The West Nile virus mutant spectrum is host-dependant and a determinant of mortality in mice

AbstractTo define the impact of mosquitoes and birds on intrahost WNV population dynamics, the mutant spectra that arose as a result of 20 serial in vivo passages in Culex pipiens and young chickens were examined. Genetically homogeneous WNV was serially passaged 20 times in each host. Genetic diversity was greater in mosquito-passaged WNV compared to chicken-passaged WNV. Changes in the viral consensus sequence occurred in WNV passaged in mosquitoes earlier and more frequently than in chicken-passaged WNV. Analysis of synonymous and nonsynonymous variation suggested that purifying selection was relaxed during passage in mosquitoes. Mortality in mice was significantly negatively correlated with the size of the WNV mutant spectrum. These studies suggest that mosquitoes serve as sources for WNV genetic diversity, that birds are selective sieves, and that both the consensus sequence and the mutant spectrum contribute to WNV phenotype

West Nile Virus Experimental Evolution in vivo and the Trade-off Hypothesis

In nature, arthropod-borne viruses (arboviruses) perpetuate through alternating replication in vertebrate and invertebrate hosts. The trade-off hypothesis proposes that these viruses maintain adequate replicative fitness in two disparate hosts in exchange for superior fitness in one host. Releasing the virus from the constraints of a two-host cycle should thus facilitate adaptation to a single host. This theory has been addressed in a variety of systems, but remains poorly understood. We sought to determine the fitness implications of alternating host replication for West Nile virus (WNV) using an in vivo model system. Previously, WNV was serially or alternately passed 20 times in vivo in chicks or mosquitoes and resulting viruses were characterized genetically. In this study, these test viruses were competed in vivo in fitness assays against an unpassed marked reference virus. Fitness was assayed in chicks and in two important WNV vectors, Culex pipiens and Culex quinquefasciatus. Chick-specialized virus displayed clear fitness gains in chicks and in Cx. pipiens but not in Cx. quinquefasciatus. Cx. pipiens-specialized virus experienced reduced fitness in chicks and little change in either mosquito species. These data suggest that when fitness is measured in birds the trade-off hypothesis is supported; but in mosquitoes it is not. Overall, these results suggest that WNV evolution is driven by alternate cycles of genetic expansion in mosquitoes, where purifying selection is weak and genetic diversity generated, and restriction in birds, where purifying selection is strong

Experimental Passage of St. Louis Encephalitis Virus In Vivo in Mosquitoes and Chickens Reveals Evolutionarily Significant Virus Characteristics

St. Louis encephalitis virus (SLEV; Flaviviridae, flavivirus) was the major cause of epidemic flaviviral encephalitis in the U.S. prior to the introduction of West Nile virus (WNV) in 1999. However, outbreaks of SLEV have been significantly more limited then WNV in terms of levels of activity and geographic dispersal. One possible explanation for these variable levels of activity is that differences in the potential for each virus to adapt to its host cycle exist. The need for arboviruses to replicate in disparate hosts is thought to result in constraints on both evolution and host-specific adaptation. If cycling is the cause of genetic stability observed in nature and arboviruses lack host specialization, then sequential passage should result in both the accumulation of mutations and specialized viruses better suited for replication in that host. Previous studies suggest that WNV and SLEV differ in capacity for both genetic change and host specialization, and in the costs each accrues from specializing. In an attempt to clarify how selective pressures contribute to epidemiological patterns of WNV and SLEV, we evaluated mutant spectra size, consensus genetic change, and phenotypic changes for SLEV in vivo following 20 sequential passages via inoculation in either Culex pipiens mosquitoes or chickens. Results demonstrate that the capacity for genetic change is large for SLEV and that the size of the mutant spectrum is host-dependent using our passage methodology. Despite this, a general lack of consensus change resulted from passage in either host, a result that contrasts with the idea that constraints on evolution in nature result from host cycling alone. Results also suggest that a high level of adaptation to both hosts already exists, despite host cycling. A strain significantly more infectious in chickens did emerge from one lineage of chicken passage, yet other lineages and all mosquito passage strains did not display measurable host-specific fitness gains. In addition, increased infectivity in chickens did not decrease infectivity in mosquitoes, which further contrasts the concept of fitness trade-offs for arboviruses

Development and Characterization of Stable Sediment-Free Anaerobic Bacterial Enrichment Cultures That Dechlorinate Aroclor 1260

We have developed sediment-free anaerobic enrichment cultures that dechlorinate a broad spectrum of highly chlorinated polychlorinated biphenyls (PCBs). The cultures were developed from Aroclor 1260-contaminated sediment from the Housatonic River in Lenox, MA. Sediment slurries were primed with 2,6-dibromobiphenyl to stimulate Process N dechlorination (primarily meta dechlorination), and sediment was gradually removed by successive transfers (10%) to minimal medium. The cultures grow on pyruvate, butyrate, or acetate plus H(2). Gas chromatography-electron capture detector analysis demonstrated that the cultures extensively dechlorinate 50 to 500 μg/ml of Aroclor 1260 at 22 to 24°C by Dechlorination Process N. Triplicate cultures of the eighth transfer without sediment dechlorinated 76% of the hexa- through nonachlorobiphenyls in Aroclor 1260 (250 μg/ml) to tri- through pentachlorobiphenyls in 110 days. At least 64 PCB congeners, all of which are chlorinated on both rings and 47 of which have six or more chlorines, were substrates for this dechlorination. To characterize the bacterial diversity in the enrichments, we used eubacterial primers to amplify and clone 16S rRNA genes from DNA extracted from cultures grown on acetate plus H(2). Restriction fragment length polymorphism analysis of 107 clones demonstrated the presence of Thauera-like Betaproteobacteria, Geobacter-like Deltaproteobacteria, Pseudomonas species, various Clostridiales, Bacteroidetes, Dehalococcoides of the Chloroflexi group, and unclassified Eubacteria. Our development of highly enriched, robust, stable, sediment-free cultures that extensively dechlorinate a highly chlorinated commercial PCB mixture is a major and unprecedented breakthrough in the field. It will enable intensive study of the organisms and genes responsible for a major PCB dechlorination process that occurs in the environment and could also lead to effective remediation applications

Serial and alternate passage experimental design.

<p>Virus derived from a WNV infectious clone was passed 20 times through chicks, 20 times through <i>Cx. pipiens</i> mosquitoes or 20 times alternating between the two (10 cycles). Each passage series was performed in triplicate and the final virus stocks were then used in <i>in vivo</i> competition assays to assess gains or losses in replicative fitness.</p

The trade-off hypothesis is not supported by results from <i>in vivo</i> competitions.

<p>After serial or alternate passage WNV strains were competed against a reference virus in chicks, <i>Cx. pipiens</i> mosquitoes (Pip.) and <i>Cx. quinquefasciatus</i> mosquitoes (Quinq.). Each of four treatments (serial passage in chicks, serial passage in mosquitoes, final chick passage of the alternate series, final mosquito passage of the alternate series) was performed in triplicate (represented by light, medium and dark shades of each color). Inocula (squares) contained approximately equal parts passed test virus and unpassed reference virus and were identical across cohorts except for 5 Pip. cohorts for which comparable inocula had to be re-created (points with white centers). Each cohort comprised 7–10 chicks or 9–11 mosquitoes with each animal represented by a circle. Mean proportions of test WNV for each cohort were compared with the inocula means in unpaired t-tests where P≤0.05 was considered significant (astrices). Bars indicate cohort mean and standard error of the mean. Dashed lines at 0.1 and 0.9 indicate the range of high accuracy for the quantitative sequencing assay used as determined by Fitzpatrick et al <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002335#ppat.1002335-Fitzpatrick1" target="_blank">[21]</a>.</p

Combined average proportions of total WNV RNA comprised of competitor RNA after competition against a marked reference virus in chicks or mosquitoes.

1<p> = input is the inoculum.</p>2<p> = output is either day 2 chick serum or day 7 whole mosquito homogenate.</p>3<p> = p-value was determined in an unpaired t-test between input and output for each cohort (significance is defined as p≤0.05 and is noted in bold).</p

Intrahost genetic diversity is associated with decreased fitness in chickens but not mosquitoes.

<p>Fitness was computed as the difference between the test:REF ratio at input and after competition, such that numbers greater than zero indicate fitness increases and numbers less than zero indicate fitness declines. Sequence diversity was computed as the proportion of nucleotides in the test virus population with mutation, as described by Jerzak et al <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002335#ppat.1002335-Jerzak3" target="_blank">[18]</a>. Fitness was measured in <i>Cx. pipiens</i> (blue circles) <i>Cx. quinquefasciatus</i> (red squares) and chickens (green triangles). Open symbols indicate passage history in mosquitoes, closed symbols indicate passage history in chickens. Sequence diversity was significantly negatively correlated with fitness in chickens (Spearman r = −0.9856, P = 0.0028).</p

Proportion of <i>Cx. pipiens</i> mosquitoes infected following inoculation of 10-fold dilutions of SLEV before (P0) or after passage in mosquitoes (MP) or chickens (CP).

<p>1Calculated using Reed-Muench formula.</p