1887

Abstract

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. 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.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/vir.0.034793-0
2011-10-01
2019-09-20
Loading full text...

Full text loading...

/deliver/fulltext/jgv/92/10/2286.html?itemId=/content/journal/jgv/10.1099/vir.0.034793-0&mimeType=html&fmt=ahah

References

  1. Adams S. C. , Broom A. K. , Sammels L. M. , Hartnett A. C. , Howard M. J. , Coelen R. J. , Mackenzie J. S. , Hall R. A. . ( 1995; ). Glycosylation and antigenic variation among Kunjin virus isolates. . Virology 206:, 49–56. [CrossRef] [PubMed]
    [Google Scholar]
  2. Allison S. L. , Schalich J. , Stiasny K. , Mandl C. W. , Kunz C. , Heinz F. X. . ( 1995; ). Oligomeric rearrangement of tick-borne encephalitis virus envelope proteins induced by an acidic pH. . J Virol 69:, 695–700.[PubMed]
    [Google Scholar]
  3. Allison S. L. , Schalich J. , Stiasny K. , Mandl C. W. , Heinz F. X. . ( 2001; ). Mutational evidence for an internal fusion peptide in flavivirus envelope protein E. . J Virol 75:, 4268–4275. [CrossRef] [PubMed]
    [Google Scholar]
  4. Bartlett P. F. , Reid H. H. , Bailey K. A. , Bernard O. . ( 1988; ). Immortalization of mouse neural precursor cells by the c-myc oncogene. . Proc Natl Acad Sci USA 85:, 3255–3259. [CrossRef]
    [Google Scholar]
  5. Beasley D. W. , Davis C. T. , Estrada-Franco J. , Navarro-Lopez R. , Campomanes-Cortes A. , Tesh R. B. , Weaver S. C. , Barrett A. D. . ( 2004a; ). Genome sequence and attenuating mutations in West Nile virus isolate from Mexico. . Emerg Infect Dis 10:, 2221–2224.[PubMed] [CrossRef]
    [Google Scholar]
  6. Beasley D. W. , Davis C. T. , Whiteman M. , Granwehr B. , Kinney R. M. , Barrett A. D. . ( 2004b; ). Molecular determinants of virulence of West Nile virus in North America. . Arch Virol Suppl 18:, 35–41.[PubMed]
    [Google Scholar]
  7. Beasley D. W. , Whiteman M. C. , Zhang S. , Huang C. Y. , Schneider B. S. , Smith D. R. , Gromowski G. D. , Higgs S. , Kinney R. M. , Barrett A. D. . ( 2005; ). Envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage 1 West Nile virus strains. . J Virol 79:, 8339–8347. [CrossRef] [PubMed]
    [Google Scholar]
  8. Bressanelli S. , Stiasny K. , Allison S. L. , Stura E. A. , Duquerroy S. , Lescar J. , Heinz F. X. , Rey F. A. . ( 2004; ). Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. . EMBO J 23:, 728–738. [CrossRef] [PubMed]
    [Google Scholar]
  9. Broom A. K. , Lindsay M. D. , Plant A. J. , Wright A. E. , Condon R. J. , Mackenzie J. S. . ( 2002; ). Epizootic activity of Murray Valley encephalitis virus in an aboriginal community in the southeast Kimberley region of Western Australia: results of cross-sectional and longitudinal serologic studies. . Am J Trop Med Hyg 67:, 319–323.[PubMed]
    [Google Scholar]
  10. Cecilia D. , Gould E. A. . ( 1991; ). Nucleotide changes responsible for loss of neuroinvasiveness in Japanese encephalitis virus neutralization-resistant mutants. . Virology 181:, 70–77. [CrossRef] [PubMed]
    [Google Scholar]
  11. Chambers T. J. , Halevy M. , Nestorowicz A. , Rice C. M. , Lustig S. . ( 1998; ). West Nile virus envelope proteins: nucleotide sequence analysis of strains differing in mouse neuroinvasiveness. . J Gen Virol 79:, 2375–2380.[PubMed]
    [Google Scholar]
  12. Chen Y. , Maguire T. , Marks R. M. . ( 1996; ). Demonstration of binding of dengue virus envelope protein to target cells. . J Virol 70:, 8765–8772.[PubMed]
    [Google Scholar]
  13. Clark D. C. , Lobigs M. , Lee E. , Howard M. J. , Clark K. , Blitvich B. J. , Hall R. A. . ( 2007; ). In situ reactions of monoclonal antibodies with a viable mutant of Murray Valley encephalitis virus reveal an absence of dimeric NS1 protein. . J Gen Virol 88:, 1175–1183. [CrossRef] [PubMed]
    [Google Scholar]
  14. Clarke D. H. , Casals J. . ( 1958; ). Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. . Am J Trop Med Hyg 7:, 561–573.[PubMed]
    [Google Scholar]
  15. Gollins S. W. , Porterfield J. S. . ( 1985; ). Flavivirus infection enhancement in macrophages: an electron microscopic study of viral cellular entry. . J Gen Virol 66:, 1969–1982. [CrossRef] [PubMed]
    [Google Scholar]
  16. Gorman B. M. , Leer J. R. , Filippich C. , Goss P. D. , Doherty R. L. . ( 1975; ). Plaquing and neutralization of arboviruses in the PS-EK line of cells. . Aust J Med Technol 6:, 65–71.
    [Google Scholar]
  17. Halevy M. , Akov Y. , Ben-Nathan D. , Kobiler D. , Lachmi B. , Lustig S. . ( 1994; ). Loss of active neuroinvasiveness in attenuated strains of West Nile virus: pathogenicity in immunocompetent and SCID mice. . Arch Virol 137:, 355–370. [CrossRef] [PubMed]
    [Google Scholar]
  18. Hall R. A. , Khromykh A. A. , Mackenzie J. M. , Scherret J. H. , Khromykh T. I. , Mackenzie J. S. . ( 1999; ). Loss of dimerisation of the nonstructural protein NS1 of Kunjin virus delays viral replication and reduces virulence in mice, but still allows secretion of NS1. . Virology 264:, 66–75. [CrossRef] [PubMed]
    [Google Scholar]
  19. Heinz F. X. , Allison S. L. . ( 2003; ). Flavivirus structure and membrane fusion. . Adv Virus Res 59:, 63–97. [CrossRef] [PubMed]
    [Google Scholar]
  20. Heinz F. X. , Auer G. , Stiasny K. , Holzmann H. , Mandl C. , Guirakhoo F. , Kunz C. . ( 1994; ). The interactions of the flavivirus envelope proteins: implications for virus entry and release. . Arch Virol Suppl 9:, 339–348.[PubMed]
    [Google Scholar]
  21. Huang C. H. , Wong C. . ( 1963; ). Relation of the peripheral multiplication of Japanese B encephalitis virus to the pathogenesis of the infection in man. . Acta Virol 7:, 322–324.
    [Google Scholar]
  22. Hurrelbrink R. J. , McMinn P. C. . ( 2001; ). Attenuation of Murray Valley encephalitis virus by site-directed mutagenesis of the hinge and putative receptor-binding regions of the envelope protein. . J Virol 75:, 7692–7702. [CrossRef] [PubMed]
    [Google Scholar]
  23. Hurrelbrink R. J. , Nestorowicz A. , McMinn P. C. . ( 1999; ). Characterization of infectious Murray Valley encephalitis virus derived from a stably cloned genome-length cDNA. . J Gen Virol 80:, 3115–3125.[PubMed]
    [Google Scholar]
  24. Kawano H. , Rostapshov V. , Rosen L. , Lai C. J. . ( 1993; ). Genetic determinants of dengue type 4 virus neurovirulence for mice. . J Virol 67:, 6567–6575.[PubMed]
    [Google Scholar]
  25. Khromykh A. A. , Kenney M. T. , Westaway E. G. . ( 1998; ). trans-Complementation of flavivirus RNA polymerase gene NS5 by using Kunjin virus replicon-expressing BHK cells. . J Virol 72:, 7270–7279.[PubMed]
    [Google Scholar]
  26. Kimura T. , Gollins S. W. , Porterfield J. S. . ( 1986; ). The effect of pH on the early interaction of West Nile virus with P388D1 cells. . J Gen Virol 67:, 2423–2433. [CrossRef] [PubMed]
    [Google Scholar]
  27. Lee E. , Lobigs M. . ( 2002; ). Mechanism of virulence attenuation of glycosaminoglycan-binding variants of Japanese encephalitis virus and Murray Valley encephalitis virus. . J Virol 76:, 4901–4911. [CrossRef] [PubMed]
    [Google Scholar]
  28. Lee E. , Leang S. K. , Davidson A. , Lobigs M. . ( 2010; ). Both E protein glycans adversely affect dengue virus infectivity but are beneficial for virion release. . J Virol 84:, 5171–5180. [CrossRef] [PubMed]
    [Google Scholar]
  29. Lobigs M. , Marshall I. D. , Weir R. C. , Dalgarno L. . ( 1988; ). Murray Valley encephalitis virus field strains from Australia and Papua New Guinea: studies on the sequence of the major envelope protein gene and virulence for mice. . Virology 165:, 245–255. [CrossRef] [PubMed]
    [Google Scholar]
  30. Lobigs M. , Usha R. , Nestorowicz A. , Marshall I. D. , Weir R. C. , Dalgarno L. . ( 1990; ). Host cell selection of Murray Valley encephalitis virus variants altered at an RGD sequence in the envelope protein and in mouse virulence. . Virology 176:, 587–595. [CrossRef] [PubMed]
    [Google Scholar]
  31. Lorenz I. C. , Kartenbeck J. , Mezzacasa A. , Allison S. L. , Heinz F. X. , Helenius A. . ( 2003; ). Intracellular assembly and secretion of recombinant subviral particles from tick-borne encephalitis virus. . J Virol 77:, 4370–4382. [CrossRef] [PubMed]
    [Google Scholar]
  32. Mackenzie J. S. , Williams D. T. . ( 2009; ). The zoonotic flaviviruses of southern, south-eastern and eastern Asia, and Australasia: the potential for emergent viruses. . Zoonoses Public Health 56:, 338–356. [CrossRef] [PubMed]
    [Google Scholar]
  33. Mackenzie J. S. , Barrett A. D. , Deubel V. . ( 2002; ). The Japanese encephalitis serological group of flaviviruses: a brief introduction to the group. . Curr Top Microbiol Immunol 267:, 1–10.[PubMed]
    [Google Scholar]
  34. May F. J. , Lobigs M. , Lee E. , Gendle D. J. , Mackenzie J. S. , Broom A. K. , Conlan J. V. , Hall R. A. . ( 2006; ). Biological, antigenic and phylogenetic characterization of the flavivirus Alfuy. . J Gen Virol 87:, 329–337. [CrossRef] [PubMed]
    [Google Scholar]
  35. McMinn P. C. , Lee E. , Hartley S. , Roehrig J. T. , Dalgarno L. , Weir R. C. . ( 1995; ). Murray valley encephalitis virus envelope protein antigenic variants with altered hemagglutination properties and reduced neuroinvasiveness in mice. . Virology 211:, 10–20. [CrossRef] [PubMed]
    [Google Scholar]
  36. McMinn P. C. , Weir R. C. , Dalgarno L. . ( 1996; ). A mouse-attenuated envelope protein variant of Murray Valley encephalitis virus with altered fusion activity. . J Gen Virol 77:, 2085–2088. [CrossRef] [PubMed]
    [Google Scholar]
  37. Monath T. P. , Cropp C. B. , Bowen G. S. , Kemp G. E. , Mitchell C. J. , Gardner J. J. . ( 1980; ). Variation in virulence for mice and rhesus monkeys among St. Louis encephalitis virus strains of different origin. . Am J Trop Med Hyg 29:, 948–962.[PubMed]
    [Google Scholar]
  38. Mukhopadhyay S. , Kuhn R. J. , Rossmann M. G. . ( 2005; ). A structural perspective of the flavivirus life cycle. . Nat Rev Microbiol 3:, 13–22. [CrossRef] [PubMed]
    [Google Scholar]
  39. Porterfield J. S. , Rowe C. E. . ( 1960; ). Hemagglutination with arthropod-borne viruses and its inhibition by certain phospholipids. . Virology 11:, 765–770. [CrossRef] [PubMed]
    [Google Scholar]
  40. Pyke A. T. , Smith I. L. , van den Hurk A. F. , Northill J. A. , Chuan T. F. , Westacott A. J. , Smith G. A. . ( 2004; ). Detection of Australasian flavivirus encephalitic viruses using rapid fluorogenic TaqMan RT-PCR assays. . J Virol Methods 117:, 161–167. [CrossRef] [PubMed]
    [Google Scholar]
  41. Randolph V. B. , Stollar V. . ( 1990; ). Low pH-induced cell fusion in flavivirus-infected Aedes albopictus cell cultures. . J Gen Virol 71:, 1845–1850. [CrossRef] [PubMed]
    [Google Scholar]
  42. Reed L. J. , Muench H. . ( 1938; ). A simple method of estimating fifty per cent endpoints. . Am J Hyg 27:, 493–497.
    [Google Scholar]
  43. Rey F. A. , Heinz F. X. , Mandl C. , Kunz C. , Harrison S. C. . ( 1995; ). The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. . Nature 375:, 291–298. [CrossRef] [PubMed]
    [Google Scholar]
  44. Shirato K. , Miyoshi H. , Goto A. , Ako Y. , Ueki T. , Kariwa H. , Takashima I. . ( 2004; ). Viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the New York strain of West Nile virus. . J Gen Virol 85:, 3637–3645. [CrossRef] [PubMed]
    [Google Scholar]
  45. Thiel H. J. , Collett M. S. , Gould E. A. , Heinz F. X. , Meyers G. , Purcell R. H. , Rice C. M. , Houghton M. . ( 2005; ). Flaviviridae . . In Virus Taxonomy, Eighth Report of the International Committee for the Taxonomy of Viruses, pp. 981–998. Edited by Fauquet C. M. , Mayo M. A. , Maniloff J. , Desselberger U. , Ball L. A. . . San Diego, CA:: Academic Press;.
    [Google Scholar]
  46. Vorovitch M. F. , Timofeev A. V. , Atanadze S. N. , Tugizov S. M. , Kushch A. A. , Elbert L. B. . ( 1991; ). pH-dependent fusion of tick-borne encephalitis virus with artificial membranes. . Arch Virol 118:, 133–138. [CrossRef] [PubMed]
    [Google Scholar]
  47. Zhang Y. , Zhang W. , Ogata S. , Clements D. , Strauss J. H. , Baker T. S. , Kuhn R. J. , Rossmann M. G. . ( 2004; ). Conformational changes of the flavivirus E glycoprotein. . Structure 12:, 1607–1618. [CrossRef] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/vir.0.034793-0
Loading
/content/journal/jgv/10.1099/vir.0.034793-0
Loading

Data & Media loading...

vol. , part 10, pp. 2286–2296

Western blot of envelope protein digested with -glycosidase F. Primers used to generate mutant constructs.

[ Single PDF file] (296 KB)



PDF

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error