Progress 09/15/04 to 09/14/08
Outputs
OUTPUTS: We have determined that both gE and Us9-deleted BHV-1 viruses established latent infection in the trigeminal ganglia (TG) of infected calves, however they do not reactivate from latency due to anterograde spread defect. We have determined that BHV-1 Us9 complements fully BHV-5 Us9 in the BHV-5 neurovirulence in rabbits. Even though it was not one of our objectives, we constructed a BHV-1 BAC (bacterial artificial chromosome) and used the infectious BHV-1 BAC clone to construct Us9 acidic domain (residues 83-90-deleted and gE cytoplasmic tail truncated BHV-1 mutant viruses. We have determined that, like the entire gE and Us9-deleted viruses, both gE cytoplasmic-tail truncated and Us9 acidic domain-deleted viruses do not reactivate from latency. These results indicated that Us9 acidic domain residues 83-90 are critical for BHV-1 anterograde neuronal transport. Although, it was not one of our objectives, results from this project also indicated that sequences within the gE cytoplasmic tail must be important for anterograde neuronal transport. Analysis of serum neutralization titers of calves infected with wild-type and mutant viruses revealed that the Us9-deleted and wild-type BHV-1 viruses infected calves had comparable serum neutralization titer after 2-3 weeks post infection. Whereas both the gE-deleted and gE cytoplasmic-tail truncated viruses induce noticeably lower serum neutralization titers although the SN titers in the case of cytoplasmic-tail truncated virus-infected animals were slightly better relative to the gE-deleted virus-infected calves. These results also indicated that sequences within the gE cytoplasmic tail must also be important in inducing better primary immune response against the virus. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Current, gE- gene-deleted vaccines are safe but are not very immunogenic when compared with other modified live virus which have safety issues. Our results indicate that regions within the Us9 and gE cytoplasmic tail regulate anterograde neuronal transport. In addition, gE cytoplasmic tail sequence may also be important for better primary immune response against the virus. Therefore, one of the most important outcomes of this project is that we are one step closer to develop a new attenuated BHV-1 vaccine strain that will be defective in anterograde neuronal transport and will not reactivate from latency, yet the vaccinated animals would have better protective immune response.
Publications
- Chowdhury, S.I., S. Mahmood, J. Simon, A. Al-Mubarak and Y. Zhou (2006). The Us9 gene encoded by bovine herpesvirus type 1 (BHV-1) effectively complements a Us9-null bovine herpesvirus 5 (BHV-5) for anterograde transport, neurovirulence and neuroinvasiveness in a rabbit model. J. Virol. 80: 4396-4405.
- Al-Mubarak-A., J. Simon, M.D. Burton and S.I. Chowdhury (2007). Glycoprotein E(gE) specified by Bovine herpesvirus type5 (BHV-5) enables trans-neuronal virus spread and neurovirulence without being a structural component of enveloped virions. Virology. 365:398-409.
- Butchi, N.B, Jones, C., Perez.S, Doster.A, and Chowdhury.S.I (2007). Role of Envelope protein Us9 in the anterograde transport of BHV-1 following reactivation in the Trigeminal Ganglia . J. Neurovirology.13:384-388.
- Liu, Z-F., Brum, M, A. Doster, C. Jones and S.I. Chowdhury (2008). A bovine herpesvirus type 1 mutant virus specifying a carboxyl-terminal truncation of glycoprotein E is defective in anterograde neuronal transport in rabbits and calves. J. Virol. 82: 7432-7442.
- Brum, M., C. Coats, S.B. Rajkumari, A. Doster, C. Jones, and S. I. Chowdhury 2008. Envelope glycoprotein gE is required for the anterograde transport of bovine herpesvirus-1 (BHV-1) from trigeminal ganglia to nose and eye upon reactivation. (In Press, J. Neurovirology)
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Progress 01/01/07 to 12/31/07
Outputs
In calves, BHV-1 Us9Δ83-90 (acidic domain mutant), virus shedding properties and serum neutralizing titers during primary infection, latency and reactivation were analyzed relative to BHV-1Us9 83-90R. BHV- 1Us9Δ83-90 was isolated from nasal and ocular swabs during primary infection but not after reactivation while BHV-1Us9 83-90R was isolated at both times. Serum neutralizing antibody titers during primary infection were similar for both BHV-1Us9 83-90R and BHV-1 Us9Δ83-90 virus infected calves. Following reactivation, seroconversion was detected only in calves infected with BHV-1Us983-90R. These results confirmed that BHV-1 Us9Δ83-90 did not reactivate from latency probably due to defective anterograde transport. Similar results were also obtained in rabbits. Additionally, analysis of VP5 transcription from the total TG RNA by RT-PCR showed that VP5-specifc transcripts are present during primary infection and reactivation but not during latency in
both BHV-1Us9 Δ83-90 and BHV-1 Us9 83-90R-infected rabbits. These results are consistent with the notion that both BHV-1 wild-type as well as BHV-1 Us9-Δ83-90 viruses are reactivated in the neuronal cell bodies of the TG in a similar fashion. However, in the absence of Us9 and in particular Us9 acidic domain sequences transport of infectious virus from neuronal cell bodies in the TG to the nerve endings in the nose and eye is defective.
Impacts
One of the most important outcomes of this work will be the generation of new attenuated BHV-1 strains that could be used for vaccine purposes. For example using a Us9 acidic domain deleted virus as a backbone, domains within the gE cytoplasmic tail could be deleted which will combine anterograde spread defect from two different genes and a serological marker based on gE to distinguish a vaccine virus from a wild-type virus.
Publications
- Butchi, N.B, Jones, C., Perez.S, Doster.A, and Chowdhury.S.I (2007). Role of Envelope protein Us9 in the anterograde transport of BHV-1 following reactivation in the Trigeminal Ganglia . J. Neurovirology.13:384-388.
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Progress 01/01/06 to 12/31/06
Outputs
We have completed the study examining the role of Us9 and gE in the anterograde transport of BHV 1 from the TG to nose and eye following reactivation from latency. The results show that both the proteins are important for anterograde transport of the virus from the TG to nose and eye. Further, We have successfully constructed a BHV-1 BAC clone, in which a mini F plasmid was inserted in the intergenic region between the gB and UL 26 genes. The BAC plasmid can be excised successfully leaving only a 40 bp loxP sequence. Additionally, using red mediated mutagenesis of the BHV-1 BAC clone, a gE cytoplasmic tail truncated BHV-1, and BHV-1 Us9 mutant with amino acid residues 83- 90 deleted. Results of calf studies show that while the reconstituted BHV-1 with the loxp sequence is as virulent as the wild-type BHV-1 virus whereas the gE cytoplasmic tail truncated virus is attenuated relative to the wild-type. Moreover, BHV-1 gE cytoplasmic tail truncated virus has defective
anterograde transport because following reactivation from latency only the reconstituted BHV-1 could be isolated in the nasal and ocular shedding while the gE cytoplasmic tail truncated virus could not be isolated. Additionally, we have developed methods to grow primary olfactory receptor neurons in culture. Characterization of the Us9 mutant virus in the primary ORN neurons is in progress.
Impacts
Retrograde and anterograde transport of BHV-1 within the trigeminal nerve is crucial for the virus transmission and maintaining virus in the cattle population. Understanding the underlying transport mechanism and identifying viral protein contributing to neuronal transport will allow designing effective modified live vaccines.
Publications
- Chowdhury, S.I., S. Mahmood, J. Simon, A. Al-Mubarak and Y. Zhou. 2006. The Us9 gene encoded by bovine herpesvirus type 1 (BHV-1) effectively complements a Us9-null bovine herpesvirus 5 (BHV-5) for anterograde transport, neurovirulence and neuroinvasiveness in a rabbit model. J. Virol. 80: 4396-4405.
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Progress 01/01/05 to 12/31/05
Outputs
Bovine herpes virus type 1(BHV-1) is a major component of bovine respiratory disease complex. During primary infection BHV-1 replicates in the nasal epithelium and then enters the sensory nerve endings of the trigeminal nerve in the nasopharynx. The nucleocapsids along with tegument structures are then transported retrogradely to the trigeminal ganglion (TG) where they establish life long latency. Corticosteroid-induced or stress- mediated reactivation of latent virus can lead to anterograde transport to the nerve endings in the nasopharynx and eye which results in virus shedding and transmission to susceptible animals. The objective of this study is to examine the role of envelope proteins gE and US9 in the anterograde transport of BHV 1 from the TG to nose and eye following reactivation from latency. During primary infection calves infected with BHV1 wild type, gE deleted or US9 deleted viruses shed similar amounts of virus in nose and eye, and similar quantities of
the genomic copy numbers were detected in TG of calves infected with all the three viruses. During latency, comparable latent genomic copy numbers were detected in TG of calves infected with all three viruses. When reactivated from latency with dexamethasone, virus shedding in the nose and eye occurred only in calves infected with wild type virus. However, the TGs of calves infected with BHV-1 WT and US9 deleted viruses contained similar/comparable genomic copy number. Compared to the copy numbers observed during latency, there was an increase of genomic copy numbers in the reactivated TG specimens for all the three viruses. These results indicate that the US9 deleted BHV1 recombinant viruses infected and grew in TG following primary infection similar to wild type virus and established latent infection but they were not transported back to the nasopharynx and eye following reactivation. Quantitation of genomic copy numbers for gE deleted virus after reactivation is in progress.
Impacts
Retrograde and anterograde transport of BHV-1 within the trigeminal nerve is crucial for the virus transmission and maintaining virus in the cattle population. Understanding the underlying transport mechanism and identifying viral protein contributing to neuronal transport will allow designing effective modified live vaccines.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Our goal in this project is: i) to determine the role of gE and Us9 in the anterograde transport of a latent BHV-1 upon reactivation from the trigeminal ganglion to the nose and ii) to understand the mechanism of anterograde transport within the neurons. A calf experiment is currently underway to determine whether gE and Us9-deleted viruses can be reactivated from latency and whether they are defective in anterograde transport from the TG to the nose.
Impacts
The project is funded since Sept 15, 2004. Therefore, it is too early to list any impact at this time.
Publications
- No publications reported this period
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