Envelope and pre-membrane protein structural amino acid mutations mediate diminished avian growth and virulence of a Mexican West Nile virus isolate

Abstract

The hallmark attribute of North American West Nile virus (WNV) strains has been high pathogenicity in certain bird species. Surprisingly, this avian virulent WNV phenotype has not been observed during its geographical expansion into the Caribbean, Central America and South America. One WNV variant (TM171-03-pp1) isolated in Mexico has demonstrated an attenuated phenotype in two widely distributed North American bird species, American crows (AMCRs) and house sparrows (HOSPs). In order to identify genetic determinants associated with attenuated avian replication of the TM171-03-pp1 variant, chimeric viruses between the NY99 and Mexican strains were generated, and their replicative capacity was assessed in cell culture and in AMCR, HOSP and house finch avian hosts. The results demonstrated that mutations in both the pre-membrane (prM-I141T) and envelope (E-S156P) genes mediated the attenuation phenotype of the WNV TM171-03-pp1 variant in a chicken macrophage cell line and in all three avian species assayed. Inclusion of the prM-I141T and E-S156P TM171-03-pp1 mutations in the NY99 backbone was necessary to achieve the avian attenuation level of the Mexican virus. Furthermore, reciprocal incorporation of both prM-T141I and E-P156S substitutions into the Mexican virus genome was necessary to generate a virus that exhibited avian virulence equivalent to the NY99 virus. These structural changes may indicate the presence of new evolutionary pressures exerted on WNV populations circulating in Latin America or may signify a genetic bottleneck that has constrained their epiornitic potential in alternative geographical locations.

Fig. 1.

Mean viral titre±sd of triplicate cultures of parental and NY99/MX03 point-mutant viruses in Vero cells inoculated at an m.o.i of 0.01, with a detection limit of 50 p.f.u. ml⁻¹.

Fig. 2.

In vitro growth profiles of parental genotype and NY99/MX03 point-mutant viruses in an avian myeloid cell line (HD11 chicken monocytes). The mean viral titre±sd was determined from triplicate cultures inoculated at an m.o.i of 0.01, with a detection limit of 50 p.f.u. ml⁻¹. The parental recombinant WN/IC-P991 (a–c) and WN-MX03/IC (b, c) were included for reference. All samples were taken from a single concurrent experiment and data were partitioned into separate graphs for presentation purposes.

Fig. 3.

(a, b) Selected viraemia profiles of American crows inoculated with 1500 p.f.u. parental genotype and NY99/MX03 point-mutant viruses. Crows were inoculated with WN/IC-P991 (n = 8), WN/IC-E (n = 8), TM171-03-pp1 (n = 8), WN/IC-prM (n = 6), WN/IC-prM.E (n = 6) and WN-MX03/IC (n = 3). The TM171-03-pp1 viraemia profile was taken from previously published data (Brault et al., 2011). Titres were determined by standard plaque assay in Vero cells with a limit of detection of 1.7 log₁₀ p.f.u. (ml serum)⁻¹. Results are shown as means±sd, and only positive or negative values are presented for clarity. The WN/IC-P991 and WN-MX03 viraemia profiles have been plotted on both graphs for reference. Viraemia profiles of additional chimeras have been omitted for clarity of presentation and are available as a Supplementary Fig. S1 (available in JGV Online).

Fig. 4.

Percentage survival of AMCRs inoculated with 1500 p.f.u. parental and NY99/MX03 point-mutant viruses and serially bled, as presented in Fig. 3 (n = 8 for WN/IC-P991, TM171-03-pp1 and WN/IC-E; n = 6 for WN/IC-prM and WN/IC-prM.E). The TM171-03-pp1 mortality plot has been reproduced for reference from previously published data (Brault et al., 2011).

Fig. 5.

(a, b) Viraemia profiles of HOSPs (n = 6; except for WN/IC-P991 where n = 5) following inoculation with 1500 p.f.u. NY99/MX03 parental genotype and point-mutant viruses. Daily mean peak titres are represented (±sd) with a detection limit of 1.7 log₁₀ p.f.u. (ml serum)⁻¹. WN/IC-P991 and WN-MX03/IC have been displayed on both graphs for comparative purposes.

Fig. 6.

Viraemia profiles of HOFIs (n = 6) following inoculation with 1500 p.f.u. parental genotype and NY99/MX03 point-mutant WNV recombinant viruses. Daily mean peak titres are represented (±sd) with a detection limit of 1.7 log₁₀ p.f.u. (ml serum)⁻¹.