Influence of Tegument Proteins of Pseudorabies Virus on Neuroinvasion and Transneuronal Spread in the Nervous System of Adult Mice after Intranasal Inoculation

Abstract

Pseudorabies virus (PrV) is a neurotropic alphaherpesvirus that, after intranasal infection of adult mice, enters peripheral neurons and propagates to the central nervous system. In recent years we have analyzed the contribution of virus-encoded glycoproteins to neuroinvasion and transneuronal spread (reviewed in T. C. Mettenleiter, Virus Res. 92:197-206, 2003). We now extend our studies to analyze the role of tegument proteins in these processes. To this end, PrV mutants unable to express the UL11, UL37, UL46, UL47, and UL48 tegument proteins, as well as the corresponding rescued viruses, were intranasally instilled into 6-to 8-weekold CD1 strain mice. First, mean survival times were determined which showed that mice infected with the UL46 deletion mutant succumbed to the disease as early as wild-type PrV-infected animals. Survival times increased in the order: PrV-⌬UL47-, PrV-⌬UL11-, and PrV-⌬UL48-infected animals, a finding which parallels the growth phenotype of these viruses in cell culture. In contrast, none of the PrV-⌬UL37-infected animals died. Upon closer histological examination, all viruses except PrV-⌬UL37 were able to infect the nasal cavity and propagate to first-and second-order neurons as shown by two-color immunofluorescence. However, neuroinvasion was delayed in PrV-⌬UL47, PrV-⌬UL11, and PrV-⌬UL48, a finding that correlated with the extended survival times. Surprisingly, whereas PrV-⌬UL48 and PrV-⌬UL37 replicated to similar titers in cell culture which were ϳ500-fold lower than those of wild-type virus, after intranasal infection of mice PrV-⌬UL48 was able to infect areas of the brain like wild-type PrV, although only after a considerably longer time period. In contrast, PrV-⌬UL37 was not able to enter neurons and was restricted to the infection of single cells in the nasal respiratory epithelium. Thus, our data demonstrate the importance of herpesviral tegument proteins in neuronal infection and show a different contribution of tegument proteins to the neuroinvasion phenotype of a neurotropic alphaherpesvirus.Pseudorabies virus (PrV) is an alphaherpesvirus that causes Aujeszky's disease, a severe neurological disorder, in a broad range of mammalian species. The pig is the natural host of PrV since it is the only animal able to survive a productive infection, and latently infected pigs serve as a virus reservoir. After oronasal infection, which is the natural route of PrV entry into the organism, adult pigs usually show only mild clinical signs of respiratory distress or reproductive failure. In contrast, young piglets are very sensitive to virus invasion of the central nervous system (CNS) (36). Pruritus, the hallmark of the syndrome in nonporcine species, is usually not observed in pigs. In contrast to pigs, the course of infection in susceptible nonporcine species is peracute with a high mortality. These species are endstage hosts, i.e., they generally do not shed the virus and are therefore less important in transmission to other species (25,29,30,45,56,60).Since PrV infects a wide range o...

Functional Analysis of the Pseudorabies Virus UL51 Protein

“…For determination of mean survival times, 10 6-to 8-week-old CD1 mice for PrV-Ka and PrV-⌬UL51F and three animals for PrV-⌬UL51FR were anesthetized and infected by bilateral intranasal instillation of a 5-l suspension containing 10 6 PFU. Animals were observed three times a day and euthanized when severe pruritus with excitations and self-mutilation ("mad-itch" syndrome) occurred (20). To analyze the kinetics of viral spread in the trigeminal circuit, mice were euthanized sequentially every 24 h after inoculation, and tissues were prepared and analyzed as described previously (20).…”

“…To analyze the kinetics of viral spread in the trigeminal circuit, mice were euthanized sequentially every 24 h after inoculation, and tissues were prepared and analyzed as described previously (20). Virus-infected cells were identified by incubation with monospecific antiserum against the major capsid protein of PrV (1:500 in Tris-buffered saline [20]), followed by biotinylated goat anti-rabbit immunoglobulin G1 (1:200; Vector, Burlingame, Calif.). Antibody binding was visualized with the avidin-biotin complex method (1:10 in Tris-buffered saline; Vector) (18) using AEC substrate (Dako, Hamburg, Germany) as chromogen.…”

“…Animals were observed three times a day and euthanized when severe pruritus with excitations and self-mutilation ("mad-itch" syndrome) occurred (20). To analyze the kinetics of viral spread in the trigeminal circuit, mice were euthanized sequentially every 24 h after inoculation, and tissues were prepared and analyzed as described previously (20). Virus-infected cells were identified by incubation with monospecific antiserum against the major capsid protein of PrV (1:500 in Tris-buffered saline [20]), followed by biotinylated goat anti-rabbit immunoglobulin G1 (1:200; Vector, Burlingame, Calif.).…”

Deletion or green fluorescent protein tagging of the pUL35 capsid component of pseudorabies virus impairs virus replication in cell culture and neuroinvasion in mice

“…(Table 1). The major capsid protein (pUL19) of PrV was detected by immunohistochemistry of skull sections of necropsied animals (Klopfleisch et al, 2004) in epithelial cells of the nasal mucosa of mice infected with either virus at 24 h p.i. At this time, PrV-Ka and PrV-DUL35R were also found in first-order neurons of the trigeminal ganglia, whereas PrV-DUL35 and PrV-UL35GFP were detectable in neurons starting at 48 h p.i.…”

“…The relevance of pUL35 for neurovirulence of PrV was investigated in a mouse model (Klopfleisch et al, 2004). Ten eight-week-old mice each were infected by bilateral intranasal instillation of 10 4 p.f.u.…”