Emerging Respiratory Viral Pathogens

Respiratory disease is arguably the most important health concern for the production animal industry [1-3]. Respiratory problems accounted for the highest mortality both in the swine and beef industries [1, 4]. Respiratory pathogens remain the most vital for swine and bovine research and disease monitoring [2, 5-7]. As pathogens, in particular viral pathogens, mutate, novel disease-causing viruses emerge, and there becomes an increasing concern and need for identification with control perimeters. One frequently identified disease syndrome is Porcine Respiratory Disease Complex (PRDC). PRDC is characterized by pneumonia of mixed respiratory infections with contributions from the environment and management practices. The main pathogens associated with PRDC include viruses, such as swine influenza virus (SIV), porcine respiratory and reproductive virus (PRRSV), and porcine circovirus 2 (PCV2), and bacteria, such as Mycoplasma hyopneumoniae, Pasteurella multocida, Streptococcus suis, Bordetella bronchiseptica, and Actinobacillus suis. Viral and bacterial pathogens can be classified as primary pathogens, capable of subverting host defense mechanisms and establishing infection on their own, or opportunistic pathogens [8]. Often, coinfections and superinfections with primary and/ or opportunistic pathogens occur with PRDC. The bovine counterpart to PRDC is Bovine Respiratory Disease (BRD). Like PRDC, BRD is a general term for a complex multi-factorial disease that encompasses upper and lower respiratory tract diseases. BRD is caused by stress, viral infection, and/ or bacterial infection with contributions from environmental factors (in particular transportation) and host characteristics (such as age, immune status, and genetics) [2, 9]. Bacterial and viral agents that are implicated in BRD include bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine herpesvirus 1 (BoHV-1), parainfluenza 3 virus (PI3V), bovine coronavirus (BCoV), Mannheimia haemolytica, Mycoplasma bovis, Pasteurella multocida, and Histophilus somni [10]. BRD is a costly disease of beef cattle with NAHMS Beef Feedlot 2011 Study reporting the direct cost of treatment for respiratory disease in feedlot cattle at $23.60 USD per case, resulting in a total cost of$54.12 million, not including production loses due to morbidity and mortality [2, 4]. With rising costs of food, the morbidity and mortality associated with respiratory diseases is economically disastrous. Many diagnostic panels are available for common respiratory pathogens for both swine and cattle. Although these panels are helpful, emerging, novel, variant, and underdiagnosed pathogens are often missed. Monitoring for these pathogens, often using metagenomic sequencing, can aid in their control and prevention. Once identified, research can begin on prevalence, pathogenesis, and control and prevention of these pathogens. This review is focused on the discovery of respiratory viral pathogens

Virus-Like Particles Generaged by Expressing Proteins of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Using the Recombinant Baculovirus Expression System

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, single stranded, positive sense RNA virus and a member of Arteriviridae. Its genome encodes 10 open reading frames for at least 7 structural proteins and 14 non-structural proteins. Membrane (M), Nuclepcapsid (N), and Glycoprotein-5 (GP5) are the major structural proteins of PRRSV, while Envelope (E), Glycoprotein-2 (GP2), Glycoprotein-3 (GP3), and Glycoprotein-4 (GP4) are the minor structural proteins of PRRSV. GP5 induces neutralizing antibodies and forms heterodimers with M, while N is the most immunogenic protein of PRRSV. Previous studies have shown viral structural proteins are able to form virus-like particles (VLPs) that can induce an immune response in respective hosts by use of the recombinant baculovirus expression system in insect cells. We sought to find if structural proteins, M, N, GP5, GP4, and E, could be expressed using the recombinant baculovirus expression system and produce VLPs. After purifying and amplifying PRRSV GP4, GP5, and E genes from PRRSV viral RNA, we inserted those genes into the pCAGEN plasmid, and expressed them in mammalian cells (cos-1). Protein expression of E and GP5 in both the supernatant and cell lysate was verified through western blot, while GP4 was not. We inserted the expressed genes into the pOET-1 plasmid for production of recombinant baculoviruses x using the flashBAC expression system in insect cells. Expression of GP5 and E proteins were verified with IFA. Viral titers were collected using bacuQUANT qRT-PCR, and used to co-infect TriEx SF9 cells with recombinant baculoviruses containing PRRSV M, N, GP5, and E at a multiplicity of infection (MOI) of 2 for N, GP5, and E proteins and a MOI of 3 for M protein for 72 hours. Protein expression was confirmed through western blot. The results of this experiment show that PRRSV M, N, GP5, and E proteins are expressed after co-infection of TriEx SF9 cells using the recombinant baculovirus system, and that VLPs of similar shape and size to the PRRSV virion are produced. These results will aid in further research of vaccine production using PRRSV VLP