Application of a Microarray-Based Assay for the Study of Genetic Diversity of West Nile Virus

Intrahost genetic diversity was analysed in naturally infected mosquitoes and birds to determine whether West Nile virus (WNV) exists in nature as a quasispecies and to quantify selective pressures within and between hosts. WNV was sampled from ten infected birds and ten infected mosquito pools collected on Long Island, NY, USA, during the peak of the 2003 WNV transmission season. A 1938 nt fragment comprising the 3′ 1159 nt of the WNV envelope (E) coding region and the 5′ 779 nt of the non-structural protein 1 (NS1) coding region was amplified and cloned and 20 clones per specimen were sequenced. Results from this analysis demonstrate that WNV infections are derived from a genetically diverse population of genomes in nature. The mean nucleotide diversity was 0·016 % within individual specimens and the mean percentage of clones that differed from the consensus sequence was 19·5 %. WNV sequences in mosquitoes were significantly more genetically diverse than WNV in birds. No host-dependent bias for particular types of mutations was observed and estimates of genetic diversity did not differ significantly between E and NS1 coding sequences. Non-consensus clones obtained from two avian specimens had highly similar genetic signatures, providing preliminary evidence that WNV genetic diversity may be maintained throughout the enzootic transmission cycle, rather than arising independently during each infection. Evidence of purifying selection was obtained from both intra- and interhost WNV populations. Combined, these data support the observation that WNV populations may be structured as a quasispecies and document strong purifying natural selection in WNV populations.

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Genetic Diversity in the Collaborative Cross Model Recapitulates Human West Nile Virus Disease Outcomes

mBio ◽

10.1128/mbio.00493-15 ◽

2015 ◽

Vol 6 (3) ◽

Cited By ~ 55

Author(s):

Jessica B. Graham ◽

Sunil Thomas ◽

Jessica Swarts ◽

Aimee A. McMillan ◽

Martin T. Ferris ◽

...

Keyword(s):

Genetic Diversity ◽

West Nile Virus ◽

Virus Infection ◽

Immune Responses ◽

West Nile Virus Infection ◽

Collaborative Cross ◽

Adaptive Immune Responses ◽

West Nile ◽

Adaptive Immune ◽

Wide Range

ABSTRACT West Nile virus (WNV) is an emerging neuroinvasive flavivirus that now causes significant morbidity and mortality worldwide. The innate and adaptive immune responses to WNV infection have been well studied in C57BL/6J inbred mice, but this model lacks the variations in susceptibility, immunity, and outcome to WNV infection that are observed in humans, thus limiting its usefulness to understand the mechanisms of WNV infection and immunity dynamics. To build a model of WNV infection that captures human infection outcomes, we have used the Collaborative Cross (CC) mouse model. We show that this model, which recapitulates the genetic diversity of the human population, demonstrates diversity in susceptibility and outcomes of WNV infection observed in humans. Using multiple F1 crosses of CC mice, we identified a wide range of susceptibilities to infection, as demonstrated through differences in survival, clinical disease score, viral titer, and innate and adaptive immune responses in both peripheral tissues and the central nervous system. Additionally, we examined the Oas1b alleles in the CC mice and confirmed the previous finding that Oas1b plays a role in susceptibility to WNV; however, even within a given Oas1b allele status, we identified a wide range of strain-specific WNV-associated phenotypes. These results confirmed that the CC model is effective for identifying a repertoire of host genes involved in WNV resistance and susceptibility. The CC effectively models a wide range of WNV clinical, virologic, and immune phenotypes, thus overcoming the limitations of the traditional C57BL/6J model, allowing genetic and mechanistic studies of WNV infection and immunity in differently susceptible populations. IMPORTANCE Mouse models of West Nile virus infection have revealed important details regarding the innate and adaptive immune responses to this emerging viral infection. However, traditional mouse models lack the genetic diversity present in human populations and therefore limit our ability to study various disease outcomes and immunologic mechanisms subsequent to West Nile virus infection. In this study, we used the Collaborative Cross mouse model to more effectively model the wide range of clinical, virologic, and immune phenotypes present upon West Nile virus infection in humans.

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An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar

10.1101/383513 ◽

2018 ◽

Cited By ~ 3

Author(s):

Nathan D Grubaugh ◽

Karthik Gangavarapu ◽

Joshua Quick ◽

Nathaniel L. Matteson ◽

Jaqueline Goes De Jesus ◽

...

Keyword(s):

Genetic Diversity ◽

West Nile Virus ◽

Virus Concentration ◽

Nanopore Sequencing ◽

Computational Tool ◽

Experimental Protocol ◽

West Nile ◽

Sequencing Coverage ◽

Virus Diversity ◽

Oxford Nanopore

AbstractHow viruses evolve within hosts can dictate infection outcomes; however, reconstructing this process is challenging. We evaluated our multiplexed amplicon approach - PrimalSeq - to demonstrate how virus concentration, sequencing coverage, primer mismatches, and replicates influence the accuracy of measuring intrahost virus diversity. We developed an experimental protocol and computational tool (iVar) for using PrimalSeq to measure virus diversity using Illumina and compared the results to Oxford Nanopore sequencing. We demonstrate the utility of PrimalSeq by measuring Zika and West Nile virus diversity from varied sample types and show that the accumulation of genetic diversity is influenced by experimental and biological systems.

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