A Single Amino Acid Substitution in the Matrix Protein (M51R) of Vesicular Stomatitis New Jersey Virus Impairs Replication in Cultured Porcine Macrophages and Results in Significant Attenuation in Pigs

Abstract

In this study, we explore the virulence of vesicular stomatitis New Jersey virus (VSNJV) in pigs and its potential relationship with the virus's ability to modulate innate responses. For this purpose, we developed a mutant of the highly virulent strain NJ0612NME6, containing a single amino acid substitution in the matrix protein (M51R). The M51R mutant of NJ0612NME6 was unable to suppress the transcription of genes associated with the innate immune response both in primary fetal porcine kidney cells and porcine primary macrophage cultures. Impaired viral growth was observed only in porcine macrophage cultures, indicating that the M51 residue is required for efficient replication of VSNJV in these cells. Furthermore, when inoculated in pigs by intradermal scarification of the snout, M51R infection was characterized by decreased clinical signs including reduced fever and development of less and smaller secondary vesicular lesions. Pigs infected with M51R had decreased levels of viral shedding and absence of RNAemia compared to the parental virus. The ability of the mutant virus to infect pigs by direct contact remained intact, indicating that the M51R mutation resulted in a partially attenuated phenotype capable of causing primary lesions and transmitting to sentinel pigs. Collectively, our results show a positive correlation between the ability of VSNJV to counteract the innate immune response in swine macrophage cultures and the level of virulence in pigs, a natural host of this virus. More studies are encouraged to evaluate the interaction of VSNJV with macrophages and other components of the immune response in pigs.

Keywords: M51R; immune response; macrophages; pathogenesis; type 1 interferon; vesicular stomatitis; virulence.

Figure 1

In vitro characterization of rNJ0612NME6 and M51R viruses in fetal porcine kidney cells (FPKC). (A) MTT assay was used to assess differences between both viruses to induce cytotoxicity in FPKC at different MOI’s, using untreated and previously treated cells with an external source of type I interferon (IFN) (2 units/ml of recombinant porcine IFN-α2A per well). Apoptosis was not detected in both controls: FPKC only treated with IFN-α2A, and untreated ones. (B) Multistep growth curves were performed in FPKC to compare virus yields between both viruses at specific times post-infection, using at initial MOI = 0.01. (C) Conventional plaque assay was conducted on FPKC to monitor differences in plaque formation between both viruses. (D) Mx-CAT reporter assay was used to test the ability of both viruses to induce the production of type I INF in FPKC. Samples were collected from multistep growth curves experiments at 24 hours post-infection (hpi). (E) Differences in the transcriptional immune gene response induced by different viruses on FPKC, were assessed by qRT-PCR. RQ values represent relative quantities of mRNA accumulation (estimated by 2^−ΔΔCt) with their corresponding SD. All experiments were conducted in triplicates.

Figure 2

In vitro characterization of rNJ0612NME6 and M51R viruses in porcine monocyte-derived macrophages cell cultures (PM-MQC). (A) Multistep growth curves were performed in PM-MQC to compare virus yields between both viruses at specific times post-infection, using at initial MOI = 0.01. Differences in the transcriptional immune gene response induced by different viruses on PM-MQC, were assessed by qRT-PCR. RQ values represent relative quantities of mRNA accumulation (estimated by 2^−ΔΔCt) with their corresponding SD in (B) type I, II and III IFN, (C) cytokines and chemokines, (D) Interferon regulatory factors, and transcription factors, and (E) Interferon stimulated genes. All experiments were conducted in triplicates.

Figure 3

Clinical evaluation in pigs. Two independent groups of pigs were inoculated with 1 × 10⁷ TCID₅₀ with either rNJ0612NME6 or M51R viruses. (A) Changes in rectal temperatures, denoting fever in pigs direct inoculated or infected by contact exposure within each group, were monitored daily during the experiments. Dashed lines represent the standard rectal temperature of an uninfected pig. (B) The development of vesicular lesions in snout, mouth, lips, feet was examined daily in both groups of pigs. In both groups, maximum clinical scores were reached at 8 dpi. In both cases values represent means and standard deviations of cumulative clinical scores within each group of pigs.

Figure 4

Clinical differences between groups of pigs infected with either rNJ0612NME6 or M51R viruses. (A) Despite the intrinsic characteristics of M51R virus to overcome the host innate immune response, clinical outcomes from different pigs infected within this group were characterized by the development of vesicular FIGURE 4lesions in both direct inoculated and contact exposure pigs. Overall pigs in this group kept a general health condition during the entire experiment (absence of fever, general depression, and lameness). (B) Contrasting differences were observed in pigs infected with rNJ0612NME6 in terms of vesicular lesion size development, systemic dissemination characterized for the development of vesicular lesions at different anatomic sites, and overall alteration of the health condition, especially during the acute phase of the infection.

Figure 5

Differences of viral shedding on nasal swabs between groups of pigs infected with either rNJ0612NME6 or M51R viruses. (A) Levels of Vesicular stomatitis virus (VSV) nucleocapsid (N) RNA in different samples were quantified by rRT-PCR. Limit of detection of qRT-PCR using for this study was previously determined to be about 1.98 VSV N copies/2.5 μl RNA. (B) Viral isolations were conducted on Vero cells, and then viral titers from positive samples were determined and expressed in TCID₅₀/ml. Values represent means and standard deviations within different groups of pigs.

Figure 6

Differences of viral shedding on oral swabs between groups of pigs infected with either rNJ0612NME6 or M51R viruses. (A) Levels of VSV nucleocapsid (N) RNA in different samples were quantified by rRT-PCR. Limit of detection of qRT-PCR using for this study was previously determined to be about 1.98 VSV N copies/2.5 μl RNA. (B) Viral isolations were conducted on Vero cells, and then viral titers from positive samples were determined and expressed in TCID₅₀/ml. Values represent means and standard deviations within different groups of pigs.

Figure 7

Differences of RNAemia levels between groups of pigs infected with either rNJ0612NME6 or M51R viruses. Levels of RNAemia were quantified by rRT-PCR from blood samples collected daily from all pigs during the course of this experiment. Limit of detection of qRT-PCR using for this study was previously determined to be about 1.98 VSV N copies/2.5μl RNA. Values represent means and standard deviations within different groups of pigs.

Figure 8

Differences in systemic levels of type I IFN induction between groups of pigs infected with either rNJ0612NME6 or M51R viruses. Mx-CAT reporter assay was used to monitor the systemic type I IFN production in pigs during the acute phase of the infection with different viruses. Values represent means and standard deviations within different groups of pigs. Asterisk at 3 dpi shows significant differences (p < 0.05) between pigs infected with rNJ0612NME6 and M51R viruses.

Figure 9

Differences in neutralizing antibody production between groups of pigs infected with either rNJ0612NME6 or M51R viruses. Viral neutralization assay was used to monitor the development of neutralizing antibody titers in pigs, using serum samples collected during the course of this experiment. Values represent means and standard deviations within different groups of pigs.

Figure 10

Differences in viral RNA accumulation in postmortem tissues between groups of pigs infected with either rNJ0612NME6 or M51R viruses. Postmortem tissues were collected at 21 dpi, and analyzed by qRT-PCR to detect VSV nucleocapsid RNA. Limit of detection of qRT-PCR using for this study was previously determined to be about 1.98 VSV N copies/2.5μl RNA. Values represent means and standard deviations within different groups of pigs. Abbreviations: tonsil of the soft palate (PTON), submandibular lymph node (SMLN), spleen (SPL), snout skin (SNT), popliteal lymph node (PopLN), and skeletal muscle (SKM).