BoMac myeloid cells express relatively less gp180 glycoprotein in comparison with epithelial cells.

<p><b>A–B</b>. MDBK and BoMac cells were infected with the BoHV-4 V.test strain at a MOI of 1. Twenty-four hours p.i., relative expressions of Bo10 spliced <i>vs</i> unspliced transcripts (<b>A</b>) and of Bo10 spliced <i>vs</i> ORF8 transcripts (<b>B</b>) were estimated as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003753#s4" target="_blank">Methods</a>. The data presented are the average ± SEMs for 3 measurements and were analyzed by Student's t-test, ** p<0.01. <b>C.</b> Detection of the gp180, encoded by the Bo10 spliced transcript, and gB glycoproteins in bovine epithelial and myeloid cells. MDBK and BoMac cells were infected with the BoHV-4 V.test strain at a MOI of 1. Twenty-four hours p.i., these cells were subjected to western blotting with anti-Bo10-c15 serum (recognizing gp180) and mAb 35 (recognizing gB) as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003753#s4" target="_blank">Methods</a>. On the gp180 blot, open and filled triangles indicate respectively the specific 180 kDa protein and a background band. On the gB blot, open and filled arrow heads indicate uncleaved gB and furin-cleaved gB C-terminus respectively. The position of a molecular mass (MM) standard (in kDa) is shown on the left.</p

Expression of the spliced form of Bo10 mRNA reverts the phenotype of myeloid virions.

<p><b>A.</b> BoHV-4 WT BAC or Bo10 Spliced virions were propagated on MDBK or BoMac cells and then added to bovine PBMCs at a MOI of 3 according to titers measured on MDBK cells. 24 hours later, percentages of eGFP positive cells were measured in CD14+ PBMCs by flow cytometry. The data presented are the average ± SEMs for 4 measurements and were analyzed by 1way ANOVA and Bonferroni posttests, ** p<0.01. <b>B.</b> BoHV-4 WT BAC or Bo10 Spliced virions propagated on MDBK or BoMac cells were incubated with sera of 3 different rabbits infected with BoHV-4 V.test strain (propagated on MDBK cells). After incubation (2 h, 37°C) the viruses were plaque assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without antibody. The data presented are the average ± SEMs for 3 measurements and were analyzed by 2way ANOVA and Bonferroni posttests, ** p<0.01.</p

Myeloid virions are more infectious for CD14+ PBMCs but are more sensitive to antibody neutralization.

<p><b>A.</b> Infection by co-cultures. MDBK or BoMac cells previously stained with the membrane dye PKH26 were infected with BoHV-4 WT BAC virions (MOI of 1). Three hours p.i., cells were washed with acidic solution (PBS pH 3) in order to inactivate and remove the inoculum, and 12 hours p.i., freshly isolated PBMCs or MDBK controls were added. After 24 hours of co-cultivation, cells were collected and the proportion of eGFP expressing cells was measured in PKH26+ and PKH26− cells. For PBMC co-cultures, the proportion of eGFP expressing cells was measured in PKH26− CD14+ cells. The data presented are the average ± SEMs for 5 measurements and were analyzed by Student's t-test, *** p<0.001. <b>B.</b> BoHV-4 WT BAC cell-free virions propagated on MDBK or BoMac cells were added to bovine PBMCs at a MOI of 1 according to titers measured on MDBK cells. 24 hours later, percentages of eGFP positive cells were measured in CD14+ PBMCs by flow cytometry. The data presented are the average ± SEMs for 5 measurements and were analyzed by Student's t-test, *** p<0.001. <b>C.</b> BoHV-4 WT BAC virions propagated on MDBK or BoMac cells were incubated with sera of 3 different rabbits infected with BoHV-4 V.test strain (propagated on MDBK cells). After incubation (2 h, 37°C), the viruses were plaque assayed for infectivity on MDBK cells. BoHV-4 titers are expressed relative to virus without antibody. The data presented are the average ± SEMs for 3 measurements and were analyzed by 2way ANOVA and Bonferroni posttests, * p<0.05, ** p<0.01, *** p<0.001.</p

Generation of the Bo10 MuDir and Spliced BoHV-4 mutant.

<p><b>A.</b> Schematic representation of the strategy followed to produce the recombinant BoHV-4 strains. We modified the BoHV-4 V.test Bo10 coding sequence (genomic coordinates 65844 to 66743) either by introducing a point mutation in the splicing donor site (Bo10 MuDir strain) or by replacing the entire Bo10 ORF by a sequence devoid of the intron (Bo10 Spliced strain). Splicing donor and acceptor sites are in red with splicing essential nucleotides in upper cases. The mutated nucleotide is in green. <b>B.</b> Verification of the molecular structure. Viral DNA was digested with <i>Bam</i>HI, resolved by agarose gel electrophoresis, and hybridized with a ³²P-labeled probe, corresponding to nucleotides 65900–66370 of the BoHV-4 V.test strain genome (coding for Bo10 Exon 1). The 7,137-bp wild-type (WT) band becomes 7060-bp (open arrow) for the Bo10 spliced mutant. Marker sizes in Kbp are indicated on the left. <b>C.</b> RT-PCR analysis of BoHV-4 Bo10 expression by the different mutants. MDBK cells were mock infected or infected with the different BoHV-4 mutant strains at a MOI of 1. Twenty-four hours p.i., expression of Bo10 was studied using different pairs of primers specific for the spliced and/or unspliced Bo10 cDNA. Uninfected cell samples (UI), reactions without reverse transcriptase (Neg. control) and amplification of viral genomic DNA are provided as controls. Sizes in bp are indicated on the right. <b>D.</b> Detection of the Bo10 encoded gp180 protein by the anti-Bo10-c15 serum. Purified virions (5*10⁵ virions per lane) were subjected to western blotting with anti-Bo10-c15 serum as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003753#s4" target="_blank">Methods</a>. The position of a molecular mass (MM) standard (in kDa) is shown on the right.</p

BoHV-4 model for switch of cell tropism through alternative splicing of the Bo10 gene.

<p>(1) Expression of the Bo10 spliced product, encoding gp180, positively regulates the infection of GAG-bearing cells. (2) Replication in epithelial cells likely allows infection of myeloid cells. (3) Myeloid infection allows infection of circulating leukocytes, such as CD14+ cells in the case of BoHV-4. (4) Infected PBMCs transmit infection to epithelium of the excretion sites by an unknown route. (5) Finally, incorporation of gp180 at the excretion site allows virions to be protected from neutralizing antibodies.</p

Bovine epithelial and myeloid cells produce different kinds of BoHV-4 virions.

<p>Detection of the gp180 and gB glycoproteins in MDBK and BoMac virions. Different amounts of MDBK and BoMac virions were compared for gp180 (with anti-Bo10-c15 serum) and gB (with mAb 35) content per PFU by immunoblotting as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003753#s4" target="_blank">Methods</a>. Anti-BoHV-4 polyserum was taken as control. For each blot, the position of a molecular mass (MM) standard (in kDa) is shown on the left.</p

Induction of WB-MCF in rabbits.

<p>A. Cumulative incidence survival curve of AlHV-1 infected rabbits. On day 0, two groups, each consisting of four rabbits, were inoculated intravenously with 3×10⁶ mock infected or AlHV-1 infected EBL cells. At day 10 post-inoculation, BrdU (25 mg/kg) was injected intravenously every day to all rabbits. Rabbit developing WD-MCF were euthanized when rectal temperature remained higher than 40°C for two consecutive days. For each euthanized infected rabbit, a contemporary mock infected rabbit was also sacrificed. Rabbits were identified according to their infected (IR) or mock infected (MR) status and according to the time of death (see numbers). B. Popliteal lymph nodes were dissected during necropsy examination. C. Relative proportions of CD11b⁺, IgM⁺, CD4⁺ and CD8⁺ cells in PBMC, popliteal lymph node and spleen of AlHV-1 infected (IR) or mock infected rabbits (MR). At time of death, cell suspensions were prepared, stained and analysed by flow cytometry.</p

AlHV-1 gene expression in secondary lymphoid organs of rabbit with WD-MCF.

<p>Mononuclear cells were isolated from the spleen and the popliteal lymph node of rabbits at the time of death. Total RNA was extracted from isolated cells. Expression of cellular β-globin gene and viral genes (ORF73, ORF50, ORF9 and ORF25) was studied using the RT-PCR approach described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001627#s2" target="_blank">Methods</a>. DNA, purified AlHV-1 genomic DNA; MR, RNA extracted from the lymph node of a mock infected rabbit, C<i>+</i>, RNA purified from infected EBL cells.</p

Load and tropism of AlHV-1 in PBMC and secondary lymphoid organs.

<p>Rabbits were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001627#pone-0001627-g001" target="_blank">Fig. 1</a>. A. Viral load in PBMC over time and in lympoid organs at the time of death. PBMC were collected at days 0, 7, 11, 15, 17, 20 and 24 post-infection, while mononuclear cells were isolated from popliteal lymph node and spleen at the time of death. DNA was extracted from cells and was subjected to viral (ORF3) and cellular (β-globin) real-time PCR quantification. The results are expressed as the estimated AlHV-1 mean genome copy number per 10⁵ cellular β-globin genomic copies. Data are the mean±SEM of triplicate measures. In the left panel, the following symbol are used: ▴, IR17/1; □, IR17/2; •, IR20; ○, IR24. B. AlHV-1 tropism amongst PBMC collected before euthanasia. Isolated cells were labelled with anti-CD11b, IgM, CD5, CD4 and CD8 mAbs as the primary antibodies, and Alexa 633-GAM, as the secondary antibody. For each mAbs, ten positive cells were sorted per well in 10 wells of a 96-well plate using an automatic cell deposit unit as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001627#s2" target="_blank">Methods</a>. After freezing at −80°C, PCR were performed in order to detect the AlHV-1 ORF50 and the cellular β-globin gene. Marker sizes (MS) are indicated on the left. The results presented in this figure were obtained with the PBMC of IR20. They are representative of the results obtained with the 4 infected rabbits.</p

Primers used in this study for PCR and RT-PCR reactions.

a<p>primers used for both PCR and RT-PCR reactions.</p