An Outbred Calf Model for Determining Innate Immune Sensing and Evolutionary Trajectories of a Cell Culture-Adapted Bovine Foamy Virus Variant

Abstract

Bovine foamy virus (BFVbta) displays a very high degree of cell-associated replication which is unprecedented even among the other known foamy viruses. Interestingly, recent studies have shown that it can in fact adapt in vitro to high-titer (HT) cell-free transmission due to genetic changes acquired during repeated rounds of cell-free BFVbta passages in immortalized bovine MDBK cells. Molecular clones obtained from the HT BFVbta Riems cell-free variant (HT BFVbta Riems) have been thoroughly characterized in MDBK cell cultures However, during recent years, it has become increasingly clear that the source of the host cells used for virus growth and functional studies of virus replication and virus-cell interactions plays a paramount role. Established cell lines, mostly derived from tumors, but occasionally experimentally immortalized and transformed, frequently display aberrant features relating, for example. to growth, metabolism, and genetics. Even state-of-the-art organoid cultures of primary cells cannot replicate the conditions in an authentic host, especially those concerning cell diversity and the role of innate and adaptive immunity. Therefore, to determine the overall replication characteristics of the cloned wt and HT BFVbta Riems variant, we conducted a small-scale animal pilot study. The replication of the original wt BFVbta Riems isolate, as well as that of its HT variant, were analyzed. Both BFVbta variants established infection in calves, with proviruses in peripheral blood mononuclear cells and induced Gag-specific antibodies. In addition, a related pattern in the host innate immune reaction was detected in the peripheral blood leukocytes of the BFV-infected calves. Surprisingly, an analysis of the Gag sequence two weeks post-inoculation revealed that the HT BFVbta variant showed a very high level of genetic reversion to the wild type (parental BFVbta genotype).

Keywords: bovine foamy virus; calves; cell-free and cell-associated variants; replication in vivo.

Figure 1

Sero-reactivity of the individual calves to BFVbta Gag following inoculation with different BFVbta Riems variants over time. The humoral response to BFVbta antigens was tested using a GST-ELISA at 16 wk p.i. in the four calves experimentally inoculated with the different variants of BFVbta Riems (calves 1 and 2: HT BFVbta Riems; calves 3 and 4: wt BFVbta Riems) and the two mock-infected calves (calves 5 and 6).

Figure 2

BFVbta viral DNA load in the PBMCs of calves determined during the 16 weeks following inoculation with the different BFVbta Riems variants (see legend). The BFVbta DNA load was determined using qPCR on the PBMCs of the four calves experimentally inoculated with the BFVbta variants during the first 16 wk p.i. No BFVbta DNA was detected in the mock-infected calves. The DNA of calf 2 was not available from 16 wk p.i.

Figure 3

Detection of BFVbta Gag protein in co-cultures using indirect immunofluorescence. The re-isolation of the virus from the PBLs of the inoculated calves was carried out via co-cultivation with permissive MDBK cells. Indirect immunofluorescence was performed after the 3rd passage using rabbit anti-BFVbta-Gag MA polyclonal serum and anti-rabbit IgG-Alexa 595 (red) as a secondary antibody, while the nuclei were stained with Hoechst 33342 (blue).

Figure 4

Phylogenetic tree inferred from the 1295 bp nucleotide sequence of the BFVbta Gag amplified from the four calves 2 wk p.i. and the HT inoculum used for the inoculation of calves no. 1 and 2. The analyses were conducted in MEGA 6 using the maximum likelihood method based on the Kimura 2-parameter model. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The reference sequences were as follows: pBFV-CMV-MDBK24_HT [17]; GenBank derived: JX307862.1_BFV Riems; JX307861.1_BFV100; NC001831.1_BFV_US; AY134750.1 BFV_3026_CN.

Figure 5

Protein interaction network created using STRING database based on Bos taurus genome. Edges represent protein–protein associations, line thickness indicates the strength of the supporting data, network nodes represent proteins, and colors indicate their functional categories: GO database—dark blue—GO:0032481 “Positive regulation of type I interferon production” (FDR = 9.69 × 10⁻¹³); green—GO:0002764 “Immune response-regulating signaling pathway” (FDR = 9.76 × 10⁻¹²); grey—GO:1901222 “Regulation of NIK/NF-kappaB signaling” (FDR = 3.33 × 10⁻⁶); KEEG-pathways—light blue—bta04620 “Toll-like receptor signaling pathway” (FDR = 1.53 × 10⁻³²); pink—bta04621 “NOD-like receptor signaling pathway” (FDR = 3.48 × 10⁻¹⁶); yellow—bta04060 “Cytokine–cytokine receptor interaction (FDR = 8.74 × 10⁻⁹); light green—bta04612 “Antigen processing and presentation (FDR = 0.0187).

Figure 6

Interleukin 6 concentration in the plasma of calves infected with different BFVbta variants during first 5 days p.i.

Figure 7

Interferon alpha concentrations in the plasma of calves infected with different BFVbta variants during first 5 days p.i.