A clinically attenuated double-mutant of porcine reproductive and respiratory syndrome virus-2 that does not prompt overexpression of proinflammatory cytokines during co-infection with a secondary pathogen.

Porcine reproductive and respiratory syndrome virus (PRRSV) is known to suppress the type I interferon (IFNs-α/ß) response during infection. PRRSV also activates the NF-κB signaling pathway, leading to the production of proinflammatory cytokines during infection. In swine farms, co-infections of PRRSV and other secondary bacterial pathogens are common and exacerbate the production of proinflammatory cytokines, contributing to the porcine respiratory disease complex (PRDC) which is clinically a severe disease. Previous studies identified the non-structural protein 1ß (nsp1ß) of PRRSV-2 as an IFN antagonist and the nucleocapsid (N) protein as the NF-κB activator. Further studies showed the leucine at position 126 (L126) of nsp1ß as the essential residue for IFN suppression and the region spanning the nuclear localization signal (NLS) of N as the NF-κB activation domain. In the present study, we generated a double-mutant PRRSV-2 that contained the L126A mutation in the nsp1ß gene and the NLS mutation (ΔNLS) in the N gene using reverse genetics. The immunological phenotype of this mutant PRRSV-2 was examined in porcine alveolar macrophages (PAMs) in vitro and in young pigs in vivo. In PAMs, the double-mutant virus did not suppress IFN-ß expression but decreased the NF-κB-dependent inflammatory cytokine productions compared to those for wild-type PRRSV-2. Co-infection of PAMs with the mutant PRRSV-2 and Streptococcus suis (S. suis) also reduced the production of NF-κB-directed inflammatory cytokines. To further examine the cytokine profiles and the disease severity by the mutant virus in natural host animals, 6 groups of pigs, 7 animals per group, were used for co-infection with the mutant PRRSV-2 and S. suis. The double-mutant PRRSV-2 was clinically attenuated, and the expressions of proinflammatory cytokines and chemokines were significantly reduced in pigs after bacterial co-infection. Compared to the wild-type PRRSV-2 and S. suis co-infection control, pigs coinfected with the double-mutant PRRSV-2 exhibited milder clinical signs, lower titers and shorter duration of viremia, and lower expression of proinflammatory cytokines. In conclusion, our study demonstrates that genetic modification of the type I IFN suppression and NF-κB activation functions of PRRSV-2 may allow us to design a novel vaccine candidate to alleviate the clinical severity of PRRS-2 and PRDC during bacterial co-infection.

GTPase activity of porcine Mx1 plays a dominant role in inhibiting the N-Nsp9 interaction and thus inhibiting PRRSV replication.

Porcine Mx1 is a type of interferon-induced GTPase that inhibits the replication of certain RNA viruses. However, the antiviral effects and the underlying mechanism of porcine Mx1 for porcine reproductive and respiratory syndrome virus (PRRSV) remain unknown. In this study, we demonstrated that porcine Mx1 could significantly inhibit PRRSV replication in MARC-145 cells. By Mx1 segment analysis, it was indicated that the GTPase domain (68-341aa) was the functional area to inhibit PRRSV replication and that Mx1 interacted with the PRRSV-N protein through the GTPase domain (68-341aa) in the cytoplasm. Amino acid residues K295 and K299 in the G domain of Mx1 were the key sites for Mx1-N interaction while mutant proteins Mx1(K295A) and Mx1(K299A) still partially inhibited PRRSV replication. Furthermore, we found that the GTPase activity of Mx1 was dominant for Mx1 to inhibit PRRSV replication but was not essential for Mx1-N interaction. Finally, mechanistic studies demonstrated that the GTPase activity of Mx1 played a dominant role in inhibiting the N-Nsp9 interaction and that the interaction between Mx1 and N partially inhibited the N-Nsp9 interaction. We propose that the complete anti-PRRSV mechanism of porcine Mx1 contains a two-step process: Mx1 binds to the PRRSV-N protein and subsequently disrupts the N-Nsp9 interaction by a process requiring the GTPase activity of Mx1. Taken together, the results of our experiments describe for the first time a novel mechanism by which porcine Mx1 evolves to inhibit PRRSV replication. IMPORTANCE: Mx1 protein is a key mediator of the interferon-induced antiviral response against a wide range of viruses. How porcine Mx1 affects the replication of porcine reproductive and respiratory syndrome virus (PRRSV) and its biological function has not been studied. Here, we show that Mx1 protein inhibits PRRSV replication by interfering with N-Nsp9 interaction. Furthermore, the GTPase activity of porcine Mx1 plays a dominant role and the Mx1-N interaction plays an assistant role in this interference process. This study uncovers a novel mechanism evolved by porcine Mx1 to exert anti-PRRSV activities.

Suppression of TRIM19 by arterivirus nonstructural protein 1 promotes viral replication.

Tripartite motif (TRIM)-containing proteins are a family of regulatory proteins that can participate in the induction of antiviral cytokines and antagonize viral replication. Promyelocytic leukemia (PML) protein is known as TRIM19 and is a major scaffold protein organizing the PML nuclear bodies (NBs). PML NBs are membrane-less organelles in the nucleus and play a diverse role in maintaining cellular homeostasis including antiviral response. Porcine reproductive and respiratory syndrome virus (PRRSV), a member virus of the family Arteriviridae, inhibits type I interferon (IFN) response during infection, and nonstructural protein 1 (nsp1) of the virus has been identified as a potent IFN antagonist. We report that the numbers of PML NBs per nucleus were significantly downregulated during infection of PRRSV. The overexpression of all six isoforms of PML suppressed the PRRSV replication, and conversely, the silencing of PML gene expression enhanced the PRRSV replication. The suppression of PML NBs by the nsp1 protein was common in other member viruses of the family, represented by equine arteritis virus, lactate dehydrogenase elevating virus of mice, and simian hemorrhagic fever virus. Our study unveils a conserved viral strategy in arteriviruses for innate immune evasion.

Reprogramming viral immune evasion for a rational design of next-generation vaccines for RNA viruses.

Type I interferons (IFNs-α/ß) are antiviral cytokines that constitute the innate immunity of hosts to fight against viral infections. Recent studies, however, have revealed the pleiotropic functions of IFNs, in addition to their antiviral activities, for the priming of activation and maturation of adaptive immunity. In turn, many viruses have developed various strategies to counteract the IFN response and to evade the host immune system for their benefits. The inefficient innate immunity and delayed adaptive response fail to clear of invading viruses and negatively affect the efficacy of vaccines. A better understanding of evasion strategies will provide opportunities to revert the viral IFN antagonism. Furthermore, IFN antagonism-deficient viruses can be generated by reverse genetics technology. Such viruses can potentially serve as next-generation vaccines that can induce effective and broad-spectrum responses for both innate and adaptive immunities for various pathogens. This review describes the recent advances in developing IFN antagonism-deficient viruses, their immune evasion and attenuated phenotypes in natural host animal species, and future potential as veterinary vaccines.

Multiple-Site SUMOylation of FMDV 3C Protease and Its Negative Role in Viral Replication.

Protein SUMOylation represents an important cellular process that regulates the activities of numerous host proteins as well as of many invasive viral proteins. Foot-and-mouth disease virus (FMDV) is the first animal virus discovered. However, whether SUMOylation takes place during FMDV infection and what role it plays in FMDV pathogenesis have not been investigated. In the present study, we demonstrated that SUMOylation suppressed FMDV replication by small interfering RNA (siRNA) transfection coupled with pharmaceutical inhibition of SUMOylation, which was further confirmed by increased virus replication for SUMOylation-deficient FMDV with mutations in 3C protease, a target of SUMOylation. Moreover, we provided evidence that four lysine residues, Lys-51, -54, -110, and -159, worked together to confer the SUMOylation to the FMDV 3C protease, which may make SUMOylation of FMDV 3C more stable and improve the host's chance of suppressing the replication of FMDV. This is the first report that four lysine residues can be alternatively modified by SUMOylation. Finally, we showed that SUMOylation attenuated the cleavage ability, the inhibitory effect of the interferon signaling pathway, and the protein stability of FMDV 3C, which appeared to correlate with a decrease in FMDV replication. Taken together, the results of our experiments describe a novel cellular regulatory event that significantly restricts FMDV replication through the SUMOylation of 3C protease. IMPORTANCE FMD is a highly contagious and economically important disease in cloven-hoofed animals. SUMOylation, the covalent linkage of a small ubiquitin-like protein to a variety of substrate proteins, has emerged as an important posttranslational modification that plays multiple roles in diverse biological processes. In this study, four lysine residues of FMDV 3C were found to be alternatively modified by SUMOylation. In addition, we demonstrated that SUMOylation attenuated FMDV 3C function through multiple mechanisms, including cleavage ability, the inhibitory effect of the interferon signaling pathway, and protein stability, which, in turn, resulted in a decrease of FMDV replication. Our findings indicate that SUMOylation of FMDV 3C serves as a host cell defense against FMDV replication. Further understanding of the cellular and molecular mechanisms driving this process should offer novel insights to design an effective strategy to control the dissemination of FMDV in animals.

Activation of NF-κB and induction of proinflammatory cytokine expressions mediated by ORF7a protein of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus disease 2019 (COVID-19) that emerged in human populations recently. Severely ill COVID-19 patients exhibit the elevation of proinflammatory cytokines, and such an unbalanced production of proinflammatory cytokines is linked to acute respiratory distress syndrome with high mortality in COVID-19 patients. Our study provides evidence that the ORF3a, M, ORF7a, and N proteins of SARS-CoV-2 were NF-κB activators. The viral sequence from infected zoo lions belonged to clade V, and a single mutation of G251V is found for ORF3a gene compared to all other clades. No significant functional difference was found for clade V ORF3a, indicating the NF-κB activation is conserved among COVID-19 variants. Of the four viral proteins, the ORF7a protein induced the NF-κB dictated proinflammatory cytokines including IL-1α, IL-1ß, IL-6, IL-8, IL-10, TNF-α, and IFNß. The ORF7a protein also induced IL-3, IL-4, IL-7, IL-23. Of 15 different chemokines examined in the study, CCL11, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL26, CCL27, and CXCL9 were significantly upregulated by ORF7. These cytokines and chemokines were frequently elevated in severely ill COVID-19 patients. Our data provide an insight into how SARS-CoV-2 modulates NF-κB signaling and inflammatory cytokine expressions. The ORF7a protein may be a desirable target for strategic developments to minimize uncontrolled inflammation in COVID-19 patients.

Inhibition of Antiviral Innate Immunity by Foot-and-Mouth Disease Virus Lpro through Interaction with the N-Terminal Domain of Swine RNase L.

Foot-and-mouth disease virus (FMDV) is the pathogen of foot-and-mouth disease (FMD), which is a highly contagious disease in cloven-hoofed animals. To survive in the host, FMDV has evolved multiple strategies to antagonize host innate immune responses. In this study, we showed that the leader protease (Lpro) of FMDV, a papain-like proteinase, promoted viral replication by evading the antiviral interferon response through counteracting the 2',5'-oligoadenylate synthetase (OAS)/RNase L system. Specifically, we observed that the titers of Lpro deletion virus were significantly lower than those of wild-type FMDV (FMDV-WT) in cultured cells. Our mechanistic studies demonstrated that Lpro interfered with the OAS/RNase L pathway by interacting with the N-terminal domain of swine RNase L (sRNase L). Remarkably, Lpro of FMDV exhibited species-specific binding to RNase L in that the interaction was observed only in swine cells, not human, monkey, or canine cells. Lastly, we presented evidence that by interacting with sRNase L, FMDV Lpro inhibited cellular apoptosis. Taken together, these results demonstrate a novel mechanism that Lpro utilizes to escape the OAS/RNase L-mediated antiviral defense pathway. IMPORTANCE FMDV is a picornavirus that causes a significant disease in agricultural animals. FMDV has developed diverse strategies to escape the host interferon response. Here, we show that Lpro of FMDV antagonizes the OAS/RNase L pathway, an important interferon effector pathway, by interacting with the N-terminal domain of sRNase L. Interestingly, such a virus-host interaction is species-specific because the interaction is detected only in swine cells, not in human, monkey, or canine cells. Furthermore, Lpro inhibits apoptosis through interacting with sRNase L. This study demonstrates a novel mechanism by which FMDV has evolved to inhibit host innate immune responses.

Recent Advances in PRRS Virus Receptors and the Targeting of Receptor-Ligand for Control.

Cellular receptors play a critical role in viral infection. At least seven cellular molecules have been identified as putative viral entry mediators for porcine reproductive and respiratory syndrome virus (PRRSV). Accumulating data indicate that among these candidates, CD163, a cysteine-rich scavenger receptor on macrophages, is the major receptor for PRRSV. This review discusses the recent advances and understanding of the entry of PRRSV into cells, viral pathogenesis in CD163 gene-edited swine, and CD163 as a potential target of receptor-ligand for the control of PRRS.

Animal coronaviruses and SARS-CoV-2.

COVID-19 is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has rapidly spread to 216 countries and territories since first outbreak in December of 2019, posing a substantial economic losses and extraordinary threats to the public health worldwide. Although bats have been suggested as the natural host of SARS-CoV-2, transmission chains of this virus, role of animals during cross-species transmission, and future concerns remain unclear. Diverse animal coronaviruses have extensively been studied since the discovery of avian coronavirus in 1930s. The current article comprehensively reviews and discusses the current understanding about animal coronaviruses and SARS-CoV-2 for their emergence, transmission, zoonotic potential, alteration of tissue/host tropism, evolution, status of vaccines and surveillance. This study aims at providing guidance for control of COVID-19 and preventative strategies for possible future outbreaks of zoonotic coronavirus via cross-species transmission.

Transcriptional Immune Signatures of Alveolar Macrophages and the Impact of the NLRP3 Inflammasome on Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Replication.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a contagious viral (PRRSV) disease in pigs characterized by poor reproductive health, increased mortality, and reductions in growth rates. PRRSV is known to implement immuno-antagonistic mechanisms to evade detection and mute host responses to infection. To better understand the cellular immunosignature of PRRSV we have undertaken transcriptome and immunomodulatory studies in PRRSV-infected porcine alveolar macrophages (PAMs). We first used genome-wide transcriptome profiling (RNA-seq) to elucidate PRRSV-induced changes in the PAM transcriptome in response to infection. We found a number of cellular networks were altered by PRRSV infection, including many associated with innate immunity, such as, the NLRP3 inflammasome. To further explore the role(s) of innate immune networks in PRRSV-infected PAMs, we used an NLRP3-specific inhibitor, MCC950, to identify the potential functionality of the inflammasome during PRRSV replication. We found that PRRSV does quickly induce expression of inflammasome-associated genes in PAMs. Treatment of PAMs with MCC950 suggests NLRP3 inflammasome activation negatively impacts viral replication. Treatment of PAMs with cell culture supernatants from macrophages subjected to NLRP3 inflammasome activation (via polyinosinic-polycytidylic acid (poly I:C) transfection), prior to PRRSV infection resulted in significantly reduced viral RNA levels compared to PAMs treated with cell culture supernatants from macrophages subjected to NLRP3 inflammasome inhibition (MCC950 treatment/poly I:C transfection). This further supports a role for NLRP3 inflammasome activation in the innate macrophagic anti-PRRSV immune response and suggests that PRRSV is sensitive to the effects of NLRP3 inflammasome activity. Taken together, these transcriptome and immunoregulatory data highlight the complex changes PRRSV infection induces in the molecular immune networks of its cellular host.

Isolation and evolutionary analyses of porcine epidemic diarrhea virus in Asia.

Porcine epidemic diarrhea virus (PEDV) is a leading cause of diarrhea in pigs worldwide. Virus isolation and genetic evolutionary analysis allow investigations into the prevalence of epidemic strains and provide data for the clinical diagnosis and vaccine development. In this study, we investigated the genetic characteristics of PEDV circulation in Asia through virus isolation and comparative genomics analysis. APEDV strain designated HB2018 was isolated from a pig in a farm experiencing a diarrhea outbreak. The complete genome sequence of HB2018 was 28,138 bp in length. Phylogenetic analysis of HB2018 and 207 PEDVs in Asia showed that most PEDV strains circulating in Asia after 2010 belong to genotype GII, particularly GII-a. The PEDV vaccine strain CV777 belonged to GI, and thus, unmatched genotypes between CV777 and GII-a variants might partially explain incomplete protection by the CV777-derived vaccine against PEDV variants in China. In addition, we found the S protein of variant strains contained numerous mutations compared to the S protein of CV777, and these mutations occurred in the N-terminal domain of the S protein. These mutations may influence the antigenicity, pathogenicity, and neutralization properties of the variant strains.

COVID-19 and veterinarians for one health, zoonotic- and reverse-zoonotic transmissions.

A novel coronavirus emerged in human populations and spread rapidly to cause the global coronavirus disease 2019 pandemic. Although the origin of the associated virus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) remains unclear, genetic evidence suggests that bats are a reservoir host of the virus, and pangolins are a probable intermediate. SARS-CoV-2 has crossed the species barrier to infect humans and other animal species, and infected humans can facilitate reverse-zoonotic transmission to animals. Considering the rapidly changing interconnections among people, animals, and ecosystems, traditional roles of veterinarians should evolve to include transdisciplinary roles.

Molecular and Cellular Mechanisms for PRRSV Pathogenesis and Host Response to Infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused tremendous amounts of economic losses to the swine industry for more than three decades, but its control is still unsatisfactory. A significant amount of information is available for host cell-virus interactions during infection, and it is evident that PRRSV has evolved to equip various strategies to disrupt the host antiviral system and provide favorable conditions for survival. The current study reviews viral strategies for modulations of cellular processes including innate immunity, apoptosis, microRNAs, inflammatory cytokines, and other cellular pathways.

African swine fever: Etiology, epidemiological status in Korea, and perspective on control.

African swine fever (ASF), caused by the ASF virus, a member of the Asfarviridae family, is one of the most important diseases in the swine industry due to its clinical and economic impacts. Since the first report of ASF a century ago, ample information has become available, but prevention and treatment measures are still inadequate. Two waves of epizootic outbreaks have occurred worldwide. While the first wave of the epizootic outbreak was controlled in most of the infected areas, the second wave is currently active in the European and Asian continents, causing severe economic losses to the pig industry. There are different patterns of spreading in the outbreaks between those in European and Asian countries. Prevention and control of ASF are very difficult due to the lack of available vaccines and effective therapeutic measures. However, recent outbreaks in South Korea have been successfully controlled on swine farms, although feral pigs are periodically being found to be positive for the ASF virus. Therefore, we would like to share our story regarding the preparation and application of control measures. The success in controlling ASF on farms in South Korea is largely due to the awareness and education of swine farmers and practitioners, the early detection of infected animals, the implementation of strict control policies by the government, and widespread sharing of information among stakeholders. Based on the experience gained from the outbreaks in South Korea, this review describes the current understanding of the ASF virus and its pathogenic mechanisms, epidemiology, and control.

Interaction of PIAS1 with PRRS virus nucleocapsid protein mediates NF-κB activation and triggers proinflammatory mediators during viral infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) activates NF-κB during infection. We examined the ability of all 22 PRRSV genes for NF-κB regulation and determined the nucleocapsid (N) protein as the NF-κB activator. Protein inhibitor of activated STAT1 (signal transducer and activator of transcription 1) (PIAS1) was identified as a cellular protein binding to N. PIAS1 is known to bind to p65 (RelA) in the nucleus and blocks its DNA binding, thus functions as a repressor of NF-κB. Binding of N to PIAS1 released p65 for NF-κB activation. The N-terminal half of PIAS1 was mapped as the N-binding domain, and this region overlapped its p65-binding domain. For N, the region between 37 and 72 aa was identified as the binding domain to PIAS1, and this domain alone was able to activate NF-κB. A nuclear localization signal (NLS) knock-out mutant N did not activate NF-κB, and this is mostly likely due to the lack of its interaction with PIAS1 in the nucleus, demonstrating the positive correlation between the binding of N to PIAS1 and the NF-κB activation. Our study reveals a role of N in the nucleus for NF-κB activation and proinflammatory cytokine production during infection.

Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion.

Porcine reproductive and respiratory syndrome virus (PRRSV) blocks host mRNA nuclear export to the cytoplasm, and nonstructural protein 1 beta (nsp1ß) of PRRSV has been identified as the protein that disintegrates the nuclear pore complex. In the present study, the molecular basis for the inhibition of host mRNA nuclear export was investigated. Nucleoporin 62 (Nup62) was found to bind to nsp1ß, and the region representing the C-terminal residues 328 to 522 of Nup62 was determined to be the binding domain for nsp1ß. The nsp1ß L126A mutant in the SAP domain did not bind to Nup62, and in L126A-expressing cells, host mRNA nuclear export occurred normally. The vL126A mutant PRRSV generated by reverse genetics replicated at a lower rate, and the titer was lower than for wild-type virus. In nsp1ß-overexpressing cells or small interfering RNA (siRNA)-mediated Nup62 knockdown cells, viral protein synthesis increased. Notably, the production of type I interferons (IFN-α/ß), IFN-stimulated genes (PKR, OAS, Mx1, and ISG15 genes), IFN-induced proteins with tetratricopeptide repeats (IFITs) 1 and 2, and IFN regulatory factor 3 decreased in these cells. As a consequence, the growth of vL126A mutant PRRSV was rescued to the level of wild-type PRRSV. These findings are attributed to nuclear pore complex (NPC) disintegration by nsp1ß, resulting in increased viral protein production and decreased host protein production, including antiviral proteins in the cytoplasm. Our study reveals a new strategy of PRRSV for immune evasion and enhanced replication during infection.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS and is known to effectively suppress host innate immunity. The PRRSV nsp1ß protein blocks host mRNA nuclear export, which has been shown to be one of the viral mechanisms for inhibition of antiviral protein production. nsp1ß binds to the cellular protein nucleoporin 62 (Nup62), and as a consequence, the nuclear pore complex (NPC) is disintegrated and the nucleocytoplasmic trafficking of host mRNAs and host proteins is blocked. We show the dual benefits of Nup62 and nsp1ß binding for PRRSV replication: the inhibition of host antiviral protein expression and the exclusive use of host translation machinery by the virus. Our study unveils a novel strategy of PRRSV for immune evasion and enhanced replication during infection.

Engineering a Live Attenuated Porcine Epidemic Diarrhea Virus Vaccine Candidate via Inactivation of the Viral 2'-O-Methyltransferase and the Endocytosis Signal of the Spike Protein.

Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets; however, effective and safe vaccines are still not available. We hypothesized that inactivation of the 2'-O-methyltransferase (2'-O-MTase) activity of nsp16 and the endocytosis signal of the spike protein attenuates PEDV yet retains its immunogenicity in pigs. We generated a recombinant PEDV, KDKE4A, with quadruple alanine substitutions in the catalytic tetrad of the 2'-O-MTase using a virulent infectious cDNA clone, icPC22A, as the backbone. Next, we constructed another mutant, KDKE4A-SYA, by abolishing the endocytosis signal of the spike protein of KDKE4A Compared with icPC22A, the KDKE4A and KDKE4A-SYA mutants replicated less efficiently in vitro but induced stronger type I and type III interferon responses. The pathogenesis and immunogenicities of the mutants were evaluated in gnotobiotic piglets. The virulence of KDKE4A-SYA and KDKE4A was significantly reduced compared with that of icPC22A. Mortality rates were 100%, 17%, and 0% in the icPC22A-, KDKE4A-, and KDKE4A-SYA-inoculated groups, respectively. At 21 days postinoculation (dpi), all surviving pigs were challenged orally with a high dose of icPC22A. The KDKE4A-SYA- and KDKE4A-inoculated pigs were protected from the challenge, because no KDKE4A-SYA- and one KDKE4A-inoculated pig developed diarrhea whereas all the pigs in the mock-inoculated group had severe diarrhea, and 33% of them died. Furthermore, we serially passaged the KDKE4A-SYA mutant in pigs three times and did not find any reversion of the introduced mutations. The data suggest that KDKE4A-SYA may be a PEDV vaccine candidate.IMPORTANCE PEDV is the most economically important porcine enteric viral pathogen and has caused immense economic losses in the pork industries in many countries. Effective and safe vaccines are desperately required but still not available. 2'-O-MTase (nsp16) is highly conserved among coronaviruses (CoVs), and the inactivation of nsp16 in live attenuated vaccines has been attempted for several betacoronaviruses. We show that inactivation of both 2'-O-MTase and the endocytosis signal of the spike protein is an approach to designing a promising live attenuated vaccine for PEDV. The in vivo passaging data also validated the stability of the KDKE4A-SYA mutant. KDKE4A-SYA warrants further evaluation in sows and their piglets and may be used as a platform for further optimization. Our findings further confirmed that nsp16 can be a universal target for CoV vaccine development and will aid in the development of vaccines against other emerging CoVs.

Inter-serotype reassortment among epizootic haemorrhagic disease viruses in the United States.

First described in 1955 in New Jersey, epizootic haemorrhagic disease (EHD) causes a severe clinical disease in wild and domestic ruminants worldwide. Epizootic haemorrhagic disease outbreaks occur in deer populations each year from summer to late autumn. The etiological agent is EHD virus (EHDV) which is a double-stranded segmented icosahedral RNA virus. EHD virus utilizes point mutations and reassortment strategies to maintain viral fitness during infection. In 2018, EHDV serotype 2 was predominantly detected in deer in Illinois. Whole genome sequencing was conducted for two 2018 EHDV2 isolates (IL41747 and IL42218) and the sequence analyses indicated that IL42218 was a reassortant between different serotypes whereas IL41747 was a genetically stable strain. Our data suggest that multiple strains contribute to outbreaks each year.

Development of a triplex real-time RT-PCR assay for detection and differentiation of three US genotypes of porcine hemagglutinating encephalomyelitis virus.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a single-stranded, positive-sense RNA virus. PHEV mainly causes two types of clinical manifestations representing vomiting and wasting and encephalomyelitis in piglets. However, our recent findings provide strong evidence that PHEV can also cause respiratory disease in older pigs. Genomic analysis of new PHEV strains identified in our former study further classifies PHEV into three genotypes. Detection and differentiation of these new mutants are critical in monitoring PHEV evolution in the field. In the present study, we report the development of a triplex real-time RT-PCR assay for detection and differentiation of three PHEV genotypes, 1, 2, and 3. Three sets of primers and probes were designed; one set of primers and probe targeting the conserved regions of the 3' end nucleocapsid for detection of all three genotypes and another two sets of primers and probes targeting the regions of NS2 with different patterns of deletions for detection of both genotypes 1 and 3, or genotype 3 only. Genotype 1 was positive when two probe dyes showed signals, genotype 2 was positive when only one probe dye showed a signal, and genotype 3 was positive when all three probes showed signals. The detection limit of the developed triplex real-time RT-PCR was as low as 8 or 9 DNA copies for three sets of primers and probes. The specificity test showed no cross reaction with other porcine viruses. Positive field-samples were correctly typed by this new assay, which was further confirmed by DNA sequencing. The triplex real-time RT-PCR provides a rapid and sensitive method to detect and differentiate all three US genotypes of PHEV from clinical samples.

Establishment and Characterization of a High and Stable Porcine CD163-Expressing MARC-145 Cell Line.

Isolation and identification of diverse porcine reproductive and respiratory syndrome viruses (PRRSVs) play a fundamental role in PRRSV research and disease management. However, PRRSV has a restricted cell tropism for infection. MARC-145 cells are routinely used for North American genotype PRRSV isolation and vaccine production. But MARC-145 cells have some limitations such as low virus yield. CD163 is a cellular receptor that mediates productive infection of PRRSV in various nonpermissive cell lines. In this study, we established a high and stable porcine CD163- (pCD163-) expressing MARC-145 cell line toward increasing its susceptibility to PRRSV infection. Indirect immunofluorescence assay (IFA) and Western blotting assays showed that pCD163 was expressed higher in pCD163-MARC cell line than MARC-145 cells. Furthermore, the ability of pCD163-MARC cell line to propagate PRRSV was significantly increased as compared with MARC-145 cells. Finally, we found that pCD163-MARC cell line had a higher isolation rate of clinical PRRSV samples and propagated live attenuated PRRS vaccine strains more efficiently than MARC-145 cells. This pCD163-MARC cell line will be a valuable tool for propagation and research of PRRSV.