫻ human G2 and human P[14] ⫻ human G2 rotavirus reassortants We generated three lapine ⫻ human G2 and two human P[14] ⫻ human G2 single gene substitution rotavirus reassortants, each of which had either (i) 10 genes from the human DS-1 strain and only the VP4 gene from lapine rotavirus (Ala ⫻ DS-1) or (ii) only the VP7 gene from the DS-1 strain and the remaining 10 genes from a lapine (C11 ⫻ DS-1 or R2 ⫻ DS-1) or human P[14] strain (PA169 ⫻ DS-1 or HAL1166 ⫻ DS-1) (Figs. 2 and 3). Each of the five reassortants was confirmed by serotyping ELISA to carry G2 specificity (data not shown). Guinea pig hyperimmune antiserum to each reassortant was also generated. Guinea pig hyperimmune antiserum to Ala ⫻ DS-1

(P[14],G2) or C11 ⫻ DS-1 (P[14],G2) neutralized homologous lapine Ala (P[14],G3) and C11 (P[14],G3) strains or VP4 genotypically identical human PA169 (P[14],G6) or HAL1166 (P[14],G8) strains to a high titer and the VP4 genotypically identical lapine R2 strain (P[14],G3) to a fourfold lower titer (Table 3), indicating that the three lapine and two human rotavirus strains shared the same VP4 neutralization specificities. The same antisera showed only a low-level cross-reactivity to certain rotavirus strains, indicating that such strains were not significantly related. Anti-R2 ⫻ DS-1 (P[14],G2) antiserum neutralized the homologous lapine R2 strain and heterologous human PA169 and HAL1166 strains to a high titer and the lapine Ala and C11 strains 16-fold lower than the homologous R2 strain (Table 3), which indicated that the Japanese lapine rotavirus (R2) VP4 was more closely related serotypically to human P[14] VP4s than to the United States lapine rotavirus (Ala and C11) VP4s.

2,560*** 1,280 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 80

⬍80 ⬍80 640 640 640 320 640 ⬍80

⬍80 ⬍80 640 640 160 640 640 ⬍80

⬍80 ⬍80 80 80 160 80 80 ⬍80

⬍80 ⬍80 80 80 640 640 640 ⬍80

⬍80 ⬍80 ⬍80 80 80 ⬍80 ⬍80 ⬍80

⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 10,240

67

⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80

* The homologous 60% PRN antibody titers ranged from 10,240 to ⬎81,920 in previous tests in this laboratory. ** VP7 homologous values in bold. *** VP4 homologous values underlined.

⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 80 ⬍80 80 80 80 160 80 80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 10,240** 10,240 10,240 40,960 10,240 5,120 10,240 10,240 80 ⬍80 80 80 ⬍80 ⬍80 80 ⬍80 2 2 2 2 2 2 2 2 10[16] [16] [14] [14] [14] [14] [14] [20] EB ⫻ DS-1 EW ⫻ DS-1 Ala ⫻ DS-1 C11 ⫻ DS-1 R2 ⫻ DS-1 PA169 ⫻ DS-1 HAL1166 ⫻ DS-1 EHP ⫻ DS-1

Wa DS-1 DS-1 ⫻ UK ST3 K8 69M Ro1845 MMU18006 NCDV UK B223 OSU EB Ala C11 R2 PA169 L338 EHP P type G [genotype] type (P1A[8],G1) (P1B[4],G2) (P1B[4],G6) (P2A[6],G4) (P3[9],G1) (P4[10],G8) (P5A[3],G3) (P5B[3],G3) (P6[1],G6) (P7[5],G6) (P8[11],G10) (P9[7],G5) (P10[16],G3) (P[14],G3) (P[14],G3) (P[14],G3) (P[14],G6) (P[18],G12) (P[20],G3) Rotavirus reassortant

Reciprocal of 60% PRN antibody titer of guinea pig hyperimmune antiserum to indicated rotavirus strain*

Characterization by Neutralization of VP4 of Selected Murine, Lapine, and Human P[14] Rotavirus Strains

TABLE 2

NEUTRALIZATION SPECIFICITIES OF VP4

FIG. 2. Electrophoretic migration patterns of genomic RNAs of lapine rotavirus Ala strain (lane 1), reassortant Ala ⫻ DS-1 (lane 2), human rotavirus DS-1 strain (lane 3), reassortant C11 ⫻ DS-1 (lane 4), lapine rotavirus C11 strain (lane 5), lapine rotavirus R2 strain (lane 6), reassortant R2 ⫻ DS-1 (lane 7), and human rotavirus DS-1 strain (lane 8) in a 10% polyacrylamide gel. Genomic RNAs were electrophoresed at 8 mA for 15 h and the resulting migration patterns were visualized by staining of the gel with silver nitrate.

Guinea pig antiserum to PA169 ⫻ DS-1 or HAL1166 ⫻ DS-1 neutralized homologous human PA169 and HAL1166 viruses to a high titer and heterologous lapine R2 virus 8-fold lower than the homologous viruses, confirming a close VP4 serotypic relationship between human P[14] viruses and lapine R2 virus (Table 3). These antisera neutralized lapine Ala and C11 viruses 64- to 128-fold less efficiently than they did against the homologous viruses, which indicated again that the human P[14] virus VP4s were more closely related serotypically to the Japanese lapine rotavirus R2 VP4 than those of the United States lapine rotaviruses (Ala and C11). It is of interest that consistent with these results, previous comparative VP4 amino acid sequence analysis suggested a closer relatedness of the Japanese lapine R2 virus to the human P[14] viruses (PA169 and HAL1166) than to the United States lapine Ala and C11 viruses (Ciarlet et al., 1997). Reassortants Ala ⫻ DS-1, C11 ⫻ DS-1, R2 ⫻ DS-1, PA169 ⫻ DS-1, and HAL1166 ⫻ DS-1 were neutralized by antiserum to the VP7-homotypic DS-1 and antiserum to VP4-homotypic Ala, C11, R2, or PA169 strains (Table 2), which confirmed a close VP4 serotypic relationship among these five P[14] viruses. Thus, this study has shown that (i) lapine rotavirus strains Ala, C11, and R2 and human rotavirus strains PA169 and HAL1166 belong to the same P serotype and (ii) human rotavirus PA169 and HAL1166 VP4s are more closely related serotypicallyto the Japanese lapine rotavirus R2 VP4 than those of

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HOSHINO, JONES, AND KAPIKIAN

FIG. 3. Electrophoretic migration patterns of genomic RNAs of human rotavirus PA169 (lane 1), reassortant PA169 ⫻ DS-1 (lane 2), human rotavirus DS-1 strain (lane 3), reassortant HAL1166 ⫻ DS-1 (lane 4), and human rotavirus HAL1166 strain (lane 5) in a 10% polyacrylamide gel. Genomic RNAs were electrophoresed at 8 mA for 15 h and the resulting migration patterns were visualized by staining of the gel with silver nitrate.

the United States lapine rotavirus Ala and C11 strains. It is of interest that human rotavirus strains bearing the P[14] genotype, which were first reported from Italy 11 years ago (Gerna et al., 1992), have been detected recently, albeit sporadically, in various parts of the world, including Thailand (Urasawa et al., 1992), Italy (Gerna et al., 1992), Finland (Gerna et al., 1994), South Africa (Mphahlele et al., 1999), Australia (Palombo et al., 1995), and Egypt (Holmes et al., 1999). As described in this study, mAbs directed to the VP4 protein often show cross-reactivity. Therefore, we have chosen to generate polyclonal hyperimmune antiserum to a reassortant with the VP4 specificity of the strain being studied because such sera have been shown to be VP4 serotype-specific (Hoshino and Kapikian, 1996). Previously, P serotypes of human rotavirus strains K8 (P3[9],G1) and Mc35 (P[14],G10) were tentatively designated P3A[9] and P3B[14], respectively (Urasawa et al., 1993). This classification was based on (i) the relatively high sequence homology of the VP4 gene of K8 and Mc35 (77.6% nucleotide and 86.0% amino acid sequence homologies, respectively) and (ii) the signifi-

cant recognition by neutralization of the Mc35 strain with a single mAb raised against the K8 VP4. However, it was shown later that polyclonal hyperimmune guinea pig antiserum raised against the VP4 of PA169 (P[14],G6) failed to neutralize significantly a P3[9] virus designated the AU-1 strain (which as noted above for the K8, a P3[9] strain, had been considered to be related to the Mc35, a P[14] strain), and thus, the VP4 serotype of PA169 was proposed to represent a new P serotype (Gerna et al., 1994). Of note is the finding that in the latter study, three of four mAbs raised against the PA169 VP4 (P[14]) were shown to demonstrate significant cross-reactivities in neutralization to various P types, including P1A[8], P2A[6], and P5B[3]. However, the observation made in that study that there was no significant VP4 serotypic relatedness between AU-1 (a P3[9] strain) and PA169 (a P[14] strain) by neutralization with hyperimmune sera was confirmed in the present study. Hence, we propose that the VP4 serotype of these strains bearing the P[14] genotype be assigned to a new P serotype (P11). Consequently, the VP4 serotype of human rotavirus strains Mc323 (P[19],G9) and Mc345 (P[19],G9), which was assigned to P serotype 11 initially (Okada et al., 2000), would be reassigned to P serotype 12. Hence, the VP4 serotype of the murine rotavirus EHP strain (P[20],G3), which has been shown in this study to be distinct, would be assigned to P serotype 13.

MATERIALS AND METHODS Rotavirus strains, cell cultures, virus titration, neutralization assay, and hyperimmune antiserum Tables 1 and 3 summarize the rotavirus strains as well as their G and P type specificities employed in the present study. Origins of murine rotavirus strains EW (P[16],G3) (isolated in the United States), EB (P10[16],G3) (isolated in the United States), and EHP (P[20],G3) (isolated in Brazil) have been described in detail previously (Greenberg et al., 1986). Lapine rotavirus strains Ala (P[14],G3) and C11 (P[14],G3) were isolated in the United States (Thouless et al., 1986) and the R2 strain (P[14],G3) in Japan (Sato et al., 1982a). Human rotavirus strains PA169 (P[14],G6) and HAL1166 (P[14],G8) were isolated in Italy (Gerna et al., 1992) and in Finland (Gerna et al., 1994), respectively. Each rotavirus strain was triply plaque-purified before use. The established monkey kidney cell line MA104 was used for virus propagation, plaque purification, plaque titration, and PRN assay. Plaque titration and the PRN assay were performed in a six-well plate as described previously (Hoshino et al.,1998). Hyperimmune antiserum to each reference rotavirus strain as well as to each reassortant was raised as described previously (Wyatt et al., 1982) in specific pathogen-free guinea pigs (National Cancer Institute, Frederick, MD) which were free of neutralizing antibod-

NEUTRALIZATION SPECIFICITIES OF VP4

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TABLE 3 Characterization by Neutralization of VP4 of Selected P[14] Lapine and Human Rotavirus Strains Reciprocal of 60% PRN antibody titer of guinea pig hyperimmune antiserum to indicated reassortant Rotavirus strain

Species of origin

P type [genotype]

G type

Ala ⫻ DS-1 (P[14],G2)

C11 ⫻ DS-1 (P[14],G2)

R2 ⫻ DS-1 (P[14],G2)

PA169 ⫻ DS-1 (P[14],G2)

HAL1166 ⫻ DS-1 (P[14],G2)

Wa DS-1 DS-1 ⫻ UK ST3 K8 69M Ro1845 SA11 MMU18006 NCDV UK B223 OSU EB Ala C11 R2 PA169 HAL1166 EHP Ala ⫻ DS-1 C11 ⫻ DS-1 R2 ⫻ DS-1 PA169 ⫻ DS-1 HAL1166 ⫻ DS-1

Human Human Reassortant Human Human Human Human Vervet monkey Rhesus monkey Bovine Bovine Bovine Porcine Murine Lapine Lapine Lapine Human Human Murine Reassortant Reassortant Reassortant Reassortant Reassortant

1A[8] 1B[4] 1B[4] 2A[6] 3[9] 4[10] 5A[3] 5B[2] 5B[3] 6[1] 7[5] 8[11] 9[7] 10[16] [14] [14] [14] [14] [14] [20] [14] [14] [14] [14] [14]

1 2 6 4 1 8 3 3 3 6 6 10 5 3 3 3 3 6 8 3 2 2 2 2 2

⬍80 40,960** ⬍80 80 80 80 80 160 320 80 ⬍80 80 80 ⬍80 10,240*** 10,240 2,560 10,240 10,240 ⬍80 40,960**** NT* NT NT NT

80 81,920 ⬍80 80 160 ⬍80 160 ⬍80 320 80 ⬍80 ⬍80 ⬍80 ⬍80 10,240 10,240 2,560 10,240 10,240 ⬍80 NT 40,960 NT NT NT

80 5,120 ⬍80 ⬍80 80 ⬍80 80 ⬍80 80 ⬍80 ⬍80 ⬍80 ⬍80 ⬍80 160 160 2,560 5,120 2,560 ⬍80 NT NT 10,240 NT NT

⬍80 81,920 ⬍80 ⬍80 160 ⬍80 160 ⬍80 160 80 ⬍80 ⬍80 80 ⬍80 160 160 1,280 10,240 10,240 ⬍80 NT NT NT 10,240 NT

⬍80 10,240 ⬍80 ⬍80 160 160 80 ⬍80 160 160 ⬍80 ⬍80 160 ⬍80 320 160 2,560 5,120 20,480 ⬍80 NT NT NT NT 40,960

* NT, not tested. ** VP7 homologous values in bold. *** VP4 homologous values underlined. **** VP7 and VP4 homologous values in bold and underlined.

ies to all the rotavirus strains employed in the study (e.g., PRN antibody titer ⬍1:20 vs Wa). Generation and characterization of reassortant rotaviruses Each reassortant rotavirus was generated as previously described (Hoshino et al., 1998). Because certain combinations of outer capsid proteins VP4–VP7 have been reported to affect the rotavirus antigen– antibody interaction (Chen et al., 1989), we utilized the identical VP7 protein (G2) in each reassortant. Briefly, tube cultures of MA104 cells were coinfected with two parental rotavirus strains at a m.o.i. approximately 1. When approximately 75% of the infected cells showed cytopathic effects, the cultures were frozen and thawed once and plated on MA104 cells in a six-well plate in the presence of (i) serotype-G3-specific mAb 159 (Greenberg et al., 1983) to generate reassortant EB ⫻ DS-1, EW ⫻ DS-1, EHP ⫻ DS-1, Ala ⫻ DS-1,

C11 ⫻ DS-1, or R2 ⫻ DS-1, (ii) serotype-G6-specific mAb UK/7 (Snodgrass et al., 1990) to generate PA169 ⫻ DS-1, or (iii) hyperimmune antiserum raised against strain 69M (P4[10],G8) to generate HAL1166 ⫻ DS-1. Desired reassortants selected in this manner were triply plaque-purified in MA104 cells. The plaquepurified reassortants were examined for G serotype by ELISA using type-specific VP7 neutralizing mAbs (Green et al., 1990). The origin of the genes of each reassortant was determined by polyacrylamide gel electrophoresis (PAGE) of its genomic RNAs. The origin of the genes undetermined by PAGE was confirmed by constant denaturant gel electrophoresis (Jones et al., in press). ACKNOWLEDGMENTS We thank Jerri Ross and Monica Bur for expert technical assistance, Dr. David Snodgrass (BioBest, Edinburgh, Scotland) for providing mAb UK/7, and Dora Kyle for editorial assistance in the preparation of the manuscript.

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HOSHINO, JONES, AND KAPIKIAN TABLE 4 Current Status of VP4 (P) Serotype Classification

VP4 (P) serotype [genotype] 1A[8] 1B[4] 2A[6] 2B[6] 3[9] 4[10] 4[12] 5A[3] 5B[3] 5B[2] 6[1] 7[5] 8[11] 9[7] 10[16] 11[14] 12[19] 13[20] Not tested Not tested Not tested Not tested Not tested

[13] [15] [17] [18] [21]

Representative rotavirus strains that have been P serotyped Human rotavirus strain (G type)

Animal rotavirus strain (G type, species)

Wa (1), KU (1), D (1), Mo (1), RV-4 (1), 89-12* (1), WI79* (1), P (3), YO (3), WI61 (9), AU32 (9) DS-1 (2), S2 (2), RV-5* (2), L26* (12) M37 (1), 1076 (2), McN (3), RV-3* (3), ST3 (4), US1205 (9) None K8 (1), AU-1 (3), PA151 (6) 57M (4), 69M (8), B37* (8) None Ro1845 (3), HCR3 (3) None None None A64* (10) 116E (9), I321* (10) None None PA169 (6), MG6 (6), HAL1166 (8) Mc323 (9) None None None None None None

LRV2c* (9, sheep), S8* (1, pig) None None Gottfried (4, pig) FRV-1* (3, cat), Cat2* (3, cat) None H-2 (3, horse), FI-14 (3, horse), FI23* (14, horse) CU-1 (3, dog), K9 (3, dog), Cat97* (3, cat) MMU18006 (3, rhesus monkey) SA11 (3, vervet monkey) NCDV (6, cow), SA11-4fM (3, vervet monkey) UK (6, cow), WC3* (6, cow), 678 (8, cow) B223 (10, cow), KK3* (10, cow), Cr* (10, cow) SB-1A (4, pig), OSU (5, pig), YM (11, pig) EB (3, mouse), EW (3, mouse), EC (3, mouse) Ala (3, rabbit), C11 (3, rabbit), R2 (3, rabbit) 4F* (3, pig) EHP (3, mouse) MDR-13* (3/5, pig) Lp14* (10, sheep) PO-13* (7, pigeon), 998/83* (7, cow) L338* (13, horse) Hg18* (15**, cow)

* The P serotype of these strains has never been determined by neutralization. ** Represents G genotype only because G serotype has not been determined by neutralization.

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