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Japanese encephalitis vaccine-specific envelope protein E138K mutation does not attenuate virulence of West Nile virus.

ABSTRACT: West Nile (WNV) and Japanese encephalitis viruses (JEV) are closely related, mosquito-borne neurotropic flaviviruses. Although there are no licensed human vaccines for WNV, JEV has multiple human vaccines, including the live, attenuated vaccine SA14-14-2. Investigations into determinants of attenuation of JE SA14-14-2 demonstrated that envelope (E) protein mutation E138K was crucial to the attenuation of mouse virulence. As WNV is closely related to JEV, we investigated whether or not the E-E138K mutation would be beneficial to be included in a candidate live attenuated WNV vaccine. Rather than conferring a mouse attenuated phenotype, the WNV E-E138K mutant reverted and retained a wild-type mouse virulence phenotype. Next-generation sequencing analysis demonstrated that, although the consensus sequence of the mutant had the E-E138K mutation, there was increased variation in the E protein, including a single-nucleotide variant (SNV) revertant to the wild-type glutamic acid residue. Modeling of the E protein and analysis of SNVs showed that reversion was likely due to the inability of critical E-protein residues to be compatible electrostatically. Therefore, this mutation may not be reliable for inclusion in candidate live attenuated vaccines in related flaviviruses, such as WNV, and care must be taken in translation of attenuating mutations from one virus to another virus, even if they are closely related.

SUBMITTER: Kaiser JA

PROVIDER: S-EPMC6895119 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Project description:Mosquito-borne flaviviruses with an enzootic transmission cycle like Japanese encephalitis virus (JEV) and West Nile virus (WNV) are a major public health concern. The circulation of JEV in Southeast Asia is well-documented, and the important role of pigs as amplification hosts for the virus is long known. The influence of other domestic animals especially poultry that lives in high abundance and close proximity to humans is not intensively analyzed. Another understudied field in Asia is the presence of the closely related WNV. Such analyses are difficult to perform due to the intense antigenic cross-reactivity between these viruses and the lack of suitable standardized serological assays. The main objective of this study was to assess the prevalence of JEV and WNV flaviviruses in domestic birds, detailed in chickens and ducks, in three different Cambodian provinces. We determined the flavivirus seroprevalence using an hemagglutination inhibition assay (HIA). Additionally, we investigated in positive samples the presence of JEV and WNV neutralizing antibodies (nAb) using foci reduction neutralization test (FRNT). We found 29% (180/620) of the investigated birds positive for flavivirus antibodies with an age-depended increase of the seroprevalence (OR = 1.04) and a higher prevalence in ducks compared to chicken (OR = 3.01). Within the flavivirus-positive birds, we found 43% (28/65) with nAb against JEV. We also observed the expected cross-reactivity between JEV and WNV, by identifying 18.5% double-positive birds that had higher titers of nAb than single-positive birds. Additionally, seven domestic birds (10.7%) showed only nAb against WNV and no nAb against JEV. Our study provides evidence for an intense JEV circulation in domestic birds in Cambodia, and the first serological evidence for WNV presence in Southeast Asia since decades. These findings mark the need for a re-definition of areas at risk for JEV and WNV transmission, and the need for further and intensified surveillance of mosquito-transmitted diseases in domestic animals.

Project description: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.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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Japanese encephalitis vaccine-specific envelope protein E138K mutation does not attenuate virulence of West Nile virus.

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