Skip to main content
PMC home page
The Journal of General Virology logoLink to The Journal of General Virology
. 2013 Apr;94(Pt 4):783–788. doi: 10.1099/vir.0.046664-0

Replacement of conserved or variable sequences of the mosquito-borne dengue virus 3′ UTR with homologous sequences from Modoc virus does not change infectivity for mosquitoes

Ebenezer Tumban 1,2,✉, Nyree E Maes 1,†, Erin E Schirtzinger 3, Katherine I Young 3, Christopher T Hanson 4, Stephen S Whitehead 4, Kathryn A Hanley 1,3
PMCID: PMC3709684  PMID: 23255623

Abstract

The genus Flavivirus includes both vector-borne and no known vector (NKV) species, but the molecular determinants of transmission mode are not known. Conserved sequence differences between the two groups occur in 5′ and 3′ UTRs. To investigate the impact of these differences on transmission, chimeric genomes were generated, in which UTRs, UTRs+capsid, or the upper 3′ UTR stem–loop of mosquito-borne dengue virus (DENV) were replaced with homologous regions from NKV Modoc virus (MODV); the conserved pentanucleotide sequence (CPS) was also deleted from the DENV genome. Virus was not recovered following transfection of these genomes in three different cell types. However, DENV genomes in which the CPS or variable region (VR) of the 3′ UTR were replaced with MODV sequences were recovered and infected Aedes aegypti mosquitoes with similar efficiencies to DENV. These results demonstrate that neither vector-borne CPS nor VR is required for vector-borne transmission.

The genus Flavivirus includes both vector-borne viruses and those with no known vector (NKV) (Billoir et al., 2000; Chambers et al., 1990; Cook & Holmes, 2006; Gaunt et al., 2001; Gritsun & Gould, 2006; Markoff, 2003). Attempts to infect ticks and mosquitoes as cultured cell lines or in vivo with NKV viruses have been unsuccessful, suggesting that this group is transmitted horizontally among vertebrates (Charlier et al., 2010; Fairbrother & Yuill, 1987; Hendricks et al., 1983; Johnson, 1967; Kuno, 2007; Lawrie et al., 2004). Vector-borne flaviviruses include important human pathogens such as dengue virus (DENV) (serotypes 1–4), yellow fever virus (YFV), tick-borne encephalitis virus (TBEV) and West Nile virus (WNV). Despite striking variation in the mechanisms of transmission within a single genus, the molecular determinants of transmission mode in the flaviviruses have not yet been identified.

Comparison of vector-borne and NKV flavivivirus genomes is facilitated by conservation of genome size, proteolytic cleavage site location and genome organization. Flaviviruses are positive-sense ssRNA viruses with a genome size of about 11 kb. The genome codes for a single ORF, which is processed by both cellular and viral proteases into three structural proteins and at least seven non-structural proteins (Chambers et al., 1990; Leyssen et al., 2002; Rice, 1996). The ORF is flanked by 5′ and the 3′ UTRs; both UTRs fold into complex stem–loop structures required for replication.

Charlier et al. (2010) investigated the role of the structural proteins in flavivirus infection of arthropod cells by generating chimeric genomes composed of the genomic backbone of one of two mosquito-borne flaviviruses, YFV or DENV, in which the structural genes were replaced with those of NKV Modoc virus (MODV). Transfection of mosquito C6/36 cells with the parent YFV or DENV genomes or the chimeric genomes yielded viable viruses, whereas MODV was incapable of infecting mosquito cells. Similarly Engel et al. (2011) have demonstrated that a chimeric DENV genome carrying the structural genes from TBEV retained the ability to infect mosquito cells in culture. Moreover, this chimeric construct failed to infect tick cells or larvae. These results suggest that the ability of a flavivirus to infect an arthropod vector is not determined by its structural proteins.

A sequence comparison suggests that key determinants of the transmission mode may reside in the UTRs of the flavivirus genome. Differences in conserved sequence motifs between vector-borne and NKV flaviviruses have been identified in the highly conserved 3′ stem–loop (3′ SL), the core region and the variable region (VR) of the 3′ UTR. For example, the conserved pentanucleotide sequence (CPS) CACAG located at the top of the 3′ SL is conserved in all vector-borne flaviviruses but differs from the most common sequence (CUCAG) found in NKV flaviviruses (Charlier et al., 2002; Leyssen et al., 2002; Markoff, 2003). Previous studies using WNV replicons have shown that replacement mutations in any but the fourth nucleotide position of the CPS abolishes replication (Tilgner et al., 2005). However, in other studies using WNV and YFV full-length genomic RNA, individual mutations of either the second, third or fourth CPS nucleotide were tolerated (Elghonemy et al., 2005; Silva et al., 2007). Some of these mutations mimicked the CPS of NKV flaviviruses but their impact on replication was tested only in mammalian cell lines. Within the VR, NKV flaviviruses have a conserved motif of 22 nt which is not present in vector-borne flaviviruses (Charlier et al., 2002). Finally, NKV and vector-borne flaviviruses each possess unique and complementary conserved sequences that circularize the genome during replication (Alvarez et al., 2005; Khromykh et al., 2001; Kofler et al., 2006). In order to investigate whether these conserved motifs in the UTRs dictate the mode of transmission, we generated chimeric constructs in which designated regions of the mosquito-borne DENV-4 genome were replaced with homologous regions of NKV MODV.

We replaced the 3′ UTR, alone or in combination with the 5′ UTR and/or capsid (C), of a full-length DENV-4 infectious clone with that of NKV MODV (Table S1, available in JGV Online). The 5′ UTR, C and 3′ UTR of MODV, synthesized by Blue Heron Biotechnology, were digested and ligated to plasmid p4, which contains the full-length cDNA genome of DENV-4 Dominica strain 814669 (GenBank accession no. AY648301) (Durbin et al., 2001). All plasmid constructs were sequenced up to and across cloning junctions to verify the authenticity of the replacement mutations.

First, we replaced the 5′ and 3′ UTRs of DENV-4 singly with those of MODV to generate p4-MODswap5′ UTR and p4MODswap3′ UTR, and then we replaced both DENV-4 UTRs together to generate p4-MODswap5′3′UTRs (Table S1). All constructs were linearized with Acc65I, which generates an authentic terminus of the rDENV-4 3′ UTR but leaves an extra G at the end of the MODV 3′ UTR. Capped mRNAs were synthesized from each linearized plasmid using Amplicap High Yield Message Maker kit (Epicentre technologies) according to the manufacturer’s instructions. Full-length RNA transcripts were purified using RNeasy kit (Qiagen) and transfection was conducted in duplicate in Aedes albopictus mosquito C6/36 cells, African green monkey kidney Vero cells or baby hamster kidney BHK-21 cells, essentially as previously described (Durbin et al., 2001; Hanley et al., 2002). MODV strain M544 (GenBank accession no. NC_003635), obtained from the World Arbovirus Reference Center, was passaged four times in Vero cells. Titre was determined by serial dilution and immunostaining (Durbin et al., 2001) on the same cell types as used for transfection; hyperimmune mouse ascites fluids against DENV-4 and MODV were used to detect recombinant DENV-4 (rDENV-4) and MODV, respectively. From a single C6/36 transfection pool of each virus, separate clones were isolated by terminal dilution as previously described (Blaney et al., 2001) in C6/36, Vero or BHK cells and working pools were prepared by amplifying terminally diluted viruses in the same cell type in which they were terminally diluted, yielding C6/36, BHK and Vero stocks of each virus.

As expected, wild-type rDENV-4 was recovered in all three cell lines, while MODV infected only the two mammalian cell lines (Charlier et al., 2010) (Table S1). No detectable virus was recovered from any of the chimeric genomes in any of the three cell types (Table S1). We hypothesized that the failure of these genomes to produce viable virus may have resulted from: (i) a mismatch in hypothetical packaging sequences in the UTRs and recognition sites in the C protein (Mandl et al., 1998; Men et al., 1996; Proutski et al., 1997) or (ii) a lack of a complementarity of the 5′ MODV cyclization sequence (5′ AAUGUGCGAAAAUAACAGG 3′, which extends into C) with the 3′ UTR cyclization sequence (3′ UUAAACCUUUUAUUGUCC 5′) (Leyssen et al., 2002) (the start codon is in bold, the C is in grey and base-paired nucleotides are not underlined).

To test these hypotheses, we next replaced the C gene in the rDENV-4-MODswap5′3′UTRs chimera with that of MODV, thus generating rDENV-4-MODswap5′C3′UTRs, which carried the complete C (including the circularization sequence above) and anchored C (Canch) sequence of MODV as well as complete C/pre-membrane (prM) signalase cleavage sites from both MODV and rDENV4 (Charlier et al., 2004). This construct also yielded no detectable virus following transfection. Finally, since previous research on chimeric flaviviruses (Huang et al., 2005; Pletnev et al., 2002) has suggested that homology between Canch, which helps to translocate the prM into the endoplasmic reticulum for post-translational maturation, and prM sequences may be critical for viability, we generated rDENV-4-MODswap5′C3′UTRs-dAn, which contained the Canch and the C/prM signallase cleavage site from rDENV-4. This construct also failed to generate viable virus (Table S1). Transfections that produced no detectable virus were repeated three times and all but MODswap5′C3′UTRs-dAn were further attempted by electroporation. In no case did a construct that failed to produce detectable virus following transfection produce any detectable virus following electroporation.

In previous work we have shown that chimeric genomes in which the 5′ UTR, C and 3′ UTR of rDENV-4 were replaced with those of tick-borne Langat virus (LGTV) also failed to generate viable virus (Tumban et al., 2011). However numerous studies have shown that structural genes of DENV can be replaced with those of either tick-borne (Lai & Monath, 2003) or NKV viruses (Charlier et al., 2010). Together with the current study, these results point to a fundamental incompatibility between the UTRs of NKV and tick-borne flaviviruses with the replication complex of mosquito-borne flaviviruses. The flavivirus replication complex includes NS1, NS2A, NS3, NS4A, NS4B, NS5, translation elongation factor 1α, La protein and polypyrimidine track-binding protein (Blackwell & Brinton, 1995; De Nova-Ocampo et al., 2002; Lai, 1998; Mackenzie et al., 1998; Umareddy et al., 2006). This replication complex interacts extensively with the 3′ UTR; for instance, NS3 and NS5 in association with NS2A have been shown to bind to the 3′ SL of WNV (Chen et al., 1997; Mackenzie et al., 1998). In addition, translation elongation factor 1α, La protein and polypyrimidine track-binding protein have been shown to bind to both the core region and the 3′ SL of flaviviruses (De Nova-Ocampo et al., 2002). More recently, host proteins such as DDX6, G3BP1 and Caprin1 have been shown to bind to the UTRs of DENV-2 (Ward et al., 2011). Thus, we propose that the failure of rDENV4-MODswap5′C3′UTR and rDENV4-MODswap5′C3′UTR-dAn to produce viable virus may have resulted from the inability of the rDENV-4 replication complex, in association with host proteins, to recognize the UTRs of MODV.

To better define which regions of the UTR may mediate compatibility with the replication complex, and to assess the impact of specific sequence motifs on the ability of DENV to infect mosquitoes, we generated replacement and deletion mutations (by standard PCR mutagenesis using the primers listed in Table S2) in both the 3′ SL and the VR. Consistent with previous studies (Silva et al., 2007), rDENV-4-ΔCPS, in which the CPS (CACAG, located at the top of 3′ SL) was deleted, did not yield detectable viruses in any of the three cell types (Table S1). rDENV-4-MODswapU3′SL containing a replacement of the upper 3′ SL of rDENV-4 with that of MODV also did not yield detectable viruses in any of the three cell types (Table S1). However, both rDENV-4-MODswapCPS (with an A→U mutation in the second nucleotide of the CPS) and rDENV-4-MODswapVR, in which the VR of rDENV-4 was replaced with that of MODV, yielded viable viruses following transfection in C6/36, Vero and BHK-21 cells (Table S1). Both constructs had post-transfection titres that were at least 1.3-fold and 100-fold lower than that of rDENV-4 in BHK-21 and Vero cells, respectively; both replicated to similar titres to their rDENV-4 parent in C6/36 cells.

Complete genomes of Vero and C6/36 stocks of rDENV-4, rDENV-4-MODswapCPS and rDENV-4-MODswapVR were sequenced (Table 1); of these, neither rDENV-4 stock possessed any mutations, both rDENV-4-MODswapCPS stocks possessed the same T1487P mutation in NS3, and both rDENV-4-MODswapVR stocks possessed a common D3308N mutation in NS5, although the C6/36-derived rDENV-4-MODswapVR stock also acquired one additional coding and one silent mutation in NS5. Common mutations probably arose during initial transfection in C6/36 cells but were not lost during terminal dilution and passage in the two different cell lines. As these mutations may represent compensatory mutations, it is not possible to unambiguously attribute the phenotypes of the mutant viruses to the MODV or VR replacements alone. However, it is possible to conclude that any phenotypes manifested by the mutant viruses were possible, despite the loss of specific DENV sequences.

Table 1. Mutations in wild-type (rDENV-4) and mutant viruses derived from a single transfection in C6/36 cells followed by separate passage in Vero or C6/36 cells.

Nucleotide and amino acid sequence numbering is from the start of the genome and the polyprotein, respectively. NA, Not applicable; Y, yes.

Virus Cell type Nucleotide mutation Amino acid mutation Gene Replacement of designated DENV sequence with MODV sequence confirmed?
rDENV-4 Vero None – – NA
rDENV-4-MODswapCPS Vero A4560C T1487P NS3 Y
rDENV-4-MODswapVR Vero G10023A D3308N NS5 Y
rDENV-4 C6/36 None – – NA
rDENV-4-MODswapCPS C6/36 A4560C T1487P NS3 Y
rDENV-4-MODswapVR C6/36 A7485C N2462H NS4B Y
A8342T Silent NS5 Y
G10023A D3308N NS5 Y
Open in a new tab

The size of 36 randomly chosen plaques (Blaney et al., 2003; Hanley et al., 2008) of Vero stocks of rDENV-4-MODswapCPS and rDENV-4-MODswapVR were compared with those of parent viruses in all three cell lines (Fig. 1). While MODV does not replicate in C6/36 cells, the mean plaque size of the two mutant viruses were similar to that of their rDENV-4 parent in this cell type. In mammalian cells, the mean plaque sizes of the two mutant viruses were similar to that of their rDENV-4 parent in both cell lines and were larger than those of MODV in Vero but not in BHK-21 cells.

Fig. 1.

Fig. 1.

Open in a new tab

Plaque sizes of rDENV-4, rDENV-4-MODswapCPS, rDENV-4-MODswapVR and MODV in C6/36, Vero and BHK-21 cells. The mean plaque diameter ±SEM (mm) of 36 randomly selected plaques is shown below each well. For each cell type, viruses that share the same letters (in parentheses) do not differ significantly from each other, while those with different letters have significantly different plaque sizes (Tukey–Kramer post-hoc test, P<0.05).

The phenotype of rDENV-4-MODswapVR is consistent with some previous studies showing that deletion of the VR of DENV and other flaviviruses did not affect viral replication in vitro or in vivo (Mandl et al., 1998; Tajima et al., 2006) but contrasts with studies, including a study that we conducted on the same rDENV-4 backbone in which the DENV VR was replaced with that of LGTV (Tumban et al., 2011), reporting that deletion or replacement of the VR decreased replication in mammalian cells but not in insect cells (Alvarez et al., 2005; Men et al., 1996; Pankhong et al., 2009; Tajima et al., 2007). These apparent discrepancies in the effect of the VR may stem from variation in the size of the deletions/replacements used in these studies. For example, the length of the VR replaced in our previous study (Tumban et al., 2011) was almost twice the size of the VR replaced in this study (225 versus 122 nt), which may explain the difference in results of VR replacement.

The ability of each of the mutant viruses to infect mosquitoes was compared with that of their two parental viruses by feeding Aedes aegypti, following previously described methods (Troyer et al., 2001), with bloodmeals containing 6.0–7.0 log10 p.f.u. ml−1 of Vero stocks of rDENV-4, MODV, rDENV-4-MODswapVR and rDENV-4-MODswapCPS, as well as C6/36 stocks of rDENV-4, rDENV-4-MODswapVR and rDENV-4-MODswapCPS. As expected, MODV did not infect any A. aegypti and both stocks of rDENV-4-infected A. aegypti (Table 2). At bloodmeal titres of approximately 6.5 log10 p.f.u. ml−1, Vero-derived rDENV-4-MODswapCPS and rDENV4-MODswapVR infected significantly fewer mosquitoes than rDENV-4 at 7.0 log10 p.f.u. ml−1 (Fisher's exact test, P<0.05 for both comparisons) but infected a similar percentage compared to rDENV-4 at 6.0 log10 p.f.u. ml−1 (Fisher's exact test, P>0.05 for both comparisons). Moreover, both mutant viruses disseminated with similar efficiency to that of rDENV-4 (statistical analysis not performed due to small sample sizes). The mean titre of both mutant viruses in the bodies of infected mosquitoes was similar to each other and to that of rDENV-4 at a bloodmeal titre of 7.0 log10 p.f.u. ml−1 (anova, df = 2, 18, F = 1.0, P = 0.39). Similar patterns were observed for C6/36-derived viruses: neither the per cent of bodies infected (contingency table analysis, Chi-squared = 2.96, P = 0.23) nor the body titre (anova, F = 2.39, P = 0.11) differed among rDENV-4, rDENV-4-MODswapVR and rDENV-4-MODswapCPS. Both rDENV-4-MODswapVR (Fisher’s exact test, P = 0.47) and rDENV-4-MODswapCPS (Fisher’s exact test, P = 0.30) disseminated with similar efficiency as rDENV-4 and head titres were similar among all three viruses (anova, F = 2.76, P = 0.10).

Table 2. Infection and dissemination in A. aegypti that were fed bloodmeals containing indicated virus.

Virus Derived from Titre of bloodmeal (log10 p.f.u. ml−1) Infection (body) Dissemination (head)*
No. No. infected (%) Mean virus titre±sem (log10 p.f.u. per body)† No. No. infected (%) Mean virus titre±sem (log10 p.f.u. per head)†
rDENV-4 Vero 7.0 21 10 (47.0 ) 3.7±0.2 10 8 (80.0) 2.6±0.3
rDENV-4 Vero 6.0 16 2 (12.5 ) 2.9±0.6 2 1 (50.0) 1.5±0
rDENV-4-MODswapCPS Vero 6.5 27 3 (11.0 ) 3.6±0.3 3 3 (100.0) 2.2±0.2
rDENV-4-MODswapVR Vero 6.6 25 3 (12.0) 3.5±0.1 3 1 (33.0) 2.4±0
MODV Vero 6.9 20 0 0 – – –
rDENV-4 C6/36 8.3 13 9 (69.2 ) 3.6±0.3 9 7 (77.8 ) 2.8±0.4
rDENV-4-MODswapCPS C6/36 7.8 18 7 (38.8) 3.1±0.4 7 3 (42.9 ) 2.3±0.3
rDENV-4-MODswapVR C6/36 7.9 14 8 (57.1 ) 4.0±0.2 8 8 (100 ) 3.5±0.2
Open in a new tab
*

Only heads from infected bodies were screened.

†

Titres from infected samples only.

The threat from vector-borne flaviviruses is increasing as the efficacy of traditional vector control measures wane (Gubler, 2006; Mackenzie et al., 2004). The development of a licensed live virus vaccine is challenging, in part, because vector transmission from vaccinees to unvaccinated populations must be prevented. Identifying regions in the genomes of flaviviruses that determine vector transmission can serve as a basis for the design of live attenuated vaccine strains that lack the capacity for vector transmission. The current study has demonstrated that neither the VR nor the CPS of mosquito-borne DENV is necessary for transmission by mosquitoes.

Acknowledgements

We would like to thank Dr Robert Tesh for providing us with Modoc virus. We would also like to thank Dr Barbara Lyons, Dr Steve Hanson and Dr Jeffrey Arterburn for their suggestions. This study was supported in part by NIH-K22-A164193, NIH-NM-INBRE P20 RR016480-05, NSF-NM-AMP HRD-0331446 and the Intramural Research Program of the NIH, NIAID.

Footnotes

Two supplementary tables are available with the online version of this paper.

References

  1. Alvarez D. E., De Lella Ezcurra A. L., Fucito S., Gamarnik A. V. (2005). Role of RNA structures present at the 3′UTR of dengue virus on translation, RNA synthesis, and viral replication. Virology 339, 200–212 10.1016/j.virol.2005.06.009 [DOI] [PubMed] [Google Scholar]
  2. Billoir F., de Chesse R., Tolou H., Gould E. A., de Micco P., Gould E. A., de Lamballerie X. (2000). Phylogeny of the genus Flavivirus using complete coding sequences of arthropod-borne viruses and viruses with no known vector. J Gen Virol 81, 2339. [DOI] [PubMed] [Google Scholar]
  3. Blackwell J. L., Brinton M. A. (1995). BHK cell proteins that bind to the 3′ stem–loop structure of the West Nile virus genome RNA. J Virol 69, 5650–5658 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blaney J. E., Jr, Johnson D. H., Firestone C. Y., Hanson C. T., Murphy B. R., Whitehead S. S. (2001). Chemical mutagenesis of dengue virus type 4 yields mutant viruses which are temperature sensitive in Vero cells or human liver cells and attenuated in mice. J Virol 75, 9731–9740 10.1128/JVI.75.20.9731-9740.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blaney J. E., Jr, Manipon G. G., Firestone C. Y., Johnson D. H., Hanson C. T., Murphy B. R., Whitehead S. S. (2003). Mutations which enhance the replication of dengue virus type 4 and an antigenic chimeric dengue virus type 2/4 vaccine candidate in Vero cells. Vaccine 21, 4317–4327 10.1016/S0264-410X(03)00487-0 [DOI] [PubMed] [Google Scholar]
  6. Chambers T. J., Hahn C. S., Galler R., Rice C. M. (1990). Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44, 649–688 10.1146/annurev.mi.44.100190.003245 [DOI] [PubMed] [Google Scholar]
  7. Charlier N., Leyssen P., Pleij C. W., Lemey P., Billoir F., Van Laethem K., Vandamme A. M., De Clercq E., de Lamballerie X., Neyts J. (2002). Complete genome sequence of Montana Myotis leukoencephalitis virus, phylogenetic analysis and comparative study of the 3′ untranslated region of flaviviruses with no known vector. J Gen Virol 83, 1875–1885 [DOI] [PubMed] [Google Scholar]
  8. Charlier N., Molenkamp R., Leyssen P., Paeshuyse J., Drosten C., Panning M., De Clercq E., Bredenbeek P. J., Neyts J. (2004). Exchanging the yellow fever virus envelope proteins with Modoc virus prM and E proteins results in a chimeric virus that is neuroinvasive in SCID mice. J Virol 78, 7418–7426 10.1128/JVI.78.14.7418-7426.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Charlier N., Davidson A., Dallmeier K., Molenkamp R., De Clercq E., Neyts J. (2010). Replication of not-known-vector flaviviruses in mosquito cells is restricted by intracellular host factors rather than by the viral envelope proteins. J Gen Virol 91, 1693–1697 10.1099/vir.0.019851-0 [DOI] [PubMed] [Google Scholar]
  10. Chen C. J., Kuo M. D., Chien L. J., Hsu S. L., Wang Y. M., Lin J. H. (1997). RNA–protein interactions: involvement of NS3, NS5, and 3′ noncoding regions of Japanese encephalitis virus genomic RNA. J Virol 71, 3466–3473 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cook S., Holmes E. C. (2006). A multigene analysis of the phylogenetic relationships among the flaviviruses (family: Flaviviridae) and the evolution of vector transmission. Arch Virol 151, 309–325 10.1007/s00705-005-0626-6 [DOI] [PubMed] [Google Scholar]
  12. De Nova-Ocampo M., Villegas-Sepúlveda N., del Angel R. M. (2002). Translation elongation factor-1α, La, and PTB interact with the 3′ untranslated region of dengue 4 virus RNA. Virology 295, 337–347 10.1006/viro.2002.1407 [DOI] [PubMed] [Google Scholar]
  13. Durbin A. P., Karron R. A., Sun W., Vaughn D. W., Reynolds M. J., Perreault J. R., Thumar B., Men R., Lai C. J. & other authors (2001). Attenuation and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3′-untranslated region. Am J Trop Med Hyg 65, 405–413 [DOI] [PubMed] [Google Scholar]
  14. Elghonemy S., Davis W. G., Brinton M. A. (2005). The majority of the nucleotides in the top loop of the genomic 3′ terminal stem loop structure are cis-acting in a West Nile virus infectious clone. Virology 331, 238–246 10.1016/j.virol.2004.11.008 [DOI] [PubMed] [Google Scholar]
  15. Engel A. R., Mitzel D. N., Hanson C. T., Wolfinbarger J. B., Bloom M. E., Pletnev A. G. (2011). Chimeric tick-borne encephalitis/dengue virus is attenuated in Ixodes scapularis ticks and Aedes aegypti mosquitoes. Vector Borne Zoonotic Dis 11, 665–674 10.1089/vbz.2010.0179 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fairbrother A., Yuill T. M. (1987). Experimental infection and horizontal transmission of Modoc virus in deer mice (Peromyscus maniculatus). J Wildl Dis 23, 179–185 [DOI] [PubMed] [Google Scholar]
  17. Gaunt M. W., Sall A. A., de Lamballerie X., Falconar A. K., Dzhivanian T. I., Gould E. A. (2001). Phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography. J Gen Virol 82, 1867–1876 [DOI] [PubMed] [Google Scholar]
  18. Gritsun T. S., Gould E. A. (2006). Origin and evolution of 3′UTR of flaviviruses: long direct repeats as a basis for the formation of secondary structures and their significance for virus transmission. Adv Virus Res 69, 203–248 10.1016/S0065-3527(06)69005-2 [DOI] [PubMed] [Google Scholar]
  19. Gubler D. J. (2006). Dengue/dengue haemorrhagic fever: history and current status. Novartis Found Symp 277, 3–16 discussion 16–22, 71–73, 251–253. 10.1002/0470058005.ch2 [DOI] [PubMed] [Google Scholar]
  20. Hanley K. A., Lee J. J., Blaney J. E., Jr, Murphy B. R., Whitehead S. S. (2002). Paired charge-to-alanine mutagenesis of dengue virus type 4 NS5 generates mutants with temperature-sensitive, host range, and mouse attenuation phenotypes. J Virol 76, 525–531 10.1128/JVI.76.2.525-531.2002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hanley K. A., Nelson J. T., Schirtzinger E. E., Whitehead S. S., Hanson C. T. (2008). Superior infectivity for mosquito vectors contributes to competitive displacement among strains of dengue virus. BMC Ecol 8, 1 10.1186/1472-6785-8-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hendricks D. A., Hardy J. L., Reeves W. C. (1983). Comparison of biological properties of St. Louis encephalitis and Rio bravo viruses. Am J Trop Med Hyg 32, 602–609 [DOI] [PubMed] [Google Scholar]
  23. Huang C. Y., Silengo S. J., Whiteman M. C., Kinney R. M. (2005). Chimeric dengue 2 PDK-53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus. J Virol 79, 7300–7310 10.1128/JVI.79.12.7300-7310.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Johnson H. N. (1967). Ecological implications of antigenically related mammalian viruses for which arthropod vectors are unknown and avian associated soft tick viruses. Jpn J Med Sci Biol 20 (Suppl.), 160–166 [PubMed] [Google Scholar]
  25. Khromykh A. A., Meka H., Guyatt K. J., Westaway E. G. (2001). Essential role of cyclization sequences in flavivirus RNA replication. J Virol 75, 6719–6728 10.1128/JVI.75.14.6719-6728.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kofler R. M., Hoenninger V. M., Thurner C., Mandl C. W. (2006). Functional analysis of the tick-borne encephalitis virus cyclization elements indicates major differences between mosquito-borne and tick-borne flaviviruses. J Virol 80, 4099–4113 10.1128/JVI.80.8.4099-4113.2006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kuno G. (2007). Host range specificity of flaviviruses: correlation with in vitro replication. J Med Entomol 44, 93–101 10.1603/0022-2585(2007)44[93:HRSOFC]2.0.CO;2 [DOI] [PubMed] [Google Scholar]
  28. Lai M. M. (1998). Cellular factors in the transcription and replication of viral RNA genomes: a parallel to DNA-dependent RNA transcription. Virology 244, 1–12 10.1006/viro.1998.9098 [DOI] [PubMed] [Google Scholar]
  29. Lai C. J., Monath T. P. (2003). Chimeric flaviviruses: novel vaccines against dengue fever, tick-borne encephalitis, and Japanese encephalitis. Adv Virus Res 61, 469–509 10.1016/S0065-3527(03)61013-4 [DOI] [PubMed] [Google Scholar]
  30. Lawrie C. H., Uzcátegui N. Y., Armesto M., Bell-Sakyi L., Gould E. A. (2004). Susceptibility of mosquito and tick cell lines to infection with various flaviviruses. Med Vet Entomol 18, 268–274 10.1111/j.0269-283X.2004.00505.x [DOI] [PubMed] [Google Scholar]
  31. Leyssen P., Charlier N., Lemey P., Billoir F., Vandamme A. M., De Clercq E., de Lamballerie X., Neyts J. (2002). Complete genome sequence, taxonomic assignment, and comparative analysis of the untranslated regions of the Modoc virus, a flavivirus with no known vector. Virology 293, 125–140 10.1006/viro.2001.1241 [DOI] [PubMed] [Google Scholar]
  32. Mackenzie J. M., Khromykh A. A., Jones M. K., Westaway E. G. (1998). Subcellular localization and some biochemical properties of the flavivirus Kunjin non-structural proteins NS2A and NS4A. Virology 245, 203–215 10.1006/viro.1998.9156 [DOI] [PubMed] [Google Scholar]
  33. Mackenzie J. S., Gubler D. J., Petersen L. R. (2004). Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat Med 10 (Suppl.), S98–S109 10.1038/nm1144 [DOI] [PubMed] [Google Scholar]
  34. Mandl C. W., Holzmann H., Meixner T., Rauscher S., Stadler P. F., Allison S. L., Heinz F. X. (1998). Spontaneous and engineered deletions in the 3′ non-coding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus. J Virol 72, 2132–2140 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Markoff L. (2003). 5′- and 3′-non-coding regions in flavivirus RNA. Adv Virus Res 59, 177–228 10.1016/S0065-3527(03)59006-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Men R., Bray M., Clark D., Chanock R. M., Lai C. J. (1996). Dengue type 4 virus mutants containing deletions in the 3′ non-coding region of the RNA genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys. J Virol 70, 3930–3937 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pankhong P., Weiner D. B., Ramanathan M. P., Nisalak A., Kalayanarooj S., Nimmannitya S., Attatippaholkun W. (2009). Molecular genetic relationship of the 3′ untranslated region among Thai dengue-3 virus, Bangkok isolates, during 1973–2000. DNA Cell Biol 28, 481–491 10.1089/dna.2008.0835 [DOI] [PubMed] [Google Scholar]
  38. Pletnev A. G., Putnak R., Speicher J., Wagar E. J., Vaughn D. W. (2002). West Nile virus/dengue type 4 virus chimeras that are reduced in neurovirulence and peripheral virulence without loss of immunogenicity or protective efficacy. Proc Natl Acad Sci U S A 99, 3036–3041 10.1073/pnas.022652799 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Proutski V., Gaunt M. W., Gould E. A., Holmes E. C. (1997). Secondary structure of the 3′-untranslated region of yellow fever virus: implications for virulence, attenuation and vaccine development. J Gen Virol 78, 1543–1549 [DOI] [PubMed] [Google Scholar]
  40. Rice C. M. (1996). Flaviviridae: the viruses and their replication. In Fields Virology, 3rd edn, pp. 931–959 Edited by Fields B. N., Knipe D. M., Howley P. M., Chanock R. M., Melnick J. L., Monath T. P., Roizman B., Straus S. E. Philadelphia: Lippincott-Raven Publishers [Google Scholar]
  41. Silva P. A., Molenkamp R., Dalebout T. J., Charlier N., Neyts J. H., Spaan W. J., Bredenbeek P. J. (2007). Conservation of the pentanucleotide motif at the top of the yellow fever virus 17D 3′ stem–loop structure is not required for replication. J Gen Virol 88, 1738–1747 10.1099/vir.0.82811-0 [DOI] [PubMed] [Google Scholar]
  42. Tajima S., Nukui Y., Ito M., Takasaki T., Kurane I. (2006). Nineteen nucleotides in the variable region of 3′ non-translated region are dispensable for the replication of dengue type 1 virus in vitro. Virus Res 116, 38–44 10.1016/j.virusres.2005.08.015 [DOI] [PubMed] [Google Scholar]
  43. Tajima S., Nukui Y., Takasaki T., Kurane I. (2007). Characterization of the variable region in the 3′ non-translated region of dengue type 1 virus. J Gen Virol 88, 2214–2222 10.1099/vir.0.82661-0 [DOI] [PubMed] [Google Scholar]
  44. Tilgner M., Deas T. S., Shi P. Y. (2005). The flavivirus-conserved penta-nucleotide in the 3′ stem–loop of the West Nile virus genome requires a specific sequence and structure for RNA synthesis, but not for viral translation. Virology 331, 375–386 10.1016/j.virol.2004.07.022 [DOI] [PubMed] [Google Scholar]
  45. Troyer J. M., Hanley K. A., Whitehead S. S., Strickman D., Karron R. A., Durbin A. P., Murphy B. R. (2001). A live attenuated recombinant dengue-4 virus vaccine candidate with restricted capacity for dissemination in mosquitoes and lack of transmission from vaccinees to mosquitoes. Am J Trop Med Hyg 65, 414–419 [DOI] [PubMed] [Google Scholar]
  46. Tumban E., Mitzel D. N., Maes N. E., Hanson C. T., Whitehead S. S., Hanley K. A. (2011). Replacement of the 3′ untranslated variable region of mosquito-borne dengue virus with that of tick-borne Langat virus does not alter vector specificity. J Gen Virol 92, 841–848 10.1099/vir.0.026997-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Umareddy I., Chao A., Sampath A., Gu F., Vasudevan S. G. (2006). Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA. J Gen Virol 87, 2605–2614 10.1099/vir.0.81844-0 [DOI] [PubMed] [Google Scholar]
  48. Ward A. M., Bidet K., Yinglin A., Ler S. G., Hogue K., Blackstock W., Gunaratne J., Garcia-Blanco M. A. (2011). Quantitative mass spectrometry of DENV-2 RNA-interacting proteins reveals that the DEAD-box RNA helicase DDX6 binds the DB1 and DB2 3′ UTR structures. RNA Biol 8, 1173–1186 10.4161/rna.8.6.17836 [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of General Virology are provided here courtesy of Microbiology Society

RESOURCES