Detection of bovine herpesvirus type 1 in blood from naturally infected cattle by using a sensitive PCR that discriminates between wild-type virus and virus lacking glycoprotein E

Abstract

In the present study, we report for the first time on the detection of bovine herpesvirus type 1 (BHV-1) in whole-blood samples derived from naturally infected cattle. Sensitive PCR assays specific for glycoprotein B (gB), gC, and gE of BHV-1 allow the detection of one BHV-1 DNA copy in 10(5) to 10(7) peripheral blood leukocytes (PBLs). The incidence of BHV-1-positive PBLs in naturally infected cattle appears to be quite high (92.2% positive PBLs among all samples tested), although in most cases only between 10(-5) and 10(-7) positive leukocytes were present. The results demonstrate that the viral DNA is detectable not only in the peripheral blood of acutely infected animals but, more importantly, also in the peripheral blood of subclinically infected cattle. The gE-specific PCR described in the report allows discrimination between wild-type (WT) virus-infected and vaccinated animals, which is of importance for control programs that use the recently introduced vaccination strategy with a gE-negative virus. The results further show that doubtful serological results can be verified or falsified and that individual animals can be monitored for the presence or absence of WT BHV-1 or gE-negative virus in cattle herds. The PCR protocols allow the detection of BHV-1 prior to seroconversion or in BHV-1-seronegative cattle. Finally, the results indicate the simultaneous presence of WT and gE-negative vaccine virus in the PBLs of several cattle. Therefore, investigations of viremia in naturally and experimentally infected cattle and on the identification of infected cell types of bovine PBLs can be now performed.

FIG. 1

Map of the BHV-1 genome showing the unique long (U_L), unique short (U_S), and inverted repeat (IR and TR) regions. The locations of the indicated glycoprotein genes (not drawn to scale) are depicted on the map. The amplified regions and the respective sizes of the different PCR products are indicated, as are the sizes of the nested PCR products gBN1/2 and gEN1/2.

FIG. 2

Specificity of the indicated PCRs. (A) DNAs (100 ng each) of BHV-1.1 LA (lanes 1), BHV-1.2 K22 (lanes 2), gE-negative vaccine virus strain Difivac (lanes 3), and PRV (lanes 4) were used as templates. (B) Results of the indicated PCRs with DNA from BHV-5 N569 compared to the results obtained with DNA from BHV-1.1 LA. BHV-5 is not detected by the gC- and gE-specific PCR. The PCR products (10 μl) were separated in a 1.0% agarose gel. The sizes of the amplified products are indicated (lane M, 1-kb ladder as a size marker).

FIG. 3

Reconstruction experiment to determine sensitivities of PCRs. Strain LA DNA was mixed together with CT DNA to obtain the indicated concentration of BHV-1 DNA per microgram of CT DNA. Southern blot hybridization of the gE1/2 and gEN1/2 PCR products with the internal probe (derived from the gEN1/2 PCR product) radioactively labelled with ³²P was performed. The sizes of the specific amplification products are indicated. (A) Exposure of X-ray film for 30 min. A total of 10 fg of strain LA DNA per μg of CT DNA is detectable after gE-1–gE-2 PCR (upper part), and at least 10 ag of viral DNA per μg of CT DNA is detectable after nested gEN-1–gEN-2 PCR (lower part). (B) An increase in the time of exposure to X-ray film to 2 h demonstrates a 10-fold increase in the limit of detection.

FIG. 4

Detection of BHV-1 DNA in blood samples from field-virus-infected cattle (animals BO-1 to BO-8 and EC-1 to EC-8) after gB-1–gB-2 PCR (upper part) and gBN-1–gBN-2 PCR (lower part). As a positive control, 10 ng of strain LA DNA was used as a template (lane LA), and as negative controls, amplifications were performed with DNA derived from noninfected cattle (lane control) and in the absence of template (lane H₂O). Lane MW, separation of molecular size markers (1-kbp ladder). The control NF1-NF2 PCR (265 bp) was performed separately, but the PCR products were separated together with the gB-1–gB-2 PCR products in the same gel slots. (A) The PCR products were analyzed by electrophoresis in an ethidium bromide-stained agarose gel (1.0%). No visible gB1/2 product (478 bp) was amplified by the first PCR, although in most cases a successful PCR could be demonstrated by a positive NF1-NF2 amplification (upper panel). Nested PCR (gBN-1–gBN-2; 385 bp) showed the presence of BHV-1 DNA in blood samples from all animals in herds BO and EC (lower part). (B) Southern blot hybridization of the gel shown in panel A with the radioactively labelled internal probe already revealed specific amplification in some of the blood specimens after the first PCR (upper part) and corroborated the positive results of the nested PCR (lower part).

FIG. 5

Different copy numbers of gB and gE were found in individual blood samples (from animals BO-1 to BO-8 and EC-1 to EC-8). The amplification products obtained by the indicated PCRs (A to E; the size of each PCR fragment is indicated to the right) were blot hybridized with the respective internal probes. The X-ray films were exposed for 2 h (A), 1 h (B), 20 h (C), 7 h (D), and 68 h (D). It can be seen that the positive samples from animals in herd BO as well as the samples from animals EC-1, EC-3, and EC-6 contained 10- to 100-fold larger amounts of gB than gE.

FIG. 6

Detection of BHV-1 DNA in blood samples (herd FO) by gE-1–gE-2 (A and B) and nested gEN-1–gEN-2 PCR (C and D). Lane ni, the result obtained with blood from noninfected cattle. The sizes of the PCR products are indicated to the right. The ELISA results for the detection of serum antibodies against BHV-1 and against gE of BHV-1 are given at the bottom for each animal. As can be seen, samples 2, 3, 5, 6, 7, 9, and 10 were clearly positive after nested gEN-1–gEN-2 PCR, but the corresponding serum samples were all negative by gE-specific ELISA.