18 October 2017
Int J Med Sci 2014; 11(1):60-64. doi:10.7150/ijms.7728
Ultra-Deep Sequencing Analysis of the Hepatitis A Virus 5
(n = 13)
|Mean age (yr)||39.9 ± 14.4||NS#||40.6 ± 11.9|
|ALT level (IU/L)||3392 ± 1886||0.024##||6940 ± 3550|
|Total bilirubin (mg/dL)||6.3 ± 3.8||NS#||5.7 ± 3.7|
|Nadir PT (%)||70.5 ± 19.1||0.031##||29.6 ± 8.3|
Abbreviations: PT, prothrombin time. Data were expressed as Mean ± SD. #Statistically not significant (NS) by Student's t test. $Statistically not significant by Chi-squared test. ##Significant difference between 13 patients with AH (acute hepatitis, non-severe form) and 3 patients with AH-S (acute hepatitis, severe form) in hepatitis A outbreak by Student's t test.
Nucleic acids were extracted from 140 μL of sera using a QIAamp Viral RNA mini kit (Qiagen, Tokyo, Japan) according to the manufacturer's instructions, and subjected to RT-PCR. For the detection of HAV RNA, two sets of amplification primers were made at the position of 5'UTR based on HAV HM175 (M59810) sequences. Complementary DNA was synthesized with primer 1 (5'-AGTACCTCAGAGGCAAACAC-3') for 1 cycle at 55oC for 30 min and at 85oC for 5 min using a Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Indianapolis, IN, USA), then amplified with primer 1 and primer 2 (5'-TCTTGGAAGTCCATGGTGAG-3') for 35 cycles at 95oC for 30 sec, 55oC for 30 sec, and 72oC for 60 sec using a FastStart high fidelity PCR system, dNTPack kit (Roche). Then, the first PCR product was further amplified with primers 3 (5'-CCACATAAGGCCCCAAAGAA-3') and 4 (5'- GGGACTTGATACCTCACCGC-3') for 35 cycles at 95oC for 30 sec, 55oC for 30 sec, and 72oC for 60 sec. Amplified products were separated by agarose gel electrophoresis and purified using a High pure PCR clean-up micro kit (Roche). Each amplicon was quantified by Nanodrop Lite spectrophotometer (Thermo Scientific, Madison, WI, USA), and all amplicons from a single viral genome were pooled together at equimolar ratios. Each pool was then quantitated, and approximately 500 ng of each was used in a fragmentation reaction mix, using a GS FLX Titanium Rapid Library Preparation Kit (Roche). Final libraries representing each genome were characterized for average size by using an Agilent High Sensitivity DNA kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Loveland, CO, USA). 4 x 107 of molecular DNA libraries were then subjected to emulsion PCR, and enriched DNA beads were loaded onto a picotiter plate and pyrosequenced with a Roche/454 GS Junior sequencer using Titanium chemistry (454 Life Sciences Corp., Branford, CT, USA) . GS Amplicon Variant Analizer Version 2.7 (Roche) was used for read mapping and calculating variant frequencies at each nucleotide position according to the reference sequence.
We cloned the PCR product from patient no. 12 into the pCR2.1-TOPO vector (Life Technologies, Tokyo, Japan). Sanger sequencing was performed using a BigDye(R) Terminator v3.1 Cycle Sequencing Kit (Life Technologies). Sequences were analyzed using Applied Biosystems 3730xl (Life Technologies).
To obtain the percentage of nucleotide variability in each sample, the total number of nucleotide substitutions was divided by the total number of nucleotides analyzed at each position. Comparison was performed using Fisher's exact test, Chi-squared test, or Student's t-test. All P-values were two-tailed, and P < 0.05 was considered statistically significant.
In order to ensure that errors introduced by PCR as well as errors inherent to the Roche/454 pyrosequencing technology were below our minimum variant frequency threshold of 1%, we sequenced the PCR products from 103 to 104 copies of control plasmid and found no mutations, indicating similar error rates lower than 1%. The average read number was 7753.
A previous study  showed one of the different hot-spots between acute hepatitis and fulminant hepatitis was located in HAV 5'UTR, according to analysis of the complete HAV genome. So we performed UDPS in 13 HAV IRES derived from patients with acute hepatitis, non-severe form, who were involved in this outbreak. The sequences were compared with the reference clonal sequence from patient no. 12, the sushi shop attendant. In these patients, 20 nucleotide substitutions at 19 positions and 3 nucleotide insertions at 3 positions were seen (Table 2), while plasmid control possessed no substitutions. In cases of acute hepatitis, non-severe form, 0-5 nucleotide substitutions and 0-1 nucleotide insertions were seen in each case. In patients no. 5 and no. 13, respectively, 97.8% nucleotide substitution (206C/T) and 18.6% nucleotide substitution (211T/C) were seen, but all other substitutions were lower than 6% at each position.
Nucleotide substitutions of HAV IRES from virus with substitutions.
|Patient No.||Locations*||Nucleotide Position||Prototype Nucleotide||Nucleotide Substitution|
|1||Between IIIc' and IIIb'||202||C||T|
|Between IVf and IVi||441||A||G|
|3||Between IIIa' and IIIe||225||T||C|
|4||Between IVa' and Va||576||G||A|
|Between IVj' and IVi||482||T||C|
|Between IVj' and IVi||484||A||G|
|10||Between IIIb' and IIIa'||212.5||-||T|
|Between IVk and IVk'||466||T||C|
|Between Vb and Vc||605||T||C|
|12||Between IVk and IVk'||471||A||G|
|Between IVj' and IVi||484||A||G|
|13||Between IIIb' and IIIa'||211||T||C|
|Between IVd and IVe||357||A||G|
|Between IIIg and IIIg'||275||A||G|
|Between IIIg and IIIg'||276||T||C|
|Between IVk and IVk'||466||T||C|
|Between IVe' and IVd'||527||G||T|
|Between IVa' and Va||578||T||C|
*Major domains of HAV 5'UTR .
We performed UDPS in 3 HAV IRES derived from patients with acute hepatitis, severe form, who were involved in this outbreak. In these patients, 4 nucleotide substitutions at 4 positions and 0 nucleotide insertions were seen (Table 2). In cases of acute hepatitis, severe form, 0-3 nucleotide substitutions were seen in each case. In patient no. 15, 0.49% nucleotide substitution (157C/T), 14.94% nucleotide substitution (378G/A) and 0.37% nucleotide substitution (450G/A) were observed. In patient no. 16, only 7.94% nucleotide substitution (242C/T) was observed. These results showed no specific mutations in HAV IRES associated with severe form existing in this outbreak.
We performed UDPS in the one HAV IRES derived from one patient with acute hepatitis, severe form, who was not involved in this outbreak. In patient no. 17, 8 nucleotides at 8 positions were different from the reference sequence (Table 2), and 64.72% (578T/C), 98.14% (208C/T), 99.71% (527G/T), 99.83% (466T/C), 100% (204A/G), 100% (275A/G), 100% (276T/C) and 100% (378G/A) nucleotide substitutions were observed. These results showed that UDPS of the sporadic case was obviously different from those of the outbreak cases.
The substitution rates were analyzed in comparison to the reference sequence . We also counted insertions as substitutions. In HAV IRES (nt. 90-620), total nucleotide substitutions of acute hepatitis, non-severe and severe forms from the outbreak, and sporadic acute hepatitis, severe form, were 0.020, 0.014, and 1.43%, respectively. In HAV IRES domain III (nt. 90-300), nucleotide substitutions of acute hepatitis, non-severe and severe forms from the outbreak, and sporadic acute hepatitis, severe form, were 0.047, 0.013, and 1.88%, respectively. In HAV IRES domain IV (nt. 301-580), nucleotide substitutions of acute hepatitis, non-severe and severe forms from the outbreak, and sporadic acute hepatitis, severe form, were 0.0034, 0.018, and 13.0%, respectively. In HAV IRES domain V (nt. 581-620), nucleotide substitutions of acute hepatitis, non-severe and severe forms from the outbreak, and sporadic acute hepatitis, severe form, were 0.0020, 0, and 0%, respectively. All nucleotide substitutions from acute hepatitis from the outbreak were transition mutations. Three nucleotide insertions were observed in only acute hepatitis, severe form, from the outbreak (Table 3).
Nucleotide substitutions of hepatitis A virus (HAV) internal ribosomal entry site (IRES).
|Type of Mutations||Insertion||Transition||Transversion||Total Numbers|
|Numbers in Outbreak Cases||3||8||4||3||9||0||27|
|AH of Outbreak Cases||3||8||2||2||8||0||23|
|AH-S of Outbreak Cases||0||0||2||1||1||0||4|
|Numbers in Patient no. 17||0||2||1||1||3||1||8|
“Numbers” equivalent to “nucleotide substitutions”.
In the present study, we analyzed the UDPSs of HAV associated with the outbreak and tried to detect specific mutations in HAV 5'UTR associated with hepatitis A, severe form. Our result showed that no specific mutations in HAV 5'UTR associated with severe form existed in this outbreak. UDPS analysis of HAV 5'UTR revealed no association between the disease severity of hepatitis A and HAV 5'UTR substitutions. It might be more interesting to perform ultra-deep sequencing of the full-length HAV genome in order to uncover possible unknown genomic determinants associated with disease severity. Further studies will be needed regarding this point. We also found minor nucleotide sequence variations, which seemed undetectable by the Sanger method. We do not yet know what these variations mean.
The sensitivity of UDPS is higher than that of Sanger sequencing. Next-generation sequencing technologies are increasingly being used to identify low-abundance minority genetic variants within a heterogeneous pool of amplified DNA molecules, such as those within a virus population, which are especially valuable for the detection of drug resistance mutations . We characterized HAV minority variants in the HAV IRES region.
In general, HAV infection risk is inversely correlated to sanitation and other socio-economic indicators . Although Japan is one of the developed countries in Asia, a universal vaccination program against HAV and HBV has not yet been initiated . Although the present study did not include acute liver failure with hepatic encephalopathy such as fulminant hepatitis A, our study is important because UDPS analysis of these HAV strains reconfirmed that a single source might have caused this outbreak as previously reported , suggesting that UDPS analysis might be a new analytical tool for the source of hepatitis A outbreaks.
The effect of mutations in 5'UTR of HAV on the severity of the disease is a long story that has never been clearly proven. Fujiwara et al. reported an association between the severity of hepatitis A and nucleotide substitutions in 5'UTR of HAV RNA [8-10]. However, there have been several contrary observations [7,11-13]. As the definition of acute liver failure is also different among different countries [11,14], it seemed difficult to compare them using different criteria. Then we compared the different HAV IRES sequences derived from a single-source outbreak, based on a single definition of liver failure. In this outbreak, the proportion of acute liver failure was very high (3/29, 10.3%), compared to the previous report , although the calculation was performed on the basis of patients admitted into two hospitals [15,16]. The reference clonal sequence from patient 12 had only one different nucleotide from the sequence of the severe hepatitis strain HAV PT (A10), which was reported by Fujiwara et al , suggesting that our present study might be conducted among specific HAV strains.
The technical approach used in the present study had the advantage of having great power in detecting mutations present at a very low frequency in the swarm of mutants. However, our results confirm the old paradigm that 5'UTR of picornaviruses and particularly of HAV is highly conserved. This reason might be associated with the function of HAV IRES, preventing the occurrence of variability.
In conclusion, there were no different HAV IRES sequences between severe and non-severe forms in this outbreak. To our surprise, HAV strains in this outbreak conserved HAV IRES sequence even if we performed analysis of UDPSs. Further analysis of HAV UDPSs could give us new information concerning the association between the disease severity of hepatitis A and HAV genome substitutions.
We are all very grateful to our colleagues at the liver unit of our hospitals who cared for the patients described herein.
This work was supported by a grant from the Chiba University Young Research-Oriented Faculty Member Development Program in Bioscience Areas (T.K.); the Japan Science and Technology Agency, Ministry of Education, Culture, Sports, Science, and Technology, Japan (SN and TK), and a grant from the Ministry of Health, Labor and Welfare of Japan (TK and OY).
The authors have declared that no competing interest exists.
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Corresponding author: Tatsuo Kanda, M.D., Ph.D., Associate Professor, Department of Gastroenterology and Nephrology, Chiba University, Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba (260-8677), Japan. Tel.: +81-43-226-2086, Fax: +81-43-226-2088; Email: kandat-cibac.jp.