West Nile virus (WNV) is found in many regions, including Africa, the Middle East, Europe, Russia, India, Indonesia, and most recently North America (
9). Phylogenetic analysis of WNV strains has revealed two distinct lineages (I and II). Lineage I strains are frequently involved in human and equine outbreaks, and lineage II strains are mostly maintained in enzootic cycles (
4,
30,
35,
36,
59). Sequence analysis showed that the strain in North America is closely related to other human epidemic strains isolated from Israel, Romania, Russia, and France, all of which belong to lineage I (
35,
36). WNV has caused significant human, equine, and avian disease since its appearance in North America in 1999 (
2,
28,
36), and the virus has quickly spread from the Northeast to the eastern seaboard and to the Midwest (
3). There were 61 human cases (7 deaths) in New York City in 1999 (
13); 21 human cases (4 deaths) in New York, New Jersey, and Connecticut in 2000 (
42); and 48 human cases (5 deaths) in New York, Florida, New Jersey, Connecticut, Maryland, Massachusetts, Georgia, and Louisiana in 2001 (
14).
WNV is a member of the
Flavivirus genus, a group of arthropod-borne viruses in the family
Flaviviridae. Besides WNV, many other members of the flaviviruses are important human pathogens, including dengue virus (DEN), yellow fever virus (YF), the tick-borne encephalitis virus complex (TBE), Japanese encephalitis virus (JE), and Murray Valley encephalitis virus (MVE) (
9). The flavivirus genome is a single plus-strand RNA of approximately 11 kb in length that encodes 10 viral proteins in a single open reading frame (
55). The encoded polyprotein is translated and co- and posttranslationally processed by viral and cellular proteases into three structural proteins (the capsid protein C; the membrane protein M, which is formed by furin-mediated cleavage of prM; and the envelope protein E) and seven nonstructural proteins (the glycoprotein NS1, NS2a, the protease cofactor NS2b, the protease and helicase NS3, NS4a, NS4b, and the polymerase NS5) (
15,
39). The 5′ and 3′ untranslated regions (UTRs) of the genomic RNA are approximately 100 and 400 to 700 nucleotides (nt) in length, respectively, and the terminal nucleotides of both the 5′ and the 3′ UTRs can form highly conserved secondary and tertiary structures (
7,
8,
55,
60).
The establishment of a reverse genetic system for the WNV strain presently circulating in the United States is a critical step in the study of the epidemic North American strains of WNV. Infectious full-length cDNA clones for a number of flaviviruses have been successfully developed for the study of viral replication and pathogenesis (
56). In several cases, assembly of full-length flavivirus clones in a plasmid vector was not straightforward because clones containing large portions of the genome were unstable and deleterious for bacterial hosts. This problem was first circumvented for YF by ligating cDNA fragments in vitro prior to RNA transcription (
54). Similar approaches were applied to develop infectious clones for JE (
62), DEN type 2 (DEN2) (
31), and TBE strain Hypr (
40). For other flaviviruses, stable full-length infectious clones were established for DEN4 (
34), Kunjin virus (
33), TBE strain Neudoerfl (
40), MVE (
27), and TBE strain Langat (
11). Although an infectious clone of the lineage II WNV strain from Nigeria was recently reported (
68), no such full-length cDNA clone has been developed for the human epidemic lineage I WNV.
MATERIALS AND METHODS
Cells and virus.
Vero (ATCC CCL-81) cells were grown in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). BHK-21/WI2 (BHK-21) (
64) and
Aedes albopictus C6/36 (C6/36) (ATCC CRL-1660) cells were grown in Dulbecco's modification of MEM with 10% FBS and 0.1 mM nonessential amino acids. Antibiotics were added to all media at 10 U/ml of penicillin and 10 μg/ml of streptomycin. Cells were maintained in 5% CO
2 at 37°C (Vero and BHK-21) or 28°C (C6/36). The parental WNV strain 3356 was isolated from the kidney of an American crow collected in October 2000 from Staten Island, New York (
18). A single viral stock was made from the second passage in Vero cells without plaque purification, stored as aliquots at −80°C, and designated as parental WNV 3356. This stock was used as parental virus in all assays. Plaque assays were performed on Vero cells as described previously (
53).
cDNA synthesis and cloning.
BHK-21 cells were infected at a multiplicity of infection (MOI) of 0.05 with parental WNV 3356, and virus was harvested from cell culture media at 36 h postinfection. Genomic RNA was extracted from the cell culture media with RNeasy (Qiagen, Valencia, Calif.). cDNA fragments covering the complete genome were synthesized from genomic RNA through ThermoScript reverse transcription (RT)-PCR according to the manufacturer's instructions (Gibco BRL, Rockville, Md.). Plasmid pBR322 was modified by replacement of the SphI-EcoRI fragment in the tetracycline resistance gene with a pair of complementary oligonucleotides to create the sequence 5′-GCATGgATCCCGTTGCGCATGCTGATTCGAACCGACTAGT-CTCGAG-TCTAGAATTC-3′ to yield plasmid pBRlinker containing the unique restriction sites BamHI, SphI, SpeI, XhoI, and XbaI (listed in order and underlined). After the modification, the original SphI site of pBR322 (italics) was mutated through a C to G substitution (lowercase italics). The modified pBR322, pBRlinker, was used as the cloning vector throughout the experiments.
Bacterial strain HB101 (Gibco BRL) was used as the host for construction and propagation of cDNA clones. Standard cloning procedures were followed (
57), except that constructs with inserts of greater than 3 kb were propagated at room temperature. Electroporation was performed to transfect plasmid into bacteria in 0.2-cm cuvettes, using a GenePulser apparatus (Bio-Rad, Hercules, Calif.) with settings of 2.5 kV, 25 μF, and 200 Ω. The virus-specific sequence of each intermediate cloning product was validated by sequence analysis (Applied Biosystems, Foster City, Calif.) before it was used in a subsequent cloning step. All restriction endonucleases were purchased from New England Biolabs (Beverly, Mass.).
RNA transcription and transfection.
Plasmid pFLWNV, containing the full-length cDNA of WNV, was amplified in Escherichia coli HB101 and purified through MaxiPrep (Qiagen). For in vitro transcription, 5 μg of pFLWNV was linearized with XbaI. Mung bean nuclease (5 U; New England BioLabs) was directly added to the XbaI digestion reaction mixture, and the reaction mixture was further incubated at 30°C for 30 min to remove the single-stranded nucleotide overhang generated by the XbaI digestion. The linearized plasmids were extracted with phenol-chloroform twice, precipitated with ethanol, and resuspended in 10 μl of RNase-free water at 0.5 μg/μl. The mMESSAGE mMACHINE kit (Ambion, Austin, Tex.) was used to in vitro transcribe RNA in a 20-μl reaction mixture with an additional 2 μl of GTP solution. The reaction mixture was incubated at 37°C for 2 h, followed by the addition of DNase I to remove the DNA template. RNA was precipitated with lithium chloride, washed with 70% ethanol, resuspended in RNase-free water, quantitated by spectrophotometry, and stored at −80°C in aliquots. A mutant RNA transcript with a deletion of the 3′-terminal 199 nt of WNV was generated from pFLWNV linearized with an internal restriction site DraI at nt position 10830. The mutant RNA was synthesized in the same manner as the full-length RNA, as described above. All procedures were performed according to manufacturer protocols.
For transfection, approximately 10 μg of RNA was electroporated to 10
7 BHK-21 cells in 0.8 ml of cold phosphate-buffered saline (PBS), pH 7.5, in 0.4-cm cuvettes with the GenePulser apparatus (Bio-Rad) at settings of 0.85 kV and 25 μF, pulsing three times, with no pulse controller. After a 10-min recovery, cells were mixed with media and incubated in a T-75 flask (5% CO
2 at 37°C) until cytopathic effects (CPE) were observed. Virus was harvested as tissue culture media, clarified by centrifugation at 10,000 ×
g, stored in aliquots at −80°C, and designated as recombinant WNV. Plaque assays were performed on Vero cells as described previously (
51).
Genetic marker analysis of the recombinant and parental virus.
Genetic markers of
StyI and
EcoRI were engineered into the cDNA clone (Fig.
1) to distinguish recombinant progeny virus from the corresponding parental virus. Recombinant virus harvested from supernatant on day 3 posttransfection and parental virus were subjected to RNA extraction with RNeasy (Qiagen). A 388-bp fragment including the genetic markers was amplified through RT-PCR from RNA extracted from either recombinant or parental virus with primers 8706V (5′-CATGGCCATGACTGACACTACTC-3′) and 9093C (5′-CTTGGCCTTTCCGAACTCTCCG-3′). The RT-PCR products were digested with
StyI or
EcoRI and analyzed on a 2% agarose gel.
IFA.
Indirect immunofluorescence assays (IFA) were used to detect viral protein expression in WNV RNA-transfected BHK-21 cells. After electroporation, approximately 105 transfected cells were spotted onto 10-mm glass coverslips. Cells on coverslips were analyzed by IFA at various times posttransfection for viral protein synthesis. Cells were fixed in 3.7% paraformaldehyde with PBS, pH 7.5, at room temperature for 30 min followed by incubation in −20°C methanol for 30 min. The fixed cells were washed with PBS, incubated at room temperature for 45 min in WNV immune mouse ascites fluid (1:100 dilution; ATCC, Manassas, Va.), and further reacted with goat anti-mouse immunoglobulin G conjugated with fluorescein isothiocyanate at room temperature for 30 min (1:100 dilution) (KPL, Gaithersburg, Md.). The coverslips were washed with PBS, mounted to a slide using fluorescent mounting medium (KPL), and observed under a fluorescence microscope equipped with a video documentation system (Zeiss, Thornwood, N.Y.).
Specific infectivity assay.
Approximately 10 μg of RNA was electroporated to 10
7 BHK-21 cells, as described above. Both transfected and untransfected BHK-21 cells were adjusted to a concentration of 6 × 10
5 cells per ml. A series of 1:10 dilutions were made by mixing 0.5 ml of transfected cells with 4.5 ml of untransfected cells. One milliliter of cells (6 × 10
5 cells total) for each dilution was seeded per individual well of six-well plates. Triplicate wells were seeded for each cell dilution. The cells were allowed to attach to the plates for 4 to 5 h under 5% CO
2 at 37°C before the first layer of agar was added, as described previously (
53). After incubation of the plates for 3 days under 5% CO
2 at 37°C, a second layer of agar containing neutral red was added. Plaques were counted after incubation of the plates for another 12 to 24 h, and the specific infectivity was calculated as the number of PFU per microgram of RNA.
Growth curves.
Subconfluent BHK-21 and C6/36 cells in 12-well plates were inoculated with either the parental or recombinant WNV at an MOI of 5 or 0.05 in triplicate wells. Virus stocks were diluted in BA-1 (M199-H [Gibco-BRL], 0.05 M Tris, pH 7.6, 1% bovine serum albumin, 0.35 g of sodium bicarbonate/liter, 100 U of penicillin/ml, 100 μg of streptomycin/ml, and 1 μg of amphotericin B [Fungizone]/ml). Attachment was allowed for 1 h under 5% CO
2 at 37°C or under 5% CO
2 at 28°C for the BHK-21 and C6/36 cells, respectively. The inocula were then removed, the monolayers were washed three times with BA-1, and 2 ml of medium was added to each well. The plates were incubated for up to 6 days under 5% CO
2 at 37°C or under 5% CO
2 at 28°C for the BHK-21 and C6/36 cells, respectively. The medium was sampled immediately after the addition of medium (1-h time point) and at 7.5, 16, 24, 32, 40, 48 and 72 h for BHK-21 and C6/36 cells, as well as at 96 and 124 h for C6/36 cells. The 10-μl samples were stored at −80°C prior to titration as previously described (
53). Cells were observed daily for CPE.
Virulence in mice.
Mice were housed in an environmentally controlled room under biosafety level 3 conditions and were given food and water ad libitum. Female outbred CD-1 mice (Charles River Laboratories, Wilmington, Mass.) were obtained at 5 weeks of age and were acclimatized for 1 week. All mice were 6 weeks of age at the start of the experiment. Eight mice per group were inoculated with diluent alone or with 102 PFU of parental or recombinant virus subcutaneously (s.c.) in the left rear footpad. Diluent was PBS (endotoxin-free) supplemented with 1% FBS. Mice were evaluated clinically and weighed daily for 2 weeks, then monitored daily and weighed thrice weekly for 2 more weeks. Observed clinical signs included ruffled fur, paresis, hindleg paralysis, and tremors. Morbidity was defined as exhibition of greater than 10% weight loss or clinical signs for 2 or more days. Mice were euthanized if they became moribund. Exposure to virus was confirmed in all surviving mice by a positive antibody titer to WNV by enzyme-linked immunosorbent assay on day 28 postinoculation.
Statistical analyses.
Microsoft Excel 97 was used for all statistical analyses. A chi-square test was used to compare the morbidity and mortality in mice for the parental and recombinant viruses, and a two-tailed Student's t test was used to evaluate the survival time for the two groups.
DISCUSSION
We report the construction of the first full-length cDNA clone of the human epidemic strain of WNV (lineage I). RNA transcripts transcribed from the cDNA clone were highly infectious upon transfection into BHK-21 cells. The identification of genetic markers engineered into the clone confirmed that the progeny virus was derived from the cDNA clone and thus was not a contaminant. The infectivity of the cDNA-derived RNA was further supported by the finding that a mutant RNA with an expected lethal deletion of the 3′-terminal 199 nt of the genome was not infectious. The recombinant virus showed biological properties indistinguishable from those of the parental virus, including plaque morphology, growth kinetics, and virulence characteristics. These results indicate that an efficient reverse genetic system has been established for lineage I WNV.
A common difficulty in assembling full-length clones of flaviviruses is that plasmids containing long flavivirus-specific inserts are unstable during propagation in bacteria. A number of approaches have been developed to assemble full-length cDNA clones of flaviviruses. (i) Full-length cDNA clone can be assembled through in vitro ligation of cDNA fragments. This approach avoids cloning and propagating full-length clones in bacteria and has been successfully applied to generate infectious RNA of YF (
54), JE (
62), DEN2 (
31), and TBE strain Hypr (
40). (ii) Genome-length cDNA containing an upstream promoter for transcription can be directly synthesized by a one-step RT-PCR without any cloning. This rapid method was successfully used to generate infectious RNA of TBE (
19). However, this approach has the limitation that the uncloned PCR-derived cDNA will produce a heterogeneous RNA population, derived from mutations during RT-PCR or from quasispecies of the original virus stock. (iii) Full-length cDNA clone can be assembled in yeast cells through homologous recombination. This method was successfully used to assemble a full-length clone of DEN2 (
49). (iv) Full-length cDNA can be cloned under the control of a eukaryotic promoter, and introns are introduced into the problematic regions of the cDNA to avoid mutations during their propagation in bacteria. This approach requires transfection of eukaryotic cells with plasmid cDNA rather than RNA. An infectious JE clone was recently developed, using this approach, in which genomic RNA was made in situ by nuclear transcription and intron splicing in transfected eukaryotic cells (
67). (v) Stable full-length cDNA clone can be constructed using low- or medium-copy-number vectors and selective bacterial hosts. This approach has been applied to a number of flaviviruses, including DEN4 (
34), Kunjin virus (
33), TBE strain Neudoerfl (
40), MVE (
27), TBE strain Langat (
38), lineage II WNV (
68), and lineage I WNV (this report).
During the construction of our WNV clone, we found that the most unstable region of the genome was within its 5′ quarter and that cDNA from this region should be assembled last in order to obtain full-length clones. These results are consistent with previous reports that cDNAs of structural regions are more likely to be unstable during cloning (
67,
68). We also found that bacterial propagation at room temperature rather than at 37°C yielded a higher success rate of cloning intact inserts. A recent report showed that cloning at room temperature was essential to the construction of infectious full-length DEN2 cDNA using high-copy-number plasmid vectors (
61).
The 3′ UTR of flaviviruses is believed to function as a promoter for initiation of minus-strand RNA synthesis. The 3′-terminal nucleotides of flavivirus genomic RNA were thermodynamically predicted and experimentally demonstrated to form distinct secondary structures, including a short stem-loop (SL) adjacent to a long SL (
8,
50,
52,
60). This secondary structure is conserved among divergent flaviviruses, although only short stretches of sequence are conserved. Structural analysis of WNV 3′ RNA reveals that the loop of the short SL interacts with the lower portion of the neighboring long SL to form a pseudoknot structure (
60). Three host proteins bind specifically to the WNV 3′ SL RNA (
5), and one of these cellular proteins is the translation elongation factor, eF1-α (
6). The NS5 (RNA-dependent RNA polymerase) and NS3 (protease and helicase) of JE were shown to bind specifically to the 3′ SL RNA (
17). The cyclization sequences in the 5′- and 3′-terminal regions of the genome were recently demonstrated to be essential for flavivirus replication, both in vivo in Kunjin virus (
32) and in vitro in DEN (
1,
69). Furthermore, the 3′ SL of WNV RNA was reported to suppress translation of mRNA (
38). All of the above reports strongly suggest that the 3′-terminal region of the flavivirus genome plays an essential role in viral replication and possibly regulates translation. Our results show that deletion of the 3′-terminal 199 nt of the genome abolishes the infectivity of WNV RNA. These results agree well with previous reports that deletions in the 3′ UTR of DEN4 (
45) and many chimeric 3′ UTRs between DEN2 and WNV (
70) are lethal for viral replication. Since the 3′-terminal 199-nt deletion in the mutant RNA included the 3′ cyclization sequence (from nt 10925 to 10932) and the downstream 3′-terminal two-SL structure (from nt 10935 to 11029), it is important to dissect their individual roles during viral replication through systematic mutagenesis analysis.
Many different genetic determinants of virulence have been identified for the flaviviruses (
43). For WNV, the studies have been limited. Chambers and coworkers (
16) found that neuroinvasion correlates with a mutation in the E gene and determinants outside the E gene. For the related viruses of the JE serocomplex, determinants of neuroinvasion and neurovirulence are in the E gene (
12,
21,
22,
37,
44,
47,
65) and the NS1 gene (
20). For other flaviviruses, many of the putative virulence determinants are in the E gene (
23-
26,
29,
48,
58), but mutations in the NS1 gene (
10,
46,
51), NS5 gene (
66), and the 5′ and 3′ UTR (
10,
41) are also associated with virulence. Site-directed mutagenesis of the cDNA clone in this report will allow identification of molecular determinants of virulence for the epidemic strain of WNV.
Lineage I WNV strains have been mostly isolated from epidemic outbreaks and epzootics in birds and equines and have a worldwide distribution. In contrast, lineage II strains have been incidentally isolated from humans with mild febrile disease or without symptoms and are restrictedly found in sub-Saharan Africa and Madagascar (
4,
35,
36,
59). Based on sequence analysis of the complete genomes, nucleotide identity between the two lineages is approximately 75% (
35). Limited information is known about the pathogenic differences between lineages I and II and among strains within each lineage. Similar growth kinetics were observed for a lineage II Nigerian strain (
68) and our lineage I New York strain for both mosquito and mammalian cells. Replacement of the 3′-terminal 1,438 nt of the Nigerian strain (lineage II) with the equivalent sequence (including the complete 3′ UTR and sequence encoding the carboxy-terminal 287 amino acids of NS5) from the prototype WNV Eg101 strain (lineage I) yielded a chimeric virus that showed growth kinetics similar to those of the wild-type Nigerian strain (
68). Others have shown differences in neuroinvasiveness in mice between viruses from lineage I and II (D. W. C. Beasley, L. Li, M. T. Suderman, and A. D. Barrett, International Conference on the West Nile Virus, New York Academy of Science Poster Section
1:5, 2001). Lineage I strains can be further divided into three clades: clade 1a includes viruses from Africa, Europe, Russia, Middle East, and United States; clade 1b includes Kunjin virus from Australia; and clade 1c includes viruses from India (
35). Within clade 1a, all U.S. isolates (including the New York crow strain 3356 used in this study) have a nucleotide identity of 99.8%, 99.7% with an Israeli 1998 strain, and 95.2 to 96.4% with strains from Europe, Russia, and Egypt. Kunjin virus in clade 1b exhibits a nucleotide identity of 86.6 to 87.2% with strains in clade 1a (
35,
36). During the recent WNV outbreaks, bird mortality was observed in the United States and Israel but not in Europe; therefore, it was speculated that genetic variability within lineage I strains could affect pathogenicity (
35,
36,
63). Lanciotti et al. (
35) recently showed that six amino acid changes are consistent with the geographic origin of these viruses and might confer the pathogenic difference among these lineage I strains. It will be very interesting to use the infectious clone described here to experimentally test these observations.
Many factors could contribute to the fact that lineage I WNV strains are frequently involved in human outbreaks, while lineage II viruses are mostly maintained in enzootic cycles (
4,
36). In addition to possible differences in virulence, differences in vector competence and transmission cycles as well as host immunity may contribute to the difference in disease pattern between the two lineages. The infectious cDNA clones of WNV will serve as a valuable tool to address many of these questions.