chronic hepatitis b virus ( hbv ) infection is a significant public health threat . approximately 3 - 4 billion people worldwide have been infected with hbv .
the natural course of chronic hbv infection is variable , ranging from an inactive hbsag carrier state to a progressive chronic hepatitis which may eventually lead to cirrhosis and hepatocellular carcinoma ( hcc ) .
previous research has shown that patients with persistent hbeag - positive state and high dna viral load ( > 10  copy / ml ) are at higher risk of hcc than the hbeag negative individuals with low dna viral load [ 1 , 2 ] . in order to have a better understanding of this phenomenon
, we obtained hbsag - positive serum samples from clinical laboratory center of the first people 's hospital of yunnan province , kunming , yunnan , china , from february to august , 2005 .
we also followed up a chronically hbv - infected patient who was hbeag positive with high hbv dna load ( 10  copy / ml ) for almost 3 years and studied the evolution of her hbv strains . by sequencing and phylogenetic analysis , we found mutations occurred within the genomes of hbv through time - course evolution . combined with clinical data , whether and how these hbv variants might contribute to viral persistence of infection or liver pathogenesis still needs to be further studied
a 31-year - old female with chronic hbv infection was followed up at the first people 's hospital of yunnan province , kunming , yunnan , china .
the subject was admitted in the hospital because of allergic purpura in may , 2005 and was diagnosed with chronic infection based on the fact of having been hbsag positive for at least 7 years .
her serum samples were collected at three time points after admission : 602 ( 1 day ) , 6022 ( 6 months ) , and 6023 ( two years and seven moths ) and stored at 80c until time of analysis .
serological markers of hbv , alt , ast , bil , alb , and glo were analyzed at each time point .
the patient was seronegative for antibodies to hav , hcv , hev , and hiv and did not have a history of alcohol abuse , drug abuse , or hepatotoxin exposure .
hbv dna was extracted from serum by proteinase k digestion , followed by phenol / chloroform extraction .
viral dna load was determined using hbv dna pcr - fluorescence quantitation kit ( kehua bio - tech .
the primers used were forward primer : 5ctttttcacctctgcctaatca3 ; reverse primer : 5agaggtgaaaaagttgcatggt3. amplification was performed in a 96-well cycler ( bio - rad , usa ) , and 25  l pcr mixture containing 2.5  mm mgcl2 , 200  m dntp , 400  nm of each primer , and 2  u of ex taq polymerase ( takara bio - tech .
the pcr reaction was performed using the following cycles : 94c predenature for 5  min , 30 cycles of 94c for 1  min , 56c for 1  min , 72c for 2  min ; 72c for 10  min as a final extension step .
then full - length amplicons were purified using a gel extraction kit ( huashun bio - engineering co. ltd . ,
the pcr products were purified and cloned in vector pmd18-t ( takara bio - tech .
white colonies were picked , correct recombinants were confirmed by pcr , and the double - restriction endonuclease digestion with ecori and hind iii .
dna sequencing analysis of the correct recombinants was performed with bigdye terminator v3.1 and 3130 genetic analyzer ( applied biosystems ) ( 7 pairs of sequencing primers can be obtained on request ) . for each time point , 8 - 9 recombinants were randomly selected and sequenced .
fourteen hbv complete genomes available in genbank ( genotypes a , b and e to h ) were applied to phylogenetic analysis .
additionally , 50 published sequences of genotype c were also used in this study for alignment analysis ( genbank accession numbers can be obtained upon request ) .
the whole genome sequences were assembled by bioedit sequence alignment editor software , version 7.0.5.2 .
phylogenetic and molecular evolutionary analysis of nucleotide differences within and between the isolate sequences were carried out by mega program , version 3.1 .
bootstrap re - sampling and reconstruction were carried out for 1,000 times to confirm the reliability of phylogenetic trees .
as showed in table 1 , hbsag and hbeag were positive at all three time points and titers changed during the course of analysis .
alt levels showed a tendency of elevation , and at the 3rd time point the level was more than 2 times of the upper limit of normal .
while no quantification could be obtained from the 1st time point due to the lack of serum , viral dna load was found to decrease from 10 to 10  copy / ml at the last two time points .
26 sequences were analyzed ( genbank accession number : dq377159 - 377165 , eu306713 - 306714 , eu306722306729 , eu439005 , eu439008439014 and eu439025 ) .
the divergence of these 26 sequences was found to be 01.4% . when compared to the 64 complete hbv genome sequences published in the genbank , all 26 sequences were determined to be genotype c and c2 subgroup with a bootstrap value of 100% ( data not shown ) .
when compared with the 50 known and complete sequences of genotype c , we found that there were 13 specific nucleotides in all sequenced clones of this patient ( data not shown ) .
further analysis of the nucleotide sequence at all three time points indicated that nucleotides t361a , c934a , c2351t / a2353 t , and c2444 t were the mutation been kept in for at least two years ' evolution .
these mutations caused nonsense mutation of pres1 and precore / core gene ( t361a and c2444 t , resp . ) , missense mutation of precore / core and pol gene ( c2351t / a2353 t and c934a , resp . ) .
nucleotides c339 t and t770c were the new and major mutation at the 3rd time point , and these mutations caused hbsag p62l and polymerase ( pol ) v560a mutation , respectively ( figure 1 ) .
premature stop codons of s gene ( g.200c > t , g.565t > a , g.361t > a , g.455c > t , and g.304_305cc >
tt ) increased during the course(1/9 versus 3/9 versus 6/8 ) ( figure 2 ) .
a1762t / g1764a double mutation existed in all clones , and g1896a existed in 4 clones obtained from all time points . performing
alignment showed that over the 2-year period , deletions across overlapping region of polymerase / pres1 ( g.2447_489del 1256 ) or pres1/pres2 ( g.3018_3202del 183 , and g.3144_56del 126 ) , and pre - s1 initiation codon mutations ( g.2846_2865del 18 ) with or without internal deletions mutations ( g.3024_3193del 168 ) were prevalent ( 22/26 , in 85% of the clones ) ( figure 2 ) .
all the defective genomes shared sequences from nucleotide position 489 to position 2447 , and common to all genomes is the presence of the complete x gene and the complete c orf .
four paires of donor / acceptor were found in the spliced genomes , besides the frequently reported donor sites 2447 and 3018 and the acceptor sites 489 , two new 5-splice donors ( nt3144 and 3024 ) and three new 3-splice acceptors ( nt56 , 3193 and 3202 ) were detected at the 3rd time .
up until now , details of the mechanism accounted for the hbv evolution remained unknown . in this study , we followed up a chronically hbv - infected patient and discovered that complex and diverse quasispecies existed in this patient for over two - year disease evolution . based on the alignment and phylogenetic analyses , the divergence and the 13 specific nucleotides of the 26 clones , as well as the phylogenic analysis
thus , these results reflect the real evolution process of homogeneous hbv strains in this patient . in the process of evolution ,
some mutations in this patient were kept in for at least 2 years , and other new mutations emerged at the 3rd time point .
these mutations resulted in amino acids substitutions and some caused premature stop codon of the coding regions . among these mutants , the t361a nonsense mutation in the small s region , which resulted in a 69aa truncated hbsag lacking the entire  a  determinant region ( aa124147 ) ,
was first detected at the 2nd time point in one clone . at the 3rd time point , however , half of the sequenced clones were found to have this mutation .
this mutant has been reported in the occult hbv - infected patients with seronegative for hbsag , and in one case hbv dna was absent from the serum after the two - year [ 7 , 8 ] . in our case , after two - year followup , the hbsag was still at a high level and could be detected by routine immunoassay - based diagnosis .
in addition , hbv dna was at a high load despite a slightly decreased level ( 2.4  10 versus 2.5  10  copy / ml ) , and the alt level showed an elevating trend over the study period . in the meanwhile , new point mutations such as c339 t and t770c were detected at the 3rd time point ; the former occurred in the hydrophilic region of hbsag and the latter was previously discovered in a hcc patient ( genbank number ay206393 ) .
we speculated that these mutants might afford hbv variants a distinct survival advantage by allowing the mutant virus to escape from the immune system .
in addition , these mutations could further transactivate certain oncogenes which in turn rendered the acceleration of the liver damage [ 913 ] . in this patient
the deletion region is in the span of hepatocellular - binding site ( aa520 ) and led to aa111 deletion in pres1 .
this deletion mutant was previously reported in different clinical cases and genotypes [ 1417 ] .
for example , this 18  bp deletion was detected in a heart transplant patient while it was not found with the donor . in our case
, retrospective study showed that the son of this patient , though infected with hbv via mother - to - infant transmission , carried none of these mutant strains in the sequenced clones .
this phenomenon may imply that host immune pressure was the primary cause of aa111 deletion in pres1 .
in addition , this mutant seemed to have reduced from the 1st time point to the 3rd time point ( 7/9 versus 7/9 versus 2/8 ) and had a tendency to be substituted by large fragments deletion located in the pres region at the 3rd time point ( figure 2 ) . whether it was a precursor of large fragment deletion mutation or
just an isolated event happened under the immune pressure still needed to be carefully studied .
the other prominent variation at the 3rd time point was the defective genomes caused by the large fragment deletion focused on the overlapping regions of polymerase / pres1 and pres1/pres2 .
up until now , there have been at least 15 types of spliced hbv dna reported in the literature ( figure 3 ) [ 20 , 21 ] , which consisted of seven 5-splice donors and six 3-splice accepters . in our case , four types of spliced hbv dna were found at the 3rd time point , the donor site and the receptor site of these splicers followed the gt - at rules . among them , the 2447/489 donor / acceptor splicer existed as described in previous reports and produced a presumptive hbv spliced protein ( hbsp ) .
in addition , positions 3144 and 3024 as the 5-donor sites and positions 3202 , 3193 , and 56 as the 3-acceptor sites were first reported in our study . during a subsequence screening , we also detected defective genomes in patients with hepatocirrhosis and chb ( genbank accesson numbers : eu439017 , eu306668 , eu306669 , eu330986 , eu306676 , eu306670 ) . though these defective genomes were different in structure , they all shared sequences from nucleotides position 489 to position 2447 .
this region contains all known signals required for conventional replication ( dr1 and dr2 ) , synthesis , and processing of pregenomic / c - mrna , prec mrna , and x mrna , and pregenomic rna encapsidation .
in contrast , all lacked the complete pre - s1 and , or pre - s2 orfs .
many surveys noted that spliced hbv dna and pre - s deletion were more frequently detected in patients with severe liver disease [ 2426 ] and often existed during the process of anti - hbe seroconversion and subsequent loss of hbsag [ 2729 ] .
however , in this case with our subsequent screening , all the patients with defective genomes were hbeag positive and with high hbv dna load ( 10  copy / ml ) . based on these findings
, we deduced that these defective genomes ( with intact x gene and all signals required for viral life cycle ) might contribute to persistent hbv replication in the patient .
other mutations such as c934a , c2351t / a2353 t , c2444 t , a1762t / g1764a , and g1896a were scattered in the whole genomes . in the present study , all clones of the mother from three time points had double mutations of nucleotide
a1762t / g1764a in basal core promoter ( bcp ) , four of which were also coupled with g1896a .
however , we did not observe any hbeag / anti - hbeag seroconversion at the three time points , nor did we detect the impact of bcp mutation on hbv dna load [ 30 , 31 ] .
in addition , multisequences alignment has showed that genotype c had a tendency for higher prevalence of the bcp mutation ( 41 versus 35 , 53.9% ) ( genbank accesson number can be obtained on request ) . combined with the clinic results ( table 1 ) ,
whether the bcp and precore mutations in this patient were related to infection with genotype c , or whether the patient was seroconverting to anti - hbeag or may develop severe disease [ 32 , 33 ] are questions that still need to be followedup . in conclusion , our data showed that viral quasispecies are very closely related to genomes but exist in an environment of mutation , selection , and competition , and thus lead to a dynamic and changing population . during this progress ,
some mutations were kept in and became the dominant strains , some new mutations emerged , and these strains may play important roles in the persistence of infection or the progression of disease .
 mutations in full - length hbv isolates obtained from a chronic hbv - infected patient were evaluated at three time points : 1 day , 6 months , and 31 months . 
 while 5 nucleotides variation , and an 18  bp deletion of pres1 have been kept in during at least the first two years , c339 t mutation occurring in the hydrophilic region of hbsag and t770c that caused polymerase v560a substitution were the new point mutations found existing in sequenced clones of the 3rd time point . 
 internal deletion of coding region obviously appeared in the 3rd time point . 
 the splicers included two new 5-splice donors and three new 3-splice acceptors besides the reported donors and acceptors and may have produced presumptive hbv - spliced proteins or truncated pres proteins . 
 alt , hbeag and viral dna load varied during the follow - up years . 
 these data demonstrated the diversity of genomes in hbv - infected patient during evolution . 
 combined with clinical data , the hbv variants discovered in this patient may contribute to viral persistence of infection or liver pathogenesis . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion