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Arthropod-Borne and Infectious Disease Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA
Correspondence
Ken E. Olson
kolson{at}colostate.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Q (histidine to glutamine) and E2-70 KE (lysine to glutamic acid). We have characterized the growth patterns of derived viruses in cell culture and determined the midgut infection rate (MIR) in A. aegypti mosquitoes. Our results clearly show that the E2-55 HQ and the E2-70 KE mutations in the TE/5'2J virus increase MIR both independently and in combination. TE/5'2J virus containing both TR339 E2 residues had MIRs similar to the parental TR339 virus. In addition, SINV propagated in a mammalian cell line had a significantly lower A. aegypti midgut 50 % infectious dose than virus propagated in a mosquito cell line. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Strauss & Strauss, 1994). The genome contains a 5' 7-methylguanosine cap with a 3' poly(A) tail and is directly translated into a polyprotein that is co- and post-translationally processed into the four non-structural proteins that make up the virus replication complex. This complex produces a negative-sense RNA molecule that is used as a template for production of genome-length as well as subgenomic RNA strands. The subgenomic RNA encodes a polyprotein that is co- and post-translationally processed into the five structural proteins which are (in genome order) the capsid, envelope (E) 3, envelope 2 (E2), 6K and envelope 1 (E1).
The SINV outer protein shell structure is an icosahedron, with a threefold axis of symmetry, composed of E1–E2 heterotrimers arranged in a T=4 lattice. The outer shell surface can be sectioned into an inner base (skirt) region composed mostly of laterally lying E1 and an outer region that is thought to be dominated by E2 trimers laced with E1 (Anthony & Brown, 1991; Paredes et al., 2004; Pletnev et al., 2001; Zhang et al., 2002). The SINV E2 glycoprotein is approximately 51 kDa and rises nearly 50 Å (5 nm) above the skirt region (Paredes et al., 2004; Pletnev et al., 2001). The E2 of many alphaviruses, including SINV, Venezuelan equine encephalitis virus (VEEV) and Ross River virus, is sensitive to neutralizing antibodies (Agapov et al., 1994; Johnson et al., 1990; Pence et al., 1990; Pereboev et al., 1996; Strauss et al., 1991; Ubol & Griffin, 1991; Vrati et al., 1996). A number of sites involved in virus neutralization have been found between aa 170 and 220 of E2 (SINV positions), which may be a putative cell receptor-binding domain (CRBD) for susceptible cells (Strauss et al., 1991). The specific cell surface receptor(s) for SINV infection have not been definitively identified, although both high-affinity laminin receptors and heparin sulphate receptors have been implicated as attachment proteins for SINV (Byrnes & Griffin, 1998; Klimstra et al., 1998; Wang et al., 1992).
SINVs are arthropod-borne viruses (arboviruses) that are maintained in nature in a transmission cycle involving mosquito species, such as Culex spp., Culiseta spp. and Aedes spp., and wild birds (Doherty et al., 1977). Aedes aegypti mosquitoes, the primary vector for yellow fever and dengue viruses, are susceptible to several SINV strains in the laboratory. Transmission of SINV from a mammalian host to a mosquito is initiated by the mosquito probing the skin of an infected host to penetrate the vasculature with their proboscis. Blood pools are then siphoned through the cannula and the arbovirus enters the lumen of the mosquito midgut. It is at this point that many arboviruses, including SINVs, encounter a mosquito midgut infection barrier (MIB), in the form of a monolayer of potentially virus-incompatible midgut epithelial cells (MEC) (Woodring, 1996). The arbovirus MIB is thought to be a result of genetic factors of both the virus and vector (Bennett et al., 2002; Black et al., 2002; Brault et al., 2002). Several VEEV and SINV studies have shown that the alphavirus genetic determinants of midgut infection are associated with the E2 glycoprotein (Myles et al., 2003; Pierro et al., 2003; Weaver et al., 2004; Woodward et al., 1991).
The prototype SINV, strain AR339, was originally isolated from a pool of Culex pipiens and Culex univittatus mosquitoes collected in Egypt (Taylor et al., 1955). A full-length cDNA infectious clone (ic), termed TR339, that represents the putative AR339 consensus sequence, has been generated (Klimstra et al., 1998; McKnight et al., 1996). We have previously characterized the infection rate and distribution of C6/36 mosquito cell-propagated TR339 virus in the midgut of A. aegypti mosquitoes and found a greater than 90 % midgut infection rate (MIR) 7 days post-infection (p.i.) (Myles et al., 2004). In contrast, the recombinant SINV strain TE/5'2J, a double subgenomic SINV often used to deliver and express genes of interest in vector species, was constructed from a chimeric mouse neurovirulent variant of AR339 called TE12 (Lustig et al., 1988), and infects less than 15 % of mosquitoes when analysed similarly (Pierro et al., 2003). Sequence comparison between the SINV TE12 and TR339 genomes predicts two amino acid differences (HQ and KE) in the E2 glycoprotein at amino acid positions 55 and 70 (E2-H55Q and E2-K70E, respectively). A. aegypti, together with these SINV, give us a unique model system to investigate more specifically the arboviral genetic determinants of midgut infection.
Here, we describe the use of SINV strain TE/5'2J with mutations at E2-55 and E2-70 to characterize the viral genetic determinants of mosquito midgut infections. Our results show that the TE/5'2J viruses with either E2-H55Q or E2-K70E mutations affected the MIR. We also found that the E2-H55Q and E2-K70E mutations, when together in TE/5'2J, had an additive affect on MIR and resulted in an MIR similar to that of the parental TR339 virus. In addition, we observed that the cell type used in virus amplification as well as the infectious dose had a significant impact on the MIR.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Infectious clones.
The cDNA ic pTE/5'2J was generously provided by Dr Charles Rice (Rockefeller University, New York, USA) and its use has been previously described (Hahn et al., 1992; Pierro et al., 2003). The cDNA ic pTR339 was generously provided by Dr Robert Johnston (University of North Carolina, Chapel Hill, NC, USA). The derived consensus sequence of TR339 and its use has been described previously (Klimstra et al., 1998; McKnight et al., 1996; Myles et al., 2004).
Following sequencing of the E2 gene of TE/5'2J, primers were engineered for site-directed mutagenesis of pTE/5'2J at nucleotides encoding the E2-55 and E2-70 residues. Using the entire pTE/5'2J plasmid as the template, the primer sets 5'-CGCCCAGTTTGGATACGACCAAAGCGGAGCCGCAAGCGC-3', 5'-GCGCTTGCTGCTCCGCTTTGGTCGTATCCAAACTGGGCG-3' and 5'-GTACCGCTACATGTCGCTTGAGCAGGATCACACCG-3', 5'-CGGTGTGATCCTGCTCAAGCGACATGTAGCGGTAC-3' were used to mutate the pTE/5'2J cDNA to contain either glutamine at E2-55 (pTE/E2-H55Q), glutamic acid at E2-70 (pTE/E2-K70E) or both (pTE/E2-55Q/70E). The site-directed mutagenesis reaction (sPCR) used a QuikChange site-directed mutagenesis kit (Stratagene). Mutation insertion was verified by sequence analysis of the plasmid DNA as well as the virus-derived RNA genome via RT-PCR. TE/E2-55Q/70E–GFP was created by insertion of enhanced green fluorescent protein (GFP) into the second subgenomic region of TE/E2-55Q/70E as previously described (Pierro et al., 2003). The amino acid alignments in Fig. 1 used the Pfam database and the colour codes used for clusters are from Jalview (Finn et al., 2006). The predictions for secondary structure and solvent access in Fig. 1 were performed by PredictProtein (Rost et al., 2004).
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; Pierro et al., 2003). Virus was generated from the infectious clone (i.e. P-0) and then passaged once (P-1) for use. RNA from P-1 virus was isolated, the E2 gene was fully sequenced and no adaptive mutations were identified. Plaque titrations of SINV were performed by infecting confluent monolayers of Vero cells as described previously (Miller & Mitchell, 1986). Plaque sizes of P-0 were analysed on Vero cells 5 days p.i. Growth curve analysis was performed using confluent monolayer of cells infected in triplicate with P-0 virus at an m.o.i. of 0.01. Indirect immunofluorescence assays (IFA) were performed on tissue culture cells, mosquito bodies and midgut tissues using an anti-SINV-E1 antibody (30.11a) as the primary antibody (1 : 150), as previously described (Myles et al., 2003).
Mosquitoes and oral infections.
A. aegypti strain Rexville D (RexD) use and the blood-meal delivery mechanism were as previously described (Myles et al., 2004). The propagation of virus used in blood meals started with confluent monolayers of either Vero or C6/36 cells that were infected with P-0 virus at an m.o.i. of approximately 0.01 in 8 % FBS/MEM and cultured in CO2 incubators for 2 days at 37 °C (Vero) or 3 days at 28 °C (C6/36). After incubation, freshly harvested P-1 cell-culture supernatant (i.e. non-frozen) containing infectious virions was, when applicable, diluted in uninfected conditioned cell-culture supernatant (final volume of 0.8 ml) to a predicted viral titre using the standardized growth curve data as a guide (Fig. 2). The pre-titrated virus was then mixed with 0.8 ml defibrinated sheep blood (Colorado Serum Co.). This infectious blood meal mixture was warmed to 37 °C and placed in a water-jacket-heated (37 °C) glass membrane feeder. Mosquitoes were allowed to probe and feed through a stretched sheet of Parafilm for no more than 30 min. Fully engorged mosquitoes were collected and maintained in the insectary with ample food and water until assayed. The viral blood meal was frozen and later quantified by plaque titration; the titre was considered acceptable at approximately 0.3 log10(p.f.u. ml–1) of the predicted viral titre. Post-blood-meal viral titres were found to be reduced by only approximately 10 % when compared with pre-blood-meal titres (data not shown).
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2 test with an 
cut off of 0.05 using SAS/STAT statistical software (SAS). Differences between slopes of the MIR were analysed by a logistic regression model fitting responses (1, 0) over dose and virus and dosexvirus with a binomial distribution and logistic link using SAS statistical software. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The second variation was a guanosine at nucleotide position 8838 in the TR339 genome, compared with an adenosine in TE/5'2J. This resulted in a glutamic acid (E) residue at position E2-70 in TR339 and a lysine (K) residue in TE5'2J.
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). The TR339 E2-55 Q residue clustered with the hydroxyl and amine groups, while the TE/5'2J H residue did not. Interestingly, none of the E2-70 residues from the panel of alphaviruses clustered together; however, a deletion region (E2 70–72) was observed. Secondary structure prediction analysis identified potential loop regions (identified as 
in Fig. 1) spanning both the E2-55 and the E2-70 residues, which were separated by a 
sheet. Interestingly, the TE/5'2J E2-55 H residue appeared to remove the predicted loop phenotype at E2-55 and add it to the serine (S) at E2-60. The predicted solvent-accessible residues (bold type in secondary structure in Fig. 1) were identified next to E2-55 and at E2-70 for both TR339 and TE/5'2J. Moreover, the predicted solvent accessibility of the S at E2-60 appears to be affected by the E2-55 H of TE/5'2J.
Generation and characterization of SINV mutants in tissue culture
Primers were designed to mutate E2-55 and E2-70 of TE/5'2J to encode the residues of TR339, resulting in the ic plasmids SINV TE/E2-H55Q, TE/E2-K70E and TE/E2-55Q/70E (Table 1). Virus replication rates were analysed by growth curve analysis in both invertebrate (C6/36 and Aag2) and vertebrate (Vero and BHK) cell lines (Fig. 2). Maximum virus titres were achieved at 60 and 36 h p.i. for viruses propagated in C6/36 and Aag2 cells, respectively, and 24–36 h for propagation in either Vero or BHK cells. The maximum virus titres [log10(p.f.u. ml–1)] ranged from 9.1 to 9.9, 7.8 to 8.9, 7.9 to 8.6 and 7.6 to 8.4 in C6/36, Aag2, Vero and BHK cells, respectively. Interestingly, the propagation characteristic of TE/5'2J in Aag2 cells had the lowest maximal titre of all five viruses and also failed to maintain a persistent high titre over time. We also observed that plaque sizes were larger for viruses that had an E at E2-70 (data not shown) as has been previously reported (McKnight et al., 1996).
Pattern of SINV infection of midguts
All of the viruses initiated an infection of the midgut with a discrete focus of infection that spread to adjacent MECs, resulting in an expanding focus (Fig. 3). One to four foci of infection were observed in SINV-infected RexD midguts 8–9 days p.i. regardless of the virus used. Neither virus nor cell type had an influence on the average number of foci per midgut (data not shown). Moreover, the spatial and temporal midgut infection pattern for mosquitoes having a disseminated infection appeared similar to those with only a midgut infection, but no dissemination.
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). The influence of infectious dose can be evaluated by comparing each of the virus’ specific MID50, which can be estimated from Fig. 4 (shaded horizontal bar in MIR); the data are presented in Table 1. A lower MID50 is an indication of an enhanced efficiency at which a virus establishes an infection in midgut cells. The MID50 was analysed for viruses propagated in C6/36 cells and in Vero cells. The TR339 virus had the lowest MID50 (7.5 and 6 log10, respectively), and TE/5'2J had the highest MID50 (>9.5 and 7.5 log10, respectively). The remaining mutants had intermediate MID50s.
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) for each virus using the MIR data from Fig. 4. We determined by logistic regression modelling that there was no significant difference between the slopes (i.e. virusxdose) of any pair of viruses for either Vero (P>0.28) or C6/36 (P>0.2) cell-derived viruses.
Effect of propagation cell type on MIR
The relative effect of cell type used for virus propagation on MIR was analysed by standardizing blood meal titres for viruses propagated in C6/36 and Vero cells (Fig. 5). We observed that at a blood meal titre of 7.5 log10 (p.f.u. ml–1), each of the viruses generated in C6/36 cells had a significantly lower mean MIR compared with the same viruses generated in Vero cells (P<0.001). Moreover, comparing the difference between the MID50s of virus propagated in Vero and C6/36 cells (represented under 
in Table 1) indicates the impact of the propagation cell type on the MIR. From Table 1, TE/5'2J had the largest MID50 (>2 log10), indicating that the cell type used in propagating this virus had at least a 2 log impact on MIR. The MID50 for the remaining viruses ranged from 1.25 to 1.75. These data indicated that at least 1.0 log10(p.f.u. ml–1) higher titre of virus in the blood meal was required for viruses propagated in C6/36 cells than in Vero cells to achieve an equivalent MID50.
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). These viruses also had the highest and the lowest MIRs, respectively, of all viruses tested, at each specific blood meal titre analysed. Changing the propagation cell type did not change the observations above.
The generation of TE/5'2J viruses containing the site-specific E2 amino acid residues of TR339 and subsequent MIR assays revealed the genetic determinants of SINV midgut infections. Specifically, the MIR for TE/E2-55Q/70E was not statistically significantly different from TR339 (P>0.5), but was higher than the MIR of TE/5'2J (P<0.001) for both Vero and C6/36 cell line-derived virus at the standardized blood meal titre of 7.5 log10(p.f.u. ml–1) (Fig. 5). Using the same titre, the Vero-derived TE/E2-K70E virus had statistically significantly higher MIRs than TE/5'2J (P=0.0105), and TE/E2-H55Q had a statistically significantly lower MIR (P=0.0152) than the TE/E2-55Q/70E virus (Fig. 5).
The three C6/36-derived mutant viruses all had significantly higher MIRs than C6/36-derived TE/5'2J (P<0.0142) at 7.5 log10(p.f.u. ml–1) (Fig. 5). Moreover, the C6/36-propagated TE/E2-55Q/70E had MIRs significantly higher than C6/36-derived TE/E2-H55Q and TE/E2-K70E viruses (P=0.0082 and P=0.0039, respectively). MIRs for TE/E2-H55Q and TE/E2-K70E did not differ statistically significantly from each other at the blood meal titre of 7.5 log10(p.f.u. ml–1) for either Vero- or C6/36-derived viruses (P=0.2564 and P=0.8617, respectively; Fig. 5). The independent effect of the two TR339 residues, E2-55Q and E2-70E, on the MIR appears to be additive.
Extrinsic viral characteristics of TE/5'2J (such as competitive exclusion, viral aggregation, defective interfering particles, etc.) may have caused the MIR to be low. Therefore, we were interested in identifying the general impact of the extrinsic viral properties of the low-MIR virus TE/5'2J on that of the high-MIR virus TE/E2-55Q/70E. The potential effect of extrinsic properties on MIR was analysed by mixing 6.5 log10(p.f.u. ml–1) TE/E2-55Q/70E–GFP (equivalent to the MID50 for this virus) together with either 7.5 or 6.5 log10(p.f.u. ml–1) of TE/5'2J (all Vero cell-derived), infecting mosquitoes, and calculating the MIR of the former by visualizing GFP in the midguts of A. aegypti mosquitoes 9 days p.i. We observed a TE/E2-55Q/70E–GFP MIR of 52.8 ±2.8 % for the non-coinfected control. For the coinfection of TE/E2-55Q/70E–GFP with 7.5 and 6.5 log10(p.f.u. ml–1) of TE5'2J, we observed a mean MIR of 48.1 and 51.9 %, respectively, both of which were not significantly different from the non-coinfected control. These data indicated that TE/5'2J extrinsic properties do not affect the MIR.
Midgut virus titre and dissemination rates
Viral titres in mosquito tissues were analysed at 3 and 9 days p.i. for Vero-propagated TR339 and TE/5'2J viruses (Fig. 6). TR339 and TE/5'2J mean titres were not statistically significantly different from each other when the virus was in the tissues of the head and thorax following a disseminated infection (P>0.07), in the midguts with disseminated infections (P>0.21) or in midguts without disseminated infection (P>0.091). The duration of the infection (i.e. 3 vs 9 days) also showed no significant difference in titre (P>0.053) within any dissected tissue type. In general, head and thorax tissue did have significantly higher virus titres than the disseminated (P<0.02) and non-disseminated (P<0.001) midguts at 3 and 9 days p.i. for the respective viruses. Titres from midguts with a disseminated infection and non-disseminated infection were not compared because the dissected midguts from mosquitoes with a disseminated infection also had infected musculature (see arrows indicating infected external musculature in Fig. 3) and tracheole cells which will skew that data.
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). Moreover, TR339 did not have a statistically significantly different MDR from TE/5'2J when derived in C6/36 cells (P=0.2688) or in Vero cells (P=0.211) at 8.5 and 7.5 log10(p.f.u. ml–1) blood meal titres, respectively. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Black et al., 2002; Brault et al., 2002). The exact mechanism for alphavirus entry into susceptible cells is not yet known. In tissue culture fibroblast cells, it is thought that the E2 glycoprotein interacts with a yet unidentified receptor(s) followed by membrane fusion via the E1 glycoprotein. In the case of alphavirus infection of mosquito midgut cells, the E2 protein sequence has been implicated as having the specific genetic determinants for viral infection (Myles et al., 2003; Pierro et al., 2003; Weaver et al., 2004; Woodward et al., 1991).
Comparison of the 423 aa of E2 from TR339 and TE/5'2J showed only two amino acid sites with sequence variation, E2-55 and E2-70. The location of both residues is unique as they do not relate to previously published alphavirus genetic determinants of midgut infection. In the case of SINV MRE16, a deletion of aa 200–229 resulted in significantly reduced MIR in RexD (Myles et al., 2003). In VEEV, a single IF change at position E2-207 significantly impacted midgut infection rates in A. aegypti midguts (Woodward et al., 1991). Also in VEEV, a SN change at aa 218 was shown to be a significant determinant of midgut infection rates in Ochlerotatus taeniorhynchus mosquitoes (Brault et al., 2004). Moreover, aa 170–220 of E2 comprise a putative CRBD and studies characterizing this domain predict that the CRBD is exposed on the viral surface (Davis et al., 1987; Smith et al., 1995; Stec et al., 1986; Strauss et al., 1991). Anti-idiotypic antibody and virus mutant studies have also established the importance of the CRBD in cell binding (Mendoza et al., 1988; Strauss et al., 1991; Tucker et al., 1993; Ubol & Griffin, 1991; Wang et al., 1991; Wang & Strauss, 1991). The location of our two TR339 sites appears to be some linear distance from all of these sites.
To further investigate these sites, we generated TE/5'2J viruses that have the two TR339 E2 residues either individually or in combination, and subsequently characterized the replication patterns of these viruses in cell culture and in A. aegypti mosquito assays. Growth curve analysis of all viruses had a similar pattern of growth (Fig. 2), with the exception of the replication pattern of TE/5'2J that did not have an equivalent sustained high titre after 72 h in Aag2 cells (A. aegypti larval cells), when compared with the other viruses. This may be an indication of the TE/5'2J virus’ limited ability to maintain an established infection in certain mosquito cells.
The MIRs for C6/36-derived TE/E2-H55Q and TE/E2-K70E were significantly higher than that of TE/5'2J, but were significantly lower than that of the TR339 clone at 7.5 log10(p.f.u. ml–1) (Fig. 5). Vero cell-derived virus followed a comparable pattern of infection. These data indicate that each of the amino acid residue sites has the ability to independently influence the infection of A. aegypti midguts, although this infection is less efficient than that of the TR339 virus. However, the clone TE/E2-55Q/70E generated an MIR higher than TE/5'2J, TE/E2-H55Q, TE/E2-K70E and an MIR equivalent to TR339. This indicated that the two sites together could have an additive effect on the virus’ ability to infect midguts.
In an effort to analyse viral receptor competitive exclusion to midgut infection by defective interfering virions, we utilized the double subgenomic promoter element of TE viruses for the expression of GFP. The TE/E2-55Q/70E–GFP virus successfully delivered GFP to approximately 50 % of A. aegypti midguts using the MID50 of the TE/E2-55Q/70E virus. We determined that the TE/E2-55Q/70E–GFP MID50 is not significantly affected by coinfection with high-titrated TE/5'2J (P>0.5). This indicates that midgut infections are not affected by receptor competition restraints from other infectious, non-infectious or defective interfering virions. We also observed that TR339 and TE/5'2J viruses that had been propagated in Vero cells were able to infect C6/36 cells at similar rates [determined by 50% tissue culture infective dose (TCID50) assays; data not shown]. The same is true for the ability of Vero cell-propagated viruses to subsequently infect Vero cells. In fact, no combination of cell types used in viral propagation (i.e. mammalian or insect) had an effect on the subsequent TCID50 infection rates. This indicated that the parental viruses had no significant advantage over the other to infect either C6/36 (invertebrate) or Vero (vertebrate) cells.
Other researchers have analysed the SINV E2-H55Q and E2-K70E residues, although for different phenotypes. Levine & Griffin (1993) found that a QH change at SINV position E2-55 contributes to increased neurovirulence in 4–6 week old BALB/cJ mice, and concluded that this mutation arose as a result of prolonged replication in host cells. Lee et al. (2002) concluded that the E2-55 Q residue reduced virus binding to a neuroblastoma cell line, but replicated similarly to an E2-55 H variant virus in BHK cells. Levine & Griffin (1993) also reported that an H at position E2-55 increased virus affinity to heparin sulphate (HS), but suggested that this residue was unlikely to interact directly with the cell surface. Moreover, they repeatedly isolated neurovirulent SINVs with the QH change at position E2-55, which suggested a strong selective advantage for neurovirulence, leading to high mortality in mice. We report here that TE/5'2J SINV having Q (and not H) at E2-55 significantly increased MIR in A. aegypti mosquitoes.
In this work we have also correlated an increased MIR with the presence of an E at position E2-70. McKnight et al. (1996) previously demonstrated that an E2-70 K residue in TR339 positively influenced BHK cell-penetration rates and reduced neurovirulence in neonatal mice. Their E2-70 EK variant was linked to higher HS-binding activity; however, the predictive linear HS interaction domain could not be found near E2-70 (Smit et al., 2002). Moreover, Klimstra et al. (1998) speculated that the TR339 virus with E2-70 E utilized a weak HS-independent attachment strategy for cell entry. It has also been observed that HS-binding proteins, when introduced into a model system, are quickly cleared from circulation by binding to tissue HS (Karlsson & Marklund, 1988; Karlsson et al., 1994). A large-plaque variant of SINV with an E2-76 KE change resulted in a virus with low HS affinity and slower host clearance (Byrnes & Griffin, 2000). Our TE/E2-K70E virus produced the larger plaque phenotype associated with TR339 and, as stated, this site was previously characterized as having a limited HS affinity. A model for midgut infection by SINV may require dissociation with HS for effective receptor recognition and/or cell entry into the midgut. The E2-70 site appeared to be an important determinant that impacted viral pathogenesis in both mosquitoes and mice.
The effect of cell type used for virus propagation on MIR points to the potential influence of viral protein processing. In particular, the late-stage processing of N-glycans in insect cells is known to diverge from that of mammalian cells by producing paucimannosidic or oligomannose structures instead of the terminal sialylated complex-type structures (Altmann et al., 1999; Marchal et al., 2001; Tomiya et al., 2003, 2004). These differences in N-glycan structure are believed to affect biological activity (Prenner et al., 1992; Takahashi et al., 2004; Tomiya et al., 2004). Moreover, insect cell membranes are populated by sterols, as opposed to cholesterols in mammalian cells, both of which are thought to affect cell rigidity and alphavirus activity (Bretscher & Munro, 1993; Clayton, 1964; Lu et al., 1999; Luukkonen et al., 1973; Marquardt et al., 1993; Vashishtha et al., 1998). Functional differences have been observed previously for SINV propagated in mammalian versus insect cell lines and, taken together, they imply that SINV maturation differs significantly between these arboviral host cell types (Lee & Brown, 1994; Li et al., 1999; West et al., 2006).
Clearly, insect cell-propagated virus is not a natural route of delivery to the mosquito midgut. Our infection model using C6/36 cells for viral propagation is intended to show efficiency of virus infection from midgut cell to midgut cell after the initial infection occurs. Our data show that insect cell-propagated virus reduces the efficiency at which midgut cells become infected. The MID50 data (Table 1), together with the phenotypic observation of SINV infection foci (Fig. 3), indicate that viruses are not only restricted in their ability to secure a primary infection of the midgut (i.e. potential receptors), but that the virus progression in the midgut may be restricted by the biochemical processing of the virus in the midgut cells.
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ACKNOWLEDGEMENTS |
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Received 19 September 2006;
accepted 13 January 2007.
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). No helices were predicted at this threshold. Bold in secondary structure indicates predicted solvent accessible residues at >36 %. The sequences used were from SINV, Western equine encephalitis virus (WEEV), Chikungunya virus (CHIK), O'nyong-nyong virus (ONNV), Semliki Forest virus (SFV), Ross River virus (RRV), Barmah Forest virus (BFV), Aura virus (AURAV), Eastern equine encephalitis virus (EEEV) and VEEV.
, TR339; 
, TE/5'2J; 
, TE/E2-K70/E; 
, TE/E2-H55Q; 
, TE/E2-H55Q/70E.
