Generation of calves persistently infected with HoBi-like pestivirus and comparison of methods for detection of these persistent infections

Abstract

The identification and elimination of persistently infected (PI) cattle are the most effective measures for controlling bovine pestiviruses, including bovine viral diarrhea virus (BVDV) and the emerging HoBi-like viruses. Here, colostrum-deprived calves persistently infected with HoBi-like pestivirus (HoBi-like PI calves) were generated and sampled (serum, buffy coat, and ear notches) on the day of birth (DOB) and weekly for 5 consecutive weeks. The samples were subjected to diagnostic tests for BVDV--two reverse transcriptase PCR (RT-PCR) assays, two commercial real-time RT quantitative PCR (RT-qPCR), two antigen capture enzyme-linked immunosorbent assays (ACE), and immunohistochemistry (IHC)--and to HoBi-like virus-specific RT-PCR and RT-qPCR assays. The rate of false negatives varied among the calves. The HoBi-like virus-specific RT-PCR detected HoBi-like virus in 83%, 75%, and 87% of the serum, buffy coat, and ear notch samples, respectively, while the HoBi-like RT-qPCR detected the virus in 83%, 96%, and 62%, respectively. In comparison, the BVDV RT-PCR test had a higher rate of false negatives in all tissue types, especially for the ear notch samples (missing detection in at least 68% of the samples). The commercial BVDV RT-qPCRs and IHC detected 100% of the ear notch samples as positive. While ACE based on the BVDV glycoprotein E(rns) detected infection in at least 87% of ear notches, no infections were detected using NS3-based ACE. The BVDV RT-qPCR, ACE, and IHC yielded higher levels of detection than the HoBi-like virus-specific assays, although the lack of differentiation between BVDV and HoBi-like viruses would make these tests of limited use for the control and/or surveillance of persistent HoBi-like virus infection. An improvement in HoBi-like virus tests is required before a reliable HoBi-like PI surveillance program can be designed.

FIG 1

(A) Aural skin biopsy specimens from a fetus infected with strain HoBi_D32/00, stained using monoclonal antibody 15C5. Positive staining in hair follicle infundibula and apocrine glands. (B) Calf 101 at 1 day of age, persistently infected with the HoBi-like virus strain Italy-1/10-1. Positive staining in epidermis, hair follicle infundibula, sebaceous glands, and dermal fibrocytes. (C) Calf 102 at 1 day of age, persistently infected with the HoBi-like virus strain HoBi_D32/00. Positive staining in epidermis, hair follicle infundibula, sebaceous glands, and arterial wall.

FIG 2

Results of testing animals persistently infected with a HoBi-like virus from day of birth (DOB) to week 5 (W5). White squares with a plus sign indicate a positive result. Positive results for the RT-qPCR are represented by C_q values. Dark squares indicate a negative result. Test identifications: BVDV RT-PCR-1, primers 324 and 326; BVDV RT-PCR-2, primers HCV 90 and 368; BVDV RT-qPCR-1, VetMAX-Gold-bovine virus diarrhea RNA test kit; BVDV RT-qPCR-2, Virotype BVDV test kit; conventional extracted samples, E^rns antigen capture ELISA, HerdCheck BVD antigen; enhanced extracted samples, E^rns antigen capture ELISA; enhanced extracted samples, NS3 antigen capture ELISA; immunohistochemistry; HoBi-like virus-specific RT-PCR, primers N2 and R5; HoBi-like virus-specific RT-qPCR, primers T134-F and T220-R and probe T155r-P. The cutoff value used for all the RT-qPCR tests was 38 cycles.

FIG 3

Sample-to-positive (S/P) ratio for each persistently infected (PI) calf (101, 102, 103, and 104). Samples from day of birth (DOB) and weekly for five consecutive weeks (W1 to W5) were tested using the commercial E^rns ACE kit, with a conventional extraction (Conv. ext.) or an enhanced extraction (Enh. ext.) process, and are represented by triangles or squares, respectively. Axis x crosses axis y at 0.3, the threshold S/P ratio for the E^rns antigen capture ELISA used.

FIG 4

Alignment of the HoBi-like virus isolates HoBi_D32/00 (AB871953.1) and Italy-1/10-1 (HQ231763.1) with the primers N2, R5, T134-F, and T220-R and the probe T155r-P, used for the specific detection of HoBi-like viruses. Reverse primers R5 and T220-R and the probe T155r-P are presented as the reverse complement of the original sequence.