Table 1.
Replication of BgV in insects and vertebrates.
Insect and vertebrate cell lines were inoculated in triplicates with BgV and WNV at MOI 1 for 1h 30min, the inoculum removed and the cells washed before incubating the replenished cultures at 28°C or 37°C. The inoculated cell culture supernatants were harvested five days p.i., titrated on C6/36 cells and analysed by fixed-cell ELISA.—represents no replication, + represents a titre over 102 TCID50IU/mL, ++ represents a titre over 104 TCID50IU/mL, +++ represents a titre over 106 TCID50IU/mL. In a few instances, not all replicates were positive, in which case the titre represented is the average of the positive replicates only.
Table 2.
BgV replication in vertebrate animal models.
Groups of three eggs were inoculated with three doses of BgV intravenously (105.7, 104.7 and 103.7 TCID50 IU/egg, respectively), incubated at 33–35°C for five days and homogenised. The homogenates were titrated on C6/36 cells and the resulting virus titres were analysed by fixed-cell ELISA. The eggs injected at the higher doses had traces of virus present but at levels suggesting they resulted from leftover inoculum rather than virus replication. The other animal models were inoculated with 106 TCID50 IU of BgV and sampled for blood on days 1–5 and 14 post-inoculation (sparrows) or on days 1, 3, 5, 7, and 28 post-inoculation (frogs, toads, snakes). The blood samples were diluted 1:5 and 1:50 and inoculated on C6/36 cells which were incubated for two days and analysed for virus by fixed-cell ELISA.
Fig 1.
A. Schematic representation of the genome composition of CPER-generated BgV-derived chimeric viruses. Black shading indicates WNVKUN genes while red shading indicates BgV genes. The prME genes were swapped for each virus, generating a chimera with BgV “backbone” sequence and WNVKUN prME: BgV/WNV-prME and the reverse chimera with WNV backbone sequence and BgV prME: WNV/BgV-prME. B. Replication kinetics of parental prototype viruses and CPER-generated chimeras and infectious clones in C6/36 cells over five days. The insect cells were inoculated with each virus at MOI 0.1 in triplicates for 1h 30min, the inoculum removed and the cells washed before incubating the replenished cultures at 28°C. The supernatants were harvested at 2, 24, 48, 72, 96 and 120 hours p.i., stored at -80°C until they were titrated on C6/36 cells and the titres determined by fixed-cell ELISA. Error bars represent the standard deviation and the dotted lines represent the lower limit of detection. C. Visualisation of virus replication in vertebrate cells by IFA. The cells were inoculated at MOI 1 with WNV, BgV, WNV/BgV-prME, BgV/WNV-prME or mock inoculated, in triplicates, the inoculum removed and the cells washed before incubating at 37°C (all vertebrate cells) or 28°C (C6/36 cells). The cells were fixed five days post-inoculation and stained with pan-flavivirus mAb 4G2 by IFA.
Fig 2.
A. Replication of BgV and WNVKUN in vertebrate cells. Cells were inoculated at MOI 1 with BgV or WNVKUN in triplicate for 1h 30min, the inoculum removed and the cells washed before incubating at 34°C (black), 37°C (red) or 28°C (blue) with fresh medium. The inoculated cell culture supernatants were harvested five days p.i., titrated on C6/36 cells and analysed by fixed-cell ELISA. The dotted line represents the lower limit of detection, * stands for p < 0.05. Error bars represent the standard deviation. B. Thermostability of BgV and WNVKUN at various temperatures over three days. Triplicates of 105 TCID50 IU of BgV and WNVKUN were held at 4°C, 28°C, 34°C and 37°C in culture medium with 2% FBS in the absence of cells, harvested at 30min, 24h, 48h and 72h after the start of incubation and stored at -80°C. These samples were titrated on C6/36 cells and the titres determined by fixed-cell ELISA. Error bars represent the standard deviation. C. Replication kinetics of BgV, BgV/WNV-prME, WNVKUN and WNV/BgV-prME, in BSR (left) and Vero cells (right) over five days at 34°C and 37°C. The cells were inoculated at MOI 0.1 in triplicates at 34°C or 37°C for 1h 30min, the inoculum removed and the cells washed before incubating at either temperature with fresh medium. The supernatants were harvested at 2, 24, 72 and 120 hours p.i., stored at -80°C until they were titrated on C6/36 cells and the titres determined by fixed-cell ELISA. Error bars represent the standard deviation and the dotted lines represent the lower limit of detection.
Fig 3.
A. Blind passages of BgV in vertebrate cells. Flasks of BSR and Vero cells were inoculated with BgV at MOI 1 and incubated at 34°C for five days. 1mL aliquots of supernatant from these flasks were blindly passaged three times to flasks of freshly seeded cells. Vero cells were split 1/10 and passaged three times, while BSR cells succumbed to cytopathic effect and could not be passaged. At each passage, one set of supernatant and cells was maintained at either 34°C or 37°C, while the third set was incubated at increasing temperatures: 35°C, 36°C and finally 37°C. The presence of BgV in the harvested supernatants was tested by titration on C6/36 cells followed by fixed-cell ELISA. The red crosses represent samples in which BgV was not detected. Three samples were selected for further passaging (circled) and RNA extracts from the resulting supernatants were deep sequenced. The samples marked with a green M carried two of three identified conserved mutations, as determined by Sanger sequencing, in NS4B and NS5. B. The three conserved mutations identified by next-generation sequencing in replication-competent BgV variants. The nomenclature adopted hereafter is BgVC-CG for prototype BgV and BgVT-AT for a BgV variant containing all three NS4A/NS4B/NS5 identified conserved mutations. C. Alignment of selected flaviviruses over two viral protease cleavage sites: NS4A/2K and NS4B/NS5. The bolded letters correspond to the identified mutation sites in BgV sequence. The underlined letters and downward arrow correspond to the viral protease cleavage site.
Fig 4.
A. Replication kinetics of BgVC-CG (prototype), BgVC-AT (double mutant NS4B/5), BgVT-AT (triple mutant NS4A/4B/5) and WNVKUN in C6/36 cells (28°C—left), BSR cells (37°C—middle) and Vero cells (37°C—right) over five days. The cells were inoculated with each virus at MOI 0.1 in triplicate, incubated 1h 30min at 28°C or 37°C, the inoculum removed and the cells washed before incubating at 28°C or 37°C with fresh medium. The supernatants were harvested at 2, 24, 48, 72, 96 and 120 hours p.i., stored at -80°C until they were titrated on C6/36 cells and the titres determined by fixed cell ELISA. Error bars represent the standard deviation and the dotted lines represent the lower limit of detection. The graph includes only representations of statistical analysis for comparisons to BgVT-AT for clarity (* = p < 0.05). The results of the comparisons to BgVC-AT were the same as those to BgVT-AT. A star can therefore be read as a statistically significant difference to the mutants at that time point. B. BgV variants and WNVKUN replication in DF-1 cells at 37°C. The cells were inoculated with each virus in triplicate at MOI 1 and incubated at 28°C (C6/36) or 37°C (DF-1). The supernatants were harvested after five days, titrated on C6/36 cells and the titres determined by fixed cell ELISA. The error bars represent the standard deviation and the dotted line represents the lower limit of detection. C. Prototype BgVC-CG, BgVC-CG/WNV-prME, and mutant BgVT-AT, BgVT-AT/WNV-prME and WNVKUN replication in vertebrate cells at 37°C. Each cell line was inoculated with each virus in triplicate at MOI 1 as above and incubated at 28°C (C6/36) or 37°C (BSR, DF-1, Vero). The supernatants were harvested, titrated on C6/36 cells and the titres determined by fixed-cell ELISA. Error bars represent the standard deviation and the dotted line represents the lower limit of detection.
Fig 5.
A. Summary of seropositive mice inoculated with BgV-derived viruses and mice with neutralising antibodies. Seroconversion was determined by fixed-cell ELISA using doubling dilutions of sera on BgV infected and fixed C6/36 cells. B. Levels of seroconversion obtained from BgV-derived viruses-injected mice. The dotted lines represent the lower and higher limits of detection. Sera from WNV/BgV-prME-injected mice were only diluted to 1/160 as opposed to the rest of the sera, diluted up to 1/512. The sample size of each group can be found in (A). C. Levels of neutralising antibodies in BgV-derived viruses-injected mice sera as determined by microneutralisation assay. The dotted lines represent the lower and higher limit of detection. The sample size of each group can be found in (A). D. Summary of virus positive mice brains three and 5 five days p.i.. The left brain hemisphere was harvested from three mice per time point in each group, stored at -80°C, weighed, homogenised in a 20% w/v mixture with RPMI, titrated on C6/36 cells and analysed by fixed-cell ELISA. E. Levels of detected virus in mice brains as determined by titration on C6/36 cells, see protocol in (D). The dotted line represents the lower limit of detection.
Fig 6.
Microphotographs of meningo-encephalitis in the brains of mice IC-injected with mutant chimera BgVT-AT/WNV-prME.
A. Brain section from mouse culled at day 3 p.i. showing widespread infiltration of neutrophils and monocytes in the neuropil accompanied by gliosis (overall marked increase in cellularity). B. Brain section from mouse culled at day 5 p.i. showing extension of inflammation into other parts of the brain distant from the injection site. C. Brain section from animal culled on day 22 p.i. where evidence of severe meningo-encephalitis is still present with perivascular cuffing and leukocyte infiltrates in the meninges (arrows). D. Brain section from mouse culled on day 22 p.i. and demonstrating persistent severe inflammation throughout the neuropil. Hematoxylin and eosin staining.
Fig 7.
Immunohistochemistry of brain sections from mice injected with BgV-derived viruses.
Virus positive cells appear with a red signal. A. Pericytes and leukocytes infiltrating in the Virchow’s space around cerebral blood vessels are positive for viral antigen in mouse culled on day 22 post-IC-inoculation of mutant chimera BgVT-AT/WNV-prME. B. Virus antigen positive cells amongst the leukocyte infiltrating the meninges in mouse culled on day 22 post-IC-inoculation with BgVT-AT/WNV-prME. C. Scattered neurons are positive for viral antigen, which is mainly located to the peri-nuclear Golgi-region (insert). Mouse culled on day 22 post-IC-inoculation of BgVT-AT/WNV-prME. D. Widespread cytoplasmic signal for viral antigen in brain of mouse IC-inoculated with WNV/BgV-prME and culled 8 days p.i..