Virus and host factors affecting the clinical outcome of bluetongue virus infection

Abstract

Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides. Here, we assessed virus and host factors influencing the clinical outcome of BTV infection using a single experimental framework. We investigated how mammalian host species, breed, age, BTV serotypes, and strains within a serotype affect the clinical course of bluetongue. Results obtained indicate that in small ruminants, there is a marked difference in the susceptibility to clinical disease induced by BTV at the host species level but less so at the breed level. No major differences in virulence were found between divergent serotypes (BTV-8 and BTV-2). However, we observed striking differences in virulence between closely related strains of the same serotype collected toward the beginning and the end of the European BTV-8 outbreak. As observed previously, differences in disease severity were also observed when animals were infected with either blood from a BTV-infected animal or from the same virus isolated in cell culture. Interestingly, with the exception of two silent mutations, full viral genome sequencing showed identical consensus sequences of the virus before and after cell culture isolation. However, deep sequencing analysis revealed a marked decrease in the genetic diversity of the viral population after passaging in mammalian cells. In contrast, passaging in Culicoides cells increased the overall number of low-frequency variants compared to virus never passaged in cell culture. Thus, Culicoides might be a source of new viral variants, and viral population diversity can be another factor influencing BTV virulence.

Importance: Bluetongue is one of the major infectious diseases of ruminants. It is caused by an arbovirus known as bluetongue virus (BTV). The clinical outcome of BTV infection is extremely variable. We show that there are clear links between the severity of bluetongue and the mammalian host species infected, while at the breed level differences were less evident. No differences were observed in the virulence of two different BTV serotypes (BTV-8 and BTV-2). In contrast, we show that the European BTV-8 strain isolated at the beginning of the bluetongue outbreak in 2006 was more virulent than a strain isolated toward the end of the outbreak. In addition, we show that there is a link between the variability of the BTV population as a whole and virulence, and our data also suggest that Culicoides cells might function as an "incubator" of viral variants.

FIG 1

In vitro replication kinetics and pathogenicity in mice of the BTV strains used in this study. (A) Replication kinetics of BTV-2_IT2000, BTV-8_NET2006, and BTV-8_IT2008 in sheep CPT-Tert cells. Cells were infected at a multiplicity of infection (MOI) of 0.05, and supernatants were collected 8, 24, 48, 72, and 96 h postinfection. Supernatants were then titrated in BSR cells by limiting dilution assays. Experiments were repeated independently three times, and data are represented as averages from the experiments. Error bars indicate standard errors. (B) Survival plots of 129sv IFNAR^−/− mice inoculated intraperitoneally with 300 PFU of BTV-2_IT2000, BTV-8_NET2006, and BTV-8_IT2008. Mice were observed for 2 weeks postinoculation for the presence of clinical signs of systemic disease. All the viruses used in this study killed all the infected mice between days 6 and 8 postinoculation. None of the five mock-infected mice showed any clinical symptoms (not shown in the figure) and survived throughout the observation period.

FIG 2

Experimental infection of goats and different sheep breeds with BTV-8_NET2006. (A) Graphs showing clinical signs recorded in BTV-infected goats and various sheep breeds, including Sardinian, mixed breed, and Dorset poll (n = 5 per each group). Animal were all approximately 2 years of age with the exception of an additional group of 8-month-old Dorset poll sheep that are indicated as “Dorset (young).” Animals were scored daily after infection using a clinical index score (shown in Table S1 in the supplemental material), taking into account general symptoms, respiratory signs, fever, need for veterinary intervention, or death. General symptoms included are depression, anorexia, and facial and feet lesions. Each group of 5 animals was infected with the same dose of BTV-8_NET2006 intradermally. Scores shown for respiratory symptoms, general symptoms, and fever represent the average values collected for each group (±standard error) during the duration of the entire experiment (28 days). Total scores are instead the cumulative values for each symptom within a group collected throughout the observation period. (B) Body temperature (average per group; values per each individual animal are shown in Fig. S1 in the supplemental material) of animals infected with BTV-8_NET2006. Physiological temperature in sheep ranges normally between 38.3 and 39.9°C (black broken lines). Fever in this study was recorded when rectal temperature was above 40°C. In experimentally infected animals, fever appeared between day 5 and 6 postinfection. (C) BTV RNA in blood samples of experimentally infected sheep and goats. Viral RNA was detected by qRT-PCR, and values are expressed as log₁₀ copy number per μg of total RNA. Note that goats reached the highest level of BTV RNA in the blood. (D) Neutralizing antibodies toward BTV in experimentally infected animals. Sera were collected at the times indicated following experimental infection (time zero) and subjected to neutralization assays as indicated in Materials and Methods. Values shown are averages ± standard deviations and represent the log₁₀ of the 50% endpoint (proportionate distance [PD]). Mock-infected goats and sheep (data shown in Fig. S2 in the supplemental material) did not show any clinical sign of bluetongue, maintained a physiological temperature throughout the experiment, and did not have any detectable BTV RNA or neutralizing antibodies.

FIG 3

Virulence of BTV-2_IT2000, BTV-8_NET2006, and BTV-8_IT2008. Clinical scores (A), rectal temperature (B), viremia (C), and neutralizing antibodies (D) of Sardinian sheep (n = 5 per group) infected with either BTV-2_IT2000, BTV-8_NET2006, or BTV-8_IT2008. Descriptions of graphs in each panel are in the legend of Fig. 2. Note that experimental infections of sheep (Dorset poll, Dorset poll young, Sardinian, or mixed breed) and goats with BTV-8_NET2006 and Sardinian sheep with BTV-2_IT2000 or BTV-8_IT2008 were carried out at the same time but are shown separately in Fig. 2 and 3 to facilitate the narrative. Consequently, the same sets of data for the Sardinian sheep infected with BTV-8_NET2006 are shown both in Fig. 2 and 3. Fever and viremia data for each individual animal are shown in Fig. S3 in the supplemental material. Note that sheep infected with BTV-8_IT2008 display very mild clinical signs, only a transitory fever, and lower levels of viremia than sheep infected with BTV-2_IT2000 and BTV-8_NET2006.

FIG 4

Genetic differences between BTV-8_NET2006 and BTV-8_IT2008. Schematic representation of the 10 genomic segments of BTV-8_NET2006 and BTV-8_IT2008. Mutations in BTV-8_IT2008 compared to BTV-8_NET2006 are indicated with red dots. Nonsynonymous mutations are highlighted with black asterisks, and the position of the mutated amino acid residue is shown. Note that the length of the schematic genome segments and the relative position of synonymous and nonsynonymous mutations in the cartoon are indicative only.

FIG 5

Experimental infection of Sardinian sheep with BTV-8_{NET2007(blood)} and BTV-8_{NET2007(1KC-2BHK)}. Clinical scores (A), rectal temperature (B), viremia (C), and neutralizing antibodies (D) of Sardinian sheep (n = 5 per group) infected with either BTV-8_{NET2007(blood)} or BTV-8_{NET2007(1KC-2BHK)}. Graphs in each panel have already been described in the legends of Fig. 2. Fever and viremia data for each individual sheep are shown in Fig. S4 in the supplemental material. Note that sheep infected with BTV-8_{NET2007(blood)} displayed more severe clinical signs and higher levels of fever and viremia than sheep infected with BTV-8_{NET2007(1KC-2BHK)}.

FIG 6

In vitro adaptation of BTV-8_{NET2007(blood)}. The effects of adaptation in vitro of BTV-8_{NET2007(blood)} were assessed by comparing the genomic sequences of BTV-8_{NET2007(blood)} with the sequences of viruses isolated in vitro after passaging in Culicoides KC cells (1 passage) and two further passages in BHK₂₁ cells. Schematic representation of the experiment is shown at the top of the figure. Two independent experiments (represented with blue or red arrows) were carried out, and sequences of the viral genome were obtained after each passage in vitro. The cartoon shows the schematic representation of individual genomic segments of BTV. Mutations found in the consensus sequences of the cell culture-passaged viruses are shown as red or blue dots, indicating the two independent experiments. Only two synonymous mutations were selected in Seg1 and Seg4 immediately after passage in KC cells in both independent experiments and were conserved after further passaging in BHK₂₁ cells.

FIG 7

Viral population diversity of BTV-8_{NET2007(blood)} before and after isolation in cell culture. Changes in nucleotide diversity of BTV-8_{NET2007(blood)} amplified directly from the spleen of an infected sheep were compared with sequences of the same virus after isolation in KC and BHK₂₁ cells. Differences were assessed by deep sequencing as described in Materials and Methods. Total reads of individual genome segments were mapped to consensus sequences, and single nucleotide polymorphisms (SNPs) were assigned above the arbitrary 0.1% frequency threshold. On the graph, each dot represents the percentage of nucleotide difference (y axis) from the consensus sequence of each nucleotide composing the individual genomic segments of the virus (x axis). The total number of variable nucleotides (>0.1%) for each sample is shown in the right corner of each plot. Dots circled in red in Seg1 and Seg4 of BTV-8_{NET2007(blood)} are those nucleotides that have been selected in the majority of the viral populations after passage in vitro.

FIG 8

Frequency distribution of variable nucleotide in BTV-8_{NET2007(blood)}, BTV-8_NET2007(1KC), BTV-8_{NET2007(1KC-1BHK)}, and BTV-8_{NET2007(1KC-2BHK)}. Histograms showing for each virus the number of nucleotides with percent variation falling within defined borders (“bins”). Panels A/B and C/D represent data from two independent experiments. Note that panels B and D have a different scale in the y axis from panels A and C, as the frequency of variants present in more than 0.4% of the total population was significantly lower than variants presented in panels A and C.