Characterization of the interactome of the porcine reproductive and respiratory syndrome virus glycoprotein-5.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry, causing reproductive failure in sows and respiratory disorders in piglets. Glycosylated protein 5 (GP5) is a major envelope protein of the virus. It is essential for virus particle assembly and involved in viral pathogenesis.

Porcine reproductive and respiratory syndrome virus envelope (E) protein interacts with tubulin.

Porcine reproductive and respiratory syndrome virus (PRRSV) encodes the small envelope (E) protein which is a minor structural component of the virion that is important for virus infectivity. To better understand the biological functions of the E protein, we studied interactions between E and PRRSV cellular proteins. Using immunoprecipitation-coupled mass spectrometry approach, we previously identified tubulin-α as an interacting partner of E.

Vaccines for porcine epidemic diarrhea virus and other swine coronaviruses.

The recent introduction of the porcine epidemic diarrhea virus (PEDV) into the North American swine herd has highlighted again the need for effective vaccines for swine coronaviruses. While vaccines for transmissible gastroenteritis virus (TGEV) have been available to producers around the world for a long time, effective vaccines for PEDV and deltacoronaviruses were only recently developed or are still in development. Here, we review existing vaccine technologies for swine coronaviruses and highlight promising technologies which may help to control these important viruses in the future.

Porcine reproductive and respiratory syndrome virus envelope (E) protein interacts with mitochondrial proteins and induces apoptosis.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses for the swine industry worldwide. The PRRSV E protein, encoded by ORF 2b, is one of the non-glycosylated minor structural proteins. In this study, we present evidence for the interaction of the E protein with mitochondrial proteins ATP5A (part of ATP synthase complex), prohibitin, and ADP/ATP translocase.

S1 domain of the porcine epidemic diarrhea virus spike protein as a vaccine antigen.

Background: Porcine epidemic diarrhea virus (PEDV) is a highly contagious virus infecting pigs of all ages with high morbidity and mortality among newborn piglets. Currently, there is no effective vaccine available to protect the pigs from PEDV. The N-terminal subunit of spike protein (S1) is responsible for virus binding to the cellular receptor and contains a number of neutralizing antibody epitopes.

Exploration of New Sites in Adenovirus Hexon for Foreign Peptides Insertion.

Adenoviral vectors are now being explored as vaccine carriers to prevent infectious diseases in humans and animals. There are two strategies aimed at the expression of a vaccine antigen by adenoviral vectors. The first includes an insertion of the foreign gene expression cassette into the E1 region.

Role of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) signalling pathways in porcine reproductive and respiratory syndrome virus (PRRSV) replication.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a positive sense, single-stranded RNA genome virus that has become a major infection in swine, exerting huge economic losses to the industry worldwide. Detailed knowledge concerning the molecular mechanisms by which the virus manipulates the host cell signals transduction machinery is not only critical to further our understanding of viral replication and pathogenesis, but also guides our efforts to design new and improved therapeutic strategies. The phosphatidylinositol-3-kinase (PI3K)-dependent Akt and the mammalian target of rapamycin (mTOR) (PI3K/Akt/mTOR) are major host cell signalling pathways that regulate protein synthesis, cell growth, proliferation, migration and survival.

Potential role of porcine reproductive and respiratory syndrome virus structural protein GP2 in apoptosis inhibition.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious threat to the pork industry, and its pathogenesis needs further investigations. To study the role of two structural proteins of PRRSV in virus-host cells interactions, two stable cell lines (MARC-2a and MARC-N) expressing GP2 and N proteins, respectively, were established. We induced apoptosis in these cells by treating them with staurosporine and found a significant reduction in the number of apoptotic cells in MARC-2a as compared to MARC-N and MARC-145 cells.

Development of a DNA-launched replicon as a vaccine for porcine reproductive and respiratory syndrome virus.

Though a modified live attenuated vaccine (MLV) is available against porcine reproductive and respiratory syndrome virus (PRRSV), its limitations in protective efficacy, safety and few others warrant the development of newer vaccines. In this study, we have constructed a propagation-defective DNA-launched PRRSV replicon as a vaccine candidate and evaluated its immunogenicity and protective efficacy in a group of pigs along with MLV vaccinated group. Our data showed that prior to the intranasal challenge with a homologous strain of PRRSV, only MLV vaccinated pigs developed antibody response measured by ELISA and none of the pigs in any group developed PRRSV neutralizing antibodies in serum.

The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses.

New insights into RNA packaging in porcine reproductive and respiratory syndrome virus.

While identifying whether the smallest packaged heteroclite subgenomic RNA (S9) of porcine reproductive and respiratory syndrome virus (PRRSV) contains a packaging signal, we found that S9 was capable of binding to the basic amino acid-rich domain (synthetic peptide of aa 34-53) of the packaging protein (N). In addition, by using truncations at the 5' and 3' ends of S9, a minimal binding region of 35 nt was found to be essential for binding to both the synthetic peptide and to the full-length N protein. Furthermore, by using cell-culture experiments, we found that S9 was capable of packaging non-viral RNA sequence into PRRSV particles and that the 35 nt region was essential for this activity.

Mucosal adenovirus-vectored vaccine for measles.

Several problems associated with the available anti-measles vaccine emphasize the need for a single shot anti-measles vaccine which is efficacious by mucosal route of administration and functional in the presence of anti-measles neutralizing antibodies. To achieve these goals, we constructed two recombinant human adenoviruses (collectively designated Ad-F/H) carrying genes for measles virus (MV) fusion (F) and haemagglutinin (H) proteins. Single intranasal or intramuscular vaccination of mice and cotton rats with Ad-F/H elicited high MV-specific serum neutralizing-antibody titers.

SARS coronavirus protein 7a interacts with human Ap4A-hydrolase.

The SARS coronavirus (SARS-CoV) open reading frame 7a (ORF 7a) encodes a 122 amino acid accessory protein. It has no significant sequence homology with any other known proteins. The 7a protein is present in the virus particle and has been shown to interact with several host proteins; thereby implicating it as being involved in several pathogenic processes including apoptosis, inhibition of cellular protein synthesis, and activation of p38 mitogen activated protein kinase.

Optimization of a DNA vaccine against SARS.

Severe acute respiratory syndrome coronavirus (SARS-CoV) first appeared in Southern China in November 2002, and then quickly spread to 33 countries on five continents along international air travel routes. Although the SARS epidemic has been contained, there is a clear need for a safe and effective vaccine should an outbreak of a SARS-CoV infection reappear in human population. In this study, we tested four DNA-vaccine constructs: (1) pLL70, containing cDNA for the SARS-CoV spike (S) gene; (2) pcDNA-SS, containing codon-optimized S gene for SARS-CoV S protein (residues 12-1255) fused with a leader sequence derived from the human CD5 gene; (3) pcDNA-St, containing the gene encoding the N-portion of the codon-optimized S gene (residues 12-532) with the CD5 leader sequence; (4) pcDNA-St-VP22C, containing the gene encoding the N-portion of the codon-optimized S protein with the CD5 leader sequence fused with the C-terminal 138 amino acids of the bovine herpesvirus-1 (BHV-1) major tegument protein VP22.

Intracellular localization of the SARS coronavirus protein 9b: evidence of active export from the nucleus.

Open reading frame 9b (ORF 9b) encodes a 98 amino acid group-specific protein of severe acute respiratory syndrome (SARS) coronavirus (CoV). It has no homology with known proteins and its function in SARS CoV replication has not been determined. The N-terminal part of the 9b protein was used to raise polyclonal antibodies in rabbits, and these antibodies could detect 9b protein in infected cells.

Construction of capsid-modified recombinant bovine adenovirus type 3.

Adenoviruses have become a popular vehicle for gene transfer into animal and human cells. However, wide prevalence of preexisting immunity to human adenovirus (HAdV) and the promiscuous nature of the virus have made the use of nonhuman adenoviruses an attractive alternative. Moreover, readministration of viral vectors is often required to maintain therapeutic levels of transgene expression, resulting in vector-specific immune responses.

Immunogenicity of a receptor-binding domain of SARS coronavirus spike protein in mice: implications for a subunit vaccine.

We studied the immunogenicity of an anti-SARS subunit vaccine comprised of the fragment of the SARS coronavirus (SARS-CoV) spike protein amino acids 318-510 (S318-510) containing the receptor-binding domain. The S protein fragment was purified from the culture supernatant of stably transformed HEK293T cells secreting a tagged version of the protein. The vaccine was given subcutaneously to 129S6/SvEv mice in saline, with alum adjuvant or with alum plus CpG oligodeoxynucleotides (ODN).

Augmentation of immune responses to SARS coronavirus by a combination of DNA and whole killed virus vaccines.

We studied the immunogenicity of a DNA SARS-vaccine, a whole killed virus, or a whole killed and DNA vaccine combination. The DNA vaccine contained a plasmid encoding the SARS coronavirus (SARS-CoV) S protein under the control of the human CMV promoter and intron A. The whole killed virus vaccine was comprised of SARS-CoV, propagated in Vero-E6 cells, with subsequent beta-propilactone inactivation and formulated with aluminum hydroxide adjuvant.

Complete genome sequence of simian adenovirus 1: an Old World monkey adenovirus with two fiber genes.

Simian adenovirus 1 (SAdV-1) is one of many adenovirus strains that were isolated from Old World monkey cells during poliomyelitis vaccine production several decades ago. Despite the availability of these viruses, knowledge of their genetic content and phylogeny is rudimentary. In the present study, the genome sequence of SAdV-1 (34,450 bp) was determined and analysed.

Severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice.

Severe acute respiratory syndrome coronavirus (SARS-CoV) has been identified as the aetiological agent of SARS. Thus, vaccination against SARS-CoV may represent an effective approach towards controlling SARS. The nucleocapsid (N) protein is thought to play a role in induction of cell-mediated immunity to SARS-CoV and thus it is important to characterize this protein.

An adenoviral type 5 vector carrying a type 35 fiber as a vaccine vehicle: DC targeting, cross neutralization, and immunogenicity.

Substituting the coat proteins of adenoviral vector serotype 5 (Ad5) can alter vector tropism and circumvent vector neutralization. Here we report that an Ad5 vector carrying a part of the fiber molecule of human subgroup B adenovirus serotype 35 (Ad5.Fib35) transduces cultured human dendritic cells (DC) and circulating myeloid derived DC with approximately 10-fold greater efficiency than Ad5 in vitro.

Bovine adenovirus type 3 containing heterologous protein in the C-terminus of minor capsid protein IX.

Earlier, we detected pIX of BAdV-3 as a 14-kDa protein in purified virions. Analysis of BAdV-3 pIX using different region antibodies revealed that the N-terminus and central domain of the pIX contain immunogenic sites and are not exposed on the surface of BAdV-3 virion. This suggested that the C-terminus of BAdV-3 pIX (125 amino acid) may be exposed on the virion and may be used as a site for incorporation of heterologous peptides or proteins.

A recombinant E1-deleted porcine adenovirus-3 as an expression vector.

Replication-defective E1-deleted porcine adenoviruses (PAVs) are attractive vectors for vaccination. As a prerequisite for generating PAV-3 vectors containing complete deletion of E1, we transfected VIDO R1 cells (fetal porcine retina cells transformed with E1 region of human adenovirus 5) with a construct containing PAV-3 E1B(large) coding sequences under the control of HCMV promoter. A cell line named VR1BL could be isolated that expressed E1B(large) of PAV-3 and also complemented PAV214 (E1A+E1B(small) deleted).