FACS analysis of peripheral blood lymphocytes. Spleen cells from infected mice (as indicated) were stained with monoclonal antibodies to the indicated cell surface markers and analyzed by FACS. Of 34 strains of BoHV-4, isolated from four different continents, all were found to contain the Bo17 gene. The most important animal γ-herpesviruses are the related Alcelaphine herpesvirus-1 and Ovine herpesvirus-2 (OvHV-2) that are associated with a fatal lymphoproliferative disease of domestic cattle and deer called malignant catarrhal fever (MCF). The detection limits of LAMP for EHV-1 gC and gE and PCR for EHV-1 gC were 1 plaque-forming unit (PFU)/tube, and those of LAMP and PCR for EHV-4 gC were 0.1 PFU/tube. The Bo17 gene acquisition was dated to around 1.5 million years ago. The results obtained can be summarized as follows.
In vivo, viruses lacking gB cleavage showed reduced lytic spread in the lungs. Given that EBV and MHV wild-type strains contain other genes that induce cIL-10 expression in latency (e.g. The recombinant vector expressing the membrane-bound form of a hybrid, rat-human Her-2 antigen was found to be the only one capable of eliciting high anti-Her-2 antibody titers in immune-tolerant, rat HER2 transgenic (BALB-neuT) mice and to afford strong protection against autochthonous Her-2+ mammary cancer development in these animals. BoHV-4 has been isolated from a variety of samples and cells (8) from healthy cattle and from cattle with abortion, metritis, pneumonia, diarrhea, respiratory infection, and mammary pustular dermatitis (reviewed by Bartha et al.  and Thiry et al. ). Therefore, it appears that, so far, Mus musculus represents a suitable host for studying gammaherpesvirus pathogenesis with MuHV-4.
However, the pathogenic role of BoHV-4 remains unclear; the direct correlation between particular strains of BoHV-4 with variable disease conditions is a delicate question, unsolved even through experimental infection. Only few investigators have successfully produced experimental disease (reviewed by Thiry et al. ), and direct inoculation of the natural host only occasionally elicited respiratory and genital disease (3, 22). Notwithstanding, no direct correlation can at present be demonstrated between BoHV-4 and specific lesions. In vivo distribution of BoHV-4 was examined by testing nasal and conjunctival exudates, peripheral blood leukocytes, and various organs of experimentally infected calves (10, 17). However, little information about excretion and transmission of BoHV-4 from naturally infected cattle has been generated. Using 10% 10 μL first PCR product each, the primer sets BS-4- P3 (5’-TGT TTC CGC CAC TCT TGA CG-3’, nucleotide positions 1857-1876) and BS-4-P4 (5’-ACT GCC TCT CCC ACC TTA CC-3’, nucleotide positions 2456-2437) were used for the secondary amplification run following the initial thermo-profile amplification program.
Herd history and sampling strategy.A herd of 100 dairy cows experiencing a high incidence of postpartum metritis, abortion, and infertility was positive by indirect fluorescent antibody testing (IFAT) (4) for BoHV-4 antibodies. Some selected clones were independently growth for 20 passages in the presence of G418. Data on the prevalence of BHV4 antibodies in Dutch cattle are also presented. Cell lines.A bovine arterial endothelial cell line (BAE-7372) (obtained from Stefano Grolli, Veterinary Biochemistry Institute, Parma University, Italy) was used, due to the high sensitivity of bovine arterial endothelial cells towards BoHV-4 and other bovine herpesviruses (12). Cells were grown at 37°C in minimum essential medium (Gibco-BRL, Paisley, United Kingdom) supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 IU/ml), and streptomycin (100 μg/ml) in a humidified atmosphere containing 5% CO2. DNA preparation. (i) Blood samples.Peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque (Pharmacia-Biotech, Uppsala, Sweden) gradient centrifugation.
They were washed with phosphate-buffered saline (PBS), and aliquots of 2 × 106 cells were made. (ii) Milk samples.Each milk sample was centrifuged at 400 × g for 10 min, and the supernatant was collected. Subsequently, the sedimented cells were washed once with PBS and resuspended in 3 ml of PBS. Aliquots of 2 × 106 cells were made. Cell-free milk supernatant (CFMS) was centrifuged at 3,500 × g for 30 min to remove cellular debris and ultracentrifuged at 90,000 × g for 2 h to collect the virus if present. Nucleic acids from PBMC aliquots, milk cell aliquots, and pellets from ultracentrifuged CFMS were extracted as suggested by standard methods (15). The LAMP reaction was carried out with a DNA amplification kitg and a fluorescent detection reagentg according to the manufacturer’s instructions.
(5). Third, as mentioned above, earlier studies have revealed the capacity of some human cell lines to support BoHV-4 replication, suggesting the ability of the virus to infect humans (11, 12, 17). DCs were grown from bone marrow progenitors of BALB/c mice in RPMI 1640 (PAA Laboratories) with 10% heat-inactivated (56°C, 30 min) FBS (PAA Laboratories or Amimed), 2 mM l-glutamine (PAA Laboratories), 50 μM 2-mercaptoethanol (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories), and 8 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Invitrogen). B cells are likely to be the means for trafficking MHV-68 from the lung to the spleen  and expression of vtRNAs, a marker for latency, has been localized to the germinal centers in the spleen [38-40]. coli clones carrying pBAC-BoHV-4 recombinants were analyzed by HindIII restriction enzyme digestion and confirmed by DNA blotting with probes specific for the three chimeric ORFs (). The primers were checked for self-complementarity by the method of Innis et al. (11).
The oligonucleotides were designated α (5′-CGAATTATAGTCTAAAGTCATCCTC-3′) and β (5′- GTAAGGACCTTTCACACTCTTAAGC-3′), and amplification led to a 2,538-bp fragment which includes the 3′ end of open reading frame 1 (ORF1) (homologous to the EBV BVRF1 gene), ORF2 (homologous to the Epstein-Barr virus BXRF1 gene), the thymidine kinase gene (ORF3), and the 5′ end of the glycoprotein H gene (ORF4). The PCR product was electrophoresed in 1% agarose gel and visualized after ethidium bromide staining. The expected, amplified 2,538-bp fragment was extracted from the agarose gel, digested with HindIII restriction endonuclease, and analyzed on 1.5% agarose gel in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA) containing ethidium bromide for DNA staining. The gel was run for 2.5 h in 1× TAE buffer. The specificity of the PCR product was determined by sequencing, using a ThermoSequenase kit (Amersham International, Amersham, United Kingdom). The resulting sequence was checked with the corresponding sequence in the GenBank database, under accession number S49773(13). Results and discussion.A herd of 100 dairy cattle was tested by IFAT for BoHV-4 antibodies, and 19 of 100 cows tested were found positive.
To assess the BoHV-4 infection of the 19 BoHV-4 IFAT-positive cows, IFAT was repeated three times at 2-month intervals during a period of 6 months. However, since the total available number of the highly similar sequence Gen Bank-derived EHV-4 reference strains aligned to EHV-4 VRLCU-412-2015 at the time conducting this investigation was eleven, a wide-based future comparative study with more reference strains is recommended to retrieve more informative phylogenic data. Blood and milk of these seven cows were collected for BoHV-4 DNA detection. Denatured probe was added to 50 mL of pre-heated 65 °C hybridization solution [7 % SDS, 0.5 M phosphate, pH 7.2 and 1 mM ethylenediaminetetraacetic acid (EDTA)] to the pre-hybridized membrane and hybridized overnight at 65 °C in a rotating hybridization oven (Techna Instruments). 1A) and in three of the seven milk cell fractions, too (Fig.2A), but none was detected in the CFMS (data not shown). The identity of the amplicon was confirmed byHindIII restriction enzyme digestion (Fig. 1C); the same fragments predicted from the published sequence were detected in the PCR products of all seven PBMC samples (Fig.
1B) and three milk fraction samples (Fig. 2B). Then, to demonstrate the infectious nature of the virus in the milk cell fraction, we cocultured the milk cell fraction with a BoHV-4-sensitive cell line. Cocultures with each of the three samples developed cytopathic effects. To confirm the specificity of the cytopathic effects, the resulting plaques were stained with an anti-BoHV-4 hyperimmune serum, and a specific positive staining was obtained (data not shown). To determine whether the milk cell fraction was productively infected or if the virus obtained after cocultivation with sensitive cells was just the reactivation of latent BoHV-4 genomes, we froze and thawed the milk cell fraction coming from the three cell milk fraction PCR-positive cows (samples 2, 3, and 6). That procedure ensured that cells were killed and only infectious virus could be recovered after cocultivation with sensitive cells.
BoHV-4 was recovered from all three cell fraction samples (data not shown). However, similar results were obtained in horse nos. This result indicates a productive infection. Immunofluorescence staining (incubation and washes) was performed in PBS containing 10% fetal calf serum (BioWhittaker, Verviers, Belgium). The amino acid and nucleotide sequences of the wild-type (WT) FCS and … Schematic of approach to generating recombinant viruses. Ten week-old BALB/c and BALB-neuT mice were anesthetized by intramuscular injection (i.m.) of 40 μL of a solution containing 5.7 μL of Zoletil 100 (Vibrac, Milano, Italia), 3.5 μL of Rompum (Bayer, Milano, Italia) and 37.5 μL of Phosphate Buffer Saline (PBS) (GIBCO, Grand Island, NY, USA).
(B) Predicted location of HindIII restriction sites and respective expected restriction fragment sizes (not shown to scale). (C) Ethidium bromide-stained gel of HindIII-digested 2,538-bp BoHV-4 amplified sequence from PBMC DNA samples (lanes 1 to 7). 1 kb, molecular size marker. (A) Specific amplification, performed with primers α and β, of DNA from milk cell fractions of milk coming from PBMC PCR-positive cows and producing a 2,538-bp product. Lanes 1 to 7 correspond to milk cell fraction samples; lanes − and + correspond to negative and positive controls respectively. 1 kb, molecular size marker. (B) Ethidium bromide-stained gel ofHindIII-digested 2,538-bp BoHV-4 amplified sequence from PBMC DNA samples (2, 3, and 6).
1 kb, molecular size marker. This study does not define the type of cell carrying the virus into the milk. However, the demonstration that BoHV-4 can establish a persistent infection in lymphoid tissues and a prolonged viremia associated with the PBMC fraction (17, 9, 10) could explain the recovery of the virus in the milk as a consequence of transport through lymphatic and circulatory system routes. We have examined BoHV-4 only from the milk of animals serologically positive for the virus and positive for the presence of the virus in PBMC. BoHV-4 presence or prevalence in milk of other populations has not been determined. 4.6. Bovine herpesviruses can generally be transmitted by horizontal and vertical routes.
The horizontal transmission occurs by close contact with moist contaminated surfaces, but droplet infections are also common. Vertical transmission via fetal infection occurs during parturition (14). Because bovine herpesviruses are highly labile once shed from the body and are readily inactivated by sunlight or drying, milk represents a good candidate as a vehicle for BoHV-4 shedding and transmission. The virus is protected by cell lipid membranes, and this could increase the probability of BoHV-4 infection of nursing animals through the oral mucosa surface. In addition, the humoral immune response following BoHV-4 infection in cattle is characterized by the production of low-avidity neutralizing antibodies (17, 22). The lack of neutralizing antibodies in milk and colostrum from infected cows could favor the transmission of BoHV-4 to nursing calves. Whether contact with infectious milk plays a role in the transmission of the virus or the stage at which shedding of BoHV-4 in milk occurs is unknown.
Although this report demonstrates the potential infectivity of BoHV-4 in milk, the importance of BoHV-4 shedding in milk in the transmission of the virus remains to be determined. However, this report emphasizes that infectious BoHV-4 can be present in milk, and nursing may be one of the transmission routes of BoHV-4.