Abdelrahman Said

An Equine Herpesvirus Type 1 (EHV-1) Expressing VP2 and VP5 of Serotype 8 Bluetongue Virus (BTV-8) Induces Protection in a Murine Infection Model

Bluetongue virus (BTV) can infect most species of domestic and wild ruminants causing substantial morbidity and mortality and, consequently, high economic losses. In 2006, an epizootic of BTV serotype 8 (BTV-8) started in northern Europe that caused significant disease in cattle and sheep before comprehensive vaccination was introduced two years later. Here, we evaluate the potential of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus, as a novel vectored DIVA (differentiating infected from vaccinated animals) vaccine expressing VP2 of BTV-8 alone or in combination with VP5. The EHV-1 recombinant viruses stably expressed the transgenes and grew with kinetics that were identical to those of parental virus in vitro. After immunization of mice, a BTV-8-specific neutralizing antibody response was elicited. In a challenge experiment using a lethal dose of BTV-8, 100% of interferon-receptor-deficient (IFNAR−/−) mice vaccinated with the recombinant EHV-1 carrying both VP2 and VP5, but not VP2 alone, survived. VP7 was not included in the vectored vaccines and was successfully used as a DIVA marker. In summary, we show that EHV-1 expressing BTV-8 VP2 and VP5 is capable of eliciting a protective immune response that is distinguishable from that after infection and as such may be an alternative for BTV vaccination strategies in which DIVA compatibility is of importance.

Equine Herpesvirus Type 4 UL56 and UL49.5 Proteins Downregulate Cell Surface Major Histocompatibility Complex Class I Expression Independently of Each Other

ABSTRACT Major histocompatibility complex class I (MHC-I) molecules are critically important in the host defense against various pathogens through presentation of viral peptides to cytotoxic T lymphocytes (CTLs), a process resulting in the destruction of virus-infected cells. Herpesviruses interfere with CTL-mediated elimination of infected cells by various mechanisms, including inhibition of peptide transport and loading, perturbation of MHC-I trafficking, and rerouting and proteolysis of cell surface MHC-I. In this study, we show that equine herpesvirus type 4 (EHV-4) modulates MHC-I cell surface expression through two different mechanisms. First, EHV-4 can lead to a significant downregulation of MHC-I expression at the cell surface through the product of ORF1, a protein expressed with early kinetics from a gene that is homologous to herpes simplex virus 1 UL56. The EHV-4 UL56 protein reduces cell surface MHC-I as early as 4 h after infection. Second, EHV-4 can interfere with MHC-I antigen presentation, starting at 6 h after infection, by inhibition of the transporter associated with antigen processing (TAP) through its UL49.5 protein. Although pUL49.5 has no immediate effect on overall surface MHC-I levels in infected cells, it blocks the supply of antigenic peptides to the endoplasmic reticulum (ER) and transport of peptide-loaded MHC-I to the cell surface. Taken together, our results show that EHV-4 encodes at least two viral immune evasion proteins: pUL56 reduces MHC-I molecules on the cell surface at early times after infection, and pUL49.5 interferes with MHC-I antigen presentation by blocking peptide transport in the ER.

Major Histocompatibility Complex Class I Downregulation Induced by Equine Herpesvirus Type 1 pUL56 Is through Dynamin-Dependent Endocytosis

ABSTRACT Equine herpesvirus type 1 (EHV-1) downregulates cell surface expression of major histocompatibility complex class I (MHC-I) in infected cells. We have previously shown that pUL56 encoded by the EHV-1 ORF1 gene regulates the process (G. Ma, S. Feineis, N. Osterrieder, and G. R. Van de Walle, J. Virol. 86:3554–3563, 2012, doi:http://dx.doi.org/10.1128/JVI.06994-11). Here, we report that cell surface MHC-I in EHV-1-infected cells is internalized and degraded in the lysosomal compartment in a pUL56-dependent fashion. pUL56-induced MHC-I endocytosis required dynamin and tyrosine kinase but was independent of clathrin and caveolin-1, the main constituents of the clathrin- and raft/caveola-mediated endocytosis pathways, respectively. Downregulation of cell surface MHC-I was significantly inhibited by the ubiquitin-activating enzyme E1 inhibitor PYR41, indicating that ubiquitination is essential for the process. Finally, we show that downregulation is not specific for MHC-I and that other molecules, including CD46 and CD63, are also removed from the cell surface in a pUL56-dependent fashion. IMPORTANCE We show that alphaherpesvirus induces MHC-I downregulation through endocytosis, which is mediated by pUL56. The dynamin-dependent endocytic pathway is responsible for MHC-I internalization in infected cells. Furthermore, we discovered that this endocytic process can be disrupted by the inhibiting ubiquitin-activating E1 enzyme, which is indispensable for ubiquitination. Finally, pUL56 action extends to a number of cell surface molecules that are significant for host immunity. Therefore, the protein may exert a more general immunomodulatory effect.

Recombinant equine herpesvirus 1 (EHV-1) vaccine protects pigs against challenge with influenza A(H1N1)pmd09.

Swine influenza virus (SIV) is not only an important respiratory pathogen in pigs but also a threat to human health. The pandemic influenza A(H1N1)pdm09 virus likely originated in swine through reassortment between a North American triple reassortant and Eurasian avian-like SIV. The North American triple reassortant virus harbors genes from avian, human and swine influenza viruses. An effective vaccine may protect the pork industry from economic losses and curb the development of new virus variants that may threaten public health. In the present study, we evaluated the efficacy of a recombinant equine herpesvirus type 1 (EHV-1) vaccine (rH_H1) expressing the hemagglutinin H1 of A(H1N1)pdm09 in the natural host. Our data shows that the engineered rH_H1 vaccine induces influenza virus-specific antibody responses in pigs and is able to protect at least partially against challenge infection: no clinical signs of disease were detected and virus replication was reduced as evidenced by decreased nasal virus shedding and faster virus clearance. Taken together, our results indicate that recombinant EHV-1 encoding H1 of A(H1N1)pdm09 may be a promising alternative for protection of pigs against infection with A(H1N1)pdm09 or other influenza viruses.

An equine herpesvirus 1 (EHV-1) vectored H1 vaccine protects against challenge with swine-origin influenza virus H1N1.

In 2009, a novel swine-origin H1N1 influenza A virus (S-OIV), antigenically and genetically divergent from seasonal H1N1, caused a flu pandemic in humans. Development of an effective vaccine to limit transmission of S-OIV in animal reservoir hosts and from reservoir hosts to humans and animals is necessary. In the present study, we constructed and evaluated a vectored vaccine expressing the H1 hemagglutinin of a recent S-OIV isolate using equine herpesvirus 1 (EHV-1) as the delivery vehicle. Expression of the recombinant protein was demonstrated by immunofluorescence and western blotting and the in vitro growth properties of the modified live vector were found to be comparable to those of the parental virus. The EHV-1-H1 vaccine induced an influenza virus-specific antibody response when inoculated into mice by both the intranasal and subcutaneous routes. Upon challenge infection, protection of vaccinated mice could be demonstrated by reduction of clinical signs and faster virus clearance. Our study shows that an EHV-1-based influenza H1N1 vaccine may be a promising alternative for protection against S-OIV infection.

Ubiquitination and degradation of the ORF34 gene product of equine herpesvirus type 1 (EHV-1) at late times of infection

The equine herpesvirus type 1 (EHV-1) open reading frame 34 (ORF34) is predicted to encode a polypeptide of 161 amino acids. We show that an ORF34 deletion mutant exhibited a significant growth defect in equine peripheral blood mononuclear cells taken directly ex vivo during early but not late times of infection. ORF34 protein (pORF34)-specific antibodies specifically reacted with a 28-kDa early polypeptide present in the cytosol of infected cells. From 10h post infection, multiple smaller pORF34-specific protein moieties were detected indicating that expression of a late viral gene product(s) caused pORF34 degradation. Proteasome inhibitors blocked pORF34 degradation as did treatment of infected cells with a ubiquitin-activating enzyme (E1) inhibitor. Finally, kinetic studies showed that pORF34 is modified by addition of multiple copies of ubiquitin. Taken together, our findings suggest that the ubiquitin proteasome pathway is required for pORF34 degradation that may modulate protein activity in the course of infection.

An equine herpesvirus type 1 (EHV-1) vector expressing Rift Valley fever virus (RVFV) Gn and Gc induces neutralizing antibodies in sheep

Rift Valley fever virus (RVFV) is an arthropod-borne bunyavirus that can cause serious and fatal disease in humans and animals. RVFV is a negative-sense RNA virus of the Phlebovirus genus in the Bunyaviridae family. The main envelope RVFV glycoproteins, Gn and Gc, are encoded on the M segment of RVFV and known inducers of protective immunity. In an attempt to develop a safe and efficacious RVF vaccine, we constructed and tested a vectored equine herpesvirus type 1 (EHV-1) vaccine that expresses RVFV Gn and Gc. The Gn and Gc genes were custom-synthesized after codon optimization and inserted into EHV-1 strain RacH genome. The rH_Gn-Gc recombinant virus grew in cultured cells with kinetics that were comparable to those of the parental virus and stably expressed Gn and Gc. Upon immunization of sheep, the natural host, neutralizing antibodies against RVFV were elicited by rH_Gn-Gc and protective titers reached to 1:320 at day 49 post immunization but not by parental EHV-1, indicating that EHV-1 is a promising vector alternative in the development of a safe marker RVFV vaccine.

Equine herpesvirus type 1 (EHV-1) open reading frame 59 encodes an early protein that is localized to the cytosol and required for efficient virus growth

Equine herpesvirus type 1 (EHV-1) ORF59 is predicted to encode a protein consisting of 180 amino acids. To determine whether ORF59 in fact encodes a protein, sequences encoding an HA epitope (YPYDVPDYA) was inserted at the carboxyterminus of the ORF59 protein in EHV-1 strain Ab4. Using anti-HA monoclonal antibodies, a 21-kDa band was specifically detected by western blot analysis in lysates of cells infected with a recombinant EHV-1 from strain Ab4 that carries the pORF59-HA but not in cells infected with parental Ab4. Further characterization of the protein using immunofluorescence and fractionation studies showed that pORF59 is an early protein that localizes to the cytosol in virus-infected cells. Recombinant EHV-1 lacking ORF59 (rAb4∆59) exhibited a small-plaque phenotype and could not be propagated. Our findings suggest that the ORF59 protein plays a major role in EHV-1 replication in vitro and likely in vivo.

Establishment and evaluation of the goose embryo epithelial (GEE) cell line as a new model for propagation of avian viruses

In this study, we report the establishment and characterization of a new epithelial cell line, goose embryonated epithelial cell line (GEE), derived from embryonic goose tissue. The purified GEE cell line can efficiently grow over 65 passages in the M199 medium supplemented with 10% fetal bovine serum at 37°C. Immunofluorescence assay was used to identify purified GEE cells as epithelial cell line by detecting expression of the Keratin-18 and -19. Further characterizations demonstrated that the GEE cell line can be continuously subcultured with (i) a high capacity to replicate for over 65 passages, (ii) a spontaneous epithelial-like morphology, (iii) constant chromosomal features and (iv) without an evidence of converting to tumorigenic cells either in vitro or in vivo study. Moreover, the GEE cell line can be effectively transfected with plasmids expressing reporter genes of different avian viruses, such as VP3, VP1 and F of goose parvo virus (GPV), duck hepatitis virus (DHV), and Newcastle disease virus (NDV), respectively. Finally, the established GEE cell line was evaluated for avian viruses infection susceptibility. Our results showed that the tested GPV, DHAV and NDV were capable to replicate in the new cell line with titers a comparatively higher to the ones detected in the traditional culture system. Accordingly, our established GEE cell line is apparently a suitable in vitro model for transgenic, and infection manipulation studies.