During in vitro propagation of a clinical strain of DENV-2 obtained from Guangzhou, China, heterogenous foci of different plaque sizes were observed in Vero cell cultures (Fig. 1A). Through limiting dilution and culture in mosquito C6/36 cells, two viral variants were separated. After subsequent passage for three rounds in C6/36 cells, two virus strains with stable foci morphology were obtained. One of them forms larger foci of about 1.25 mm diameter after 80 h culture, and it was termed as DENV-2 8H2-7-LP (Fig. 1A); the other forms smaller foci of about 0.25 mm diameter and was termed as DENV-2 1D4-5-SP (Fig. 1A).
Figure 1. Different replication capacity of two viral variants DENV-2 1D4-5-SP and DENV-2 8H2-7-LP in Vero cells. A Foci of the parental clinical isolate, and DENV-2 1D4-5-SP, DENV-2 8H2-7-LP in infected Vero cells on day 4 post infection; One-step growth curve of DENV-2 1D4-5-SP, DENV-2 8H2-7-LP in Vero cells (B), and C6/36 cells (C). Viruses were used to infect cells at MOI 2.5, supernatant of infected cells were collected at the indicated timepoints. Titers of infectious virus in supernatant were measured using foci forming assay in Vero cells. Detection limits of infectious virus titer were indicated with dashed lines. Significance of the differences was calculated with Two-way ANOVA test, ***P < 0.001, n.s., not significant.
We next compared the replication abilities of these two viruses in both Vero cells and C6/36 cells using the one-step growth curve. DENV-2 8H2-7-LP had a higher replicating efficiency in Vero cells than DENV-2 1D4-5-SP during the first 72 h after infection, followed by a decline afterwards, due to the probability of running out target cells for infection; in comparison, DENV-2 1D4-5-SP rose slowly to a peak at 96 h post infection (hpi), and kept its plateau until 120 hpi (Fig. 1B). Distinct from what was observed in Vero cells, there was no obvious growth difference between the two viruses in mosquito cells (Fig. 1C).
Taken together, these data demonstrated that two viral variants with different foci sizes are isolated from one clinical virus isolate. And these two variants have different replication abilities in Vero but not mosquito C6/36 cells.
To determine whether the different replication phenotypes reflect underlying genetic divergence between the LP and SP viruses, we sequenced the whole coding sequence of DENV-2 8H2-7-LP and DENV-2 1D4-5-SP, and aligned their envelope sequence with those of representative viruses belonging to different DENV-2 genotypes, including Asian genotype, American Asian genotype, Cosmopolitan genotype and Sylvatic genotype. Additionally, DENV-2 strains isolated in Guangdong, China, from 2005 to 2014 were also included for comparison. The phylogenetic analysis showed that DENV-2 8H2-7-LP and DENV-2 1D4-5-SP belonged to the Cosmopolitan genotype and were clustered together with many strains isolated in West pacific regions, with percentages of homology ranging from 99.5% to 100% (Fig. 2).
Figure 2. Phylogenetic analysis of DENV-2 1D4-5-SP and DENV-2 8H2-7-LP with representative serotype-2 dengue viruses of different genotypes isolated from different geographical regions. The phylogenetic tree was obtained after analyzing envelope sequences of DENV-2 with MEGA X software, using the Maximum Likelihood method and Kimura 2-parameter model. Members of six reported genotypes of DENV-2 were included. DENV-2 1D4-5-SP (MN952967) and DENV-2 8H2-7-LP (MN952966) were denoted by red dots in the tree. The scale bar denotes an evolutionary distance of 0.02 nucleotides per position in the sequence.
Further comparison of genomic information between these two viruses also indicated that they had sequence variations in coding regions as illustrated in Table 1. Collectively, we found 14 sequence variations in E, NS3, NS4B and NS5 regions, including 7 synonymous mutations and 7 amino acid changes. Among the 7 nonsynonymous mutations, two are in domain Ⅰ/Ⅱ regions of E protein, two are in peptidase domain and helicase domain of NS3 respectively, another is located in a transmembrane helix of NS4B, and the rest two are in NS5 RdRp domain. All of these domains are components of viral binding, replication or processing machinery, and highly related to viral antagonisms to host immunity. The conservation of mutations was also analyzed in comparison with representative strains from different genotypes, interestingly, though most synonymous mutations were also observed in other dengue-2 viruses, the seven nonsynonymous mutations were unique, and these sites seem more conserved in dengue-2 viruses (Table 1). These genomic characteristics may contribute to the different infectivity both in vitro and in vivo.
Genotype Segment E NS2A NS2B NS3 NS4A NS4B NS5 Domain aDI DII / / Pro Hel / TM MTase RdRp / bLocation(nt) 1399 1434 1540 4080 4125 4429 4876 5065 5817 6390 7144 8182 9112 9207 cLocation(aa) 155 166 202 191 206 97 119 182 332 5 107 151 461 492 Cosmopolitan 1D4-5-SP d(A)Thr (A)P (A)Lys (C)L (G)K (C)R (A)Asn (A)Ile (G)V (T)N (C)Leu (T)L (C)Leu (C)Asp 8H2-7-LP (G)Ala (G)P (G)Glu (T)L (A)K (A)R (G)Asp (G)Val (C)V (C)N (T)Phe (C)L (A) Ile (A)Glu eAY037116 – (C)P (G)Glu – – (A)R – – (C)V – – – – – Asian I DQ181801 – – (G)Glu – – (A)R – – (T)V (C)N – – – – Asian Ⅱ MK506263 – – (G)Glu – – (A)R – – (C)V (C)N – – – – Asian/American M29095 – – (G)Glu – – (A)R – – (T)V (C)N – – – – American AY702040 – – (G)Glu – – (A)R – – (T)V (C)N – – – (T)Asp Sylvatic EF105378 – (C)P (G)Glu – (A)K (A)R – – (T)V – – (C)L – (T)Asp aDⅠ, DⅡ domain Ⅰ, Ⅱ of E protein ectodomain, Pro Protease, Hel Helicase, TM Transmembrane, MTase, methyltransferase; RdRp, RNA-dependent RNA polymerase;
bLocation of nucleotide variation in the whole viral genomes;
cLocation of both synonymous and nonsynonymous variation sites on amino acid sequences of each viral protein;
dNonsynonymous variations were presented with three-letter amino acid codes;
eRepresentative strains from each genotype were selected from sequences analyzed in Fig. 2, and listed in table with their access number for comparison. "–" means the nucleotide and residue are identical to the first strain 1D4-5-SP.
Table 1. Sequence variation between 8H2-7-LP and 1D4-5-SP.
As type Ⅰ/Ⅱ interferon deficient mice are more susceptible to dengue virus infection, we determined the in vivo infectivity of these two viruses in TypeⅠ and Type Ⅱ IFN receptors double knock-out AG6 mice. AG6 mice between 6 and 8 weeks old were infected through s.c. injection with 103 PFU, 104 PFU and 105 PFU DENV-2 8H2-7-LP or DENV-2 1D4-5-SP (Fig. 3). It is observed that infection of either virus led to 100% mortality within 7–11 days, even at a dose as low as 103 PFU, though DENV-2 8H2-7-LP caused more weight loss and faster death than DENV-2 1D4-5-SP at the same dosage. Specifically, weight loss began on 3 dpi, 4 dpi and 5 dpi in mice infected with 105, 104, and 103 PFU DENV-2 8H2-7-LP, respectively (Fig. 3A, 3B, 3C), and death began on 7 dpi, 8 dpi and 9 dpi correspondingly (Fig. 3D, 3E, 3F). In contrast, weight loss was first observed 1–3 days later in mice infected with DENV-2 1D4-5-SP, on 5 dpi or 6 dpi with inoculation of 104–105 PFU or 103 PFU, respectively. The death was also delayed, began on 8 dpi or 9 dpi correspondingly. As a mixture of two types of viral variants, the parental strain was also able to cause weight loss and lethal diseases in AG6 mice. Comparing to the two purified variants, the parental quasispecies presented intermediate level of pathology, which was most obvious in the dose groups of 105 PFU. Through Evans blue staining, we also detected plasma leakage in sick mice on day 7 post infection by either DENV-2 8H2-7-LP or DENV-2 1D4-5-SP viruses (Fig. 4). Consistent with the progress of weight loss and survival curves, leakage of Evans blue in liver or digestive tract was more severe in mice infected with 8H2-7-LP on 7 dpi.
Figure 3. Weight loss and survival curve of AG6 mice after infection with DENV-2 8H2-7-LP and DENV-2 1D4-5-SP. AG6 mice between 6-8 weeks old were infected with two viral variants and the parental isolate through s.c. injection. Three different dose of viruses, 103 PFU (A, D; n = 3), 104 PFU (B, E; n = 3), and 105 PFU (C, F; LP and SP n = 4, Parental n = 3), were inoculated into mice. Mice injected with PBS were included within each panel for control (CT, n = 6). Weight change was recorded and presented as a ratio to the initial weight on day 0. Moribund mice with weight loss over 20% were euthanatized and recorded as death. Two-way ANOVA was used in statistical analysis of differences among groups, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001, n.s., not significant.
Figure 4. Vascular leakage in AG6 mice induced by infection of DENV-2 8H2-7-LP and DENV-2 1D4-5-SP. AG6 mice of 8 weeks old were infected with 105 PFU DENV-2 8H2-7-LP or 1D4-5-SP through s.c. injection. Seven days post infection, mice were injected intravenously with Evans blue and DENV-induced vascular leakage was visualized in different tissues. PBS infected mice were used as negative controls.
The evidence above demonstrated that the two viral isolates show differences on the pathology and disease kinetics, though they both have capabilities for causing lethal infection in IFN deficient mice.
Although both viruses can cause lethal infection in TypeⅠ and Type Ⅱ double knock-out mice, whether the LP and SP viruses behave differently in Type Ⅱ IFN competent host is unknown. We have recently developed a ZIKV infection model in Balb/c mice with transient blockage of TypeⅠ IFN receptor (IFNAR) (Liang et al. 2018), we thus used the same strategy to examine these two dengue viruses for further comparison.
WT Balb/c mice were injected with antibody specific for IFNAR at one day prior to s.c. infection with 106 PFU of either viral strain, and monitored for seven days. The viral RNA of DENV-2 8H2-7-LP could be detected throughout the week after infection, and the peak RNA level of about 106 genomic copy per microgram total RNA was reached on day 3 post infection, followed by gradually dropping to 104 copy on the 7th day (Fig. 5A). In contrast, viral RNA of DENV-2 1D4-5-SP was not detected until day 4 post infection, when merely 104 copy per microgram of total RNA was transiently detected for 2 days. Consistently, infectious virus was detected in sera of DENV-2 8H2-7-LP infected mice from 2 to 4 dpi, during which peak viremia of 103 PFU/mL appeared on day 3 post infection. Meanwhile, infectious virus was not detectable in mice infected by DENV-2 1D4-5-SP (lower than detection limit of 20 PFU/mL) (Fig. 5B). Moreover, 8H2-7-LP viral RNA was also detected in liver, spleen and eyes on 3 dpi (Fig. 5C), further confirming replication of 8H2-7-LP in susceptible organs. The parental strain also replicated in Balb/c mice (Fig. 5A and 5B), and the kinetics of viremia were closer to that of 8H2-7-LP, except for the delay of peak viremia to the 4th day post infection in both assays.
Figure 5. Viral replication of DENV-2 8H2-7-LP and DENV-2 1D4-5-SP in Balb/c mice pretreated with IFNAR blocking antibodies. Female Balb/c mice of 6–8 weeks old were injected through i.p. with 2 mg antibody to TypeⅠ IFN receptor (MAR1-5A3) at one day prior to infection. On the next day, 106 PFU of viruses were inoculated into mice through s.c. route. Then, blood samples were collected daily after infection. A Viral RNA copies in blood were measured using qRT-PCR, Two-way ANOVA was used in statistical analysis of the differences among groups, **P < 0.01, ****P < 0.0001, and B Titers of infectious virus in serum samples were determined through foci forming assay in Vero cells; n = 5. T test was used in statistical analysis of the differences among groups, *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001. C Eye, liver and spleen were collected from 8H2-7-LP virus-infected mice, and viral RNA loads were determined, n = 4. Detection limits of RNA copy and infectious virus titer were indicated with dotted lines. N.D. not detected.
To examine whether the above observed different virological properties of the two DENV variants affect adaptive immunity in host, we evaluated adaptive immune responses induced by their infection in Balb/c mice. DENV-2 8H2-7-LP infection elicited antibodies cross-neutralizing the reference strain DENV-2 16681 (Fig. 6A) and it also stimulated T cell responses specific to peptides of DENV-2 NS1 and ZIKV NS3 (Fig. 6B). Similarly, neutralizing antibody and T cell responses were also detected in mice which were previously infected by DENV-2 1D4-5-SP, though both responses were weaker than those elicited by DENV-2 8H2-7-LP. Considering genome similarity between the two variants, such different magnitudes of adaptive immune responses are mainly related to the different viral replication of two variants and different expression level of immunogens in vivo.
Figure 6. DENV-2 specific adaptive immune responses elicited by DENV-2 8H2-7-LP and DENV-2 1D4-5-SP infection. At 40 days post infection, FRNT (Foci Reduction Neutralization Test) A and IFN-γ Elispot assay (B) were performed with mouse sera and splenocytes, respectively. Reference strain DENV-2 16681 was used in FRNT assay, peptides and proteins derived from DENV-2 16681 (D2-NS1 265, D2-NS1, D2-E80) and Zika virus (Z-NS3 43, Z-NS3 291) were included in Elispot assay to measure specific and cross-reactive responses; n = 4. Significance of the differences was calculated with t test, ***P < 0.001.
In summary, we isolated from the same viral quasispecies two DENV variants that showed distinct in vitro replication phenotype in Vero cells, and different in vivo infectivity and pathogenicity in mice. Using either of these two viral variants, we have established a lethal infection model in immune deficient AG6 mice. With the variant DENV-2 8H2-7-LP, we have also developed a self-limited infection model in WT mice pretreated with Type-Ⅰ IFN receptor antibodies. These two closely-related viruses not only offer new tools for in vivo studies, but also provide resources to investigate the fundamental molecular basis that modulate viral replication.