Role of All of the PRRSV Glycoproteins in Protective Immune Response

Porcine reproductive and respiratory syndrome virus (PRRSV) contains the major glycoprotein, GP5, as well as three other minor glycoproteins, namely, GP2a, GP3, and GP4, on the virion envelope, all of which are required for generation of infectious virions. To study their interactions amongst each other and with the cellular receptor for PRRSV, we have cloned each of the viral glycoproteins and CD163 receptor in expression vectors and examined their expression and interaction with each other in transfected cells by co-immunoprecipitation (co-IP) assay using monospecific antibodies. Our results show that strong interaction exists between GP4 and GP5 proteins, although weak interactions among the other minor envelope glycoproteins and GP5 have been detected. Both GP2a and GP4 proteins were found to interact with all the other GPs resulting in the formation of multiprotein complex. Our results further show that GP2a and GP4 proteins also specifically interact with the CD163 molecule. The carboxy-terminal 223 residues of CD163 molecule are not required for interactions with either the GP2a or the GP4 protein, although these residues are required for conferring susceptibility to PRRSV infection in BHK-21 cells. Overall, we conclude that the GP4 protein is critical for mediating interglycoprotein interactions and along with GP2a, serves as the viral attachment protein that is responsible for mediating interactions with CD163 for virus entry into susceptible host cell. Additionally, using a series of glycosylation-site mutants of GPs, we have examined the ability of the hypoglycosylated forms of the protein to generate infectious PRRSV. Our results show that mutations at certain sites in various GPs are critical for production of infectious virus. Using several mutant PRRSVs with hypoglycosylated minor GPs on the envelope, we have found that these viruses do not induce higher titers of neutralizing antibody response, contrary to our previous observations with the major glycoprotein, GP5

Molecular Structures of PRRSV that Contribute to PRRSV Protective Immunity

One of the objectives of the proposal is to delineate the regions of the two glycoproteins (GP2 and GP4) of PRRSV that interact with CD163. Other objectives of the proposal are to generate antibodies to these small regions of the glycoproteins as well as to the full-length proteins for future studies to determine if any of these antibodies possess PRRSV neutralizing activity. To carry out the studies in the proposed objectives, we generated a series of mutants of GP2 as well as GP4 proteins in which various regions were specifically removed by manipulating the plasmids encoding these proteins. We then examined these proteins for their ability to interact with CD163 to ascertain the regions important for such interactions. Our results identified the regions of GP2 and GP4 that appear to interact with CD163. Furthermore, we generated recombinant baculoviruses that expressed these viral GPs. The viral proteins were purified form the cells and have been used to generate antibodies. Further studies will be conducted to characterize these antibodies in the future. Part of our studies supported by the NPB grant (#09-248) has been recently published in Virology (Das et al., Virology, 410: 385-394, 2011; a copy of the paper has also been forwarded to B. L. Everitt)

Enhancement of efficacy of PRRSV vaccines by altering the glycosylation pattern of viral glycoproteins

PRRSV, the causative agent of PRRS is of major economic significance to the pork industry in the USA and around the world. Current commercial vaccine does not provide adequate protection against PRRSV outbreaks. Therefore, there is an urgent need for development of more efficacious vaccine to combat PRRS. Our previous studies have suggested that (i) induction of neutralizing antibody response is an important correlate of evaluating the efficacy of a vaccine; (ii) neutralizing antibodies can be enhanced by hypoglycosylation of the major surface glycoprotein (GP5). While it is known that GP5 plays a prominent role in neutralizing antibody induction, it has been suggested that other PRRSV glycoproteins, such as GP4 (which is known to be the target of a PRRSV-neutralizing monoclonal antibody) may also have a role. On the other hand, nothing is known about the possible role of PRRSV GP2 and GP3 in neutralization of PRRSV. We hypothesized that PRRSV neutralizing antibody response can be enhanced by hypoglycosylation of GP2, GP3, and GP4 proteins. Towards this goal, using the infectious cDNA clone (FL12) of PRRSV prepared in our laboratory, we generated a series of mutant PRRSVs containing hypoglycosylated forms of these minor glycoproteins. These viruses possessed different growth potential in vitro. When these viruses were inoculated into pigs and their neutralizing antibody response was examined, we observed that neutralizing antibody response in most of the mutant virus-infected pigs was lower than the wt PRRSV infected pigs. These results indicate that interactions of the wild-type minor glycoproteins with GP5 may be critical for neutralizing antibody response and that altering the glycosylation pattern of the minor glycoproteins may have negatively affected their interactions with GP5 resulting in lower neutralizing antibody response

Current Status of Zika Virus Vaccines: Successes and Challenges

The recently emerged Zika virus (ZIKV) spread to the Americas, causing a spectrum of congenital diseases including microcephaly in newborn and Guillain-Barré syndrome (GBS) in adults. The unprecedented nature of the epidemic and serious diseases associated with the viral infections prompted the global research community to understand the immunopathogenic mechanisms of the virus and rapidly develop safe and efficacious vaccines. This has led to a number of ZIKV vaccine candidates that have shown significant promise in human clinical trials. These candidates include nucleic acid vaccines, inactivated vaccines, viral-vectored vaccines, and attenuated vaccines. Additionally, a number of vaccine candidates have been shown to protect animals in preclinical studies. However, as the epidemic has waned in the last three years, further development of the most promising vaccine candidates faces challenges in clinical efficacy trials, which is needed before a vaccine is brought to licensure. It is important that a coalition of government funding agencies and private sector companies is established to move forward with a safe and effective vaccine ready for deployment when the next ZIKV epidemic occurs

Current Status of Zika Virus Vaccines: Successes and Challenges

The recently emerged Zika virus (ZIKV) spread to the Americas, causing a spectrum of congenital diseases including microcephaly in newborn and Guillain-Barré syndrome (GBS) in adults. The unprecedented nature of the epidemic and serious diseases associated with the viral infections prompted the global research community to understand the immunopathogenic mechanisms of the virus and rapidly develop safe and efficacious vaccines. This has led to a number of ZIKV vaccine candidates that have shown significant promise in human clinical trials. These candidates include nucleic acid vaccines, inactivated vaccines, viral-vectored vaccines, and attenuated vaccines. Additionally, a number of vaccine candidates have been shown to protect animals in preclinical studies. However, as the epidemic has waned in the last three years, further development of the most promising vaccine candidates faces challenges in clinical efficacy trials, which is needed before a vaccine is brought to licensure. It is important that a coalition of government funding agencies and private sector companies is established to move forward with a safe and effective vaccine ready for deployment when the next ZIKV epidemic occurs

Manipulation of Cellular Processing Bodies and Their Constituents by Viruses

The processing bodies (PBs) are a form of cytoplasmic aggregates that house the cellular RNA decay machinery as well as many RNA-binding proteins and mRNAs. The PBs are constitutively present in eukaryotic cells and are involved in maintaining cellular homeostasis by regulating RNA metabolism, cell signaling, and survival. Virus infections result in modification of the PBs and their constituents. Many viruses induce compositionally altered PBs, while many others use specific components of the PBs for their replication. PB constituents are also known to restrict virus replication by a variety of mechanisms. Further, continuing studies in this rapidly emerging field of PB-virus interactions will undoubtedly provide important clues to the understanding of the role of PBs in cellular homeostasis as well as their role in virus infections and innate immune signaling

Overview of Rhabdo- and Filoviruses

Enveloped viruses with a negative-sense, single-stranded monopartite RNA genome have been classified into the order Mononegavirales. Five families of viruses that constitute the order are: Rhabdoviridae, Filoviridae, Paramyxoviridae, Bornaviridae and Nyamiviridae. Members of these families possess a helical nucleocapsid core containing the viral genome and a host-derived lipid envelope containing viral proteins. This introductory chapter provides a brief overview of the Rhabdoviridae and the Filoviridae, the two families of viruses that are the subject of this book. Many members of these two families are highly significant human and animal pathogens. The rationale and goal of the book is to provide the reader with the most recent information on the structure, genome organization and replication strategy, epidemiology, evolution and emergence, host response to infection, viral countermeasures as well as vaccines and antivirals against these pathogens. More detailed descriptions of these topics are presented in individual chapters of this book

Cells that express all five proteins of vesicular stomatitis virus from cloned cDNAs support replication, assembly, and budding of defective interfering particles

An alternative approach to structurefunction analysis of vesicular stomatitis virus (VSV) gene products and their interactions with one another during each phase of the viral life cycle is described. We showed previously by using the vaccinia viruslT7 RNA polymerase expression system that when cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with defective interfering (DI) particles, rapid and efflicient replication and amplification of DI particle RNA occurred. Here, we demonstrate that all five VSV proteins can be expressed simultaneously when cells are cotransfected with plasmids containing the matrix protein (M) gene and the glycoprotein (G) gene of VSV in addition to plasmids containing the genes for the N, NS, and L proteins. When cells coexpressing all five VSV proteins were superinfected with DI particles, which because of their defectiveness are unable to express any viral proteins or to replicate, DI particle replication, assembly, and budding were observed and infectious DI particles were released into the culture fluids. Omission of either the M or G protein expression resulted in no DI particle budding. The vector-supported DI particles were similar in size and morphology to the authentic DI particles generated from cells coinfected with DI particles and helper VSV and their infectivity could be blocked by anti-VSV or anti-G antiserum. The successful replication, assembly, and budding of DI particles from cells expressing all five VSV proteins from cloned cDNAs provide a powerful approach for detailed structure-function analysis of the VSV gene products in each step of the replicative cycle of the virus

Manipulation of Cellular Processing Bodies and Their Constituents by Viruses

The processing bodies (PBs) are a form of cytoplasmic aggregates that house the cellular RNA decay machinery as well as many RNA-binding proteins and mRNAs. The PBs are constitutively present in eukaryotic cells and are involved in maintaining cellular homeostasis by regulating RNA metabolism, cell signaling, and survival. Virus infections result in modification of the PBs and their constituents. Many viruses induce compositionally altered PBs, while many others use specific components of the PBs for their replication. PB constituents are also known to restrict virus replication by a variety of mechanisms. Further, continuing studies in this rapidly emerging field of PB-virus interactions will undoubtedly provide important clues to the understanding of the role of PBs in cellular homeostasis as well as their role in virus infections and innate immune signaling

Overview of Rhabdo- and Filoviruses

Enveloped viruses with a negative-sense, single-stranded monopartite RNA genome have been classified into the order Mononegavirales. Five families of viruses that constitute the order are: Rhabdoviridae, Filoviridae, Paramyxoviridae, Bornaviridae and Nyamiviridae. Members of these families possess a helical nucleocapsid core containing the viral genome and a host-derived lipid envelope containing viral proteins. This introductory chapter provides a brief overview of the Rhabdoviridae and the Filoviridae, the two families of viruses that are the subject of this book. Many members of these two families are highly significant human and animal pathogens. The rationale and goal of the book is to provide the reader with the most recent information on the structure, genome organization and replication strategy, epidemiology, evolution and emergence, host response to infection, viral countermeasures as well as vaccines and antivirals against these pathogens. More detailed descriptions of these topics are presented in individual chapters of this book