A Putative Lipid-Associating Motif in the West Nile Virus NS4A Protein Is Required for Efficient Virus Replication

Flavivirus replication is intimately associated with re-organized cellular membranes. These virus-induced changes in membrane architecture form three distinct membranous “organelles” that have specific functions during the flavivirus life cycle. One of these structures is the replication complex in which the flaviviral RNA is replicated to produce progeny genomes. We have previously observed that this process is strictly dependent on cellular cholesterol. In this study we have identified a putative cholesterol recognition/interaction amino acid consensus (CRAC) motif within the West Nile virus strain Kunjin virus (WNVKUN) NS4A protein. Site-directed mutagenesis of this motif within a WNVKUN infectious clone severely attenuated virus replication and the capacity of the mutant viruses to form the replication complex. Replication of the mutant viruses also displayed reduced co-localization with cellular markers recruited to replication sites during wild-type virus replication. In addition, we observed that the mutant viruses were significantly impaired in their ability to remodel cytoplasmic membranes. However, after extensive analysis we are unable to conclusively reveal a role for the CRAC motif in direct cholesterol binding to NS4A, suggesting additional complex lipid-protein and protein-protein interactions. We believe this study highlights the crucial role for this region within NS4A protein in recruitment of cellular and viral proteins to specialized subdomains on membrane platforms to promote efficient virus replication.

Evidence for current circulation of an ancient West Nile virus strain (NY99) in Brazil

IntroductionIn Brazil, West Nile virus (WNV) was first detected in 2018 from horses with neurological disease.AimHere we present the first reported case in Ceará state and complete genome sequence from an isolated in Espírito Santo state from 2019.MethodsThe virus was isolated from a horse that died with neurological signs in Espírito Santo and was sequenced by MiSeq.ResultsPhylogenetic analysis reveals that this isolate belongs to lineage 1a, clustering with NY99 strain, that disappeared from the USA since 2005.ConclusionsOur findings reinforce the hypothesis that WNV has been silently circulating in Brazil for many years.

Inflammatory responses to a pathogenic West Nile virus strain

Background West Nile virus (WNV) circulates across Australia and was referred to historically as Kunjin virus (WNVKUN). WNVKUN has been considered more benign than other WNV strains circulating globally. In 2011, a more virulent form of the virus emerged during an outbreak of equine arboviral disease in Australia. Methods To better understand the emergence of this virulent phenotype and the mechanism by which pathogenicity is manifested in its host, cells were infected with either the virulent strain (NSW2012), or less pathogenic historical isolates, and their innate immune responses compared by digital immune gene expression profiling. Two different cell systems were used: a neuroblastoma cell line (SK-N-SH cells) and neuronal cells derived from induced pluripotent stem cells (iPSCs). Results Significant innate immune gene induction was observed in both systems. The NSW2012 isolate induced higher gene expression of two genes (IL-8 and CCL2) when compared with cells infected with less pathogenic isolates. Pathway analysis of induced inflammation-associated genes also indicated generally higher activation in infected NSW2012 cells. However, this differential response was not paralleled in the neuronal cultures. Conclusion NSW2012 may have unique genetic characteristics which contributed to the outbreak. The data herein is consistent with the possibility that the virulence of NSW2012 is underpinned by increased induction of inflammatory genes.

RESULTS OF VIROLOGICAL EXAMINATION OF THE CLINICAL MATERIALS COLLECTED FROM PATIENTS WITH ACUTE FEBRILE INFECTIONS UNKNOWN ORIGIN

In 2012-2018, 243 samples of blood, serum, urine and saliva of patients with acute febrile diseases hospitalized in medical institutions of Astrakhan, Astrakhan Region and Infectious Diseases Hospital №1 of Moscow were examined in experiments on intracerebral inoculation of newborn white mice. As a result, 8 strains of dengue 1 virus were isolated from patients infected in Thailand (7 strains) and Vietnam (1 strain); eight strains of dengue 2 virus from patients infected in Thailand (5 strains), Indonesia (2 strains) and the Maldives (1 strain); one strain of dengue 3 and one strain of Chikungunya virus (Asian genotype) from patients hospitalized after returning to Moscow from trips to the Philippines; 2 strains of Zika virus (Asian genotype) from patients visiting the Dominican Republic; 6 strains of Crimean-Congo hemorrhagic fever virus genotype Europe 1 and one West Nile virus strain of genotype 1a from patients of the Astrakhan region.

RESULTS OF VIROLOGICAL EXAMINATION OF THE CLINICAL MATERIALS COLLECTED FROM PATIENTS WITH ACUTE FEBRILE INFECTIONS UNKNOWN ORIGIN

In 2012-2018, 243 samples of blood, serum, urine and saliva of patients with acute febrile diseases hospitalized in medical institutions of Astrakhan, Astrakhan Region and Infectious Diseases Hospital №1 of Moscow were examined in experiments on intracerebral inoculation of newborn white mice. As a result, 8 strains of dengue 1 virus were isolated from patients infected in Thailand (7 strains) and Vietnam (1 strain); eight strains of dengue 2 virus from patients infected in Thailand (5 strains), Indonesia (2 strains) and the Maldives (1 strain); one strain of dengue 3 and one strain of Chikungunya virus (Asian genotype) from patients hospitalized after returning to Moscow from trips to the Philippines; 2 strains of Zika virus (Asian genotype) from patients visiting the Dominican Republic; 6 strains of Crimean-Congo hemorrhagic fever virus genotype Europe 1 and one West Nile virus strain of genotype 1a from patients of the Astrakhan region.