Partial Genetic Characterization of West Nile Virus Strains, New York State, 2000.We analyzed nucleotide sequences from the envelope gene of 11 West Nile (WN) virus strains collected in New York State during the 2000 transmission season to determine whether they differed genetically from each other and from the initial strain isolated in 1999. The complete envelope genes of these strains were amplified by reverse transcription-polymerase chain reaction. The resulting sequences were aligned, the genetic distances were computed, and a phylogenetic tree was constructed. Ten (0.7%) of 1,503 positions in the envelope gene were polymorphic in one or more sequences. The genetic distances were 0.003 or less. WN virus strains circulating in 2000 were homogeneous with respect to one another and to a strain isolated in 1999. The first outbreak of West Nile (WN) virus infection in North America (1) was apparently the result of single introduction and subsequent amplification of WN virus among Culex Culex /Cu·lex/ (ku´leks) a genus of mosquitoes found throughout the world, many species of which are vectors of disease-producing organisms. Cu·lex n. pipiens mosquitoes and their avian hosts (2-4). Human disease was accompanied by an epizootic ep·i·zo·ot·ic adj. Affecting a large number of animals at the same time within a particular region or geographic area. Used of a disease. ep in which high death rates from severe meningoencephalitis meningoencephalitis /me·nin·go·en·ceph·a·li·tis/ (me-ning?go-en-sef?ah-li´tis) inflammation of the brain and meninges. toxoplasmic meningoencephalitis and myocarditis Myocarditis Definition Myocarditis is an inflammatory disease of the heart muscle (myocardium) that can result from a variety of causes. While most cases are produced by a viral infection, an inflammation of the heart muscle may also be instigated by were reported in some avian hosts, notably American Crows (Corvus brachyrhynchos) (5). RNA virus populations are subject to high mutation rates and may evolve rapidly under certain conditions (6-8). To determine whether WN virus genotypes circulating in New York during the 2000 transmission season differed from those isolated there in 1999, WN virus strains were collected from mosquito pools and dead vertebrates, the complete nucleotide sequences of the envelope genes were determined, and the sequences of these strains were compared with one another and with a strain isolated during 1999. Materials and Methods WN virus was isolated from pools of infected mosquitoes collected throughout New York State and from vertebrate tissues submitted by the N.Y. State Wildlife Pathology Unit. Mosquitoes were collected overnight in standard miniature light traps or gravid gravid /grav·id/ (grav´id) pregnant. grav·id adj. Carrying eggs or developing young. gra·vid traps, and they were pooled and sent to the New York State Arbovirus arbovirus Any of a large group of viruses that develop in arthropods (chiefly mosquitoes and ticks). The name derives from “arthropod-borne virus.” The spheroidal virus particle is encased in a fatty membrane and contains RNA; it causes no apparent harm to the Laboratories. Pools of mosquitoes and vertebrate tissues were homogenized ho·mog·e·nize v. ho·mog·e·nized, ho·mog·e·niz·ing, ho·mog·e·niz·es v.tr. 1. To make homogeneous. 2. a. To reduce to particles and disperse throughout a fluid. b. in 2 mL of mosquito diluent diluent /dil·u·ent/ (dil´oo-int) 1. causing dilution. 2. an agent that dilutes or renders less potent or irritant. dil·u·ent adj. Serving to dilute. n. (20% heat-inactivated fetal bovine serum Fetal bovine serum ( or foetal bovine serum) is serum taken from the fetuses of cows. Fetal Bovine Serum (or FBS) is the most widely used serum in the culturing of cells. In some papers the expression foetal calf serum is used. [FBS FBS abbr. fasting blood sugar FBS Fasting blood sugar. See Fasting glucose. ] in Dulbecco's phosphate-buffered saline plus 50 [micro]g/mL penicillin/streptomycin, 50 [micro]g gentamicin gentamicin /gen·ta·mi·cin/ (jen?tah-mi´sin) an aminoglycoside antibiotic complex isolated from bacteria of the genus Micromonospora, , and 2.5 [micro]g/mL fungizone) or 350 [micro]L lysis buffer, respectively, by using an SPEX SPEX Special Licensing Examination Medtalk An examination administered to ±1500 physicians/yr who seek relicensure and/or want to practice in a different state yrs after initial licensure mixer-mill (Spex CertiPrep, Metuchen, NJ) and glass beads; 500 [micro]L of the resulting suspension was transferred to 1.5-mL microcentrifuge tubes and centrifuged at 16,000 RCF for 10 min; 100 [micro]L of the clarified solution was applied to confluent con·flu·ent adj. 1. Flowing together; blended into one. 2. Merging or running together so as to form a mass, as sores in a rash. monolayers of African Green Monkey Kidney (Vero) cells in T-25 flasks, and virus was allowed to adsorb adsorb /ad·sorb/ (ad-sorb´) to attract and retain other material on the surface; to conduct the process of adsorption. ad·sorb v. To take up by adsorption. for 1 hr at 37 [degrees] C, 5% [CO.sub.2]. After adsorption, 5 mL of minimum essential medium (MEM) containing 2% FBS and antibiotics (as above) was applied to the cells, and they were returned to the incubator. Cultures were checked for signs of cytopathic effect (CPE (Customer Premises Equipment) Communications equipment that resides on the customer's premises. CPE - Customer Premises Equipment ) daily. When [is greater than] 50% of cells in a culture flask displayed CPE, the culture was harvested, and clarified aliquots of the culture media supernatant were supplemented with FBS (20% of final volume) and stored in 1.5-mL cryovials at -80 [degrees] C until further use. Virus stocks were passed by applying 100 [micro]L of the initial culture supernatant to a second confluent monolayer mon·o·lay·er n. 1. A film or layer one molecule thick formed at the interface between water and either oil or air by a substance such as a partially esterified fatty acid that contains both hydrophobic and hydrophilic groups in the same of Vero cells, as above. When CPE was evident in [is greater than] 50% of the cells in the culture, the cells were scraped from the flask and centrifuged with the media in 15-mL conical tubes at 3,000 x g for 20 minutes. RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic was extracted from the resulting cell pellet by using RNeasy columns (Qiagen, Valencia, CA) as directed by the manufacturer. The complete envelope sequences were amplified by reverse transcription-polymerase chain reaction (RT-PCR RT-PCR reverse transcriptase-polymerase chain reaction. See PCR1. ) with primers (Forward [5-CATCGAATTCGTTACCCTCTCTAACTTCCAAGGGAAGGTG-3] Reverse [5-GTATGGATCCTGATGCTCCAGTCTGGAAACTGATCGTA-3]) designed to amplify the genomic sequence covering the coding region of the complete prM/M, E, and the N-terminal NS1. These primers also contain engineered restriction sites for use in other experiments that will be described elsewhere. Reaction products were electrophoretically separated on 2% agarose gel, and bands of the predicted size were excised and purified by using the Qiaquick gel extraction kit (Qiagen, Valencia, CA). Purified DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. fragments were sequenced on an ABI Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother. (Application Binary Interface) A specification for a specific hardware platform combined with the operating system. PRISM 377XL automated DNA sequencer (Applied Biosystems, CA) with six forward and six reverse primers (Table 1). Table 1. Primers used in West Nile virus sequencing Primer Sequence WNSE1F CTC TCT AAC TTC CAA GGG AAG WNSE2F CAC TCT AGC GAA CAA GAA GG WNSE3F TCT CCA CCA AAG CTG CGT GC WNSE4F TAC TAC GTG ATG ACT GTT GGA A WNSE5F CCT TGC AAA GTT CCT ATC TC WNSE6F TCC TGT TGT GGA TGG GCA TC WNSE1R TGT CTT CTG GAT CAT TAC CAG C WNSE2R GCC ACC AGG GCA TAT CCA GG WNSE3R TTC AAG ATG GTT CTT CCT ATT GC WNSE4R GGA ATG GCT CCA GCC AAA GC WNSE5R TGT TCT CCT CTG CCC ACC AC WNSE6R TCC ATC CAA GCC TCC ACA TC Two of the vertebrate strains (3282 and 3356) were processed differently. RT-PCR was conducted directly on RNA isolated from infected tissues. The primers used for the amplification and sequencing steps will be described elsewhere (Lanciotti et al., manuscript in preparation). Sequences were aligned with a WN virus strain collected in 1999 (GenBank Accession #AF260967) and a distantly related St. Louis encephalitis St. Louis encephalitis see St. Louis encephalitis. virus sequence (AF205490) by using the clustal method on the DNAStar software package. Initial analysis was done by the distance method using MEGA (9). Evolutionary distances were computed by the Kimura 2 parameter method including both transitions and transversions. Distance trees were constructed by the neighborjoining method, and their robustness was estimated by performing 500 bootstrap replicates. Results WN virus strains from diverse locations, times, and host types were assembled for this study (Table 2). The strains were isolated from avian and mosquito hosts collected from midsummer through autumn at the epicenter and at the periphery of the 2000 epizootic. Strains were thus a representative sample of WN virus circulating in New York during 2000.
Table 2. Characteristics of strains studied
Collection
Strain date County/borough Site/town
3000017 Jul-2000 Staten Island Richmond
3000259 Jul-2000 Suffolk Calhoun
3000548 Jul-2000 Queens Country Farm Museum
3000622 Jul-2000 Westchester Twin Lake Stable
3100271 Jul-2000 Rockland Unknown
3100352 Jul-2000 Staten Island Saw Mill Marsh
3100365 Jul-2000 Staten Island Fresh Kills Landfill
842 Jul-2000 Staten Island Amboy Rd.
2741 Sep-2000 Albany SUNY
3282 Oct-2000 Oswego New Haven
3356 Oct-2000 Staten Island Mariner's Harbor
Passage GenBank
Strain Source history(*) Accession No.
3000017 Cx. pipiens V2 AF346309
3000259 Cx. pip/restuans V2 AF346316
3000548 Cx. pip/res V2 AF346311
3000622 Cx. pip/res V2 AF346313
3100271 Cx. pip/res V2 AF346312
3100352 Cx. salinarius V2 AF346314
3100365 Cx. pipiens V2 AF346310
842 American Crow V2 AF346317
2741 American Crow V2 AF346315
3282 Ruffed Grouse P AF346319
3356 American Crow P AF346318
(*) V2=Two vero passages, P=primary RNA tissue extract
Nucleotide substitutions occurred at 10 (0.7%) of the 1,503 positions in the envelope gene (Table 3). Of these substitutions, all were transitions, two (0.4%) of which resulted in amino acid changes. The C to U substitution at position 2321 (position numbers refer to Lanciotti et al. [1]) results in a serine serine (sĕr`ēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein. to leucine leucine (l  `sēn), organic compund, one of the 20 amino acids commonly found in animal proteins. change in envelope
amino acid number 452, and the A to G substitution at position 2386
results in an isoleucine isoleucine (ī'səl`sēn), organic compound, one of the 20 amino acids commonly found in animal proteins. to valine valine (văl`ēn), organic compound, one of the 22 α-amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein. change in envelope amino acid
position 474. The mean pairwise Kimura 2-parameter distances between the
isolates were 0.003 or less. The phylogenetic tree of the nucleotide
sequences studied showed similarly minimal distances between the
isolates, with low bootstrap confidence values at the nodes separating
the branches. WN virus strains circulating in New York State during the
2000 transmission season were relatively homogeneous at both the
nucleotide and amino acid levels.
Table 3. Nucleotide substitutions in strains studied
Position
Strain 1285 1332 1449 1899 1974
NY99-EQHS C U C U C
3000017 -- -- -- -- U
3000259 -- -- -- -- --
3000548 -- C -- -- --
3000622 -- -- U -- --
3100271 -- -- -- -- --
3100352 -- -- -- C U
3100365 -- -- -- -- U
842 -- -- -- -- --
2741 -- -- -- -- --
3282 U -- -- -- --
3356 U -- -- -- U
Position
Strain 2280 2321 2359 2386 2424
NY99-EQHS U C C A C
3000017 C -- -- -- U
3000259 -- -- -- -- --
3000548 -- -- -- -- --
3000622 -- -- -- -- --
3100271 -- -- -- -- --
3100352 -- -- -- -- --
3100365 -- -- -- -- --
842 -- -- U G --
2741 -- -- -- -- --
3282 -- -- -- -- --
3356 -- U -- -- --
Position numbers correspond to Lanciotti et al.(1).
Conclusion These data represent the first population study of WN virus in North America since its introduction in 1999. The envelope sequences of these virus strains establish a baseline sequence dataset against which strains isolated during future transmission seasons may be compared. Only the envelope sequences were studied, which were analyzed by using distance matrices and neighbor-joining methods. Although complete genome sequences may have provided additional information, short sequence fragments have often been used in population studies of arboviruses arboviruses (ar´bōvī´r  sz),n. (1013). Additional criteria (maximum parsimony and maximum likelihood) may have provided corroboration for the close relationships observed; however, the sequences are so similar and the nodes on the neighbor-joining tree so poorly supported that additional analyses seemed unwarranted. Given the close relationship of the strains, it is unlikely that additional nucleotide data or analytic methods would have greatly enhanced our understanding of WN virus population structure in this hemisphere. Mosquito- and vertebrate-derived sequences appeared to be distributed randomly in the phylogenetic tree. Date of isolation of the strains was similarly unimportant in the clustering of sequences. Additionally, passage history seemed not to affect the gene sequences. RT-PCR amplification directly from infected mosquito pools often failed or produced amplicons that did not conform to size expectations, necessitating Vero cell passage of many strains before amplification. The two sequences obtained from RNA extracts of infected tissue without Vero passage were not different from those passed through these cells twice. The WN virus sequences in this study were homogeneous with respect to passage history, host, and time. A single nucleotide substitution, a C to U change at position 1974, was present in four of five strains isolated from Staten Island but not from other locations. This mutation caused these strains (3000017, 3100352, 3100365, and 3356) to cluster in the phylogenetic tree, but bootstrap confidence in this clustering pattern, as for all the relationships displayed (Figure), was low. The utility of this particular substitution for molecular epidemiologic studies of WN virus in North America is difficult to ascertain, but in principle, findings of this type may provide useful information in determining the mode or modes of spread of particular WN virus strains in North America. The envelope sequences studied are highly conserved. RNA viruses are well known to exist as quasispecies, composed of a swarm of competing viral genotypes (14,15). This mode of existence, because of the lack of proofreading Proofreading traditionally means reading a proof copy of a text in order to detect and correct any errors. Modern proofreading often requires reading copy at earlier stages as well. and mismatch-repair mechanisms of most viral encoded RNA-dependent RNA polymerases (16), may allow rapid evolution under certain circumstances. Dengue virus, another mosquito-borne flavivirus, is thought to have diversified as the viral population expanded with human and mosquito populations (17). WN virus, having entered a naive ecosystem and vastly expanded its range, may evolve similarly. Many arboviruses, however, are remarkably conserved across time and space, implying stringent constraints on viral structural proteins and replicative machinery (18). Fitness of vesicular stomatitis virus vesicular stomatitis virus A rhabdovirus which replicates in the cytoplasm of infected cells; most VSV victims were in direct contact with oral secretions of infected livestock Clinical Fever, chills, malaise, myalgia, N&V, pharyngitis. , an animal RNA virus, has been shown to drop precipitously as the virus passed through a series of population bottlenecks (19). Whether WN virus will follow a pattern of diversification or conservation is unclear. The viruses in this study are likely the result of a single introduction of WN virus, primary expansion during 1999, overwintering o·ver·win·ter·ing n. The persistence of an infectious agent in its vector for an extended period, as in the cooler winter months, during which the vector has no opportunity to be reinfected or to infect another host. , and secondary expansion during the 2000 transmission season. Determining the genetic structure of WN virus populations in subsequent transmission seasons may advance our understanding of WN virus perpetuation, selection, and evolution. Acknowledgments We thank Robert Lanciotti and Amy Kerst for supporting the sequencing of 3282 and 3356; Harry Taber and the Wadsworth Center Molecular Genetics Core for institutional support at the Wadsworth Center; the Wadsworth Center Molecular Genetics Core; and the New York State Department of Health Division of Epidemiology, particularly Dennis White and Millicent Eidson, for guiding and supporting collection of the mosquito pools and vertebrates from which WN virus strains were isolated and for thoughtful comments on this manuscript. Dr. Ebel is a research scientist in the Arbovirus Laboratories of the Wadsworth Center, New York State Department of Health. His research focuses on the population structure, biology, and molecular evolution of arthropod-borne zoonoses Zoonoses Infections of humans caused by the transmission of disease agents that naturally live in animals. People become infected when they unwittingly intrude into the life cycle of the disease agent and become unnatural hosts. . References (1.) Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K. et al. Origin of the West Nile virus West Nile virus, microorganism and the infection resulting from it, which typically produces no symptoms or a flulike condition. The virus is a flavivirus and is related to a number of viruses that cause encephalitis. responsible for an outbreak of encephalitis in the northeastern United States. Science 1999;286:2333-7. (2.) Baqar S, Hayes CG, Murphy JR, Watts DM. Vertical transmission of West Nile virus by Culex and Aedes species mosquitoes. Am J Trop Med Hyg 1993;48:757-62. (3.) Rappole JH, Derrickson SR, Hubalek Z. Migratory birds and spread of West Nile virus in the Western Hemisphere. Emerg Infect Dis 2000;6:319-28. (4.) Komar N. West Nile viral encephalitis. Rev Sci Tech 2000;19:166-76. (5.) Steele KE, Linn MJ, Schoepp RJ, Komar N, Geisbert TW, Manduca RM, et al. Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City New York City: see New York, city. New York City City (pop., 2000: 8,008,278), southeastern New York, at the mouth of the Hudson River. The largest city in the U.S. , New York. Vet Pathol 2000;37:208-24. (6.) Holland JJ, Spindler K, Horodyski F, Grabau E, Nichol S, VandePol S. Rapid evolution of RNA genomes. Science 1982;215:1577-85. (7.) Steinhauer DA, Holland JJ. Rapid evolution of RNA viruses. Annu Rev Microbiol 1987;41:409-33. (8.) Garmendia AE, Van Kruiningen HJ, French RA, Anderson JF, Andreadis TG, Kumar A, et al. Recovery and identification of West Nile virus from a hawk in winter. J Clin Microbiol 2000;38:3110-1. (9.) Kumar S, Tamura K, Nei M. MEGA: molecular evolutionary genetics analysis. University Park (PA): Pennsylvania State University Pennsylvania State University, main campus at University Park, State College; land-grant and state supported; coeducational; chartered 1855, opened 1859 as Farmers' High School. Press; 1993. (10.) Kramer LD, Presser SB, Hardy JL, Jackson AO. Genotypic and phenotypic variation of selected Saint Louis encephalitis Saint Lou·is encephalitis n. A viral encephalitis occurring in parts of North America and transmitted by a mosquito of the genus Culex. viral strains isolated in California. Am J Trop Med Hyg 1997;57:222-9. (11.) Harris E, Roberts TG, Smith L, Selle J, Kramer LD, Valle S, et al. Typing of dengue viruses in clinical specimens and mosquitoes by single-tube multiplex reverse transcriptase PCR RT-PCR is a one or two-step process for converting RNA to DNA and the subsequent amplification of the reversely-transcribed DNA. In the first step of RT-PCR, called the “first strand reaction,” complementary DNA (cDNA) is made from an mRNA template using . J Clin Microbiol 1998;36:2634-9. (12.) Powers AM Oberste MS, Brault AC, Rico-Hesse R, Schmura SM, Smith JF, et al. Repeated emergence of epidemic/epizootic Venezuelan equine encephalitis Venezuelan equine encephalitis An alphavirus infection first identified in a sick horse in Venezuela in 1938, which occurs as an epizootic infection in central and northern South America; most exposed humans develop flu-like Sx; ±4%, especially adolescents, from a single genotype of enzootic en·zo·ot·ic adj. Prevalent among or restricted to animals of a specific geographic area. Used of a disease. n. An enzootic disease. enzootic peculiar to or present constantly in a location. See also endemic. subtype ID virus. J Virol 1997;71:6697-705. (13.) Ebel GD, Foppa I, Spielman A, Telford SR. A focus of deer tick virus transmission in the northcentral United States. Emerg Infect Dis 1999;5:570-4. (14.) Duarte EA, Novella IS, Weaver SC, Domingo E, Wain-Hobson S, Clarke DK, et al. RNA virus quasispecies: significance for viral disease and epidemiology. Infect Agents Dis 1994;3:201-14. (15.) Holland JJ, de la Torre JC, Steinhauer DA. RNA virus populations as quasispecies. Curr Top Microbiol Immunol 1992;176:1-20. (16.) Steinhauer DA, Domingo E, Holland JJ. Lack of evidence for proofreading mechanisms associated with an RNA virus polymerase. Gene 1992;122:281-8. (17.) Zanotto PMDA PMDA Plastics Machinery Distributors' Association (United Kingdom) PMDA Plutonium Management and Disposition Agreement (US-Russia) PMDA Pharmaceuticals and Medical Device Agency (Japan) , Gould EA, Gao GF, Harvey PH, Holmes EC. Population dynamics of flaviviruses revealed by molecular phylogenies. Proc Natl Acad Sci U S A 1996;93:548-53. (18.) Pisano MR, Tolou H. The topotype top·o·type n. Biology A specimen of an organism taken from the type locality of that species. notion and the quasispecies concept: The yellow fever virus yellow fever virus n. An arbovirus of the genus Flavivirus that causes yellow fever and is transmitted by mosquitoes. as example. Travaux Scientifiques des Chercheurs du Service de Sante des Armees 1996;69-70. (19.) Duarte E, Clarke D, Moya A, Domingo E, Holland J. Rapid fitness losses in mammalian RNA virus clones due to Mueller's ratchet. Proc Natl Acad Sci U S A 1992;89:6015-9. Gregory D. Ebel, Alan P. Dupuis II, Kiet Ngo, David Nicholas, Elizabeth Kauffman, Susan A. Jones, Donna Young, Joseph Maffei, Pei-Yong Shi, Kristen Bernard, and Laura D. Kramer New York State Department of Health, Slingerlands, New York Slingerlands is a hamlet in the Town of Bethlehem, Albany County, New York, USA. The community is in the Eastern Standard time zone. The latitude of Slingerlands is 42.629N. The longitude is -73.865W. The zip code for Slingerlands is 12159. , USA Address for correspondence: Gregory Ebel, Arbovirus Laboratories, 5668 State Farm Road, Slingerlands, NY 12159, USA; fax: 518-869-4530; e-mail: ebel@wadsworth.org |
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