Deciphering the Role of Bovine Viral Diarrhea Virus Non-Structural NS4B Protein in Viral Pathogenesis

Abstract

Bovine viral diarrhea virus (BVDV) is a (+) ssRNA virus that belongs to the family Flaviviridae. BVDV is a significant animal pathogen causing substantial economic losses to the cattle industry worldwide through respiratory and gastrointestinal infections and abortion or birth of persistently infected calves. While the immunogenic profile of some of the BVDV proteins (i.e., E^rns, E2 and NS3) is well established during viral pathogenesis, very little information is available about most of BVDV's non-structural proteins in this regard. In recent times, the NS4B protein has emerged as an interesting target of diagnostic, vaccination and therapeutic value in viral infections of other members of the family Flaviviridae due to its key scaffold-like contribution in the viral replication complex. Although, BVDV-NS4B has a membrane topology alongside its role in induction of autophagosomes in vitro. However, information on its immunogenicity during BVDV pathogenesis and vaccination is scarce. To characterize the immunogenic profile of the NS4B, five cows were vaccinated with the live attenuated BVDV vaccine Bovela^® and blood samples were taken pre- and post-immunization for serum isolation. Virus neutralization assay (VNA) confirmed the presence of anti-BVDV antibodies in the sera of vaccinated cows. VNA also revealed pre-existing antibodies against BVDV in the pre-immunization sera of two cows. To identify BVDV-NS4B specific antibodies, the NS4B protein was expressed in mammalian cells by using the pCI-neo vector system. The sera from BVDV vaccinated cows were evaluated for the presence of BVDV-NS4B specific antibodies through western blot and indirect ELISA. Interestingly, t sera from cows with pre-existing immunity against BVDV were able to detect NS4B in western blot and ELISA, suggesting the presence of NS4B-specific antibodies. The obtained results provide the first indication of the immunogenic nature of BVDV-NS4B protein in sero-converted animals. These findings are consistent with the observation made for NS4B in other Flaviviridae members and confirm this protein as an interesting target with diagnostic, vaccination and therapeutic value.

Keywords: NS4B; antibodies; bovine viral diarrhea virus; immunogenic; western blot.

Figure 1

Experimental timeline for the study. Pre-immunization blood collection and Bovela^® vaccination was performed on day 0 with booster vaccination on day 21. The second blood collection was performed on day 42 which served as a source of post-immunization sera.

Figure 2

Western blot analysis for detection of NS4B protein with serum from Bovine viral diarrhea virus (BVDV)-vaccinated cows. NS4B was expressed in CHO cells, as outlined in Supplementary Figure S1. After 72 h of transfection, membrane protein fractions were retrieved by the Mem-PER™ Plus kit and separated on 12% SDS-PAGE and transferred to nitrocellulose membrane. (A) Western blot with pre-immune sera; NS4B recognized by pre-immune sera of cow 3 and 5. Lane 1 and 5) CHO-empty pCI-neo vector lysate, Lane 2 and 6) Madin Darby bovine Kidney (MDBK) cell lysate, Lane 3 and 7) 38 kDa NS4B recognized by the sera of the cow 3 and 5, respectively, Lane 4 and 8) NADL infected MDBK cell lysate. (B) western blot with post-immune sera; Lane 1, 5 and 12) CHO-empty pCI-neo vector lysate, Lane 2, 6 and 11) MDBK cell lysate, Lane 3, 8 and 9) 38kD NS4B protein recognized by the sera of the cow 3, 4 and 5, respectively, Lane 4, 7 and 10) NADL-infected MDBK cell lysate.

Figure 3

Indirect ELISA for detection of NS4B by using BVDV vaccinated cow sera. Results are being expressed as the difference between the OD405nM obtained with the NS4B and with negative control. Pre- and post-immune sera of cow 3 and 5 are positive for detection of NS4B.