Age-Dependent Differences in Pseudorabies Virus Neuropathogenesis and Associated Cytokine Expression

Abstract

The severity of clinical symptoms induced by pseudorabies virus (PRV) infection of its natural host is inversely related to the age of the pig. During this study, 2- and 15-week-old pigs were inoculated with PRV strain NIA3. This resulted in important clinical disease, although the associated morbidity and mortality were lower in older pigs. Quantitative PCR analysis of viral DNA in different organs confirmed the general knowledge on PRV pathogenesis. Several new findings and potential explanations for the observed age-dependent differences in virulence, however, were determined from the study of viral and cytokine mRNA expression at important sites of neuropathogenesis. First, only limited viral and cytokine mRNA expression was detected in the nasal mucosa, suggesting that other sites may serve as the primary replication site. Second, PRV reached the trigeminal ganglion (TG) and brain stem rapidly upon infection but, compared to 2-week-old pigs, viral replication was less pronounced in 15-week-old pigs, and the decrease in viral mRNA expression was not preceded by or associated with an increased cytokine expression. Third, extensive viral replication associated with a robust expression of cytokine mRNA was detected in the olfactory bulbs of pigs from both age categories and correlated with the observed neurological disease. Our results suggest that age-dependent differences in PRV-induced clinical signs are probably due to enhanced viral replication and associated immunopathology in immature TG and the central nervous system neurons of 2-week-old pigs and that neurological disease is related with extensive viral replication and an associated immune response in the olfactory bulb.

Importance: It is well known that alphaherpesvirus infections of humans and animals result in more severe clinical disease in newborns than in older individuals and that this is probably related to differences in neuropathogenesis. The underlying mechanisms, however, remain unclear. Pseudorabies virus infection of its natural host, the pig, provides a suitable infection model to study this more profoundly. We show here that the severe neurological disease observed in 2-week-old pigs does not appear to be related to a hampered innate immune response but is more likely to reflect the immature development state of the trigeminal ganglia (TG) and central nervous system (CNS) neurons, resulting in an inefficient suppression of viral replication. In 15-week-old pigs, viral replication was efficiently suppressed in the TG and CNS without induction of an extensive immune response. Furthermore, our results provide evidence that neurological disease could, at least in part, be related to viral replication and associated immunopathology in the olfactory bulb.

Keywords: neuropathogenesis; olfactory bulb; pseudorabies virus; trigeminal ganglion; virulence.

FIG 1

Distribution of viral DNA in different tissues after NIA3 infection. Several tissues were collected from domestic 2- and 15-week-old pigs at different time points after intranasal inoculation with the NIA3 reference strain and tested by qPCR directed against gB. Viral DNA concentrations for different primary and secondary sites of replication for 2- and 15-week-old pigs (a and c, respectively) and for peripheral and central nervous system tissues for 2- and 15-week-old pigs (b and d, respectively) are shown.

FIG 2

Nasal mucosa. Domestic 2- and 15-week-old pig were intranasally inoculated with the NIA3 strain and euthanized at different time points postinfection. mRNA expression of several PRV genes in the nasal mucosa was studied by RT-qPCR in 2- and 15-week-old pigs (a and d, respectively). Viral gene levels were expressed relative to the lowest positive sample for each age category. Furthermore, cytokine-related mRNA expression in de nasal mucosa was tested by RT-qPCR in 2- and 15-week-old pigs (panels b and c and panels e and f, respectively). Individual cytokine levels for all animals are expressed relative to the average cytokine expression in the control group (separately for 2- and 15-week-old animals).

FIG 3

PRV immunofluorescence staining. Immunofluorescence images of the nasal mucosa (a) and olfactory bulb (b) of the NIA3-inoculated 2-week-old piglet N08 euthanized at 3 days p.i. PRV antigens (green) were stained with a combination of mouse monoclonal anti-gB and -gD antibodies (1:100 in PBS) followed by an incubation step with FITC-labeled goat anti-mouse antibodies (1:200). Nuclei (blue) were counterstained with Hoechst 33342. Images were taken with a 63× objective on a confocal microscope.

FIG 4

Trigeminal ganglion. Domestic 2- and 15-week-old pigs were intranasally inoculated with the NIA3 strain and euthanized at different time points postinfection. The mRNA expression of several PRV genes in the trigeminal ganglia was studied by RT-qPCR in 2- and 15-week-old pigs (a and d, respectively). Viral gene levels were expressed relative to the lowest positive sample for each age category. Furthermore, cytokine-related mRNA expression in the trigeminal ganglion was tested by RT-qPCR in 2- and 15-week-old pigs (panels b and c and panels e and f, respectively). Individual cytokine levels for all animals are expressed relative to the average cytokine expression in the control group (separately for 2- and 15-week-old animals).

FIG 5

Pons. Domestic 2- and 15-week-old pigs were intranasally inoculated with the NIA3 strain and euthanized at different time points postinfection. The mRNA expression of several PRV genes in the pons was studied by RT-qPCR in 2- and 15-week-old pigs (a and d, respectively). Viral gene levels were expressed relative to the lowest positive sample for each age category. Furthermore, cytokine-related mRNA expression in the pons was tested by RT-qPCR in 2- and 15-week-old pigs (panels b and c and panels e and f, respectively). Individual cytokine levels for all animals are expressed relative to the average cytokine expression in the control group (separately for 2- and 15-week-old animals).

FIG 6

Olfactory bulb. Domestic 2- and 15-week-old pigs were intranasally inoculated with the NIA3 strain and euthanized at different time points postinfection. The mRNA expression of several PRV genes in the olfactory bulb was studied by RT-qPCR in 2- and 15-week-old pigs (a and d, respectively). Viral gene levels were expressed relative to the lowest positive sample for each age category. Furthermore, cytokine-related mRNA expression in the olfactory bulb was tested by RT-qPCR in 2- and 15-week-old pigs (panels b and c and panels e and f, respectively). Individual cytokine levels for all animals are expressed relative to the average cytokine expression in the control group (separately for 2- and 15-week-old animals).