the patient , a 55-year - old man , sought treatment at a hospital in the southeast of scotland in april 2012 for nausea , vomiting , anorexia , jaundice , headache , and abdominal pain . at admission ,
the patient s blood sample was positive for anti - hev igm and igg and raised levels of alanine aminotransferase ( alt ) ( 4,023
iu / l ) and bilirubin ( 91 mol / l ; reference range 317 mol / l ) . over the next 10 days , alt declined steadily to 404 iu / l and returned to normal levels 1 month later .
the patient s abdominal pain had largely resolved by 6 weeks after seeking treatment , but fatigue and lethargy persisted for > 8 weeks .
there was no evidence that the patient was immunocompromised or was co - infected with other hepatitis viruses .
the patient had unexplained jaundice at age 10 years and a history of alcohol abuse as an adult .
virus rna was extracted from 140 l of serum and sequenced at limiting dilution in the hvr and orf2 regions as described ( 6 ) .
analysis of the orf2/orf3 overlap region used the primers 5-cgggtggaatgaataacatgt-3 ( outer sense nucleotides 50985118 relative to m73218 ) , 5-gcrgtyarcggcgmrgccccagctg-3 ( outer antisense , 54815457 ) , 5-tytgcctatgctgcccgcgccaccg-3 ( inner sense , 51845209 ) and 5-ggcgctgggmytggtcrcgccaag-3 ( inner antisense , 54265403 ) .
genbank accession numbers for the orf2 and orf2/3 sequences described here are jx516004jx516053 and for the hvr sequences are jx270882jx270902 .
nucleotide sequence analysis of the hvr from individual virus genomes sampled by limiting dilution of cdna revealed 2 distinct genotype 3 populations ( populations a and b , figure ) .
members of these populations all differed by the same 18 nucleotide substitutions , of which 13 were synonymous and 5 nonsynonymous . two populations ( a and b ) were also observed among 25 orf2 sequences ( the 2 populations differed by 5 synonymous substitutions ) and 26 orf2/orf3 sequences ( the 2 populations differed by a single substitution , which was nonsynonymous in orf2 but synonymous in orf3 ) .   phylogenetic tree of hepatitis e virus hypervariable region variants .
a neighbor - joining tree of hypervariable region sequences was constructed by using mega 4 ( 15 ) . a and b indicate sequences for virus populations a and b. sequences from the patient studied here ( patient 21 ) belonging to virus population a are indicated by a solid square , and those belonging to virus population b are indicated by a solid circle .
1 of the virus populations represented 80%90% of the sequences : in hvr , 17 sequences were population a and 4 population b ; in orf2 , 21 sequences were population a , 2 population b , and 1 mixed ; and in orf2/3 , 20 sequences were population a , 4 population b , and 2 mixed .
the 3 mixed sequences contained ambiguities at positions where the 2 populations differed , implying that templates from both populations were present in the limiting dilution reaction .
the expected number of such mixed - template reactions can be calculated by using the poisson distribution and the number of pcr - negative replicate reactions , which were 47% ( hvr ) , 50% ( orf2 ) , and 43% ( orf2/3 ) .
thus , 17.5% , 15.4% , and 20.8% , respectively , of reactions would be expected to contain > 1 template , of which , given the ratio of populations a and b in each region , 31% , 17.2% , and 28% , respectively , would contain templates from different populations , equivalent to 5.4% , 2.6% , and 5.8% , respectively , of positive reactions , compared with the 4.2% ( 3/71 ) observed .
all other substitutions in these sequence sets were unique to a single sequence . a trivial explanation for the observation of mixed populations in this sample would be contamination during sample collection , processing , or analysis .
however , this appears unlikely , given the low frequency of hev infection in the united kingdom , the lack of pcr products in negative controls , and the distinctness of the sequences obtained from those previously studied in our laboratory .
infection of the patient in this study with 2 hev variants could arise for several different reasons .
first , the person could have been exposed to a homogeneous source of hev , which then diversified during infection .
assuming that infection occurred 1 month before the first symptoms and 6 weeks before sampling , a divergence of hvr sequences at 18 ( 7.4% ) of 243 nucleotide positions equates to a rate of nucleotide substitution from a presumed ancestor of both populations of 0.32/site / year .
not only were these substitutions mostly synonymous , and therefore unlikely to represent selection during infection , but the inferred rate of nucleotide substitution would be several orders of magnitude higher than those previously reported for hev ( 0.0014/site / year ) ( 7 ) or for the hcv hvr ( 0.0043/site / year ) ( 8) .
in addition , the divergence between the 2 virus populations contrasts with the homogeneity observed among 7 other acutely infected patients and between viruses transmitted from 1 person or host to another ( 6 ) .
a second possibility is that this person had a subclinical chronic hev infection and then became superinfected with a second virus that induced jaundice .
no previous blood samples were available for testing , but a prior subclinical chronic infection seems unlikely , given the absence of immunosuppression ; the presence of anti - hev igm and igg ; the decline and increase , respectively , in these antibody titers in a sample collected 7 days later ; and the decline in alt and bilirubin levels in the weeks following the patient s hospital visit .
last , the clinical features and our immunologic and virologic findings are consistent with the patient having been infected from a single source containing > 1 variant , such as pig - derived figatellu sausage ( 9 ) .
alternatively , he might have been multiply infected from different sources within in a short period and before the development of protective immunity .
follow - up interview revealed no occupational exposure to animals , apart from a domestic dog , and no recent foreign travel ; he spent 3 days in a beach resort in southeastern scotland 12 days before onset of symptoms .
the patient ate supermarket - bought prewashed salad vegetables and fresh fruit and drank only bottled or tap water .
the patient ate fish , shellfish , chicken , pork , bacon , sausages , ham , brussels pat , and eggs but not venison or pig liver .
hev has been found on the hands and gloves of 17% of persons selling pork products ( 10 ) .
this study suggests that mixed infection with hev can occur in immunocompetent persons with no obvious high - risk exposure to hev - infected food or sewage .
only about 300 cases of acute hev are reported each year in england and wales ( 11 ) , and the prevalence of hev pcr - positive blood donors in england is 0.014% ( 12 ) .
however , the seroprevalence of anti - hev igg is 1,000 higher ( 16% ) ( 13 ) , and in this context , the identification of sources of autochthonous hev infection remains an important goal .
our results are also relevant to the suggestion that hev may undergo recombination ( 14 ) .
the case described here suggests that the conditions required for virus recombination may occasionally arise in immunocompetent persons .
 we detected 2 hepatitis e virus ( hev ) strains in an acutely infected immunocompetent patient . 
 two populations of genotype 3 virus were observed in the hypervariable regions and open reading frames 2 and 3 , indicating multiple infection with hepatitis e virus . 
 persons with mixed infections may provide the opportunity for virus recombination . 
The Study
Conclusions