Bovine Herpesvirus 5 (BHV-5) Us9 Is Essential for BHV-5 Neuropathogenesis

Abstract

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alphaherpesvirus that causes fatal encephalitis in calves.In a rabbit model, the virus invades the central nervous system (CNS) anterogradely from the olfactory mucosa following intranasal infection. In addition to glycoproteins E and I (gE and gI, respectively), Us9 and its homologue in alphaherpesviruses are necessary for the viral anterograde spread from the presynaptic to postsynaptic neurons. The BHV-5 Us9 gene sequence was determined, and the predicted amino acid sequence of BHV-5 Us9 was compared with the corresponding Us9 sequences of BHV-1.1. Alignment results showed that they share 77% identity and 83% similarity. BHV-5 Us9 peptide-specific antibody recognized a doublet of 17-and 19-kDa protein bands in BHV-5-infected cell lysates and in purified virions. To determine the role of the BHV-5 Us9 gene in BHV-5 neuropathogenesis, a BHV-5 Us9 deletion recombinant was generated and its neurovirulence and neuroinvasive properties were compared with those of a Us9 rescue mutant of BHV-5 in a rabbit model. Following intranasal infection, the Us9 rescue mutant of BHV-5 displayed a wild-type level of neurovirulence and neural spread in the olfactory pathway, but the Us9 deletion mutant of BHV-5 was virtually avirulent and failed to invade the CNS. In the olfactory mucosa containing the olfactory receptor neurons, the Us9 deletion mutant virus replicated with an efficiency similar to that of the Us9 rescue mutant of BHV-5. However, the Us9 deletion mutant virus was not transported to the bulb. Confocal microscopy of the olfactory epithelium detected similar amounts of virus-specific antigens in the cell bodies of olfactory receptor neuron for both the viruses, but only the Us9 rescue mutant viral proteins were detected in the processes of the olfactory receptor neurons. When injected directly into the bulb, both viruses were equally neurovirulent, and they were transported retrogradely to areas connected to the bulb. Taken together, these results indicate that Us9 is essential for the anterograde spread of the virus from the olfactory mucosa to the bulb.

Experimental infection of rabbits with a recombinant bovine herpesvirus type 5 (BoHV-5) gI, gE and US9-negative

“…Infectious virus, viral antigens and DNA were found in limited portions of the CNS of one out of six calves infected with the recombinant (Hübner et al 2005). These results are in contrast with previous experiments performed with different gE and US9 BoHV-5 recombinants (Chowdhury et al 2000(Chowdhury et al , 2002. A BoHV-5 gE -recombinant was able to induce neurological signs in about 10 % of the infected rabbits and no deaths, while the wild type virus caused apparent neurological disease in 70 to 80 % of the infected animals.…”

“…On that occasion, and contrasting with the findings presented herein, the gE -recombinant was not found in the CNS of infected rabbits (Chowdhury et al 2000(Chowdhury et al , 2002. In another experiment, a US9 negative BoHV-5 was virtually apathogenic, did not invade the CNS and caused no death when inoculated in rabbits (Chowdhury et al 2000(Chowdhury et al , 2002. We believe that differences in age and breed of the infected rabbits could not justify such discrepancies, since these were comparable in both experiments.…”

“…A BoHV-5 gE -recombinant was able to induce neurological signs in about 10 % of the infected rabbits and no deaths, while the wild type virus caused apparent neurological disease in 70 to 80 % of the infected animals. On that occasion, and contrasting with the findings presented herein, the gE -recombinant was not found in the CNS of infected rabbits (Chowdhury et al 2000(Chowdhury et al , 2002. In another experiment, a US9 negative BoHV-5 was virtually apathogenic, did not invade the CNS and caused no death when inoculated in rabbits (Chowdhury et al 2000(Chowdhury et al , 2002.…”

“…As gI, gE and US9 were previously reported to play a role in neurovirulence of BoHV-5 (Chowdhury et al 2000(Chowdhury et al , 2002, the present study was carried out to examine the neurovirulence and the potential for reactivation of a recombinant BoHV-5 lacking the gI, gE and US9 genes. When the recombinant BoHV-5 gI -/gE -/US9 -was inoculated intranasally in four weeks-old rabbits, it was less virulent than the wild type BoHV-5.…”

“…In an experimental infection of rabbits with a BoHV-5 gE negative recombinant, the virus dissemination in CNS and induction of neurological signs were significantly impaired (Chowdhury et al 2000). In addition, also for BoHV-5, US9 was shown to play an important role in viral transport from the olfactory receptor neurons to second order neurons within the olfactory bulb of rabbits, and a US9 negative BoHV-5 recombinant was virtually nonpathogenic and failed to invade the CNS (Chowdhury et al 2002).…”