erythrovirus ( formally parvovirus ) b19 ( b19 ) causes a wide range of diseases in humans .
b19 is a currently the only accepted member of the erythrovirus genus and the only erythrovirus known to be pathogenic in humans .
several workers reported cases of children with fulminant or acute hepatitis with acute b19 infection and suggested that b19 could be the cause of the hepatitis 1 - 3 .
their diagnosis was based on the detection of the serum b19 dna by pcr assay .
however , the cause of fulminant hepatitis and biliary atresia in many patients is unexplained and this is a serious issue , especially in newborns and infants .
more than 80% of these cases ultimately require liver transplantation or die of hepatic failure .
b19 has been proposed as the cause of the fulminant hepatic failure in patients with or without aplastic anemia , on the basis of the presence of b19 dna in liver specimens .
an immunologically - mediated mechanism of b19-induced hepatocyte destruction has been postulated , but not documented .
thus , there could be an unsuspected cofactor that is associated with b19 infection and is deleterious to the liver .
to address these important issues , we conducted a molecular characterization of b19 genomes isolated from patients with fulminant hepatitis and biliary atresia .
patients : we compared 2 different groups consisted with 47 japanese patients ( aged from newborns to 66 years old ; 24 males and 23 females ) . group a consisted of 28 patients with fulminant hepatitis ( 14 males and 14 females ; 9 infants less than one year old , 8 children 1 - 10 years old , 3 adolescents 11 - 20 years old and 8 adults over 21 years old ) . on the other hand ,
group b consisted of 19 patients with biliary atresia ; 7 males and 12 females ; 4 infants less than one year old , 10 children 1 - 10 years old , 4 adolescents 11 - 20 years old and one adult over 21 years old ) .
all patients examined had no evidence of hepatitis virus infections and diagnosed as non - b , non - c hepatitis .
most of the patients tested underwent living - donor liver transplantation at kyoto university hospital from 1994 to 2000 .
majority of patients had history of blood transfusion or blood product infusion in their course of treatment .
the liver tissues and serum samples from recipients obtained at operation stored at -80c until used .
informed consent for participation in this study was obtained from each individual . nucleic acid extraction , pcr and sequencing analysis :
nucleic acids ( dna / rna ) were extracted from frozen liver tissues and serum using sepagene rv - r kit ( sanko - junyaku , tokyo , japan ) .
the resulting pellet was resuspended in 100 l rnase - free water , following the manufacturer 's instruction .
b19 dna was screened by nested pcr using primers designed from vp1 region ; pv1 ( sense , 5'-gct gtt aag gat gtt aca ga-3 ' , nucleotide ( nt ) 3520 - 3521 ) and pv1r ( antisense , 5'-gga tcc gta taa ggg att gt-3 ' , nt 3882 - 3901 ) for the 1st pcr and pv2 ( sense , 5'-cag gtt act gac agc act ac-3 ' , nt 3541 - 3560 ) and pv2r ( antisense , 5'-tgt tga ctg cag ccc tct aa-3 ' , nt
the sensitivity of this pcr assay allowed detection of as few as 10 copies of b19 genome .
in addition , we sequenced the b19 isolates consisted of 4561 bp covering ns1 , vp1 and vp2 regions obtained from liver tissues of patients . for this purpose ,
three overlapping pcr products ( fragments a to c ) were generated ; for fragment a ( 1944 bp ) , pv3 ( sense , 5'-ttt ccc gcc tta tgc aaa tgg gca g-3 ' , nt 393 - 417 ) and pv5r ( antisense , 5'-agc tcc cac atg gca gct ac-3 ' , nt 2533 - 2552 ) for the 1st pcr and pv4 ( sense , 5'-tgt aac ggt taa aat ggg cgg agc g-3 ' , nt 457 - 481 ) and pv6r ( antisense , 5'- ccc ctt aca ccg tcc cac ac-3 ' , nt 2382 - 2401 ) for the 2nd pcr ; for fragment b ( 1835 bp ) , pv5 ( sense , 5'-gca gca gtg gtg gtg aaa gc-3 ' , nt 2143 - 2162 ) and pv1r for the 1st pcr and pv6 ( sense , 5'-ggc gcc tgg aac act gaa ac-3 ' , nt 2208 - 2227 ) and pv2r for the 2nd pcr ; for fragment c ( 1297 bp ) , pv1 and pv3r ( sense , 5'-tac agt ctg ggt ggt act ggt ggg c-3 ' , nt 5010 - 5034 ) for the 1st pcr and pv2 and pv4r ( antisense , 5'-ctg gtg ggc gtt tag tta cgc atc c-3 ' , nt 4994 - 5018 ) .
the pcr was done with amplitaq gold ( perkin elmer , norwalk , conn . ) and blend - taq - plus dna polymerase ( toyobo co. , tokyo , japan ) .
the amplicons were separated by 1% agarose gel electrophoresis and purified using the qiaquick gel extraction kit ( qiagen inc . , chatsworth , calif . ) .
recovered amplicons were subjected to direct sequencing from both directions using the abi prismtm big dye terminator cycle sequencing ready reaction kit ( perkin elmer ) .
sequences of amplified dna were determined using a sequencer ( abi model 373a ; applied biosystems , foster city , calif . ) . additionally , hepatitis a , b , c and e genomes were also determined by the nested pcr .
detection of b19 mrna : the purified rna obtained from liver tissues as described above , was digested by rq i dnase ( promega co. , madison , wis . ) to remove any dna .
b19 cdna was synthesized from the pretreated rna by reaction with 100 units of moloney murine leukemia virus reverse transcriptase ( promega co. ) and pv1r antisense primer .
phylogenetic analysis : characterization of b19 isolates was examined by the phylogenetic analysis as described previously 4 .
the data were analyzed using a standard statistical software package ( stat view ; brain power inc . , calabases , ca , u.s.a . ) .
statistic analyses : statistic analyses were performed by the chi - square test . a difference with a p value of less than 0.05
b19 dna in liver tissues was detected in 10 of 28 ( 35.7% ) patients with fulminant hepatitis and 7 of 19 ( 36.8% ) patients with ba , respectively ( statistically not significant ) .
except for two infants with fulminant hepatitis who were hbv dna - positive , all cases tested were negative for hav rna , hcv rna and hev rna .
importantly , among the hepatic b19 dna - positive 8 patients who had paired samples of liver tissues and serum , the serum b19 sequence was detectable in only one case ( table 1 ) . to determine the form of viral replication , the presence of b19 mrna in the liver tissue was examined .
the results showed that b19 mrna was present in all cases with fulminant hepatitis to be hepatic b19-positive , but only 1 of 7 ( 14.3% ) cases in biliary atresia tested .
amplification of b19 dna did not take place in the absence of reverse transcriptase , indicating minimal dna contamination .
in addition to the above - described examinations , to characterize the b19 genomes , we obtained 14 isolates of the b19 genome ( 10 isolates with nearly full - length sequences ( 4561 bp ) that covered the complete genes of ns1 , vp1 and vp2 and 4 isolates with only one short sequence in the ns1 ) .
all b19 isolates showed an overall identity of 98% at the nt level among each isolate with 4561 bp recovered in this study and to prototypes of b19 hv ( f162273 ) and b19 au ( m13178 ) , and an identity of 87% to the b19 v9 isolate ( ay083234 ) .
interestingly , phylogenetic analysis based on the ns1 gene revealed three different clusters : two ( genotypes 1 and 3 ) for isolates from fulminant hepatitis and the other ( genotype 2 ) for isolates from biliary atresia ( fig .
, specific sites of the deduced amino acid sequence change were identified in the ns1 gene ( fig .
the amino acid at position 181 and 201 showed methionine and aspartic acid , respectively .
furthermore , in 5 out of 6 isolates belonging to group 3 , the amino acid at position 183 substituted to alanine .
nucleotide sequence data reported are available in the ddbj / genbank / embl database under the accession numbers : ab126262 for b19-an23 , ab126263 for b19-an28 , ab126264 for b19-an30 , ab126265 for b19-an34 , ab126266 for b19-an40 , ab126267 for b19-an41 , ab126268 for b19-an56 , ab126269 for b19-an66 , ab126270 for b19-an85 and ab126271 for b19-an87 .
the etiology of hepatitis remains obscure in 3 - 10% of cases in europe 5 and the united states 6 and up to 30% of cases in asia 7 , 8 .
particularly , the cause of fulminant hepatitis in children is unexplained in up to 50% of cases in our hospital .
once well - known hepatotropic agents and metabolic , toxic , and immunological causes have been excluded , an infectious origin remains a possibility .
on the other hand , the concept that biliary atresia consists of two major types based on the clinical aspects has been accepted 9 , 10 .
one is the fetal , embryonic or prenatal type , and the other is the acquired or perinatal type .
the perinatal type is believed to account for 70 - 80% of all biliary atresia .
the first is called the ductal plate malformation theory , which assumes a congenital anomaly of the intrahepatic bile trees . according to the other theory ,
the progressive occlusion of the extrahepatic bile duct is probably triggered by viral infection and worsened by the subsequent immunopathological process .
reovirus , rotavirus 11 , 12 , cytomegalovirus 13 , 14 and others have been proposed as likely causative viruses , but no clear relation has been found . to the best of our knowledge , there are only a few reports 1 , 15 , 16 on the analysis of viral infections based on direct detection of viral genomes from liver tissues .
b19 was discovered serendipitously in the sera of normal blood bank donors while screening for the hepatitis b virus .
b19 , a member of the family parvoviridae , subfamily parvovirinae , genus erythrovirus , is a small single stranded dna virus .
this virus is responsible for various clinical manifestations including erythema infectiosum in children , polyarthropathy syndrome in adults , transient aplastic crisis inpatients with chronic anemia , persistent infections manifesting as chronic anemia in immunocompromised patients , transplant recipients , and hydrops fetalis and fetal death by intrauterine infection .
aplastic anemia , which occurs in 33% of children and 5% of adult patients who undergo orthotopic liver transplantation ( olt ) for non - a , non - b , non - c fulminant hepatitis is pointed out as a complication with a high rate of mortality 17 .
the cause of aplastic anemia after olt may include factors such as posthepatitic aplasia , b19 , and drugs .
1 reported the possibility of b19 as the cause of non - a - c fulminant hepatitis - associated aplastic anemia .
actually , b19 may be an etiologic agent for hepatitis - associated aplastic anemia , because of the known tropism of b19 for erythroid precursors 18 .
19 reported no evidence for hepatitis e or parvovirus b19 infection in patients with acute liver failure . to investigate the possibility that b19 infection of the liver might cause fulminant hepatitis and biliary atresia
, we conducted a study to obtain direct evidence of a viral sequence in the liver from patients who underwent liver transplantation . in this retrospective study of 47 patients with liver diseases of unknown etiology ,
we postulated that the virus might even be detected in liver that is serologically negative for the virus .
in fact , our results showed that only one case was sero - positive for the b19 sequence among 8 cases with intrahepatic - b19 dna .
these results suggest that seronegativity for the b19 sequence does not exclude the existence of present infection of b19 within the liver .
they showed b19 dna is frequently present in livers of anti - b19 seropositive adults suggesting persistence of b19 in the liver .
our results also indicate that direct detection of viral genomes in tissues by a highly sensitive method is as important as serology . in the present study
, we found a high prevalence of b19 infection not only in fulminant hepatitis patients , but also in biliary atresia patients .
however , all fulminant hepatitis patients infected with b19 had the replicative form of b19 mrna . while , low rate of b19 mrna detection in biliary atresia patients who had intra - hepatic b19 dna suggested silent infection of b19 .
this suggests that b19 may be a cause of severe hepatic diseases although this remains to be investigated .
b19 encodes three major viral proteins : vp1 and np2 , the viral capsid proteins , and ns1 , a nonstructural protein .
both vp1 and vp2 are derived from overlapping reading frames and share substantial amino acid sequences .
ns1 is known to be implicated in viral replication , the activation of viral gene transcription , and target cell cytotoxicity 21 - 25 . among the sequence records deposited in the database ,
only 16 isolates of b19 have been sequenced in the full or nearly full - genome so far .
here we reported that the nearly full - length sequence of b19 was isolated from 10 patients with liver diseases .
it is known that b19 has a genome length of 5.4 kb with hairpin structures at each extremity ; in addition , a partial deletion and rearrangement of the sequence exist in the extremity regions .
these findings indicate the difficulty in amplifying the sequences in these regions . in our study
interestingly , phylogenetic analysis based on the ns1 gene revealed three different clusters : two for isolates from fulminant hepatitis and the other for isolates from biliary atresia cases .
viral genotype has diagnostic and clinical implications , including use for assessing responses to anti - viral therapy and future vaccine development .
the possibility that such variation is involved in the severity of associated liver disease , and that the difference between symptomatic and asymptomatic infection , may also occur as well as hbv and hcv .
furthermore , among each genotype , specific sites of the deduced amino acid sequence change were identified in the ns1 gene .
clarification of the relation between the genotypes or variants of b19 and its pathogenicity in hepatic failure including hepatitis and biliary atresia is awaited with great interest . to solve these important issues ,
a geographical study of the b19 genomes and various diseases including liver diseases in different countries is now planning and we shall report on further investigations in the future . in conclusion , we found a high prevalence of b19 infection in livers from patients with fulminant hepatitis and biliary atresia .
interestingly , all fulminant hepatitis patients infected with b19 had the replicative form of b19 mrna .
it is noteworthy that serum b19 dna was not detected in 7 of 8 cases that were hepatic b19 dna - positive .
our results presented here suggested that b19 may serve as an etiologic agent for severe hepatitis .
phylogram generated by neighbor - joining analysis of genetic distances in the ns1 region of erythrovirus b19 .
alignment of amino acid sequences ( upper ) and nucleic acid sequences ( lower ) in the ns1 region of erythrovirus b19 .
 background : fulminant hepatitis and biliary atresia are serious problems and their causes have not been explained well . 
 we investigated whether or not erythrovirus b19 is a candidate etiologic agent in such liver disease patients who had undergone liver transplantation.methods : liver tissues from 47 patients consisted of 28 fulminant hepatitis and 19 biliary atresia were examined to detect b19 genes by pcr and further analyzed their genomic characterization.results : b19 dna was detected by nested pcr in 10 of 28 cases ( 35.7% ) livers in the fulminant hepatitis group and 7 of 19 ( 36.8% ) livers in the biliary atresia group , respectively ( statistically not significant ) . 
 importantly , among the 8 hepatic b19 dna - positive patients who had paired samples of liver and serum , the serum b19 genome was detectable in only one case . 
 b19 mrna was identified in all of 10 fulminant hepatitis cases with hepatic b19 dna , but only 1 out of 7 ( 14.3% ) cases in biliary atresia tested . 
 furthermore , we obtained ten isolates having the b19 genome with nearly full - length sequences . 
 interestingly , phylogenetic analysis based on the ns1 gene revealed three different clusters : two for isolates from fulminant hepatitis and the other for isolates from biliary atresia.conclusions : our results presented here suggested that b19 may be an etiologic agent of fulminant hepatitis . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
Figures and Tables