doi: 10.1016/j.virol.2012.01.028. Epub 2012 Feb 23.
Internally deleted WNV genomes isolated from exotic birds in New Mexico: function in cells, mosquitoes, and mice
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- PMID:22365325
- PMCID: PMC3312038
- DOI: 10.1016/j.virol.2012.01.028
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Internally deleted WNV genomes isolated from exotic birds in New Mexico: function in cells, mosquitoes, and mice
Kendra N Pesko et al. Virology..
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Abstract
Most RNA viruses exist in their hosts as a heterogeneous population of related variants. Due to error prone replication, mutants are constantly generated which may differ in individual fitness from the population as a whole. Here we characterize three WNV isolates that contain, along with full-length genomes, mutants with large internal deletions to structural and nonstructural protein-coding regions. The isolates were all obtained from lorikeets that died from WNV at the Rio Grande Zoo in Albuquerque, NM between 2005 and 2007. The deletions are approximately 2kb, in frame, and result in the elimination of the complete envelope, and portions of the prM and NS-1 proteins. In Vero cell culture, these internally deleted WNV genomes function as defective interfering particles, reducing the production of full-length virus when introduced at high multiplicities of infection. In mosquitoes, the shortened WNV genomes reduced infection and dissemination rates, and virus titers overall, and were not detected in legs or salivary secretions at 14 or 21 days post-infection. In mice, inoculation with internally deleted genomes did not attenuate pathogenesis relative to full-length or infectious clone derived virus, and shortened genomes were not detected in mice at the time of death. These observations provide evidence that large deletions may occur within flavivirus populations more frequently than has generally been appreciated and suggest that they impact population phenotype minimally. Additionally, our findings suggest that highly similar mutants may frequently occur in particular vertebrate hosts.
Copyright © 2012 Elsevier Inc. All rights reserved.
Figures
Identification of deletion mutants. A) PCR amplicons produced by primers to the structural coding regions of WNV genome to each of three samples, WT target size is 2.5 kb, actual size observed from isolates is 0.5–0.7 kb, B) Northern blot of RNAs from cells infected with infectious clone derived virus (WT) or isolate 2774 from infected birds, and probe to envelope. C) Northern analysis of RNAs as in B but with probe to NS-5. D) Genomic locations of deletions found in isolates 2774, 3336, and 3337. At top, diagram of West Nile virus genome, showing structural protein coding regions for a nucleocapsid (C), membrane precursor (prM), and envelope (E) towards the 5′ end, and nonstructural proteins (NS1–5) towards the 3′ end. Locations of deletions found in isolates 2774, 3337, and 3336 are shown by grey boxes, with numbers at start and finish indicating distance in nucleotide bases from start of genome where deletions begin and end. Three letter codes are given for the resultant amino acid sequence at the deletion sites. For isolates 3337 and 3336 the deletion mutant population was mixed evenly between two variants (a and b).
50% majority-rule consensus phylogram is shown based on partial genome sequences (nt 789–10395) with posterior probabilities given as values at nodes. Strain designations are given in table 3.
A) Titers produced at 72 h post infection of Vero cells with infectious clone derived virus (WT, black circles) or deletion mutant containing virus (2774, grey squares) at indicated multiplicities of infection. Individual replicates are represented, with the mean and 95% confidence interval given as accompanying bars. B) Deletion mutant genomes detected by qRT-PCR in supernatants at 72 h post infection of Vero cells with deletion mutant containing virus.
A) Infection, dissemination, and transmission rates forCulex pipiens quinquefasciatus mosquitoes fed on full length only (FL, grey) or deletion mutant containing virus (DM+FL, white). Data from days 14 and 21 are combined, as day of sampling had no significant influence on rates, by chi-squared comparison. **p<0.005, *p<0.05. Infection was calculated as the percentage of mosquitoes exposed with positive bodies, dissemination rates were calculated as the percentage of mosquitoes with positive bodies containing positive legs, transmission rates were calculated as the percentage of mosquitoes with positive legs containing positive salivary secretions. B) Body titers of infected mosquitoes, as estimated by plaque assay in Vero cells.
(A–C) Survivorship curves for C3H mice inoculated with 102 pfu/mouse (DM+FL, red, n=8; FL, green, n=8; WT, blue, n=8) (A), C3H mice inoculated with 105 pfu/mouse (DM+FL, red, n=8; FL, green, n=8; WT, blue, n=5) (B), and C57/Bl6 mice inoculated with 105 pfu/mouse (DM+FL, red, n=9; FL, green, n=9; WT, blue, n=8)(C), with deletion mutant containing isolate (red), plaque purified isolate containing full length virus only (green), infectious clone derived full length virus only (blue), or mock inoculated (black). (D–F) Percent weight change in mice measured daily, shown as the average daily percent difference from starting weight for C3H mice inoculated with 102 pfu/mouse (D), C3H mice inoculated with 105 pfu/mouse (E), and C57/Bl6 mice inoculated with 105 pfu/mouse (F) with deletion mutant containing isolate (red), plaque purified isolate containing full length virus only (green), infectious clone derived full length virus only (blue), or mock inoculated (black).
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