The West Nile virus-like flavivirus Koutango is highly virulent in mice due to delayed viral clearance and the induction of a poor neutralizing antibody response.

UNLABELLED: The mosquito-borne West Nile virus (WNV) is responsible for outbreaks of viral encephalitis in humans, horses, and birds, with particularly virulent strains causing recent outbreaks of disease in eastern Europe, the Middle East, North America, and Australia. Previous studies have phylogenetically separated WNV strains into two main genetic lineages (I and II) containing virulent strains associated with neurological disease. Several WNV-like strains clustering outside these lineages have been identified and form an additional five proposed lineages. However, little is known about whether these strains have the potential to induce disease. In a comparative analysis with the highly virulent lineage I American strain (WNVNY99), the low-pathogenicity lineage II strain (B956), a benign Australian strain, Kunjin (WNVKUN), the African WNV-like Koutango virus (WNVKOU), and a WNV-like isolate from Sarawak, Malaysia (WNVSarawak), were assessed for neuroinvasive properties in a murine model and for their replication kinetics in vitro. While WNVNY99 replicated to the highest levels in vitro, in vivo mouse challenge revealed that WNVKOU was more virulent, with a shorter time to onset of neurological disease and higher morbidity. Histological analysis of WNVKOU- and WNVNY99-infected brain and spinal cords demonstrated more prominent meningoencephalitis and the presence of viral antigen in WNVKOU-infected mice. Enhanced virulence of WNVKOU also was associated with poor viral clearance in the periphery (sera and spleen), a skewed innate immune response, and poor neutralizing antibody development. These data demonstrate, for the first time, potent neuroinvasive and neurovirulent properties of a WNV-like virus outside lineages I and II. IMPORTANCE: In this study, we characterized the in vitro and in vivo properties of previously uncharacterized West Nile virus strains and West Nile-like viruses. We identified a West Nile-like virus, Koutango virus (WNVKOU), that was more virulent than a known virulent lineage I virus, WNVNY99. The enhanced virulence of WNVKOU was associated with poor viral clearance and the induction of a poor neutralizing antibody response. These findings provide new insights into the pathogenesis of West Nile virus.

Determinants of attenuation in the envelope protein of the flavivirus Alfuy.

Murray Valley encephalitis virus (MVEV) is a mosquito-borne flavivirus endemic to Australia and Papua New Guinea. Most strains of MVEV cause potentially fatal cases of encephalitis in humans and horses, and have been shown to be highly neuroinvasive in weanling mice. In contrast, the naturally occurring subtype Alfuy virus (ALFV) has never been associated with human disease, nor is it neuroinvasive in weanling mice, even at high doses. To identify viral factors associated with ALFV attenuation, a chimeric infectious clone was constructed containing the structural genes premembrane (prM) and envelope (E) of ALFV swapped into the MVEV genome. The resulting virus (vMVEV/ALFVstr) was no longer neuroinvasive in mice, suggesting that motifs within prM-E of ALFV confer attenuation. To define these motifs further, mutants were constructed by targeting divergent sequences between the MVEV and ALFV E proteins that are known markers of virulence in other encephalitic flaviviruses. MVEV mutants containing a unique ALFV sequence in the flexible hinge region (residues 273-277) or lacking the conserved glycosylation site at position 154 were significantly less neuroinvasive in mice than wild-type MVEV, as determined by delayed time to death or increased LD(50). Conversely, when the corresponding MVEV sequences were inserted into the vMVEV/ALFVstr chimera, the mutant containing the MVEV hinge sequence was more neuroinvasive than the parental chimera, though not to the same level as wild-type MVEV. These results identify the hinge region and E protein glycosylation as motifs that contribute to the attenuation of ALFV.