In order to understand the phylogenetic status of XJ11129, the complete sequence information of 28 WNV strains isolated at different times and locations was analyzed to construct a phylogenetic tree. These WNV strains are classified into eight major lineages. XJ11129 belongs to lineage 1 and is further clustered into clade 1a, which clusters most closely with viruses isolated from Russia and India, followed by strains isolated from the US (NY99) and Israel (Fig. 1A).
Figure 1. The phylogeny and genetic diversity of the West Nile virus lineages. A Phylogenetic tree of XJ11129 strains compared with reference strains. Each strain is listed by GenBank accession number, geographic origin, and collection date. Scale bar indicates substitutions per site. Abbreviations of geographic origin are list in Supplementary Table S1. XJ11129 was highlighted with a red solid circle and NY99 was highlighted with a blue solid box. B Pairwise percent identity between nucleotide (lower left) and amino acid (upper right) sequences of the polyprotein. Sequences are labeled in the following format: accession number, 2-letter country code, and year of isolation. C Amino acid differences between XJ11129 and NY99. A total of 23 amino acids were different between these two strains. The numeral subscript after amino acid is the location of each gene.
Further, the genetic identity of XJ11129 was compared at the gene and protein levels with that of strains representing 8 lineages. Since both XJ11129 and NY99 belong to lineage 1 and clade 1a, XJ11129 exhibited the highest nucleotide identity (97.1%) and amino acid identity (99.4%) with NY99. In addition, XJ11129 shared 75.3% –87.5% nucleotide similarity and 88.5% –97.3% amino acid similarity with the strains from the other lineages (Fig. 1B).
Next, the amino acid differences between XJ11129 and NY99 were compared. A total of 23 amino acids differed between the two strains (Fig. 1C). Six amino acid differences were located in the E protein: Y90F, T126I, T159V, F167L, S277N, and S467A. NS1, NS2A, and NS5 each contained three amino acid differences. NS3 and NS4A each contained two amino acid differences. C, M, NS2B and NS4B each had one amino acid difference.
The above results show that XJ11129 is classified in clade 1a of lineage 1 and showed high sequence identity with the pathogenic strain NY99. However, whether XJ11129 exhibits a high virulence level similar to that of NY99 needs further experimental verification.
Infectious clones are powerful tools for flavivirus study. Here, a modified GA method was employed to rapidly rescue XJ11129. The viral genome was divided into three fragments, as shown in Fig. 2A. The vector fragment contained the CMV promoter, the hepatitis delta virus ribozyme sequence and the SV40 poly (A) signal and was used for the GA reaction, which ensured the transcription of XJ11129 in eukaryotic cells. All four fragments were obtained by PCR amplification and used for the GA reaction. Although no expected DNA bands were seen, the amounts of the four DNA fragments were significantly reduced, and large quantities of the DNA samples remained in the wells of the gel, confirming the occurrence of the GA reaction (Fig. 2B, 2C). To rescue the virus, the GA products were transfected directly into BHK-21 cells instead of into bacteria. Seventy-two hours post transfection, obvious CPEs were observed in the cells of the GA group, while no CPEs were observed in the cells of the control group (Fig. 3A). The plaque morphology of rXJ11129 was examined in BHK-2 cells. rXJ11129 formed plaques with an average size similar to that of the parental virus (Fig. 3B). The IFA results showed that the rescued virus could effectively infect BHK-21 cells and successfully express virus-specific proteins (Fig. 3C). A sequencing assay was conducted and showed that only one silent mutation emerged at position 4968 in NS3 (T to C) (Fig. 3D). These results indicate that GA is a rescue strategy for rapid acquisition of viruses and that the rXJ11129 clone rescued by the GA method has high genetic similarity to the parental viruses.
Figure 2. Strategy for rapid rescue of XJ11129 by GA. A Full-length preparation strategy of the XJ11129 genome by RT-PCR with overlapping primers. Full length of XJ11129 genome was divided into three fragments, named F1, F2 and F3. B PCR-amplified fragments. M: DNA Ladder maker DL10000 (TAKARA). V: vector fragment, which used pCMV/EV71 plasmid as the template and contained the CMV promoter, the hepatitis delta virus ribozyme sequence followed by the SV40 polyA signal. Length of vector fragment is 3510 bp. F1, F2, F3 are the WNV genome fragments. Length of the three fragments is 4031 bp, 4329 bp and 2719 bp, respectively. C GA reaction of the four fragments. M: DNA Ladder maker DL10000 (TAKARA). Lane 1, 2: GA reaction of the four fragments; Lane 3: Mixture of the four fragments without GA reaction.
Figure 3. Rescue and identification of XJ11129 in vitro. A Visible cytopathic effect (CPE) of BHK-21 cells in 2–3 days after infection with recued virus. B Plaque morphology of rescued XJ11129 and the parental XJ11129. Plaques were observed 3 days post-infection after staining with 0.2% crystal violet. C The GA products were inoculated to BHK-21 cells for incubation of 96 h. The infected cells were observed with a fluorescence microscope. D Differences between the sequences of rXJ11129 with its parental virus XJ11129. The numeral subscript after nucleotide is the location of whole viral genome. E Growth kinetics of rXJ11129. The experiments were performed with BHK-21 cells, Vero cells, C6/36 cells and DF-1 cells. Viral titres were determined on BHK-21 cells by plaque assay at the indicated times. The dotted lines represent the limit of sensitivity of the plaque assay. The error bars indicate the range in values of two independent experiments.
WNV has a wide range of hosts, including mosquitoes, birds, mammals and so on. To verify whether XJ11129 can infect these hosts, the growth kinetics of rXJ11129 were assessed in four cell lines: mouse cells (BHK-21), avian cells (DF-1), primate cells (Vero) and mosquito cells (C6/36). rXJ11129 exhibited efficient replication ability in all four cell lines. In BHK-21, Vero and C6/36 cells, the rXJ11129 titer peaked at 72 h, at up to 105.81, 106.39, and 105.42 PFU/mL, respectively. However, in DF-1 cells, rXJ11129 replicated to a peak titer of 106.49 PFU/mL at 48 h post infection (Fig. 3E). Taken together, these results clearly show that rXJ11129 can replicate in the above four types of cells and demonstrates the highest replication efficiency in avian cells (DF-1).
XJ11129 and NY99 belong to clade la of lineage 1. To characterize the virulence of XJ11129, a virulence assay was conducted in a mouse model, and NY99 was used as the control strain.
To determine the neuroinvasiveness of rXJ11129, groups of 6-week-old BALB/c mice were inoculated intraperitoneally with the corresponding dose of rXJ11129 or NY99. As shown in Table 1, 105 PFU of rXJ11129 resulted in 100% mortality in mice, with an average survival time (AST) of 10.6 days. However, inoculation of 105 PFU of NY99 resulted in 80% mortality, with an AST of 9.75 days. Neither morbidity nor mortality was observed in mice following inoculation with 103 PFU of rXJ11129 or NY99. The LD50 of rXJ11129 was slightly higher than that of NY99 (6310 PFU vs 5623 PFU), but the difference between the two viruses was not statistically significant.
Virus Dose (log10 PFU) Mortality (No. survival/No. text) ASTa (Day) LD50 (PFU) rXJ11129 5 0(0/5) 10.6 6310 4 20 (1/5) 10.75 3 100(5/5) - NY99 5 20 (1/5) 9.75 5623 4 20 (1/5) 11 3 100(5/5) - AST average survival time, - not available.
Table 1. Comparison of rXJ11129 with NY99 in nerveinvasiveness.
To characterize the neurovirulence of rXJ11129, groups of 6-week-old BALB/c mice were inoculated intracranially with 10 PFU or 1 PFU of each virus via the i.c route. All mice in every group showed symptoms such as a hunched posture, mental distress, and hind limb paralysis and died within 8 days (Fig. 4). No significant difference was found between the two WNV strains. The above results indicate that rXJ11129 exhibits both neuroinvasiveness and neurovirulence in mice, and its virulence in mice is equivalent to that of NY99.