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. 2016 Apr 14;90(9):4757-4770.
doi: 10.1128/JVI.02861-15. Print 2016 May.

Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence

Alexander J McAuley  1   2 , Maricela Torres  1 , Jessica A Plante  2   3 , Claire Y-H Huang  4 , Dennis A Bente  1   2   5   6 , David W C Beasley  7   2   5   6
Affiliations

Recovery of West Nile Virus Envelope Protein Domain III Chimeras with Altered Antigenicity and Mouse Virulence

Alexander J McAuley et al. J Virol. .

Abstract

Flaviviruses are positive-sense, single-stranded RNA viruses responsible for millions of human infections annually. The envelope (E) protein of flaviviruses comprises three structural domains, of which domain III (EIII) represents a discrete subunit. The EIII gene sequence typically encodes epitopes recognized by virus-specific, potently neutralizing antibodies, and EIII is believed to play a major role in receptor binding. In order to assess potential interactions between EIII and the remainder of the E protein and to assess the effects of EIII sequence substitutions on the antigenicity, growth, and virulence of a representative flavivirus, chimeric viruses were generated using the West Nile virus (WNV) infectious clone, into which EIIIs from nine flaviviruses with various levels of genetic diversity from WNV were substituted. Of the constructs tested, chimeras containing EIIIs from Koutango virus (KOUV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), and Bagaza virus (BAGV) were successfully recovered. Characterization of the chimeras in vitro and in vivo revealed differences in growth and virulence between the viruses, within vivo pathogenesis often not being correlated within vitro growth. Taken together, the data demonstrate that substitutions of EIII can allow the generation of viable chimeric viruses with significantly altered antigenicity and virulence.

Importance: The envelope (E) glycoprotein is the major protein present on the surface of flavivirus virions and is responsible for mediating virus binding and entry into target cells. Several viable West Nile virus (WNV) variants with chimeric E proteins in which the putative receptor-binding domain (EIII) sequences of other mosquito-borne flaviviruses were substituted in place of the WNV EIII were recovered, although the substitution of several more divergent EIII sequences was not tolerated. The differences in virulence and tissue tropism observed with the chimeric viruses indicate a significant role for this sequence in determining the pathogenesis of the virus within the mammalian host. Our studies demonstrate that these chimeras are viable and suggest that such recombinant viruses may be useful for investigation of domain-specific antibody responses and the more extensive definition of the contributions of EIII to the tropism and pathogenesis of WNV or other flaviviruses.

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Figures

FIG 1
FIG 1
Schematic overview of chimera insert sizes and locations and sequence alignment of donor EIII sequences. (A) Donor EIII sequences were cloned into the WNV infectious clone using four combinations of up- and downstream restriction sites. The four resulting constructs were labeled A, B, C, and D. The A forms had the largest insertion size, with the donor virus EIII sequence being flanked by the donor virus EI sequence upstream and the first stem-helix structure downstream. The B forms contained donor virus EIII without the additional sequences, while the C and D forms contained the extra up- and downstream sequences, respectively. EH, E helical domain; TM, transmembrane domain. (B) Donor virus EIII amino acid sequences were aligned to determine the differences present in the chimeric viruses. Amino acid position numbers correspond to those of the WNV NY99 E sequence. Alignments were performed using the Geneious (v9.0.2) program (Biomatters, Auckland, New Zealand).
FIG 2
FIG 2
Antigenicity of chimeric viruses. (A) Reactivity of chimeric E proteins with monoclonal antibodies against flavivirus EII (MAb 4G2), WNV EIII (MAb 3A3), and JEV EIII (MAb ab81193); (B) reactivity of chimeric E proteins with various MIAFs.
FIG 3
FIG 3
Plaque morphology and particle stability of chimeric viruses. (A) After 72 h of incubation, the plaque morphologies of WNV/KOUV-EIII and WNV/BAGV-EIII in Vero cells were significantly smaller than those of WNV NY99, whereas those of WNV/JEV-EIII and WNV/SLEV-EIII remained similar to those of the NY99 control. (B) Ten representative plaques were measured for each chimera, with the mean and standard deviation (Std Dev) plaque sizes being determined for each. Plaque sizes statistically significantly different from those of WNV NY99 (P < 0.05, as determined by one-way ANOVA with the Bonferroni correction) are in bold. (C) Duplicate virus (WNV NY99 and EIII chimeras) samples were held at 37°C, and aliquots were harvested at 0, 12, 24, 36, 48, and 72 h. Samples were titrated, and the values were normalized to the titer at 0 h. One-phase decay curves were fitted to the data using GraphPad Prism (v6.0g) software.
FIG 4
FIG 4
In vitro growth kinetics for chimeric viruses. Growth kinetics were determined for WNV NY99 and each of the four chimeras following infection of Vero (A) and C6/36 (B) cells in triplicate flasks at a low MOI of 0.0005. Statistical significance was determined by ANOVA with the Bonferroni correction for all viruses by comparison to the results for WNV NY99 (*, P < 0.05; **, P < 0.01; ***, P < 0.001; the different colors correspond to individual chimeras). The graphs show mean values with standard errors.
FIG 5
FIG 5
Virulence of chimeric viruses following i.p. and i.c. inoculation. (A) Three- to 4 week-old female Swiss Webster mice were infected with 100 PFU of virus i.p. (n = 10 or 14). (B) Swiss Webster mice were infected with WNV NY99 (n = 5 per group) or an EIII chimera (n = 5 per group for WNV/JEV-EIII, n = 10 per group for WNV/KOUV-EIII, WNV/SLEV-EIII, and WNV/BAGV-EIII) at 100 or 10 PFU via the i.c. route. For the mice that succumbed to infection, average survival times (ASTs) with standard deviations were calculated and are shown on the right. Comparison of the mortality curves for mice infected with the chimeric viruses and those for mice infected with WNV NY99 for statistically significant differences was performed using a log-rank (Mantel-Cox) test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Comparison of the average survival times of mice infected with the chimeric viruses and those of mice infected with WNV NY99 for statistically significant differences was performed using a two-tailed Student's t test, with statistically significant differences being indicated in bold (P < 0.05).
FIG 6
FIG 6
Temperature and weight changes following i.p. inoculation with EIII chimeras. Groups of five 3- to 4-week-old female Swiss Webster mice were infected with 100 PFU of WNV NY99 or an EIII chimera via the i.p. route. Temperatures (A) and weights (B) were measured daily for 21 days following infection. Three different weight profiles emerged: one for the WNV NY99- and WNV/JEV-EIII-infected mice (Bi), a second one for the WNV/KOUV-EIII- and WNV/SLEV-EIII-infected mice (Bii), and a third for the WNV/BAGV-EIII-infected mice (Biii).
FIG 7
FIG 7
Organ titers following i.p. inoculation. Three- to 4-week-old female Swiss Webster mice were inoculated with either WNV NY99 or an EIII chimera at 100 PFU i.p. Three mice per group were euthanized on days 1, 3, 5, 7, and 9, and blood and organs were removed for titration. Plasma (A) and brain (B) titers were determined at all time points, while liver (C), spleen (D), lung (E), and kidney (F) titers were determined on days 1, 3, and 5. The graphs show individual values with means and standard errors.
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Alexander McAuley was supported by a James W. McLaughlin predoctoral fellowship and a Jeane B. Kempner predoctoral scholarship.

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