Journal of General Virology (1990), 71, 983-986. Printed in Great Britain
983
The genome type of human parvovirus B19 strains isolated in Japan during 1981 differs from types detected in 1986 to 1987: a correlation between genome type and prevalence Kenichi Umene 1. and Tadasu Nunoue 2 1Department of Virology, Faculty of Medicine, Kyushu University 60 and ZSchool of Health Sciences, Kyushu University, Fukuoka 812, Japan.
The genome DNAs of 12 strains of human parvovirus B19, isolated in Japan at two different times, 1981 and 1986 to 1987, were molecularly cloned in the plasmid pUC18. The cloned B19 DNAs were analysed by cleaving with restriction endonucleases, and were classified into several groups by genome type. The restriction endonuclease cutting patterns ofB 19 strains isolated during 1981 were similar to that of the group
IV genome type, and the patterns of those isolated later were similar to that of group II, suggesting a correlation between the genome type and the prevalence. We conclude that the prevalences of B 19 infection in Japan during 1981 and in 1986 to 1987 were caused by viruses differing in genome type, and that B19 viruses with similar genome types disseminated widely in Japan during each prevalence.
The human parvovirus B19, originally discovered in blood-bank donors by Cossart et al. (1975), is now known to be an aetiological agent of several clinical disorders (Ware, 1989). The B19 viral particles contain a singlestranded DNA of either polarity (Clewley, 1984; Summers et al., 1983), and the genome is a 5.54 kilobase linear molecule with hairpin structures at each extremity (Cotmore & Tattersall, 1984; Mori et al., 1987). B19 has been successfully propagated in suspension cultures of human erythroid bone marrow ceils but such cells are not suitable for use in large-scale propagation of B19 (Mortimer et al., 1983; Ozawa et al., 1986). Molecular cloning of the B19 genome has facilitated analyses of genome structures and functions (Blundell et al., 1987; Clewley, 1985; Cotmore & Tattersall, 1984). Viruses with a DNA genome can be analysed with restriction endonucleases (REs) for differentiation and classification, and Morinet et al. (1986) first investigated the genetic variability of B19. Mori et al. (1987) mapped DNAs of 48 B 19 strains with 13 REs and classified them into several genome types. Four B19 strains isolated in Japan in 1981 were all classified as having the group IV genome type, and no other strain isolated outside of Japan belonged to group IV (Mori et al., 1987). Thus, questions were raised as to whether B19 strains in Japan were restricted to group IV, and whether Japan was sequestered from other areas with respect to the prevalence of B19 infection. To elucidate this question, we collected sera of patients suspected of having B 19 infection and attempted to clone
the BI9 genome DNA, with the intention of obtaining enough DNA for analyses. Single-stranded B19 DNAs extracted from these sera were treated with Escherichia coli DNA polymerase I Klenow fragment, to synthesize double-stranded DNA, utilizing as a primer the hairpin structure at the 3' end of each molecule. Then, the DNAs were either digested with AatII which has a cleavage site in the terminal repeats of the B19 genome (Shade et al., 1986) or with nuclease S1, to remove the terminal 'turn around' form. The DNAs were treated with the Klenow fragment to convert the staggered ends to blunt ends and then joined to vector plasmid DNAs of pUC18 at the SmaI site. The ligated mixture was then transformed into E. coli JM83 (Yanisch-Perron et al., 1985). Hybrid plasmids containing a sufficient length of B19 genome DNA were constructed from 12 sera (Table 1). The structures of the B19 DNAs were analysed by cleavage with 13 REs, i.e. BamHI, BgllI, BstEII, EcoRI, HindlII, HpaI, KpnI, PstI, PvulI, SalI, Sinai, XbaI, and XhoI, as described by Mori et al. (1987) (Fig. 1, 2). No RE sites for EcoRI, Sail and XhoI were detected in the cloned DNAs, as was the case for other B19 strains. One BamHI site, one BstEII site, one KpnI site and one SmaI site were detected in all 12 of the cloned B19 DNAs (Fig. 1). Differences among the B19 strains were detected in the cleavage patterns for BgllI, HindlII, HpaI, PstI, PvulI and XbaI. Four strains (N8, N11, N14, N15) had the same RE cutting pattern as the group II genome type proposed by Mori et al. (1987). Two B19 strains (N2 and N4) had the same pattern as group IV. The cutting
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Table 1. Human parvovirus B19 strains analysed Source of B19 strains Case no. 1 2 3 4 5 6 7 8 9 10 11 12
Clinical symptom*
Place
Date
Aget
Sex~
Strain no.
AC/HS
Osaka
Mar. 1981
11 y
F
N25
AC/HS Blood donor Blood donor AC/HS AC/HS AC/HS AC/HS AC/HS AC/HS AC/HS Asymptomatic
Niigata Fukuoka Fukuoka Tokyo Ibaragi Tokyo Kobe Kobe Fukuoka Fukuoka Saga
Jun. 1981 Dec. 1981 Dec. 1981 Oct. 1986 Nov. 1986 Dec. 1986 Jan. 1987 Jan. 1987 Apr. 1987 May 1987 Jul. 1987
34 y 16 y 17 y 6y 8y 4y 34 y 5y 6y 10 m 10 y
F F M M M M F F F M M
N5 N2 N4 N14 Nll N8 N9 N10 NI5 N16 N22
Hybrid plasmid containing B19 DNA UK295-1 UK295-2 UK284-6 UK294-4 UK283-4 UK289-1 UK287-4 UK281-2 UK285-3 UK292-13 UK290-1 UK291-4 UK293-2
Genome type§
(Iv) IVb IV IV II II II IIb lib II IIb IIc
* AC, Aplastic crisis; HS, hereditary spherocytosis. I" Y, Years; m, months. F, Female, M, male. § Definition of groups is based on that by Mori et al. (1987), and this work (see text).
patterns of four strains (N9, N10, N16 and N22) were similar to but not the same as that of the group II genome type (Fig 1, 2)i The three strains N9, N10 and N16 did not have HindlII sites in the position marked on line 2 of Fig. 1 by a parenthesized HindlII. The absence of this site caused the loss of two fragments of 0.94 kb and 0.90 kb and the gain of a 1-8 kb fragment, in comparison with the group II genome type (lanes 2, 4, 5 and 6 of part B of Fig. 2). Strains N9, NI0 and N16 were classified as group IIb in this work (Table 1). The BgllI/EcoRI/SalI cleavage products of hybrid plasmid UK293-2 (containing genome DNA from strain N22) contained only two fragments of vector DNA and the B19 genome insert (lane 3 of part A of Fig. 2), indicating the absence of a BgllI site in the N22 strain (line 3 of Fig. 1). N22 was classified as group IIc (Table 1). The central one-fifth of the N25 genome was not cloned. The other four-fifths were cloned and had the same structure as that of group IV genome type (line 5 of Fig. 1). We provisionally assigned N25 to group IV (Table 1). The RE cutting patterns of strain N5, except for PvulI, were the same as those of the group IV genome type. The recombinant plasmid DNA containing the N5 genome was cleaved into two fragments of vector DNA and insert DNA after digestion with PvuII/EcoRI/SalI (lane 7 of part C of Fig. 2), thereby indicating that N5 has no PvuII site (line 6 of Fig. 1). N5 was classified as group IVb (Table 1). Mori et al. (1987) classified 48 B19 strains into five types, defined by having the same RE cutting pattern, and an unassigned group. The five groups of genome types can be distinguished by differences in one to five
RE cutting sites. Group IV is different from groups Ilia and IIIb in five RE cutting sites, from group II in four sites, and group I in three sites, suggesting a remote relationship between group IV and the other four groups. Groups lib and IIc are apparently more closely related to group II than group IV. The twelve B19 strains analysed in this work could be classified into two clusters, with regard to their RE cleavage pattern (Table 1). One cluster, having a pattern similar to that of group IV, consisted of B19 strains isolated in 198I. The other cluster, similar to group II, consisted of B19 strains isolated in 1986 to 1987. The results show a correlation between the genome type and the time of isolation (prevalence), suggesting an epidemiological relationship among strains. The prevalence of B 19 infection is recognized by the presence of erythema infec'tiosum (El) (Anderson et al., 1983; Nunoue et al., 1985). Surveillance of infectious diseases in Japan showed two major prevalences of El in 1981 to 1982 and 1986 to 1987 (Nunoue et al., 1987). The genome types of the BI9 strains correlated with the time of isolation but not with the area of isolation (Table 1). We assume that a nation-wide B 19 prevalence occurred in 1981 to 1982 and 1986 to 1987 in Japan, and that each was caused by a group of viruses with a similar genome type, hence indicating highly efficient transmission of B19 viruses. B19 strains of the group II genome type have been isolated from different areas in the world, i.e. France, Norway, U.K. and U.S.A. (Mori et al., 1987). It is probable that a group II B 19 virus entered Japan around 1986 and caused the new epidemic of B19 infection in 1986 to 1987.
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2
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5 kb
4
I
(a)
A 1
1 2
C
B
!
i
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!
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!
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M --23-1 --9-4 --6-6
1
--4.4 w
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-v--2.3 --2"0 i
6
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--0-56
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Fig. 1. Restriction endonuclease map of B19 DNA. The enzyme cutting sites on the group II genome type are shown at line 1. The restriction maps of group IIb genomes (line 2), group IIc genomes (line 3), group IV genomes (line 4), strain N25 from case 1 (line 5), and group IVb genomes (line 6) are shown. At lines 2 to 6, addition and loss of restriction sites are shown in comparison with the map of group II. Additional sites are entered in the maps, and lost sites are indicated by parentheses. The genome DNA of N25 was molecularly cloned as two hybrid plasmids, one containing the left-side two-fifths region (UK295-1) and the other containing the right-side two-fifths region (UK295-2). The central one-fifth of the N25 genome (indicated by parentheses in line 5) was not cloned. The locations of fragments used as probe DNAs are shown in line 7. The EcoRI site of the 0.7 kb PtrulIEcoRI fragment is derived from the EcoRI linker flanking cloned BI9 DNA.
Sources of an outbreak and the mode of transmission among cases of viral infection could be investigated by analysing the genome type of the viruses. Of the cases analysed in this work, the case 9 patient is the daughter of the case 8 patient (Table 1). They developed aplastic crisis (AC) at the same time, on the 6th day after the onset of AC in a 9 year old boy with hereditary spherocytosis (HS) in the family, presumably due to the same source of infection. Both B19 D N A s from the two belonged to the IIb genome type (Table 1). The results obtained by analyses of the genome type are m keeping with the deduction from the clinical observations, i.e. the source of infection was the same. The genome types of virus strains can be used to examine whether a virus strain causes a specific clinical manifestation. Morinet et al. (1986) reserved speculation concerning a possible association of B19 variants with
2
3
C
i
I
1
2 4
5
6
I
I
1
2
7
M --23.1 --9-4 --6.6 --4"4
--2.3 ~2.0
--0-56
Fig. 2. Variations of restriction endonuclease cleavage patterns found in B19 strains analysed in this work. Hybrid plasmid DNAs of UK283-4 (as a representative of group IV) (lane 1), UK281-2 (as a representative of group II) (lane 2), UK293-2 (lane 3), UK285-3 (lane 4), UK292-13 (lane 5), UK291-4 (lane 6) and UK284-6 (lane 7) were cleaved with BgllI (part A), HindlII (part B) or PvulI (part C), after digestion with EcoRI and Sail. The cleaved DNAs were electrophoresed in 0.8% agarose gels, and stained with ethidium bromide (a). The lane designated M contains a marker mixture (kb) of HindlII fragments of 2 phage DNA. The DNAs in the agarose gels were then transferred to nylon membranes (Umene, 1985). The DNAs cleaved with BgllI and PvulI (parts A and C of a) were hybridized with a 32p. labelled probe, the 0.7 kb PvulI-EcoRI fragment of pJB (see line 7 of Fig. 1) (parts A and C of b). The hybrid plasmid pJB, constructed by inserting a 5.2 kb B19 DNA flanked with EcoRI linkers into the EcoRI site of vector pGEM-1, was provided by J. P. Clewley (Mori et al., 1987). HindlII digests (part B of a) were hybridized with the 32p. labelled 0.90 kb HindlII-HindlII fragment ofpJB (see line 7 of Fig. 1) (part B of b). Bands marked with a black dot in (a) correspond to those detected by Southern hybridization in (b). The positions of vector DNA fragments are indicated by a v in (a).
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d i s t i n c t clinical s y m p t o m s . M o r i et al. (1987) e x a m i n e d w h e t h e r one g e n o m e t y p e could be c o r r e l a t e d w i t h a p a r t i c u l a r clinical m a n i f e s t a t i o n o f infection, b u t f o u n d n o correlation. N i n e B19 strains a n a l y s e d in our w o r k were isolated f r o m p a t i e n t s w i t h H S at A C ( T a b l e l ) a n d six o f t h e m were a s s o c i a t e d w i t h s k i n r a s h e s ( N u n o u e et al., 1987). T h e m o d e o f the r a s h e s v a r i e d , h e n c e we could n o t correlate the g e n o m e t y p e w i t h t h e m o d e o f rashes a n d w i t h t h e severity o f A C . W e a s s u m e t h a t clinical m a n i f e s t a t i o n s o f B19 infection are m o d i f i e d greatly b y host factors. H o w e v e r , v a r i a t i o n in a viral g e n o m e m a y p o s s i b l y cause v a r i a t i o n o f a m i n o a c i d c o m p o n e n t s o f v i r u s - e n c o d e d p r o t e i n a n d also v a r i a t i o n in cis-acting e l e m e n t s in the virus genome. T h e s e v a r i a t i o n s m a y affect v i r u s - h o s t i n t e r a c t i o n . W e were i n t e r e s t e d in p o s s i b l e c o r r e l a t i o n s b e t w e e n the g e n o m e t y p e a n d the scale o f p r e v a l e n c e , as the r e p o r t e d n u m b e r s o f cases o f E I a n d A C in 1986 to 1987 (group I I genorne type) were h i g h e r t h a n those in 1981 to 1982 (group I V g e n o m e type). T h e r e were seven cases o f foetal d e a t h a s s o c i a t e d w i t h B19 d u r i n g 1986 to 1987 b u t n o n e in 1981 to 1982. T h e isolation o f g r o u p I V strains was r e s t r i c t e d to c e r t a i n a r e a s (in J a p a n ) a n d in t i m e (in 1981), a n d the g r o u p I V g e n o m e t y p e is d i s t a n t f r o m o t h e r t y p e s w i t h r e s p e c t to its RE cutting pattern. We thank Jonathan P. Clewley for generously providing the hybrid plasmid pJB and M. Ohara for helpful comments. Part of this study was supported by grants from the Ministry of Education, Science and Culture of Japan.
References ANDERSON,M. J., JONES,S. E., FISHER-HOCH,S. P., LEWIS,E., HALL, S. M., BARTLETt, C. L. R., COHEN, B. J., MORTIMER, P. P. & PEREmA, M. S. (1983). Human parvovirus, the cause of erythema infectiosum (fifth disease)? Lancet i, 1378.
BLUNDELL, M. C., BEARD, C. & ASTELL, C. R. (1987). In vitro identification of a B19 parvovirus promoter. Virology 157, 534-538. CLEWLEY, J. P. (1984). Biochemical characterization of a human parvovirus. Journal of General Virology 65, 241-245. CLEWLEY, J. P. (1985). Detection of human parvovirus using a molecularly cloned probe. Journal of Medical Virology 15, 173-181. COSSART,Y. E., FIELD, A. M., CANT, B. & WIDDOWS,D. (1975). Parvovirus-like particles in human sera. Lancet i, 72-73. COl'MORE, S. F. & TATTERSALL,P. (1984). Characterization and molecular cloning of a human parvovirus genome. Science226, 11611165. MORI,J., BEATTIE,P., MELTON,D. W., COHEN,B. J. & CLEWLEY,J. P. (1987). Structure and mapping of the DNA of human parvovirus B19. Journal of General Virology 68, 2797-2806. MORII'~T, F., TRATSCmN, J.-D., PEROL, Y. & SIEGL, G. (1986). Comparison of 17 isolates of the human parvovirus B19 by restriction enzyme analysis. Archives of Virology 90, 165-172. MORTIMER,P. P., HUMPHRIES,R. K., MOORE,J. G., PURCELL,R. H. & YOUNG, N. S. (1983). A human parvovirus-like virus inhibits haematopoietic colony formation in vitro. Nature, London 302, 426429. NUNOUE, T., OKOCHI,K., MORTIMER,P. P. & COHEN,B. J. (1985). Human parvovirus (B19) and erythema infectiosum. Journal of Pediatrics 107, 38-40. NUNOUE, T., KOIKE, T., KOIKE, R., SANADA,M., TSUKADA,T., MORTIMER, P. P. & COHEN, B. J. (1987). Infection with human parvovirus (B19), aplasia of the bone marrow and a rash in hereditary spherocytosis. Journal of Infectian 14, 67-70. OZAWA,K., KURTZMAN,G. & YOUNG, N. (1986). Replication of the B19 parvovirus in human bone marrow cell cultures. Science 233, 883-886. SHADE,R. O., BLUNDELL,M. C., COTMORE,S. F., TATrERSALL,P. & ASTELL,C. R. (1986). Nucleotide sequence and genome organization of human parvovirus B19 isolated from the serum of a child during aplastic crisis. Journal of Virology 58, 921-936. SUMMERS,J., JONES,S. E. & ANDERSON,M. J. (1983). Characterization of the genome of the agent of erythrocyte aplasia permits its classification as a human parvovirus. Journalof General Virology64, 2527-2532. UMENE,K. (1985). Variabilityof the region of the herpes simplex virus type 1 genome yielding defective DNA: Sinai fragment polymorphism. Intervirology 23, 131-139. WARE, R. (1989). Human parvovirus infection. Journal of Pediatrics 114, 343-348. YANISCH-PERRON,C., VIEIRA,J. & MESSINGJ. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103-119. (Received 14 August 1989; Accepted 4 December 1989)
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The genome type of human parvovirus B19 strains isolated in Japan during 1981 differs from types detected in 1986 to 1987: a correlation between genome type and prevalence Kenichi Umene 1. and Tadasu Nunoue 2 1Department of Virology, Faculty of Medicine, Kyushu University 60 and ZSchool of Health Sciences, Kyushu University, Fukuoka 812, Japan.
The genome DNAs of 12 strains of human parvovirus B19, isolated in Japan at two different times, 1981 and 1986 to 1987, were molecularly cloned in the plasmid pUC18. The cloned B19 DNAs were analysed by cleaving with restriction endonucleases, and were classified into several groups by genome type. The restriction endonuclease cutting patterns ofB 19 strains isolated during 1981 were similar to that of the group
IV genome type, and the patterns of those isolated later were similar to that of group II, suggesting a correlation between the genome type and the prevalence. We conclude that the prevalences of B 19 infection in Japan during 1981 and in 1986 to 1987 were caused by viruses differing in genome type, and that B19 viruses with similar genome types disseminated widely in Japan during each prevalence.
The human parvovirus B19, originally discovered in blood-bank donors by Cossart et al. (1975), is now known to be an aetiological agent of several clinical disorders (Ware, 1989). The B19 viral particles contain a singlestranded DNA of either polarity (Clewley, 1984; Summers et al., 1983), and the genome is a 5.54 kilobase linear molecule with hairpin structures at each extremity (Cotmore & Tattersall, 1984; Mori et al., 1987). B19 has been successfully propagated in suspension cultures of human erythroid bone marrow ceils but such cells are not suitable for use in large-scale propagation of B19 (Mortimer et al., 1983; Ozawa et al., 1986). Molecular cloning of the B19 genome has facilitated analyses of genome structures and functions (Blundell et al., 1987; Clewley, 1985; Cotmore & Tattersall, 1984). Viruses with a DNA genome can be analysed with restriction endonucleases (REs) for differentiation and classification, and Morinet et al. (1986) first investigated the genetic variability of B19. Mori et al. (1987) mapped DNAs of 48 B 19 strains with 13 REs and classified them into several genome types. Four B19 strains isolated in Japan in 1981 were all classified as having the group IV genome type, and no other strain isolated outside of Japan belonged to group IV (Mori et al., 1987). Thus, questions were raised as to whether B19 strains in Japan were restricted to group IV, and whether Japan was sequestered from other areas with respect to the prevalence of B19 infection. To elucidate this question, we collected sera of patients suspected of having B 19 infection and attempted to clone
the BI9 genome DNA, with the intention of obtaining enough DNA for analyses. Single-stranded B19 DNAs extracted from these sera were treated with Escherichia coli DNA polymerase I Klenow fragment, to synthesize double-stranded DNA, utilizing as a primer the hairpin structure at the 3' end of each molecule. Then, the DNAs were either digested with AatII which has a cleavage site in the terminal repeats of the B19 genome (Shade et al., 1986) or with nuclease S1, to remove the terminal 'turn around' form. The DNAs were treated with the Klenow fragment to convert the staggered ends to blunt ends and then joined to vector plasmid DNAs of pUC18 at the SmaI site. The ligated mixture was then transformed into E. coli JM83 (Yanisch-Perron et al., 1985). Hybrid plasmids containing a sufficient length of B19 genome DNA were constructed from 12 sera (Table 1). The structures of the B19 DNAs were analysed by cleavage with 13 REs, i.e. BamHI, BgllI, BstEII, EcoRI, HindlII, HpaI, KpnI, PstI, PvulI, SalI, Sinai, XbaI, and XhoI, as described by Mori et al. (1987) (Fig. 1, 2). No RE sites for EcoRI, Sail and XhoI were detected in the cloned DNAs, as was the case for other B19 strains. One BamHI site, one BstEII site, one KpnI site and one SmaI site were detected in all 12 of the cloned B19 DNAs (Fig. 1). Differences among the B19 strains were detected in the cleavage patterns for BgllI, HindlII, HpaI, PstI, PvulI and XbaI. Four strains (N8, N11, N14, N15) had the same RE cutting pattern as the group II genome type proposed by Mori et al. (1987). Two B19 strains (N2 and N4) had the same pattern as group IV. The cutting
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Table 1. Human parvovirus B19 strains analysed Source of B19 strains Case no. 1 2 3 4 5 6 7 8 9 10 11 12
Clinical symptom*
Place
Date
Aget
Sex~
Strain no.
AC/HS
Osaka
Mar. 1981
11 y
F
N25
AC/HS Blood donor Blood donor AC/HS AC/HS AC/HS AC/HS AC/HS AC/HS AC/HS Asymptomatic
Niigata Fukuoka Fukuoka Tokyo Ibaragi Tokyo Kobe Kobe Fukuoka Fukuoka Saga
Jun. 1981 Dec. 1981 Dec. 1981 Oct. 1986 Nov. 1986 Dec. 1986 Jan. 1987 Jan. 1987 Apr. 1987 May 1987 Jul. 1987
34 y 16 y 17 y 6y 8y 4y 34 y 5y 6y 10 m 10 y
F F M M M M F F F M M
N5 N2 N4 N14 Nll N8 N9 N10 NI5 N16 N22
Hybrid plasmid containing B19 DNA UK295-1 UK295-2 UK284-6 UK294-4 UK283-4 UK289-1 UK287-4 UK281-2 UK285-3 UK292-13 UK290-1 UK291-4 UK293-2
Genome type§
(Iv) IVb IV IV II II II IIb lib II IIb IIc
* AC, Aplastic crisis; HS, hereditary spherocytosis. I" Y, Years; m, months. F, Female, M, male. § Definition of groups is based on that by Mori et al. (1987), and this work (see text).
patterns of four strains (N9, N10, N16 and N22) were similar to but not the same as that of the group II genome type (Fig 1, 2)i The three strains N9, N10 and N16 did not have HindlII sites in the position marked on line 2 of Fig. 1 by a parenthesized HindlII. The absence of this site caused the loss of two fragments of 0.94 kb and 0.90 kb and the gain of a 1-8 kb fragment, in comparison with the group II genome type (lanes 2, 4, 5 and 6 of part B of Fig. 2). Strains N9, NI0 and N16 were classified as group IIb in this work (Table 1). The BgllI/EcoRI/SalI cleavage products of hybrid plasmid UK293-2 (containing genome DNA from strain N22) contained only two fragments of vector DNA and the B19 genome insert (lane 3 of part A of Fig. 2), indicating the absence of a BgllI site in the N22 strain (line 3 of Fig. 1). N22 was classified as group IIc (Table 1). The central one-fifth of the N25 genome was not cloned. The other four-fifths were cloned and had the same structure as that of group IV genome type (line 5 of Fig. 1). We provisionally assigned N25 to group IV (Table 1). The RE cutting patterns of strain N5, except for PvulI, were the same as those of the group IV genome type. The recombinant plasmid DNA containing the N5 genome was cleaved into two fragments of vector DNA and insert DNA after digestion with PvuII/EcoRI/SalI (lane 7 of part C of Fig. 2), thereby indicating that N5 has no PvuII site (line 6 of Fig. 1). N5 was classified as group IVb (Table 1). Mori et al. (1987) classified 48 B19 strains into five types, defined by having the same RE cutting pattern, and an unassigned group. The five groups of genome types can be distinguished by differences in one to five
RE cutting sites. Group IV is different from groups Ilia and IIIb in five RE cutting sites, from group II in four sites, and group I in three sites, suggesting a remote relationship between group IV and the other four groups. Groups lib and IIc are apparently more closely related to group II than group IV. The twelve B19 strains analysed in this work could be classified into two clusters, with regard to their RE cleavage pattern (Table 1). One cluster, having a pattern similar to that of group IV, consisted of B19 strains isolated in 198I. The other cluster, similar to group II, consisted of B19 strains isolated in 1986 to 1987. The results show a correlation between the genome type and the time of isolation (prevalence), suggesting an epidemiological relationship among strains. The prevalence of B 19 infection is recognized by the presence of erythema infec'tiosum (El) (Anderson et al., 1983; Nunoue et al., 1985). Surveillance of infectious diseases in Japan showed two major prevalences of El in 1981 to 1982 and 1986 to 1987 (Nunoue et al., 1987). The genome types of the BI9 strains correlated with the time of isolation but not with the area of isolation (Table 1). We assume that a nation-wide B 19 prevalence occurred in 1981 to 1982 and 1986 to 1987 in Japan, and that each was caused by a group of viruses with a similar genome type, hence indicating highly efficient transmission of B19 viruses. B19 strains of the group II genome type have been isolated from different areas in the world, i.e. France, Norway, U.K. and U.S.A. (Mori et al., 1987). It is probable that a group II B 19 virus entered Japan around 1986 and caused the new epidemic of B19 infection in 1986 to 1987.
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2
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5 kb
4
I
(a)
A 1
1 2
C
B
!
i
3
!
I
1
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5 6
!
!
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M --23-1 --9-4 --6-6
1
--4.4 w
m
--V
-V
-v--2.3 --2"0 i
6
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I
J
II
I
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--0-56
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A
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[
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1
Fig. 1. Restriction endonuclease map of B19 DNA. The enzyme cutting sites on the group II genome type are shown at line 1. The restriction maps of group IIb genomes (line 2), group IIc genomes (line 3), group IV genomes (line 4), strain N25 from case 1 (line 5), and group IVb genomes (line 6) are shown. At lines 2 to 6, addition and loss of restriction sites are shown in comparison with the map of group II. Additional sites are entered in the maps, and lost sites are indicated by parentheses. The genome DNA of N25 was molecularly cloned as two hybrid plasmids, one containing the left-side two-fifths region (UK295-1) and the other containing the right-side two-fifths region (UK295-2). The central one-fifth of the N25 genome (indicated by parentheses in line 5) was not cloned. The locations of fragments used as probe DNAs are shown in line 7. The EcoRI site of the 0.7 kb PtrulIEcoRI fragment is derived from the EcoRI linker flanking cloned BI9 DNA.
Sources of an outbreak and the mode of transmission among cases of viral infection could be investigated by analysing the genome type of the viruses. Of the cases analysed in this work, the case 9 patient is the daughter of the case 8 patient (Table 1). They developed aplastic crisis (AC) at the same time, on the 6th day after the onset of AC in a 9 year old boy with hereditary spherocytosis (HS) in the family, presumably due to the same source of infection. Both B19 D N A s from the two belonged to the IIb genome type (Table 1). The results obtained by analyses of the genome type are m keeping with the deduction from the clinical observations, i.e. the source of infection was the same. The genome types of virus strains can be used to examine whether a virus strain causes a specific clinical manifestation. Morinet et al. (1986) reserved speculation concerning a possible association of B19 variants with
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C
i
I
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2 4
5
6
I
I
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M --23.1 --9-4 --6.6 --4"4
--2.3 ~2.0
--0-56
Fig. 2. Variations of restriction endonuclease cleavage patterns found in B19 strains analysed in this work. Hybrid plasmid DNAs of UK283-4 (as a representative of group IV) (lane 1), UK281-2 (as a representative of group II) (lane 2), UK293-2 (lane 3), UK285-3 (lane 4), UK292-13 (lane 5), UK291-4 (lane 6) and UK284-6 (lane 7) were cleaved with BgllI (part A), HindlII (part B) or PvulI (part C), after digestion with EcoRI and Sail. The cleaved DNAs were electrophoresed in 0.8% agarose gels, and stained with ethidium bromide (a). The lane designated M contains a marker mixture (kb) of HindlII fragments of 2 phage DNA. The DNAs in the agarose gels were then transferred to nylon membranes (Umene, 1985). The DNAs cleaved with BgllI and PvulI (parts A and C of a) were hybridized with a 32p. labelled probe, the 0.7 kb PvulI-EcoRI fragment of pJB (see line 7 of Fig. 1) (parts A and C of b). The hybrid plasmid pJB, constructed by inserting a 5.2 kb B19 DNA flanked with EcoRI linkers into the EcoRI site of vector pGEM-1, was provided by J. P. Clewley (Mori et al., 1987). HindlII digests (part B of a) were hybridized with the 32p. labelled 0.90 kb HindlII-HindlII fragment ofpJB (see line 7 of Fig. 1) (part B of b). Bands marked with a black dot in (a) correspond to those detected by Southern hybridization in (b). The positions of vector DNA fragments are indicated by a v in (a).
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d i s t i n c t clinical s y m p t o m s . M o r i et al. (1987) e x a m i n e d w h e t h e r one g e n o m e t y p e could be c o r r e l a t e d w i t h a p a r t i c u l a r clinical m a n i f e s t a t i o n o f infection, b u t f o u n d n o correlation. N i n e B19 strains a n a l y s e d in our w o r k were isolated f r o m p a t i e n t s w i t h H S at A C ( T a b l e l ) a n d six o f t h e m were a s s o c i a t e d w i t h s k i n r a s h e s ( N u n o u e et al., 1987). T h e m o d e o f the r a s h e s v a r i e d , h e n c e we could n o t correlate the g e n o m e t y p e w i t h t h e m o d e o f rashes a n d w i t h t h e severity o f A C . W e a s s u m e t h a t clinical m a n i f e s t a t i o n s o f B19 infection are m o d i f i e d greatly b y host factors. H o w e v e r , v a r i a t i o n in a viral g e n o m e m a y p o s s i b l y cause v a r i a t i o n o f a m i n o a c i d c o m p o n e n t s o f v i r u s - e n c o d e d p r o t e i n a n d also v a r i a t i o n in cis-acting e l e m e n t s in the virus genome. T h e s e v a r i a t i o n s m a y affect v i r u s - h o s t i n t e r a c t i o n . W e were i n t e r e s t e d in p o s s i b l e c o r r e l a t i o n s b e t w e e n the g e n o m e t y p e a n d the scale o f p r e v a l e n c e , as the r e p o r t e d n u m b e r s o f cases o f E I a n d A C in 1986 to 1987 (group I I genorne type) were h i g h e r t h a n those in 1981 to 1982 (group I V g e n o m e type). T h e r e were seven cases o f foetal d e a t h a s s o c i a t e d w i t h B19 d u r i n g 1986 to 1987 b u t n o n e in 1981 to 1982. T h e isolation o f g r o u p I V strains was r e s t r i c t e d to c e r t a i n a r e a s (in J a p a n ) a n d in t i m e (in 1981), a n d the g r o u p I V g e n o m e t y p e is d i s t a n t f r o m o t h e r t y p e s w i t h r e s p e c t to its RE cutting pattern. We thank Jonathan P. Clewley for generously providing the hybrid plasmid pJB and M. Ohara for helpful comments. Part of this study was supported by grants from the Ministry of Education, Science and Culture of Japan.
References ANDERSON,M. J., JONES,S. E., FISHER-HOCH,S. P., LEWIS,E., HALL, S. M., BARTLETt, C. L. R., COHEN, B. J., MORTIMER, P. P. & PEREmA, M. S. (1983). Human parvovirus, the cause of erythema infectiosum (fifth disease)? Lancet i, 1378.
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