Higher Cytopathic Effects of a Zika Virus Brazilian Isolate from Bahia Compared to a Canadian-Imported Thai Strain

Abstract

Zika virus (ZIKV) is an emerging pathogen from the Flaviviridae family. It represents a significant threat to global health due to its neurological and fetal pathogenesis (including microcephaly and congenital malformations), and its rapid dissemination across Latin America in recent years. The virus has spread from Africa to Asia, the Pacific islands and the Americas with limited knowledge about the pathogenesis associated with infection in recent years. Herein, we compared the ability of the Canadian-imported Thai strain PLCal_ZV and the Brazilian isolate HS-2015-BA-01 from Bahia to produce infectious ZIKV particles and cytopathic effects in a cell proliferation assay. We also compared the intracellular viral RNA accumulation of the two strains by quantitative RT-PCR (reverse transcription polymerase chain reaction) analyses. Our observations show that HS-2015-BA-01 is more cytopathic than PLCal_ZV in proliferation assays in Vero, Human Embryonic Kidney HEK 293T and neuroblastoma SH-SY5Y cells. Quantitative RT-PCR shows that the level of viral RNA is higher with HS-2015-BA-01 than with PLCal_ZV in two cell lines, but similar in a neuroblastoma cell line. The two strains have 13 amino acids polymorphisms and we analyzed their predicted protein secondary structure. The increased cytopathicity and RNA accumulation of the Brazilian ZIKV isolate compared to the Thai isolate could contribute to the increased pathogenicity observed during the Brazilian epidemic.

Keywords: Zika virus; cytopathicity; predicted protein structure; qRT-PCR; viral titer.

Figure 1

Zika virus (ZIKV) genome representation. Stars through the genome represent the position of aa differences between the Canadian-imported Thai strain PLCal_ZV and the Brazilian strain HS-2015-BA-01.

Figure 2

ZIKV HS-2015-BA is more cytopathic than PLCal_ZV in Vero, HEK 293T and SH-SY5Y cells. (A) Kinetics of cytopathicity of ZIKV in Vero cells. Vero cells were infected at multiplicity of infection (MOI) 0.1 with mock, PLCal_ZV or HS-2015-BA-01 as indicated. Live cells were photographed under the microscope after 6, 12, 24 and 48 h post-infection, scale bar = 63 μm. White arrows show the cytopathic effect of HS-2015-BA on Vero cells; (B) plaque assay in Vero cells of ZIKV PLCal_ZV and HS-2015-BA-01 after infection with 10⁻¹, 10⁻⁴ and 10⁻⁶ dilution of virus from Vero cell supernatants, scale bar = 1.74 cm in the plates and 1 mm in the zoomed pictures; (C) quantification of ZIKV plaque forming units (PFUs) formed by PLCal_ZV and HS-2015-BA-01 produced in Vero, HEK 293T and SH-SY5Y cell lines. Supernatant from each infected cell line at MOI of 0.01, 0.1 or 0.5 was collected and filtered on a 0.45 μm pore membrane. The generated supernatant was used to infect new batches of Vero cells and calculate the PFUs for every cell line. Detection limit is 400 PFUs/mL. ND = not detected. The graphs represent the average of three independent experiments ± standard error of the mean (SEM).

Figure 3

ZIKV intracellular RNA accumulation. Vero (red), HEK 293T (blue) and SH-SY5Y (orange) cells were mock infected or infected with PLCal_ZV (A) or HS-2015-BA-01 (B) at MOI 0.01, 0.1 or 0.5 as indicated. RNA accumulation was quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) at 24 h post-infection. The amount of ZIKV RNA was analyzed by the threshold cycle (Ct) comparative method and normalized to the reference genes TATA-box binding protein (TBP) and Peptidylprolyl isomerase A (PPIA) RNAs using Bio-Rad CFX software. The graphs represent an average of three independent experiments ± standard error of the mean (SEM). GraphPad Prism was used to calculate P-values for effects on RNA accumulation between PLCal_ZV and HS-2015-BA-01 in the different cell lines. Results from a two-way ANOVA were: p < 0.0001 for Vero cells, p < 0.05 for HEK293T cells and not significant for SH-SY5Y cells.

Figure 4

Cellular viability assays in different cells infected with PLCal_ZV or HS-2015-BA-01. Cells were infected with PLCal_ZV (blue) or HS-2015-BA-01 (red). (A–C) Cell viability was measured at 24 h in Vero (A); HEK 293T (B) and SH-SY5Y (C); (D–F) Cell viability assays at 48 h in Vero (D); HEK 293T (E) and SH-SY5Y (F). The OD 450 nm of the mock was set as 100% viability, and each condition was expressed as a percentage of the mock. The values are the average of three independent experiments ± standard error of the mean (SEM). GraphPad Prism was used to calculate p-values for effects on cell viability between PLCal_ZV and HS-2015-BA-01. Results from a two-way ANOVA are shown on each graph, A–F.