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Journal of Clinical Microbiology, February 2000, p. 898-901, Vol. 38, No. 2
Clinical Microbiology and Public Health
Laboratory, Addenbrooke's Hospital, Cambridge CB2
2QW,1 and Enteric and Respiratory Virus
Laboratory, Virus Reference Division, Central Public Health Laboratory,
Colindale, London NW9 5HT,2 United
Kingdom
Received 28 June 1999/Returned for modification 20 September
1999/Accepted 1 November 1999
A degenerate version (1T1-D) of the rotavirus P[8]-specific
primer (1T-1) allowed strains previously untypeable due to the accumulation of point mutations at the primer binding site to be P
typed by reverse transcription-PCR. Sequencing of the cDNA followed by
sequence alignment and phylogenetic analysis identified lineages and
sublineages within the rotavirus P[8] types, while the use of 1T-1 or
1T-1D primers did not yield viral clusters in any particular lineage.
Rotaviruses are the major cause of
gastroenteritis in infants and young children worldwide (9).
They are members of the Reoviridae family and contain a
genome consisting of 11 segments of double-stranded RNA (dsRNA)
enclosed in a triple-layered capsid (2). The outer layer of
rotavirus particles is composed of two proteins, VP7 and VP4, encoded
by RNA segments 9 (or 7 or 8, depending on the strain) and 4, respectively. Sequencing and phylogenetic analysis of the VP7 and VP4
genes has revealed a considerable genetic diversity within G types
(VP7-specific) and P types (VP4-specific), and distinct lineages have
been described within G1, G2, G3, and P[8] types (5, 8, 10, 12,
14, 15).
In 25 of 648 (3.9%) rotavirus-positive fecal specimens collected from
different geographical locations in the United Kingdom in 1995 and 1996 for which the G type was successfully determined by reverse
transcription (RT)-PCR, a P type could not be assigned by using
previously described oligonucleotide primers and methods (3, 4,
7). However, amplicons were obtained using the VP4 consensus
primers, Con2 and Con3, in first-round RT-PCR (3). Partial
sequencing of the VP4 genes was performed, and the data allowed us both
to explain failure to type and to improve the typing method.
VP4-specific amplicons of 876 bp (spanning nucleotides [nt] 11 to
887) from 11 isolates that could not be P typed were selected for
partial sequencing of the VP4 gene. Twenty-two isolates typed by RT-PCR
were also selected as controls: 3 P[6], 1 P[4], and 18 other P[8]
strains. Approximately 75 to 100 ng of PCR amplicons was purified with
the QIAquick PCR purification kit (Qiagen) and sequenced in both
directions using the same primer pairs as in the VP4 first-round PCR
reaction (3, 7) and the ABI PRISM Dye Terminator or Big Dye
Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer Applied
Biosystems, Foster City, Calif.). Sequence data were analyzed using
SeqManII (DNASTAR, Inc., Madison, Wis.), and sequences were aligned by
the CLUSTAL method. Phylogenetic analyses were carried out using the
MegAlign program (DNASTAR, Inc.).
The following VP4 gene sequences available from GenBank and EMBL and
comprising P[8], P[6], P[4], P[13], P[14], and an unusual P
type were used for the comparative alignments. The GenBank and EMBL
accession numbers are indicated in Fig.
1.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Diversity within the VP4 Gene of Rotavirus P[8]
Strains: Implications for Reverse Transcription-PCR
Genotyping
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FIG. 1.
Phylogenetic tree constructed with partial nucleotide
sequences of the VP4 gene (nt 11 to 887) using the CLUSTAL method and
MegAlign. Sequence nomenclature indicates the P type followed by the
laboratory number or accession number or strain designation for
sequences obtained from GenBank or EMBL; the rotavirus seasons (1995 to
1996, 1996 to 1997, and 1997 to 1998 are indicated as -96, -97, and
-98, respectively). Strains that could not be typed with the 1T-1
primer are marked with *. The calibration scale indicates the percent
divergence among nucleotide sequences.
The sequences derived from the strains typed by RT-PCR clustered with
GenBank and EMBL sequences of their corresponding P types (Fig. 1). All
the sequences derived from strains that were untypable by RT-PCR
clustered with P[8] sequences (
95% homology at the nucleotide
level). The nucleotide sequence alignment showed a series of point
mutations at the binding region of the P[8]-specific primer 1T-1
(3) (Fig. 2). Mismatches
between the P[8]-specific primer and its complementary region of the
VP4 cDNA in some of the P[8] isolates would explain the failure of
the typing RT-PCR. The oligonucleotide primer used for P[8] typing
(1T-1) was designed using the VP4 sequence of the strain KU (G1P
[8]) as a template. The primer 1T-1 showed 100%
complementarity only with the VP4 gene of the KU strain. Sequences from
all other strains, including those obtained from GenBank and EMBL,
showed two consistent mismatches at nt 9 and 12 from the primer's 3'
end (A:A and T:C, respectively). The majority of strains also showed
mismatches with primer 1T-1 at nt 7 and 10 from the 3' end (A:C and
G:T, respectively). Those strains that could not be typed by RT-PCR
using primer 1T-1 showed a substitution at nt 1 (C:A) or 4 (G:A or G:T)
from the 3' end and/or the accumulation of a total of five to six
mismatches between the primer and template cDNA (Fig. 2).
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A new degenerate primer, designated 1T-1D, was designed (Fig. 2) to take account of the variability at the P[8]-type defining region. All the previously untypable strains (25 in total) and 15 strains typed by RT-PCR using the original primer (1T-1) were amplified in the presence of the 1T-1D primer in a multiplex-typing PCR, and all were successfully typed. Strains typed as P[8] using the degenerate 1T-1D primer were sequenced in order to confirm the specificity of the primer. Alignment of the sequence data obtained and phylogenetic analysis indicated that they were all P[8] strains (Fig. 1).
The sensitivity of the typing PCR with the new degenerate primer (1T-1D) and with the original primer (1T-1) was compared by testing serial 10-fold dilutions of the first-round amplicons in the multiplex-typing PCR. The sensitivity of the P-typing PCR was increased by 10-fold to 10,000-fold in the presence of three and four mismatches, respectively, when the degenerate primer was included in the reaction. The yield of PCR product determined by densitometry was doubled when the 1T-1D primer was used (data not shown). There was a significant correlation between typing efficiency and calculated melting temperatures between the templates and the P[8] specific primers (data not shown).
Mismatches at any of the first three nucleotides at the 3' end have been shown to prevent the amplification of templates with primers between 17 and 20 nt long (13). Although certain mismatches between primer and template are tolerated depending on their position, number, and nature, they can also have a very detrimental effect on the efficiency and product yield of the PCR, especially when the primer is <20 nt long (6, 11, 13). Failure to type rotavirus isolates can be explained by the accumulation of mismatches (five or more, or three when one of the mismatches was closer to the 3' end), which in combination with the relatively short length of the P[8]-specific primer (18 nt) would destabilize the primer-template duplex sufficiently to prevent primer elongation or drastically reduce the efficiency of the PCR. A comparison of the multiplex PCR using the original P[8]-specific primer to type strains with three or four mismatches showed that the accumulation of one extra mismatch considerably reduced the typing efficiency (data not shown).
The deduced amino acid sequence alignments of the P[8] strains showed that the variability was greater at the P[8]-specific binding site than at the positions for other type-specific primers (data not shown). The fact that all the strains that could not be typed with the original set of primers were P[8] strains as well as that no problems have so far been encountered in typing other P types by PCR could suggest that the type-specific sites for other P types are more conserved. Sequence data obtained from seven P[6] strains isolated in the United Kingdom showed that the P[6]-specific primer binding site was conserved in all the strains and that there was 100% complementarity with the primer used for typing (data not shown). However, as P[8] is much more prevalent than any other P type, greater numbers of all other P types need to be examined. Alternatively, the greater diversity within P[8] strains may explain the much higher prevalence and continuing circulation of this type across different parts of the world, season after season, as is speculated for G1 strains (12).
The sequence diversity within the VP8* subunit of P[8] strains may suggest that the current typing system is oversimplified and may not fully reflect geographical or temporal differences. There was no clustering according to the G type of the strains, supporting further the evidence that the VP4 and VP7 genes segregate independently. The rotavirus strains isolated in the United Kingdom during two consecutive seasons form three distinct lineages. Similar lineages (P[8]-1, P[8]-2, and P[8]-3), have been described by Maunula and von Bonsdorff (12) and appear to have been maintained across different countries during different seasons. The lineage-defining amino acid substitutions are concentrated in the hypervariable region of the VP8* fragment. Analysis of the amino acid sequences of short peptides spanning amino acids 78 and 195 were found to differentiate the same groups as the phylogenetic analysis of nucleotide sequences of >800 bp. An analysis of the nucleotide sequences revealed a greater number of point mutations defining the lineages and sublineages, many of which were silent. Maunula and von Bonsdorff (12) described three sublineages of P[8]-1 based on the phylogenetic analysis of the nucleotide sequences of VP8*; however, sublineages defining amino acid substitutions were not identified. In our study, two sublineages were distinguished in lineages P[8]-1 and P[8]-2 on the basis of the phylogenetic analysis of the sequences. These sublineages were found to be defined by amino acid substitutions in the same region of VP8* as those defining the lineages, at amino acid positions 120, 149, and 195 in P[8]-1 and positions 120 and 189 in P[8]-2.
The use of a degenerate primer in the multiplex-typing PCR allowed all the previously untyped P[8] strains to be successfully typed. Use of the degenerate primer did not affect the specificity of the PCR or the ability to type strains previously characterized with the original P[8]-specific primer.
Failure to type due to natural variation in primer binding sites has been reported previously for G8 strains (1) and should be constantly considered as a possibility for problems in large surveillance studies to assess the molecular epidemiology of rotaviruses.
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ACKNOWLEDGMENTS |
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This work is part of a rotavirus surveillance study supported by a grant from the Public Health Laboratory Service, London, United Kingdom.
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FOOTNOTES |
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* Corresponding author. Mailing address: Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Hills Rd., Cambridge CB2 2QW, United Kingdom. Phone: 44-1223-257028. Fax: 44-1223-242775. E-mail: jg2{at}mole.bio.cam.ac.uk.
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Journal of Clinical Microbiology, February 2000, p. 898-901, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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