Pseudorabies Virus Propagated in Rabbit Kidney-Derived RK13 Cells is Neutralized by Natural IgM Antibodies in Normal Swine Serum which Specifically Lyse Host Cells

Abstract

ABSTRACT. Pseudorabies virus (PRV) propagated in rabbit kidney-derived RK-13 cells (PRV-RK) was neutralized by serum obtained from specific pathogen-free pigs through the activation of complement. The virus-neutralizing activity of swine serum was lost after treatment with ethylene glycol-bis-aminoethylether-N,N,N',N'-tetraacetic acid (EGTA) or ethylenediaminetetraacetic acid (EDTA). Anti-C1q and anti-IgM antibodies also inhibited virus-neutralizing activity. Though IgG-depleted swine serum neutralized PRV, IgM and IgG-free swine serum lost virus-neutralizing activity. Pre-incubation of swine serum with RK-13 cells, but not with swine kidney-derived CPK cells, at 4°C eliminated the virus-neutralizing activity to PRV-RK. Results indicated that swine serum contained natural IgM against an antigen(s) on the RK-13 cell surface and that this surface antigen was integrated into the PRV envelope during the budding process. Thus the natural IgM in swine serum reacted with the RK-13 antigen on the viral envelope, activated the complement cascade and neutralized the PRV-RK. KEY WORDS: complement, natural antibodies, PRV.J. Vet. Med. Sci. 67(3): 229-234, 2005 We previously reported that pseudorabies virus (PRV) grown in rabbit kidney-derived RK-13 cells was neutralized by swine serum while the same virus grown in porcine kidney-derived CPK cells was resistant [17]. However, the virus-neutralizing activity of swine serum was lost when serum was heat-inactivated at 56°C for 30 min. We speculated that neutralization of PRV was caused by activation of complement via the alternative pathway because of the apparent absence of anti-PRV antibodies in swine serum [17]. We also found that a similar phenomenon was observed when PRV grown in several cell lines was incubated with human serum [13]. PRV grown in several cell lines also showed different resistant levels against rabbit serum [13].It has been known that human immunodeficiency virus type 1 acquires cell membrane complement regulatory factors on its envelope from cells during the budding process, thereby escaping from complement attack [18]. Therefore it was assumed that RK-13-grown PRV acquired complement regulatory factors from the host rabbit cells and these were less effective against swine complement attack. On the other hand PRV grown in CPK cells acquired swine complement regulatory factors which are more effective regulators against swine complement and thus were resistant to swine complement attack [17].However, further study revealed that RK-13 cells were lysed by swine serum via the activation of complement classical pathway (Hayashi et al., in preparation). It was found that swine serum contained natural IgM antibodies that specifically reacted with an antigen or antigens on the RK-13 cell surface. Therefore a question arose whether the anti-RK-13 natural IgM in swine serum is responsible for the neutralization of PRV grown in RK-13 cells.In this study, using swine serum and PRV propagated in RK-13 cells (PRV-RK), we investigated the mechanism for the virus-neu...

Robust expression of p27Kip1 induced by viral infection is critical for antiviral innate immunity

“…MDRV and HSV‐1 virus was propagated in Vero cells as previously described (Ouyang et al, 2014). PRV strain Min‐A was propagated in Madin–Darby Canine Kidney (MDCK) cells, as previously described (Hayashi, Takashima, & Otsuka, 2005; Wei et al, 2017). The following antibodies were used in this study: monoclonal anti‐p27Kip1, anti‐phospho STAT1 (Cell signalling, CST); anti‐influenza A NS1, anti‐RIG‐I, anti‐β‐actin, anti‐STAT1, anti‐Myc (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal p27Kip1, anti‐IFITM3 (Proteintech, Chicago, IL); anti‐IAV NP polyclonal antibody was obtained by immunising rabbits with GST‐tagged NP protein as previously described (Wang et al, 2014).…”

An amphioxus gC1q protein binds human IgG and initiates the classical pathway: Implications for a C1q‐mediated complement system in the basal chordate

“…The C1q‐, IgG‐, and IgM‐depleted human serum was prepared from the C1q‐depleted serum (CALBIOCHEM, USA) as previously described . Briefly, 500 μL of goat anti‐human IgM serum (Bioss) was incubated with 50 μL of protein A/G PLUS‐Agarose beads (Santa Cruz Biotechnology) at 4°C for 1 h. After washed twice with TBS, 50 μL of protein A/G beads coupled with goat anti‐human IgM Abs was incubated with 500 μL of C1q‐depleted human serum at 4°C for 2 h. The mixture was centrifuged at 3 000 × g for 10 min, and the supernatant was pooled and applied to a prepacked protein A column (HiTrapTM protein A HP, Amersham Biosciences, Sweden).…”

Identification of an Interferon-Stimulated Long Noncoding RNA (LncRNA ISR) Involved in Regulation of Influenza A Virus Replication

“…Sendai virus (SeV) were propagated in specific pathogen free (SPF) embryonated chicken eggs, as previously described [27]. Pseudorabies virus (PRV) strain Min-A was propagated in Madin–Darby canine kidney cells (MDCK) cells, as previously described [35,36]. Herpes simplex virus 1 (HSV-1) was propagated in Vero cells as previously described [27].…”