Abstract
An attenuated Australian strain of West Nile virus (WNV), Kunjin (KUN), shares ~98% amino acid homology with the pathogenic New York 99 NY99 strain (NY99). To investigate the viral factors involved in NY99 virulence we generated an infectious cDNA clone of the WNV NY99 4132 isolate from which virus was recovered and was shown to be indistinguishable from the parental isolate. We then introduced the regions of the NY99 non-structural (NS) proteins and/or untranslated regions (UTRs) into the KUN backbone. Chimeric KUN viruses containing NY99 5′UTR and the parts of NS coding region were more virulent in mice than parental KUN virus. Chimeric NY99 viruses, containing KUN NS2A protein with alanine 30 to proline substitution were significantly less cytopathic in cells and less virulent in mice. Our results identify the 5′UTR and NS proteins as WNV virulence determinants and confirm a role for the NS2A in WNV cytopathicity and virulence.
Keywords: Flavivirus, West Nile virus, Kunjin virus, virulence factors, infectious clone, chimeric viruses
INTRODUCTION
West Nile virus (WNV) is a member of the Japanese encephalitis serogroup of flaviviruses which includes other medically important, neuro-invasive viruses, such as Japanese encephalitis virus (JEV), Murray Valley encephalitis (MVE), and St Louis encephalitis virus (SLEV) (Calisher et al., 1989). The virus is endemic throughout Africa, the Middle East, parts of Asia, and Europe, however since a 1999 outbreak in the USA, WNV has emerged as the most common cause of arboviral encephalitis in North and Middle America (Petersen, 2009). An unusually high percentage of neurological infections associated with WNV circulated in the Americas compared to the relatively low neuro-invasiveness of WNV strains previously circulated in the Old World initiated studies on identifying viral determinants of the high neuro-invasiveness of the American strain. Based on serological and genetic data, WNV strains have been grouped into two distinct lineages, lineage 1 and lineage 2 (Lanciotti et al., 2002). The highly neuro-invasive New York 99 strain (NY99) belongs to lineage 1 which also contains a relatively benign Australian strain Kunjin (KUN) (Lanciotti et al., 2002). KUN virus was first isolated in 1960 in North Queensland (Doherty et al., 1963) and since then has been found to be endemic in Australia (Hall et al., 2002). KUN causes mainly asymptomatic infection and was associated with only a handful number of cases of mild encephalitis and no death (Hall et al., 2002). Immunization of mice with KUN virus or plasmid DNAs encoding a full-length cDNA copy of the KUN genome provides highly effective protection against NY99 (Hall et al., 2003) and therefore KUN has been considered as potential vaccine candidate against NY99. Although both NY99 and KUN are lethal after peripheral injection in weanling (less than 21 day old) mice, the 50% lethal dose (LD50) for NY99 is substantially lower than for KUN. In contrast, only NY99 and not KUN is lethal after peripheral injection in adult (more than 21 day old) mice, thus providing a convenient model for identifying virulence determinants.
Studies comparing the neuro-invasiveness of NY99 with other WNV strains as well as with genetically engineered viral mutants in mice identified a number of virulence determinants residing in both structural and non-structural genes as well as in the 3′ untranslated region (UTR) (Beasley et al., 2004; Beasley et al., 2001, 2002; Beasley et al., 2005; Davis et al., 2007; Wicker et al., 2006; Zhang et al., 2006). Analysis of chimeric WNV viruses between NY99 strain and lineage 2 attenuated W956 strain showed that high cytopathicity and high virulence of NY99 strain was associated with determinants in the non-structural coding region (Borisevich et al., 2006). Previous studies with KUN demonstrated a role for NS2A in virus-induced cytopathicity in cells and virulence in mice and showed that a single point mutation from alanine to proline at position 30 (A30P) in NS2A significantly reduced virus-induced cytopathicity in cells and virulence in mice (Liu et al., 2004; Liu et al., 2006). Similar studies with NY99 virus also showed a key role for NS2A in virus-induced cytopathicity and virulence, however A30P mutation was not shown to have such a dramatic effect on NY99 viral properties as it had in KUN studies (Rossi et al., 2007) suggesting a potential contribution of other residues in NS2A different between NY99 and KUN viruses to the WNV-induced cytopathicity and virulence. Here we report the generation and characterization of infectious clone of NY99 isolate 4132 and of a number of chimeric viruses containing various parts of NY99 4132 genomes on KUN background and show that the majority of viral determinants responsible for high virulence of NY99 and low virulence of KUN reside in the 5′UTR and nonstructural coding region. In addition, by analyzing the properties of chimeric NY99 viruses containing wild type and A30P-mutated KUN NS2A we provide further evidence for the role of NS2A protein in WNV-induced cytopathicity and virulence.
RESULTS
Generation of two plasmid system for infectious RNA for NY99 bird isolate 4132 and characterization of recovered virus
In order to initiate studies on identifying virulence determinants between NY99 and KUN strains, a two plasmid system for the generation of an infectious RNA for NY99 4132 isolate was constructed. Viral RNA was reverse transcribed and fragments WN1/2, WN3/4, WN5/6, WN7/10 and WN8/9 spanning the entire genome were PCR amplified (Fig. 1A). PCR fragments were then cloned individually and assembled into two plasmids, pWNV1 covering nucleotides 1–3324 and 5713–11029 in pBR322 vector and pWN2 covering nucleotides 2710–5139 of NY99 4312 genome in pBluescript II KS+ vector.
Figure 1.
Construction of a WNV NY99 clone and recovery of infectious NY99 virus. (A) Schematic representation of the cloning strategy. NY99 viral RNA was reverse transcribed and the cDNA fragments were ligated to form pWN NY99 1 (fragments 1–2880 and 5781–11029nt) and pWN NY99 2 (fragment 2881–5780). To create infectious virus pWN NY99 1 and −2 were digested with BspEI and BsiWI, in vitro ligated, digested with SalI and in vitro transcribed to form full-length viral RNA. The RNA was then electroporated into BHK-21 cells, and virus was recovered. (B) Comparison of plaque morphology of BHK, vero, and A549 cells infected at an MOI of ~1 with wt NY99 and virus recovered from in vitro ligated pWN NY99. The cells were then overlaid with 0.75% LMT agarose in DMEM containing 2% FCS. At the indicated time, the cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet. (C) Growth kinetics of wt NY99 and cloned NY99 in A549 cells infected at an MOI of ~1. At the indicated time post-infection culture supernatants were collected and viral titres were determined by plaque assay on Vero cells. (E) Survival of adult (4–5-week-old) Swiss outbred mice infected with NY99-4132 isolate and virus recovered from NY99-4132 infectious clone. Groups of 10 mice were i.p. injected with 10-, 100- or 1000 pfu of each virus. The mice were monitored for 14 days post-infection for signs of encephalitis at which point the mice were sacrificed. All mouse experiments in this were conducted with approval from the University of Queensland Animal Experimentation Ethics Committee in accordance with the guidelines for animal experimentation as set out by the National Health and Medical Research Council, Australia.
Sequence differences between KUN and NY99 genomes
cDNA derived from RNA isolated from virus recovered from the NY99 4132 infectious clone and cDNA generated from viral RNA isolated from the initial stock of NY99 4312 virus were sequenced and shown to be identical (not shown). Sequence comparison between published NY99 prototype 382-99 isolate (Lanciotti et al., 2002) and the sequence of NY99 4132 virus recovered from infectious RNA generated from two plasmid system revealed 7 nucleotide differences with four of them leading to the amino acid changes including two non-conservative changes from C to R at position 168 of NS2A protein and from Q to R at position 110 of NS3 protein (Table 1). Sequence comparison between NY99 4312 and KUN strains revealed 88.6% homology at the nucleotide level and 97.8% homology at the amino acid level. A total of 77 amino acid substitutions with 34 of them being non-conservative were different between these two strains (Table 2). The structural genes accounted for 25 amino acid changes (13 non-conservative) while the remaining 52 changes (21 non-conservative) were located in the nonstructural region. Interestingly, amino acids at positions 168 in NS2A (C) and position 110 in NS3 (Q) in KUN were the same as in published NY99 382-99 sequence. In addition to changes in the coding region, 3 nucleotides were different in the 5′UTR (nts 50–52, AAC in NY99 4312 and TTG in KUN). There were also 52 nucleotide substitutions plus a 7- nucleotide insertion in the 3′UTR. None of these substitutions in the 3′UTR however, were located in the last 108 nucleotides containing highly conserved cyclization sequence and stem-loop structure.
Table 1.
Differences between the sequences of published NY99 382-99 isolate (AF404756) and NY99 4132 isolate (HQ596519)
| Nucleotide position | Gene | aa position in polyprotein and encoded protein | NY99(AF404756) | NY99 (HQ596519) | ||
|---|---|---|---|---|---|---|
| Aa code | Amino acid | aa code | Amino acid | |||
| 450 | prM | - | ATC | I | ATT | - |
| 2321 | E | 742 (452) | TTA | L | TCA | S |
| 4027 | NS2A | 1311(168) | TGC | C | CGC | R |
| 4195 | NS2A | 1367(224) | ACA | T | GCA | A |
| 4939 | NS3 | 1615(110) | CAG | Q | CGG | R |
| 7011 | NS4B | - | TTT | F | TTC | - |
| 10624 | 3′UTR | - | C | T | ||
Nucleotides in bold in the corresponding codons indicate different nucleotides
Sequence of NY99 4132 isolate have been deposited to GeneBank and obtained accession numbers HQ596519.
Table 2.
Amino acid differences between KUN FLSDX and NY99 4132 infectious cDNA clones
| Gene | aa position in polyprotein (corresponding protein) | KUN-FLSDX | NY99 4132 | |
|---|---|---|---|---|
| Amino acid | Amino acid | Number of substitutions in each gene (number of non-conservative changes) | ||
| C | 28 | T | I | 4 (2) |
| 41 | R | K | ||
| 44 | T | I | ||
| 71 | S | G | ||
|
| ||||
| prM | 113(8) | F | V | 7 (3) |
| 120(15) | G | S | ||
| 145(40) | I | V | ||
| 166(61) | H | Y | ||
| 195(90) | L | S | ||
| 228(123) | S | A | ||
| 279(174) | A | V | ||
|
| ||||
| E | 356(66) | E | D | 14 (8) |
| 379(89) | S | A | ||
| 382(92) | K | R | ||
| 416(126) | T | I | ||
| 446(156) | F | S | ||
| 449(159) | T | V | ||
| 452(162) | A | T | ||
| 489(199) | S | N | ||
| 519(229) | E | G | ||
| 521(231) | N | T | ||
| 600(310) | R | K | ||
| 628(338) | I | V | ||
| 655(365) | S | A | ||
| 700(410) | A | T | ||
|
| ||||
| NS1 | 879(88) | I | V | 9 (0) |
| 893(102) | R | K | ||
| 926(135) | I | V | ||
| 961(170) | R | K | ||
| 997(206) | F | L | ||
| 1027(236) | V | I | ||
| 1036(245) | I | V | ||
| 1055(264) | S | N | ||
| 1118(327) | N | S | ||
|
| ||||
| NS2A | 1255(112) | A | V | 6 (2) |
| 1262(119) | Y | H | ||
| 1272(129) | M | I | ||
| 1311(168) | C | R | ||
| 1355(212) | F | L | ||
| 1366(223) | V | I | ||
|
| ||||
| NS2B | 1438(64) | G | S | 2 (1) |
| 1477(103) | A | V | ||
|
| ||||
| NS3 | 1520(15) | R | K | 11 (6) |
| 1615(110) | Q | R | ||
| 1680(174) | V | I | ||
| 1754(249) | A | P | ||
| 1809(303) | R | K | ||
| 1836(331) | A | S | ||
| 1861(356) | I | T | ||
| 1889(383) | I | V | ||
| 1912(406) | V | I | ||
| 1991(486) | C | F | ||
| 2115(610) | S | A | ||
|
| ||||
| NS4A | 2129(5) | F | L | 5 (1) |
| 2209(85) | V | A | ||
| 2213(89) | A | V | ||
| 2265(141) | L | M | ||
| 2269(145) | G | S | ||
|
| ||||
| NS4B | 2288(15) | G | S | 7 (3) |
| 2296(23) | T | V | ||
| 2302(29) | I | M | ||
| 2387(114) | S | A | ||
| 2449(176) | V | I | ||
| 2459(186) | L | V | ||
| 2518(245) | V | I | ||
|
| ||||
| NS5 | 2553(25) | I | T | 12 (8) |
| 2561(33) | T | I | ||
| 2575(47) | R | G | ||
| 2577(48) | I | V | ||
| 2690(162) | L | I | ||
| 2705(177) | K | R | ||
| 2775(247) | K | R | ||
| 2840(567) | E | D | ||
| 3059(531) | R | K | ||
| 3181(653) | S | F | ||
| 3259(731) | T | V | ||
| 3405(877) | S | A | ||
|
| ||||
| Total | 77 (33) | |||
NY99 4132 isolate and virus derived from its infectious cDNA clone replicate with similar efficiencies in cells and mice
We compared the plaque morphology of virus recovered from the NY99 4132 cDNA clone with the parental isolate in Vero cells (Fig. 1B) and the virus growth kinetics in Vero and A549 cells (Fig. 1C). Both the plaque size and replication kinetics of the cloned NY99 4132 were virtually identical to the parental NY99 4132 isolate. We then compared the virulence of the wt NY99 4132 and the cloned NY99 4132 in 4–5-week old Swiss outbred mice (Fig 1D) and observed no difference in survival at 10-, 100-, or 1000 pfu (p= 0.9, 0.6 and 0.3, respectively). Thus the two plasmid system for recovery of infectious RNA from the NY99 4132 isolate produces virus indistinguishable from the original virus isolate and therefore is suitable for genetic manipulations in further studies.
5′UTR contributes to the higher virulence of NY99 4132 isolate
KUN infectious clone FLSDX was chosen for generating the first set of chimeric viruses to see whether the substitution of various regions in KUN genome with those derived from NY99 genome will provide an advantage in replication and increase in virulence. Given significant number of substitutions in the UTRs, we first generated KUN chimeric viruses containing either NY99 5′UTR, 3′UTR or 5′UTR and 3′UTRs together (Fig. 2A). Plaque assay of recovered viruses in BHK, Vero and A549 cells showed that neither 5′UTR or 3′UTR alone nor 5′UTR and 3′UTR together provided a noticeable advantage in viral spread, as the plaque sizes of chimeric viruses in all 3 cell lines were similar to those of parental KUN virus (Fig 2B). Multiple growth curve analysis of KUN-NY99 UTR chimeric viruses in Vero and C6/36 cells also showed little differences in the replication kinetics and virus titres between parental KUN and chimeric viruses containing NY99 UTRs (Fig 2C). Surprisingly, when the chimeric viruses were examined for virulence in weanling 19 day old Swiss outbred mice, the KUN-NY99 5′UTR chimeric virus was significantly more virulent than wt KUN virus at 10 pfu dose (Fig, 2D; p<0.01). In contrast, the virulence of KUN-NY99 3′UTR or KUN-NY99 5′UTR/3′UTR chimera was not significantly different from the wt KUN virus at the same dose (Fig 2D, p=0.8 and p=0.4, respectively). The results suggest a role for the 3 nucleotide difference in the 5′UTR in the higher virulence of WNV in mice.
Figure 2.
Charaterization of chimeric KUN-NY99 viruses containing NY99 UTRs in KUN genome. (A) Schematic representation of the chimeric construct showing KUN sequence in grey and NY99 sequence in white. The 5′ and 3′UTRs of NY99 (open boxes) were inserted into the KUN FLSDX clone instead of KUN UTRs (solid lines) to produce KUN-NY99 5′UTR, KUN-NY99 3′UTR, and KUN-NY99 5′UTR/3′UTR chimeric constructs. (B) Comparison of plaque morphology in BHK-21 (3d.p.i), Vero (4d.p.i.) and A549 cells (5d.p.i.) infected with the KUN-NY99 chimeric viruses. Cells in 6-well plates were infected at an MOI of 1 and overlaid with 0.75% LMT agarose in DMEM containing 2% FCS. At the indicated time, the cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet. (C) Growth kinetics of wt and KUN-99 UTR chimeric viruses in Vero and C6/36 cells infected at an MOI of ~1. Viral titres were determined by plaque assay on vero cells. Error bars represent standard deviation of duplicate samples (D) Survival of weanling (18–19 days old) Swiss-outbred mice inoculated i.p. with 10pfu of wt or chimeric virus. The mice were monitored for 14 days post-infection for signs of encephalitis at which point the mice where sacrificed.
Nonstructural coding region provides significant contribution to the virulence of NY99 4132 strain
To assess the contribution in virulence of the nonstructural genes we extended the regions for substitution first to the NS5-3′UTR region and then to the NS2A-3′UTR region (Fig 3A). Both chimeras produced larger plaques than parental KUN in BHK, Vero and A549 cells, with plaques in BHK and Vero having sizes similar to those produced by the NY99 4312 virus (Fig 3B). The plaques in A549 cells on average were smaller for chimeric viruses compared with NY99 4312 virus, although some of them, particularly for the KUN-99 5′/NS2A-3′UTR chimera, were very similar to NY99 4132 plaques (Fig 3B). Replication of KUN-99 NS5-3′UTR chimera in Vero cells was slightly more efficient than that of KUN but less efficient than that of NY99 4132 virus, while replication efficiency of KUN-99 5′/NS2A-3′UTR chimera was indistinguishable from NY99 4132 virus early in infection (24h p.i.) with some decline in efficiency compared to NY99 4132 virus observed at later time points (48 and 72h p.i.) (Fig. 3C). Both chimeras replicated with efficiency similar to KUN and lower than NY99 in C6/36 cells while their replication efficiency in A549 cells was intermediate between KUN and NY99 (Fig 3C). Although replication efficiency of KUN-99 NS5-3′UTR and KUN-99 5′/NS2A-3′UTR chimeras appeared to vary in different cells, the common trend was that these chimeras replicated with efficiencies which were intermediate between parental KUN and NY99 viruses.
Figure 3.
Characterization of chimeric KUN-NY99 viruses containing NY99 non-structural proteins in KUN genome. (A) Schematic representation of the chimeric constructs showing KUN sequences in grey and NY99 sequences in while. The NS5-3′UTR and the 5′UTR/NS2A-3′UTR regions of NY99 were inserted into KUN FLSDX clone to produce KUN-NY99 NS5-3′UTR and KUN-NY99 5′UTR/NS2A-3′UTR chimeric constructs. (B) Comparison of plaque morphology in BHK-21 (3d.p.i), vero (4d.p.i.) and A549 cells (5d.p.i.) infected with the KUN-NY99 chimeric viruses. (C) Growth kinetics of wt and KUN-99 chimeric viruses in Vero, C6/36 and A549 cells infected at an MOI of ~1. Viral titres were determined by plaque assay on vero cells. Error bars represent standard deviation of duplicate samples (D) Survival of weanling (18–19 days old) mice inoculated i.p. with 10pfu and adult (4-week old) mice i.p. inoculated with 1000pfu of wt or chimeric virus. The mice were monitored for 14 days post-infection for signs of encephalitis at which point the mice where sacrificed.
We then examined virulence of these chimeric viruses after intraperitoneal injection with 10pfu virus in weanling (19 day old) Swiss outbred mice. KUN-99 5′/NS2A-3′UTR and KUN-99 NS5-3′UTR were clearly more virulent than parental KUN (p<0.001 and p<0.05, respectively) (Fig 3D). The average survival time for both chimeras was shorter than for KUN, and LD50 for KUN-99 5′/NS2A-3′UTR chimera was approaching that of NY99 (0.6 pfu and 0.4 pfu, respectively, Table 3).
Table 3.
Virulence of chimeric KUN-NY99 viruses in 18–19 day old Swiss outbred mice after i.p. injection
| Virus | Dose (pfua) | Mortality (%) | A.S.T.b (Days) | LD50c |
|---|---|---|---|---|
| NY99 | 100 | 10/10 (100%) | 5.5 | 0.4 pfu |
| 10 | 10/10 (100%) | 5.8 | ||
| 1 | 9/10 (90%) | 6.5 | ||
| 0.1 | 0/10 (0%) | 14 | ||
|
| ||||
| KUN | 1000 | 5/5 (100%) | 7.8 | 17.8 pfu |
| 100 | 4/5 (80%) | 8.8 | ||
| 10 | 2/5 (40%) | 8.5 | ||
| 1 | 2/5 (40%) | 9 | ||
|
| ||||
| KUN-NY99 NS5-3′UTR | 1000 | 6/6 (100%) | 5.7 | 2.7 pfu |
| 100 | 10/10 (100%) | 6.3 | ||
| 10 | 9/10 (90%) | 7.2 | ||
| 1 | 2/10 (20%) | 9 | ||
| 0.1 | 0/10 (0%) | 14 | ||
|
| ||||
| KUN-NY99 5′/NS2A-3′UTR | 1000 | 6/6 (100%) | 5.5 | 0.6 pfu |
| 100 | 10/10 (100%) | 5.9 | ||
| 10 | 10/10 (100%) | 6.5 | ||
| 1 | 6/10 (60%) | 6.8 | ||
| 0.1 | 2/10 (20%) | 7 | ||
pfu – plaque forming unit
A.S.T. – Average survival time
LD50 – Lethal dosage (50%)
We then examined virulence of chimeras in adult (4 week old) Swiss outbred mice. Our previous experiments showed that intraperitoneal infection with 1000 pfu of NY99 4132 virus resulted in 80–90% mortality, while the same dose of KUN virus did not kill any mice (Hall et al., 2003). Thus 1000 pfu was used in this experiment. KUN-99 5′/NS2A-3′UTR chimera killed 40% of mice by day 11, while KUN-99 NS5-3′UTR killed 20% of mice by day 11 with 10% more mice dying at day 14, which was significantly less than NY99 (p<0.01 and p<0.05, respectively) (Fig. 3D). As expected, none of the mice died after KUN infection, and 90% mice died by day 12 after NY99 infection (Fig. 3D). The results in adult mice correlated with the results in weanling mice in terms of increased virulence of the chimeras compared to KUN, however, even KUN-99 5′/NS2A-3′UTR chimera was still significantly less virulent than NY99 virus in adult mice.
KUN NS2A in combination with A30P substitution significantly decrease cytopathicity and virulence of NY99 virus
Our previous studies with NS2A A30P mutant of KUN virus identified NS2A as one of the major determinants of viral cytopathicity in cells and virulence in mice (Liu et al., 2004; Liu et al., 2006). The increased virulence of NS2A-3′UTR chimera over NS5-3′UTR chimera (Fig. 3) also indicated a potential role for NY99 NS2A together with other NS proteins in virus virulence. Other studies with WNV also showed an important role for NS2A in virus-induced cytopathicity and virulence (Rossi et al., 2007). Therefore it was logical to assume that NS2A is likely to provide a certain contribution to the difference in virulence between KUN and NY99 4312 viruses. To test this hypothesis, we generated chimeric virus NY99-KUN NS2A containing KUN NS2A on the background of NY99 4132 genome (Fig. 4A). However, plaque assays in BHK, Vero and A549 cells showed that this chimera was indistinguishable from the parental NY99 4312 virus (Fig. 4B), and replicated with similar efficiency and was as cytopathic as parental NY99 4132 virus in A549 cells (Fig. 4C). However, a statistically significant attenuation of NY99-KUN NS2A virulence was observed in 4-week old mice compared to that of NY99 4132 virus (p<0.001, for all doses, Fig. 4D, Table 4) suggesting that NS2A is indeed contributing to the difference in virulence between KUN and NY99 4132 viruses.
Figure 4.
Characterisation of NY99-KUN chimeras viruses containing wt and A30P-mutaed KUN NS2A gene in NY99 backbone. (A) Schematic representation of the chimeric viruses showing NY99 sequence in white and KUN sequence in grey. The KUN wt and A30P-mutated NS2A genes were inserted were inserted into the backbone of NY99 4132 infectious clone to produce NY99-KUN/NS2A and NY99-KUN NS2A/A30P chimeric constructs. In addition a NY99-NS2A/A30P virus containing A30P mutation in the NY99 NS2A was generated. (B) Comparison of plaque morphology in, Vero (6d.p.i.) and A549 cells (4d.p.i.) infected with the NY99-NS2A mutant viruses. (C) Growth kinetics and cytopathicity of NY99-NS2A mutant viruses in A549 cells infected at an MOI of ~1. Viral titres were determined by plaque assay on Vero cells. A549 cells in 96 well plate were fixed 5d.p.i with 4% formaldehyde and stained with 0.2% crystal violet. Methanol was added to each well and the amount of crystal violet released was determined at OD620nm (shown as percentage CPE where control represents 0% CPE). The error bars indicate the standard deviation of the duplicate wells. Error bars represent standard deviation of duplicate samples (D) Survival of adult (4-week-old) Swiss outbred mice infected with wt NY99 and chimeric/mutant viruses. Groups of 10 mice were i.p. injected with 10-, 100- or 1000 pfu of virus. The mice were monitored for 14 days post-infection for signs of encephalitis at which point the mice were sacrificed.
Table 4.
Virulence of chimeric NY99-KUN viruses in 4-week old Swiss outbred mice after i.p. injection
| Virus | Dose (pfua) | Mortality | A.S.T.b (Days) | LD50 |
|---|---|---|---|---|
| NY99 | 1000 | 10/10 (100%) | 5.6 | <1 pfu |
| 100 | 10/10 (100%) | 5.6 | ||
| 10 | 10/10 (100%) | 6.2 | ||
| 1 | 7/10 (70%) | 8.6 | ||
|
| ||||
| NY99/NS2A/A30P | 100 000 | 5/5 (100%) | 6.4 | 21.4 pfu |
| 10 000 | 9/10 (90%) | 7.3 | ||
| 1000 | 8/10 (80%) | 8.0 | ||
| 100 | 7/10 (70%) | 9.7 | ||
| 10 | 4/10 (40%) | 11.2 | ||
|
| ||||
| NY99-KUN/NS2A | 100 000 | 9/10 (90%) | 7.9 | <10pfu |
| 10 000 | 6/10 (60%) | 7.3 | ||
| 1000 | 8/10 (80%) | 8.5 | ||
| 100 | 7/10 (70%) | 8.6 | ||
| 10 | 6/9 (66.7%) | 10.2 | ||
|
| ||||
| NY99-KUN/NS2A/A30P | 100 000 | 4/5 (80%) | 7.6 | 1000 pfu |
| 10 000 | 6/10 (60%) | 10.1 | ||
| 1000 | 5/10 (50%) | 11.5 | ||
| 100 | 4/10 (40%) | 11.8 | ||
| 10 | 3/10 (30%) | 12.1 | ||
pfu – plaque forming unit
A.S.T. – Average survival time
LD50 – Lethal dosage (50%)
To further clarify a role in WNV virulence for the Ala at position 30 in NS2A, which has been previously shown to significantly attenuate KUN virus (Liu et al., 2006), we generated two NY99 viruses, one with Ala 30 to Pro substitution in NY99 NS2A (NY99-NS2A/A30P), and another one in which Ala 30 to Pro substitution was introduced on the background of KUN NS2A in the NY99-KUN NS2A chimeric virus (NY99-KUN NS2A/A30P) (Fig. 4A). Both A30P-mutant viruses produced smaller plaques than parental NY99 4132 or chimeric NY99-KUN NS2A viruses in Vero and A549 cells (Fig. 4B). NY99-KUN NS2A/A30P virus also replicated relatively less efficiently and was significantly less cytopathic than NY99 4132 in A549 cells (Fig 4C). Although the replication efficiency of NY99-NS2A/A30P was similar to that of parental NY99 virus, the mutant virus was less cytopathic in A549 cells (Fig 4C). NY99/NS2A/A30P and NY99-KUN/NS2A/A30P were then examined for virulence in adult 4 week old Swiss outbred mice after intraperitoneal infection. Both A30P-mutant viruses were attenuated compared to the parental NY99 4132 virus (NY99/NS2A/A30P: 10pfu p<0.05, 100pfu p<0.001, 1000pfu p<0.01, NY99-KUN/NS2A/A30P p<0.0001 for all doses) with NY99-KUN NS2A/A30P virus showing >1000-fold increase in LD50 value (Fig. 4D, Table 4). The LD50 of NY99/NS2A/A30P mutant was ~20–40-fold higher that of wt NY99 4132 virus (Table 4). Thus, A30P mutation alone in the NY99 NS2A lead to noticeable decrease in cytopathicity and virulence, while combination of A30P with other substitutions present in the KUN NS2A lead to a significant decrease in the cytopathicity and virulence of NY99 virus. As KUN NS2A differs from NY99 4132 NS2A by 6 amino acids (Table 2) it is not clear at this stage which of these 6 amino acids act in accord with A30P in making the NY99-KUN/NS2A/A30P virus significantly less cytopathic and less virulent.
DISCUSSION
Despite belonging to the same lineage I of West Nile viruses, the circulating in North America NY99 strain and circulating in Australia KUN strain of WNV differ greatly in their virulence in mice and their ability to cause severe encephalitis in birds, horses and humans. Although a number of studies identified genetic determinants of virulence in the various parts of genome in pathogenic WNV strains (Beasley et al., 2002; Beasley et al., 2005; Davis et al., 2007; Wicker et al., 2006; Zhang et al., 2006), the viral determinants of the less pathogenic KUN strain compared to the NY99 strain have not been identified. In order to identify these determinants we first constructed a two plasmid system for generation of an infectious RNA of the NY99 4132 isolate and showed that recovered virus has properties identical to those of the original isolate (Fig 2). Previously we also generated an infectious clone of KUN strain (Khromykh et al., 1998; Khromykh and Westaway, 1994) which was used as a backbone for creating KUN-NY99 chimeras. We have found six nucleotide changes in the coding region and one in the 3′UTR of NY99 4132, compared with the published sequence of NY99 382-99, of which four result in amino acid changes. In particular, the Cys to Arg substitution in NS2A and Gln to Arg in NS3 may be of some interest due to non-conservative nature of these substitutions. Despite these differences, virus recovered from infectious RNA for NY99-4132 isolate showed similar virulence in mice to previously published data for other NY99 isolates (Beasley et al., 2001). The appearance of these substitutions as the result of two passages in C6/36 and two passages in Vero cells prior to isolating viral RNA used for generation of cDNA fragments can also not be excluded. Further detailed comparison of NY99-4132 isolate with other previously and currently circulating North American WNV isolates in mosquito, bird, horse and mouse models may prove to be useful to determine if any differences in virulence exists between them.
To initiate studies on identifying virulence determinants between NY99 and KUN strains, we first swapped the UTR regions in KUN virus with those of NY99 strain. Surprisingly, the three nucleotide differences in the NY99 5′UTR resulted in a significantly more pathogenic virus compared with KUN, despite similar replication kinetics and cell spread in mammalian and mosquito cell lines (Fig 2). M-fold predictions of the NY99 and KUN 5′UTR structures show little difference in structure stability, suggesting the AAC nucleotides present in NY99 may enhance the binding of viral and/or unknown host proteins which promote viral pathogenicity. Alignment of the 5′UTR of complete WNV genomes available on Flavitrack (http://carnot.utmb.edu/flavitrack/) show that KUN is the only sequence with TTG at nucleotides 50–52. The vast majority of sequences contain AAC at nucleotides 50–52, suggesting that the TTG sequence in KUN contributes to its attenuation, although other viral factors must also be involved since other attenuated WNV isolates (such as B959 (Yamshchikov et al., 2004)) do contain the AAC sequence at nucleotides 50–52. Surprisingly, the introduction of both the NY99 5′UTR and 3′UTRs together into KUN did not lead to increased virulence compared to KUN in weanling mice (Fig 3). Comparison of NY99 with an attenuated American strain (Bird 1153) found that mutations within NS4B, NS5 and 3′UTR were responsible for smaller plaque sizes, temperature sensitivity and virulence in mice (Davis et al., 2007). The temperature-sensitive phenotype was the result of the 4 nucleotide changes in the 3′UTR, of which, KUN encodes one of these nucleotide changes (T10767 matching T10774 from Bird 1153). However, no difference in virulence in mice was detected between NY99 and the 3′UTR only mutant (Davis et al., 2007) which is also the case for KUN-NY99/3′UTR chimeric virus. This suggests that any increase in virulence attributed to AAC in the 5′UTR is eliminated by the differences in the 3′UTR when both of them are replaced. One potential explanation could be that long range interactions between homologous and heterologous 5′UTRs and 3′UTRs may alter the overall RNA structure leading to different exposure of AAC/TTG region for interactions with viral and/or host proteins.
To investigate the influence of the non-structural proteins in the virulence of NY99, we created chimeras KUN-99 5′/NS2A-3′UTR and KUN-99 NS5-3′UTR (Fig 1B). Both chimeras showed intermediate replication, cell spread and virulence compared with NY99 and KUN (Fig 4), suggesting that the non-structural proteins are involved in the increased pathogenicity of NY99. Moreover, the NS5 protein appears to play a key role the increased virulence of chimeras as the differences in mouse survival between KUN-99 5′/NS2A-3′UTR and KUN-99 NS5-3′UTR were not significant at any dosage in weanling or adult mice. Given that 8 of the 12 amino acid differences between NY99 and KUN are non-conserved (Table 2), it is reasonable to assume that NS5 provides a large contribution to the increased virulence of NY99. This is in agreement with our recent findings showing higher resistance of NY99 NS5 to anti-viral activity of IFN compared to that of KUN virus (Laurent-Rolle et al., 2010). However, the presence of other NY99 non-structural proteins (NS2A-NS4B) in KUN-99 5′/NS2A-3′UTR chimera was also beneficial to the increased virulence in weanling mice as the LD50 of this chimera was nearly the same as wt NY99 virus (0.6 and 0.4 pfu, respectively, Table 3), while the LD50 of KUN-99 NS5-3′UTR chimera was somewhat higher (2.7pfu).
The individual contribution of NS2A to the WNV virulence was evaluated by creating chimeras containing KUN NS2A and its A30P mutant (shown previously for KUN virus to significantly decrease cytopathicity in cells and virulence in mice) on the background of NY99 4132 virus. A dramatic decrease in cytopathicity and significant (>1000-fold) attenuation in mice of the NY99-KUN/NS2A/A30P chimera thus confirms our previous observations with KUN virus on the crucial role for the NS2A in WNV-induced cell cytopathicity (Liu et al., 2004; Liu et al., 2006). Surprisingly, chimeric NY99 virus containing wt KUN NS2A was as cytopathic in cells and only mildly attenuated in mice indicating that NS2A alone is likely to provide only minor contribution to the higher virulence of NY99 strain. Interestingly, introducing A30P mutation alone in the NY99 background lead only to moderate decrease in cytopathicity in cells and only ~20-fold attenuation in adult mice, similar to the previously published results by others (Rossi et al., 2007). However when this mutation was combined with 6 other amino acid substitutions present in KUN NS2A, the viral attenuation was much more pronounced. It would therefore be interesting to investigate which of these 6 substitutions provide such a profound cumulative effect with A30P mutation. This may provide further insight into the structure and functions of this multifunctional protein and its role in viral pathogenesis.
The results determine the differences in the genome sequence between a naturally attenuated Australian WNV KUN and highly pathogenic WNV NY99-4132 isolate and identify the virulence determinants between these two viruses in the 5′UTR and the non-structural proteins, in particularly NS5. In addition, the results further confirm a role for the NS2A protein in WNV-induced cytopathicity and pathogenicity.
MATERIALS AND METHODS
Cells and viruses
BHK cells were maintained in Dulbecco’s modification of minimum essential medium (DMEM; Invitrogen) supplemented with 5% FBS.Vero cells were maintained in DMEM containing 10% FBS. A549 cells were maintained in DMEM/F12 medium (Invitrogen) containing 10% FBS. BHK, Vero and A549 cells were grown at 37°C in a CO2 incubator. C6/36 cells were maintained in RPMI (Invitrogen) containing 10% FBS and grown at 28°C.
NY99-4132 isolate of West Nile virus was originally isolated on 27 August 1999 from infected American crow in Queens, New York, passaged once in CrowVIC6 and once in C6/36 cells followed by two passages in Vero cells and one passage in C6/36 cells to generate virus used for RNA isolation.
Construction of two plasmid system for generation of infectious WNV NY99 RNA
WNV NY99 4232 virus from culture fluid of infected cells was concentrated using Centricon column (Millipore) and viral RNA was isolated using Nucleospin RNA virus isolation kit (Macherey-Nagel, Dueren, Germany). Primers for RT-PCR of viral RNA were designed based on published WNV NY99 sequence (GeneBank ID number AF260967). The RNA was reverse transcribed with AMV reverse transcriptase (Promega) using the primers WN2, WN4, WN6, WN8, and WN10 (Table 1). The resultant cDNA was amplified using PfuUltra (Invitrogen) and the primer pairs WN1 and WN2 (WN1/2, 3124bp; Fig 1A), WN3 and WN4 (WN3/4, 3209 bp), WN5 and WN6 (WN5/6, 2239bp), WN7 and WN10 (WN7/10, 2900bp), and WN8 and WN9 (WN8/9, 696bp). The cDNA fragments were sequenced to determine a consensus sequence, and then cloned into EcoRV-digested pBluescript II KS+ vector (Clontech) (referred to as pWN1/2, pWN3/4, pWN5/6, pWN7/10, pWN8/9). pWN1 was created by assembling WN1/2, WN5/6, WN7/10 and WN8/9 fragments in pBR322 vector (Fig 1A). Quikchange PCR was performed on pWN3/4 using the primer BspEImut. This removed a BspEI restriction site and introduced a marker mutation resulting in a silent change in NS2A gene. This plasmid was renamed as pWN2 (Fig 1A). All the plasmids were grown in DH5α E.coli, and all but pWN1 plasmid were grown at 37°C. pWN1 plasmid was not stable at 37°C and was grown at 22°C for 2–3 days.
Cloning strategy for KUN-NY99 chimaeras
KUN-NY99 5′UTR
To create KUN-NY99 5′UTR, a smaller cloning vector of KUN FLSDX (Khromykh et al., 1999) was created by digestion with XmaI (NEB) and extracting the band corresponding to 5136bp. This fragment was self-ligated with T4 ligase (NEB) overnight at 4°C. Quikchange PCR was performed on this cloning vector to mutate 3 nucleotides different between KUN and NY99 using the primers KUN-NY99 5′UTR-F and KUN-NY99 5′UTR-R (Table 1). The resultant KUN-NY99 5′UTR cloning vector was linearlised with XmaI and ligated with the 9978bp fragment of XmaI-digested FLSDX, creating a full-length KUN-NY99 5′UTR plasmid (Fig 2A).
KUN-NY99 5′UTR/3′UTR
The 3′UTR region of pWN1 was amplified using KUN-NY99 3′UTR-F and KUN-NY99 3′UTR-R primers, digested with XmaI and XhoI and cloned into the smaller (XmaI-deleted) KUN-NY99 5′UTR plasmid digested with XmaI and XhoI. This also introduced a mutation into the FLSDX sequence of NS5 amino acid 877. Quikchange PCR was then performed using the primers NY99 NS5 877-F and NY99 NS5 877-R to convert this mutation back to the wildtype FLSDX sequence at NS5 amino acid 877. This smaller cloning plasmid of KUN-NY99 5′UTR/3′UTR was linearised with XmaI and ligated with the 9978bp fragment of XmaI digested FLSDX, creating a full-length KUN-NY99 5′UTR/3′UTR plasmid (Fig 2A).
KUN-NY99 3′UTR
KUN-NY99 5′UTR/3′UTR was digested with AgeI and XhoI. The bp fragment was ligated into AgeI-XhoI digested FLSDX to create KUN-NY99 3′UTR.
KUN-NY99 NS5-3′UTR
AgeI –NdeI fragment from pWN1 containing most of the NY99 NS5, complete 3′UTR and part of pBR322 vector was cloned into AgeI and NdeI digested FLSDX to produce KUN-NY99 NS5-3′UTR plasmid (Fig 3A).
KUN-NY99 5′UTR/NS2A-3′UTR
A full-length cloning vector of NY99 was constructed by cloning BspEI-BsiWI fragment from pWN2 plasmid into pWN1 vector. This full length clone pFL-NY99 was found to have a single nucleotide deletion in NS1 leading to a frameshift and was therefore not used for producing infectious virus. However, pFL-NY99 plasmid was used to generate KUN-NY99 5′UTR/NS2A-3′UTR chimeric clone. SphI-NdeI fragment from pFL-NY99 containing NS2A-3′UTR region of NY99 genome and a part of pBR322 vector was cloned into FLSDX digested with SphI and NdeI to produce intermediate chimeric plasmid with deletion of SpHI-SpHI fragment containing most of the NS1 gene. SphI-SphI fragment from FLSDX containing NS1 gene was then cloned into the resultant intermediate plasmid to obtain final KUN-NY99 5′UTR/NS2A-3′UTR plasmid (Fig 3A).
All chimeric plasmids were confirmed by DNA sequencing, propagated overnight at 37°C and extracted from DH5α E. coli.
Construction of NY99-KUN NS2A chimeras
NY99-NS2A/A30P
To introduce the NS2A A30P mutation into pWN2, Quikchange PCR was performed using the primers WNA30P-F and WNA30P-R. The mutated pWN2 was then used to generate NY99-NS2A/A30P mutant virus (Fig 4A) in conjunction with pWN1.
NY99-KUN/NS2A and NY99-KUN/NS2A/A30P
These plasmids (Fig 4A) were generated by cloning BsmI-PshAI fragments containing KUN wt and A30P-mutated NS2A genes from FLSDX and FLSDX-NS2A/A30P, respectively, into pWN2 vector digested with BsmI and PshAI.
All plasmids were confirmed by DNA sequencing and propagated at 22°C for 3 days.
Generation of NY99 and NY99-KUN NS2A chimeric viruses from 2-plasmid system
pWN1 was digested with BspEI for 2h at 37°C followed by BsiWI for 2h at 55°C. The band corresponding to 10926bp was extracted from the gel using Wizard SV gel cleanup kit (Promega). pWN2, pWN2-NS2A/A30P, pWN2-KUN/NS2A and pWN2-KUN/NS2A/A30P were digested with BspEI and BglI (for easier separation of fragments in the gel) for 2h at 37°C followed by BsiWI for 2h at 55°C. The band corresponding to 2900bp was extracted as above. The isolated fragments were ligated overnight at 16°C using T4 DNA Ligase (NEB). The ligated DNAs were then linearized with SalI located 20 nucleotides downstream of the 3′-termini of NY99 genome (Fig 1A) and purified by phenol:chloroform extraction. The linearised DNA was then used for in vitro transcription with T7 RNA polymerase. Approximately 10μg of RNA was electroporated into 2×106 BHK21 cells suspended in 400μl of cold PBS using a 0.2cm electrode gap cuvette (Bio-Rad) and a Bio-Rad gene pulser II apparatus. The cells were incubated at 37°C for 48–96h until cytopathic effect (CPE) was evident. Culture supernatants were harvested and the virus titres were determined by plaque assay on BHK21 cells.
In vitro transcription and electroporation of KUN-NY99 chimeric viruses
FLSDX, KUN-NY99 5′UTR, KUN-NY99 3′UTR, and KUN-NY99 5′UTR/3′UTR were digested overnight with XhoI at 37°C. KUN-NY99 NS5-3′UTR and KUN-NY99 5′UTR/NS2A-3′UTR were digested overnight with SalI at 37°C. The DNA was purified by phenol:chloroform extraction and approximately 1μg of DNA was used for in vitro transcription with Sp6 RNA polymerase (Roche). Approximately 10μg of RNA was electroporated into BHK21 cells as above.
Plaque assay
BHK, Vero and A549 cells were seeded into 6-well plates and infected with wt or mutant viruses for 2h at 37°C. The cells were overlaid with 0.75% LMT agarose in DMEM containing 2% FBS. At the indicated times post-infection, the cells were fixed with 4% formaldehyde and stained with 0.2% crystal violet.
Viral growth kinetics
Vero, A549 and/or C6/36 cells were seeded into 6-well plates and infected with wt or chimeric virus at an MOI of ~1 in DMEM (Vero), DMEM:F12 (A549) or RPMI (C6/36) supplemented with 2% FBS. The cells were incubated at 37°C (Vero/A549) or 28°C (C6/36) and culture supernatants were harvested at the indicated times post-infection. Viral titres were determined using standard plaque assay on Vero cells.
Virulence in mice
Groups of 5–10 weanling (18–19 days old) or adult (4-week old) Swiss outbred mice were infected intraperitoneally (i.p.) with 100μl of 10-fold serial dilutions of wt or chimeric viruses and monitored for 14 days post-infection for the signs of paralysis or encephalitis. The lethal dose (50% - LD50) was calculated for each virus by the method of Reed and Muench (Reed and Muench, 1938). The experiments were conducted with approval from the University of Queensland Experimentation Ethics Committee in accordance with the guidelines for animal experimentation as set by the National Health and Medical Research Council, Australia.
Acknowledgments
We thank Nick Komar and Robert Lanciotti for providing NY99-4132 isolate. We thank Anneke Funk for helpful discussions and Ruth Lee, Karen Shiels and Rebecca West for assistance with the animal experiments.
This work was supported by the grants to A.A.K. from the National Health and Medical Research Council of Australia and the U.S. National Institutes of Health
Footnotes
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