Laboratory of Public Health Entomology, Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115, USA1
Authors for correspondence: Gregory Ebel (Present address: Arbovirus Unit of the Wadsworth Centers Griffin Laboratory, 5668 State Farm Rd., Slingerlands, NY 12159, USA; Fax +1 518 869 4530; e-mail ebel{at}wadsworth.org) and Sam Telford (Fax +1 617 432 1796; e-mail stelford@hsph.harvard.edu).
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Abstract |
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Introduction |
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In 1996 a virus closely related to POW was discovered in deer ticks (Ixodes dammini or I. scapularis of some authors) in Massachusetts and Connecticut (Telford et al., 1997 ). A similar isolate was subsequently established from deer ticks infesting distant sites where other deer tick-transmitted agents perpetuate (Ebel et al., 1999
). This virus was provisionally designated deer tick virus (DTV) based on apparent differences in vertebrate reservoir (Johnson, 1987
; Main et al., 1979
; McLean & Donahue, 1959
; McLean et al., 1966
), arthropod vector (Artsob, 1989
; Ebel et al., 1999
; Telford et al., 1997
), the lack of clinically apparent cases of encephalitis among human residents of transmission foci, and molecular genetic data indicating that DTV strains may form a distinct clade within the TBE complex (Ebel et al., 1999
; Telford et al., 1997
).
Molecular genetic studies have contributed greatly to our understanding of the epidemiology and evolutionary history of flaviviruses and other zoonotic viral agents (Heinz & Kunz, 1982 ; Ksiazek et al., 1997
; Kuno et al., 1998
; Lanciotti et al., 1994
, 1997
; Monroe et al., 1999
; Zanotto et al., 1995
, 1996
). Our previously published analyses of POW and DTV (Ebel et al., 1999
; Telford et al., 1997
), however, seem severely limited because gene sequence data from only one POW strain was available (Mandl et al., 1993
). To determine whether POW and DTV constitute distinct tick-borne flaviviral populations co-circulating in North America, we analysed nucleotide sequence data from eleven POW strains and five DTV strains. Specifically, we constructed phylogenetic trees based on fragments of the envelope (env) and nonstructural protein 5 (ns5) genes, and an untranslated region. We then determined whether the pattern of polymorphism observed within POW nucleotide sequences supports the observation that strains isolated from deer ticks seem to perpetuate in a cycle different from that of other POW strains (Ebel et al., 2000
).
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Methods |
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RNA amplification and sequencing.
To obtain gene sequence data, RNA samples were reverse transcribed using random hexamers, and nucleotides 10271548 (env) and 86728966 (nucleotide numbering after Mandl et al., 1993 ) (ns5) genes were amplified using primers env-A, POW-6, DTVNS5-F and DTVNS5-2 as previously described (Ebel et al., 1999
; Telford et al., 1997
). Nucleotides 1035610806 (3' noncoding region) were amplified using primers [utr-f (5' to 3' gcactgggagctcaagctagagagc) and utr-r (5' to 3' agcgggtgtttttccgagtcacaca)] designed to amplify approximately 500 bp from this region. Reaction products from all regions were electrophoretically separated on a 2% agarose gel, excised, purified and sequenced as described (Ebel et al., 1999
).
Phylogenetic analyses.
To construct phylograms, alignments were generated with the PILEUP program of the Wisconsin Genetics Computer Group and initially analysed by the distance method using MEGA (Kumar et al., 1993 ). Evolutionary distances were computed using the Kimura 2-parameter method including both transitions and transversions. Distance trees were constructed using the neighbour-joining method, and their robustness was estimated by performing 500 bootstrap replicates. Maximum parsimony and maximum likelihood analyses were performed using PAUP version 4.0b3a for Windows (Sinauer Associates). Parsimony and likelihood analyses were performed with 500 bootstrap replicates of a heuristic (maximum parsimony) or fast-heuristic (maximum likelihood) search. Trees generated using PAUP were displayed using TreeView version 1.5.2 (Roderick D. M. Page). MEGA was used to analyse predicted amino acid sequences derived from nucleotide sequences aligned as above. Amino acid distances were computed using the number of differences algorithm and trees were constructed by the neighbour-joining method. The robustness of these trees was estimated as above. McDonaldKreitman tests (McDonald & Kreitman, 1991
) were performed using DnaSP 3.00 (Rozas & Rozas, 1999
) and the P-values reported are Fishers exact test values. For comparison, we used this same method to compare the TBE subtypes central European encephalitis virus (CEE), Russian spring-summer encephalitis virus (RSSE) and louping ill virus (LI). GenBank accession numbers for the sequences used are: U27491, U39292, U27495, X60286 (CEE); X07755, M38310, M97356 (RSSE); X69975, M59376, M94956 (LI). The neutrality index reports the degree of amino acid polymorphism departure from the neutral expectation (Kimura, 1983
).
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Results |
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Geographical- and host-associations may define each of the two lineages
We then evaluated the substructure within each major lineage. Within lineage one, analysis of the nucleotide sequences of all three regions recognized two minor clades separating strains isolated in Canada from New York-derived strains. The neighbour-joining tree constructed from the envelope protein, however, failed to resolve these two sub-lineages. In this protein analysis, isolates from lineage one are virtually identical. Other substructure within lineage one was poorly conserved between trees constructed using different genes, and was not strongly supported by bootstrap analysis. Within lineage two, two minor groups similarly distinguish isolates from Wisconsin from those isolated on the eastern seaboard (Fig. 1). In contrast to lineage one, these minor clades are well supported by bootstrap testing and were recognized by envelope protein analysis. In this analysis, the fox-derived isolate from Virginia was not placed within either of the clades constituting lineage two, but alone between groups of New England and Wisconsin isolates. Although the nucleotide and amino acid sequences of New York and Canadian POW isolates are highly conserved across time and space, those deriving from deer ticks and a fox are more variable.
We then quantified the divergence between lineages by computing the mean pairwise percent nucleotide and amino acid differences between and within each of the two main lineages. For all genes studied, the percent nucleotide differences between lineages were at least 11% and were at least 3-fold greater than the corresponding values within each lineage (Table 2). The percent amino acid sequence difference was also greater between lineages than within either lineage. Between lineages one and two the variation in the envelope protein sequence was 13-fold greater and the variation in the ns5 protein sequence was 8-fold greater than the difference within lineage one. We conclude that the divergence between lineages is much greater than the variation within either lineage, and that lineage two appears to be more genetically diverse than lineage one.
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Discussion |
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Only a small portion (approximately 12%) of the virus genome was analysed in this study. Although the complete sequences for the ns5 and env genes might provide more information, the homogeneity of our findings across three functionally diverse regions suggests that the availability of extended sequence data would be unlikely to significantly alter our conclusions. Phylogenetic studies of other zoonotic viruses have relied on analyses of single gene sequences or gene sequence fragments, and our tree-building approach is consistent with many of these studies (Childs et al., 1994 ; Ecker et al., 1999
; Lanciotti et al., 1994
, 1997
; Plyusnin et al., 1996
; Weaver et al., 2000
). Analysis of nucleotide sequences from each of three regions of the virus genome produced similar tree topologies showing that two distinct POW lineages circulate in North America. Lineage one comprises solely isolates from New York and Canada, while lineage two includes strains isolated from the Atlantic coast of the United States and from Wisconsin. This result was not contingent upon the function of the sequence studied or on the analytical method employed. These observations establish unambiguously that two main POW lineages perpetuate in cycles involving ixodid ticks in North America
The mode of perpetuation of each isolate appears to be associated with membership of a lineage. Strains in lineage one appear to have been isolated from I. cookei (subgenus Pholeoixodes) and members of the Sciuridae, and were obtained mainly before the range expansion of deer ticks (subgenus Ixodes) that occurred during the 1970s. Strains in lineage two, on the other hand, appear to be associated with deer ticks. Five of the six strains constituting this lineage were isolated from deer ticks, and the remaining strain was isolated from a region and at a time where deer ticks may have been present. Strains in lineage one seem to be mainly associated with a groundhogI. cookei cycle, and strains in lineage two appear to be associated with deer ticks and the white-footed mice upon which they most frequently feed (Ebel et al., 2000 ).
Nucleotide variation within natural POW populations appears to be maximal within the envelope gene, but the protein sequences are much less variable than are ns5 protein sequences, both within and between lineages. The conservation of the amino acid sequences within each lineage is consistent with proposed functions of the envelope protein in host cell binding and membrane fusion. It may be, then, that the observed changes in the env gene between lineage one and lineage two are not due to immune-mediated selection (antigenic drift), but to functional constraints arising from different modes of perpetuation, specifically the reliance of each lineage on a different tick vector. This mode of virusvector association was proposed when strains of RSSE virus were recovered from Ixodes persulcatus ticks where CEE was the sole virus expected (Ecker et al., 1999 ). Further field and laboratory observations are required to determine whether specific vectorvirus relationships have evolved between TBE complex viruses and their ixodid tick vectors.
MK tests on envelope gene- and protein-derived sequence data support the observation that the two POW lineages perpetuate differently by rejecting the hypothesis that isolation and random genetic drift have been the most important factors in producing the extant taxa. The envelope protein sequences studied contained more molecular evidence for differentiating the two POW lineages than for differentiating CEE and RSSE [now considered as subtypes of a single virus species (Ecker et al., 1999 )] or CEE and LI, virus pairs known to differ in mode of perpetuation and outcome of human infection. Based on the envelope gene and protein sequences, it seems likely that the two POW lineages perpetuate differently.
The ns5 data, however, do not add support to the adaptive evolution hypothesis. It may be that a longer sequence, or one spanning a different region of the ns5 gene might have produced a different result in the MK test, but the precise effect is difficult to predict in the absence of this data. Alternatively, it may be that due to its role in virus replication, the ns5 gene, which codes a protein with RNA-dependent RNA polymerase activity (Khromykh et al. 2000 ; Lai et al., 1999
; Steffens et al., 1999
), is subject to different selective pressures than the env protein, and would not necessarily provide evidence for an evolutionary history that is concordant to one derived from the env sequences.
These results clearly illustrate the division of North American POW strains into two genetic lineages. Within each lineage, the virus nucleotide and amino acid sequences are highly conserved. The biological significance of the genetic differences between and within POW subtypes requires further investigation, but is likely to be great. Only one subtype (lineage one) has been associated with human disease, while only the other (lineage two) appears to have epidemic potential through its apparently unique association with aggressively human-biting deer ticks. The genetic diversity within our POW samples is well within that which has been reported within other flavivirus species (Ecker et al., 1999 ; Lanciotti et al., 1994
, 1997
; Lepiniec et al., 1994
; Nam et al., 1996
), indicating that the two lineages do not constitute distinct species. The two POW lineages circulating in North America constitute distinct POW subtypes that may perpetuate in distinct enzootic cycles.
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Acknowledgments |
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This work was supported in part by NIH grants AI 39002, AI37993 and AI19693. Dr Ebel was supported while conducting this work by NIH AI 07350 Doctoral Training Program in Tropical Public Health.
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