Classifying genotype F of hepatitis B virus into F1 and F2 subtypes

Table 1 Demographic characteristics and liver diseases of the 11 patients from whom HBV isolates of genotype F were recovered

HBV strains	Accession number	Age (yr)	Gender	Ethnicity	Clinical diagnosis
USF10	AB059659	46	Female	Caucasian	Liver cirrhosis
USF1122	AB064315	46	Male	Hispanic	Liver cirrhosis
USF1778	AB059660	44	Male	Hispanic	Liver cirrhosis
USF2065	AB059661	47	Male	Hispanic	Acute hepatitis
USF2573	AB064316	67	Male	Hispanic	Liver cirrhosis
JPF1130	AB086397	43	Male	Asian	Chronic hepatitis
JPNTAKA	AB116654	NA	Male	Asian	NA
VENEZ2	AB116549	35	Male	Hispanic	Acute hepatitis
VENEZ4	AB116550	35	Male	Hispanic	Acute hepatitis
PANAM5	AB116551	NA	NA	NA	NA
PANAM6	AB116552	NA	NA	NA	NA

NA: not available.

Table 2 Primers used for PCR and sequencing

Primer1	Polarity	Sequence (5’–3’)	Positions (nt)
HBV/Ff1	Sense	GGG TCA CCA TAT TCT TGG GAA	2 814–2 834
HBV/Fr1	Antisense	CGT TGC CGA GCA ACG GGG TAA AGG	1 163–1 140
HBV/Ff2	Sense	GTT TCT CCT GGC TCA GTT TA	660–679
HBV/Fr2	Antisense	AAA AAG TTG CAT GGT GCT GG	1 825–1 806
HBV/Ff3	Sense	ACG TCG CAT GGA GAC CAC CG	1 601–1 620
HBV/Fr3	Antisense	GAA CTG GAG CCA CCA GCA GG	75–56
HBV/Ff4	Sense	CCT CCT GCT TCC ACC AAT CG	3 124–3 143
HBV/Fr4	Antisense	AGA TGA GGC ATA GCA GCA GGA TG	431–409
HBV/Ff5	Sense	GTC TAG ACT CGT GGT GGA CTT CTC	246–269
HBV/Fr5	Antisense	AAG CCA GAC AGT GGG GGA AAG C	730–709
HBV/Ff6	Sense	CTC GCC AAC TTA CAA GGC CTT T	1 098–1 119
HBV/Fr6	Antisense	GAT TCA GCG CCG ACG GGA CGT A	1 447–1 426
HBV/Ff7	Sense	CTC TGC CGA TCC ATA CTG CGG AA	1 256–1 278
HBV/Fr7	Antisense	TGA GAT CTT CTG CGA CGC GGC	2 431–2 410
HBV/Ff8	Sense	TCA GGC AAG CTA TTT TGT GCT GG	2 064–2 086
HBV/Fr8	Antisense	TGT TCA CAT TTG TGT ATC AAA T	2 587–2 565
HBV/Ff9	Sense	GCC GCG TCG CAG AAG ATC TCA A	2 410–2 431
HBV/Fr9	Antisense	AAA ATG AGG CGC TAT GTG TGG ATT	2 811–2 788

¹The first three pairs of primers (HBV/Ff1 to HBV/Fr3) were used for PCR and sequencing, while the remaining six pairs (HBV/Ff4 to HBV/Fr9) for sequencing only.

Table 3 Sequence homology of seven HBV/F2 isolates among themselves and to seven HBV isolates representative of HBV/F1 as well as the other genotypes (A, B, C, D, E, and G)

Genomic regions	Homology within seven HBV/F2 isolates and that of HBV/F2 to seven representative of HBV/F1 as well as genotypes A, B, C, D, E, and G1
	F2	F1	A	B	C	D	E	G
Full genome	97.5	91.6	86	85.9	85.7	85.7	86.1	84.5
	(96.3–99.2)	(91.0–92.1)	(85.4–86.5)	(85.5–86.2)	(85.2–86.0)	(85.1–86.3)	(85.6–86.4)	(84.1–84.8)
PreS1 region	98.1	90	77.3	75.7	78.9	79.3	81.4	80.6
	(97.2–99.4)	(89.1–90.5)	(76.8–78.2)	(75.1–76.5)	(77.9–80.1)	(78.3–80.2)	(80.7–82.0)	(80.4–81.1)
PreS2 region	97.4	90.9	82.9	79.2	82.6	81.2	83.3	81.3
	(92.7–100)	(86.1–92.7)	(78.0–84.2)	(75.8–80.6)	(79.4–83.6)	(77.6–82.4)	(79.4–84.2)	(77.0–83.0)
S gene	99.4	96.4	92.2	92.6	91.3	92.7	92.6	92.8
	(98.7–99.9)	(95.9–96.8)	(91.8–92.5)	(92.5–92.8)	(91.0–91.6)	(92.4–93.0)	(92.2–92.8)	(92.4–93.0)
C gene	95.8	91.9	84.8	87.3	86.6	84.9	85.8	87.2
	(90.9–100)	(88.8–93.5)	(83.0–85.8)	(85.1–88.6)	(84.8–87.5)	(82.8–86.6)	(84.1–86.8)	(84.9–88.2)
P gene	97.9	91.7	86.2	85.8	86.3	86.1	86.5	86.1
	(96.6–99.1)	(90.9–92.0)	(85.7–86.6)	(85.4–86.0)	(86.1–86.6)	(85.5–86.4)	(86.2–86.8)	(85.7–86.4)
X gene	97.1	91.1	87.7	88.7	85.4	89.2	88.3	84.2
	(94.2–99.8)	(89.3–92.3)	(86.5–88.7)	(86.8–90.1)	(84.7–86.8)	(88.0–90.2)	(87.1–89.5)	(85.0–83.7)

¹The seven representatives of each genotype were: F1 (AB064316 [USF2573 isolate]), A (AB064314), B (D23678), C (S75184), D (D23681), E (X75657) and G (AF160501).

Table 4 Homology in the entire nucleotide sequence of the seven HBV/F2 isolates to HBV/F1 isolates and among themselves

	F2 subtype (this study)				F2 subtype (reported)			F1 subtype (this study)
	USF10	USF1122	USF1778	USF2065	1853Nic	2928Nic	LAS2523	USF2573	JPF1130	JPNTAKA	PANAM5	PANAM6	VENEZ2	VENEZ4
HBV/F1 isolates
(reported)
AB036905	91.7	91.9	92.3	92.6	92.3	92.7	92.5	94.6	94.1	93.6	99.2	97.9	99	94.7
AB036910	91.7	91.9	92.3	92.7	92.2	92.6	92.6	94.7	94.1	93.6	99.2	98	99.5	94.8
AB036920	91.7	91.9	92.2	92.5	92.2	92.7	92.5	94.6	94	93.6	99.1	97.8	98.8	94.7
X69798	91.1	91.4	91.9	92.2	91.6	92.1	92.2	94.3	93.9	93.6	96.2	95.2	96.1	94.5
X75659	90.5	90.7	91.2	91.6	91	91.4	91.4	93.6	92.9	92.6	95.4	94.3	95.5	93.7
X75663	91	91.2	91.8	92.2	91.7	92.1	91.9	94	93.3	92.8	97.2	96	97.6	94.1
AY090455	91	91.1	91.5	91.9	91.4	91.9	91.9	94.2	93.8	93.4	96	94.9	95.8	94.3
AY090458	90.8	90.9	91.5	92	91.6	91.9	91.6	97.9	97.3	96.9	94.9	93.8	94.9	98.1
AY090459	90.8	90.8	91.5	91.9	91.6	91.9	91.5	98.2	97.5	97.2	95	93.9	94.9	98.3
AY090461	90.9	91	91.6	92	91.6	91.9	91.7	98.1	97.5	97.1	95	93.9	94.8	98.3
HBV/F1 isolates
(this study)
USF2573	91	91.1	91.6	92	91.8	92.1	91.8	–	98.5	97.9	94.8	93.7	94.7	99.6
JPF1130	90.7	90.6	91.3	91.5	91.3	91.6	91.3	98.5	–	97.8	94.3	93.4	94.2	98.4
JPNTAKA	90.5	90.5	91.1	91.3	91.0	91.3	91.1	97.9	97.8	–	93.9	92.9	93.7	97.9
PANAM5	91.9	92	92.4	92.8	92.3	92.8	92.7	94.8	94.3	93.9	–	98.6	99.2	95
PANAM6	91.1	91.2	91.7	91.7	91.4	91.9	91.7	93.7	93.4	92.9	98.6	–	98	93.8
VENEZ2	91.8	92.1	92.5	92.8	92.3	92.8	92.7	94.7	94.2	93.7	99.2	98	–	94.9
VENEZ4	91.1	91.2	91.7	92.1	91.9	92.2	92	99.6	98.4	97.9	95	93.8	94.9	–
F2 isolates
(this study)
USF10	–	97	96.9	97.2	96.3	96.6	97.9	91.0	90.7	90.5	91.9	91.1	91.8	91.1
USF1122	97	–	97.1	97.6	96.5	96.9	98.2	91.1	90.7	90.5	92	91.2	92.1	91.2
USF1778	96.9	97.1	–	98.6	97.2	97.4	98	91.6	91.3	91.1	92.4	91.7	92.5	91.7
USF2065	97.2	97.6	98.6	–	97.6	97.8	98.4	92	91.5	91.3	92.8	91.7	92.8	92.1
F2 isolates
(reported)
1853Nic	96.3	96.5	97.2	97.6	–	99.2	97.5	91.8	91.3	91.0	92.3	91.4	92.3	91.9
2928Nic	96.6	96.9	97.4	97.8	99.2	–	97.7	92.1	91.6	91.3	92.8	91.9	92.8	92.2
LAS2523	97.9	98.2	98	98.4	97.5	97.7	–	91.8	91.3	91.1	92.7	91.7	92.7	92

Values less than 92, clearing genotype difference, are shown in boldface.

Table 5 Genotype- or subtype-specific nucleotides

HBV isolates		Nucleotide positions
	Genotype						P gene
	/subtype
		22	1 467	1 552	1 754	2 495	2 558	2 579	2 596	2 603	2 606	2 622	2 628	2 634	2 636	2 660	3 121	3 129
X70185	A	T	G	T	T	G	T	T	T	C	T	A	A	A	A	T	A	T
D00329	B	–	–	–	–	–	–	–	–	A	C	–	–	–	–	–	G	–
D50520	C	–	–	–	–	A	–	–	A	–	–	–	–	–	–	–	G	–
D23681	D	–	–	–	–	–	A	C	–	–	A	–	–	C	–	–	G	G
X75657	E	–	–	–	–	–	–	–	–	T	–	–	–	–	C	–	G	G
AB056515	G	–	–	–	C	–	C	A	–	–	–	–	–	–	–	C	G	G
X68798	F1	–	–	–	–	–	–	–	–	–	A	T	–	–	–	–	G	G
X75658	F1	–	–	–	C	–	–	A	–	–	A	T	–	–	–	–	G	G
X75663	F1	–	–	–	C	–	–	A	–	–	G	T	–	–	–	–	G	G
AB036905	F1	–	–	–	C	–	–	A	–	–	G	T	–	–	–	–	G	G
AB036910	F1	–	–	–	C	–	–	A	–	–	G	T	–	–	–	–	G	G
AB036920	F1	–	–	–	C	–	–	A	–	–	G	T	–	–	–	–	G	G
USF2573	F1	–	–	–	–	–	–	A	–	G	–	T	–	–	–	–	G	G
JPF1130	F1	–	–	–	–	–	–	A	–	–	–	T	–	–	–	–	G	G
JPNTAKA	F1	–	–	–	–	–	–	A	–	–	–	T	–	–	–	–	G	G
PANAM5	F1	–	–	–	C	–	–	A	–	–	G	T	–	–	–	–	G	G
PANAM6	F1	–	–	–	C	–	–	A	–	–	G	T	–	–	–	–	G	G
VENEZ2	F1	–	–	–	C	–	–	A	–	–	G	T	–	–	–	–	G	G
VENEZ4	F1	–	–	–	T	A	–	A	–	–	–	T	–	–	–	–	G	G
USF10	F2	A	T	C	G	T	–	A	–	–	A	–	C	–	–	–	C	A
USF1122	F2	A	T	C	G	T	–	A	–	–	A	–	C	–	–	–	C	A
USF1778	F2	A	T	C	G	T	–	A	–	–	A	–	C	–	–	–	C	A
USF2065	F2	A	T	C	G	T	–	A	–	–	A	–	C	–	–	–	C	A
AY090454	F2	A	T	C	G	T	–	A	–	–	A	G	C	–	–	–	C	A
AY090457	F2	A	T	C	G	T	–	A	–	–	A	G	C	–	–	–	C	A
AY090460	F2	A	T	C	G	T	–	A	–	–	A	–	C	–	–	-	C	A

Genotype- or subtype-specific nucleotides are shown in boldface, and a dash (–) indicates the nucleotide identical to that of X70185.