Preparation of monoclonal antibody and identification of two novel B cell epitopes to VP1 protein of porcine sapelovirus.

Porcine sapelovirus (PSV) is an important emerging swine pathogen that causes diarrhoea, respiratory distress, severe reproductive system and neurological disorders in pigs, posing huge threat to swine industry. However, there are no effective serological diagnostic products and the epitope characterization of PSV VP1 protein is still largely unknown. In current study, we successfully expressed recombinant His-VP1 protein by prokaryotic expression system and the recombinant VP1 protein had good immunogenicity. BALB/C mice were then selected and immunized with purified recombinant VP1 protein, and two monoclonal antibodies (Mabs) 9F10 and 15E4 against VP1 were successfully prepared by hybrioma technology. The isotype of these two Mabs were identified and showed that Mab 9F10 with the heavy chain subtype was IgG1 and the light chain subtype was kappa. Mab 15E4 was identified as IgG2 for the heavy chain subtype and Kappa for the light chain subtype. The antigen epitopes of prepared two VP1 Mabs were clearly identified. The minimal unit of B cell specific epitope recognized by Mab 15E4 was 203YDGDG207 and conserved in different strain genotypes of PSV, indicating this epitope may be a good target for serological detection of PSV. However, the epitope recognized by Mab 9F10 was 8QAIVNRT14 and varied greatly among different PSV strains. Structural modeling analysis showed that the identified two novel B cell epitopes were located on the surface of VP1. Our study provides useful tool for the establishment the serological detection methods of PSV and may support the study of VP1 protein function.

Identification of a B-Cell Epitope in the VP3 Protein of Senecavirus A.

Senecavirus A (SVA) is a member of the genus Senecavirus of the family Picornaviridae. SVA-associated vesicular disease (SAVD) outbreaks have been extensively reported since 2014-2015. Characteristic symptoms include vesicular lesions on the snout and feet as well as lameness in adult pigs and even death in piglets. The capsid protein VP3, a structural protein of SVA, is involved in viral replication and genome packaging. Here, we developed and characterized a mouse monoclonal antibody (mAb) 3E9 against VP3. A motif 192GWFSLHKLTK201 was identified as the linear B-cell epitope recognized by mAb 3E9 by using a panel of GFP-tagged epitope polypeptides. Sequence alignments show that 192GWFSLHKLTK201 was highly conserved in all SVA strains. Subsequently, alanine (A)-scanning mutagenesis indicated that W193, F194, L196, and H197 were the critical residues recognized by mAb 3E9. Further investigation with indirect immunofluorescence assay indicated that the VP3 protein was present in the cytoplasm during SVA replication. In addition, the mAb 3E9 specifically immunoprecipitated the VP3 protein from SVA-infected cells. Taken together, our results indicate that mAb 3E9 could be a powerful tool to work on the function of the VP3 protein during virus infection.

Innate immune evasion by picornaviruses.

The family Picornaviridae comprises a large number of viruses that cause disease in broad spectrum of hosts, which have posed serious public health concerns worldwide and led to significant economic burden. A comprehensive understanding of the virus-host interactions during picornavirus infections will help to prevent and cure these diseases. Upon picornavirus infection, host pathogen recognition receptors (PRRs) sense viral RNA to activate host innate immune responses. The activated PRRs initiate signal transduction through a series of adaptor proteins, which leads to activation of several kinases and transcription factors, and contributes to the consequent expression of interferons (IFNs), IFN-inducible antiviral genes, as well as various inflammatory cytokines and chemokines. In contrast, to maintain viral replication and spread, picornaviruses have evolved several elegant strategies to block innate immune signaling and hinder host antiviral response. In this review, we will summarize the recent progress of how the members of family Picornaviridae counteract host immune response through evasion of PRRs detection, blocking activation of adaptor molecules and kinases, disrupting transcription factors, as well as counteraction of antiviral restriction factors. Such knowledge of immune evasion will help us better understand the pathogenesis of picornaviruses, and provide insights into developing antiviral strategies and improvement of vaccines.

Identification of linear B cell epitopes on VP1 and VP2 proteins of Senecavirus A (SVA) using monoclonal antibodies.

Senecavirus A (SVA), previously called Seneca Valley virus, belongs to the family Picornaviridae, species Senecavirus A, in the Senecavirus genus, and can cause vesicular lesions in sows and acute death in piglets. In this study, recombinant VP1 and VP2 proteins were expressed in prokaryotic expression system and used to generate eight monoclonal antibodies (mAbs) against VP1 or VP2 protein. And all of the mAbs reacted specifically with SVA virus by both Western blot and indirect immunofluorescence assay (IFA). The resurts showed that all of the epitopes aganist these mAbs were B cell linear epitopes. To map the epitopes, both Western blot and indirect enzyme-linked immunosorbant assay (indirect ELISA) were performed. The epitope 21GELAAP26 recognized by mAb 1G9, was likely to be a significant B cell epitope due to the high antigenic index and the fully exposure on the surface of the VP1. Other mAbs were recognized by VP2 protein. MAbs 1E7 and 8E8 recognized the same epitope at 12DRVITQT18, 1A5 recognized the epitope at 71WTKAVK76, 1G2 recognized the epitope at 98GGAFTA103, 9D2 and 6B11 recognized the same epitope at 150KSLQELN156, and 7E4 recognized the epitope at 248YKEGAT253. Alignment of amino acids revealed that four epitopes were completely conserved among all SVA strains, including 21GELAAP26, 71WTKAVK76, 98GGAFTA103, and 248YKEGAT253. Interestingly, there were some amino acid mutations in 12DRVITQT18 and 150KSLQELN156, but no significant difference was detected on the reaction intensity between epitopes and the corresponding mAbs. This is the first report about the SVA epitopes, which will benefit to the study of viral pathogenic mechanism, vaccine design, as well as the establishment of detection methods.

Comprehensive review on immunopathogenesis, diagnostic and epidemiology of Senecavirus A.

Senecavirus A (SVA), formerly known as Seneca Valley virus, is a single-strand, positive-sense RNA virus in the family Picornaviridae. This virus has been associated with recent outbreaks of vesicular disease (SVA-VD) and epidemic transient neonatal losses (ETNL) in several swine-producing countries. The clinical manifestation of and lesion caused by SVA are indistinguishable from other vesicular diseases. Pathogenicity studies indicate that SVA could regulate the host innate immune response to facilitate virus replication and the spread of the virus to bystander cells. SVA infection can induce specific humoral and cellular responses that can be detected within the first week of infection. However, SVA seems to produce persistent infection, and the virus can be shed in oral fluids for a month and detected in tissues for approximately two months after experimental infection. SVA transmission could be horizontal or vertical in infected herds of swine, while positive animals can also remain subclinical. In addition, mice seem to act as reservoirs, and the virus can persist in feed and feed ingredients, increasing the risk of introduction into naïve farms. Besides the pathological effects in swine, SVA possesses cytolytic activity, especially in neoplastic cells. Thus, SVA has been evaluated in phase II clinical trials as a virotherapy for neuroendocrine tumors. The goal of this review is summarize the current SVA-related research in pathogenesis, immunity, epidemiology and advances in diagnosis as well as discuses current challenges with subclinical/persistent presentation.

Natural antiviral compound silvestrol modulates human monocyte-derived macrophages and dendritic cells.

Outbreaks of infections with viruses like Sars-CoV-2, Ebola virus and Zika virus lead to major global health and economic problems because of limited treatment options. Therefore, new antiviral drug candidates are urgently needed. The promising new antiviral drug candidate silvestrol effectively inhibited replication of Corona-, Ebola-, Zika-, Picorna-, Hepatis E and Chikungunya viruses. Besides a direct impact on pathogens, modulation of the host immune system provides an additional facet to antiviral drug development because suitable immune modulation can boost innate defence mechanisms against the pathogens. In the present study, silvestrol down-regulated several pro- and anti-inflammatory cytokines (IL-6, IL-8, IL-10, CCL2, CCL18) and increased TNF-α during differentiation and activation of M1-macrophages, suggesting that the effects of silvestrol might cancel each other out. However, silvestrol amplified the anti-inflammatory potential of M2-macrophages by increasing expression of anti-inflammatory surface markers CD206, TREM2 and reducing release of pro-inflammatory IL-8 and CCL2. The differentiation of dendritic cells in the presence of silvestrol is characterized by down-regulation of several surface markers and cytokines indicating that differentiation is impaired by silvestrol. In conclusion, silvestrol influences the inflammatory status of immune cells depending on the cell type and activation status.

Construction of eGFP-Tagged Senecavirus A for Facilitating Virus Neutralization Test and Antiviral Assay.

Senecavirus A (SVA), also known as Seneca Valley virus, is an emerging virus that causes vesicular disease in pigs. This virus belongs to the genus Senecavirus in the family Picornaviridae. The SVA CH-LX-01-2016 was isolated from Guangdong Province of China in 2016. In this study, a recombinant SVA CH-LX-01-2016 was constructed using reverse genetics, and proven to be able to express efficiently an enhanced green fluorescent protein (eGFP) in vitro. This eGFP-tagged recombinant SVA (rSVA-eGFP) exhibited a high capacity for viral replication. Its fluorescence-tracked characteristics greatly facilitated both virus neutralization test (VNT) and antiviral assay. The rSVA-eGFP-based VNT was used to detect eight porcine serum samples, out of which four were determined to be neutralization titer-positive. Subsequently, two antiviral drugs, ribavirin and apigenin, were assayed for evaluating both effects against the rSVA-eGFP in vitro. The result showed that only the ribavirin exhibited an anti-SVA activity.

Cryo-EM Studies of Virus-Antibody Immune Complexes.

Antibodies play critical roles in neutralizing viral infections and are increasingly used as therapeutic drugs and diagnostic tools. Structural studies on virus-antibody immune complexes are important for better understanding the molecular mechanisms of antibody-mediated neutralization and also provide valuable information for structure-based vaccine design. Cryo-electron microscopy (cryo-EM) has recently matured as a powerful structural technique for studying bio-macromolecular complexes. When combined with X-ray crystallography, cryo-EM provides a routine approach for structurally characterizing the immune complexes formed between icosahedral viruses and their antibodies. In this review, recent advances in the structural understanding of virus-antibody interactions are outlined for whole virions with icosahedral T = pseudo 3 (picornaviruses) and T = 3 (flaviviruses) architectures, focusing on the dynamic nature of viral shells in different functional states. Glycoprotein complexes from pleomorphic enveloped viruses are also discussed as immune complex antigens. Improving our understanding of viral epitope structures using virus-based platforms would provide a fundamental road map for future vaccine development.

A Novel Live Attenuated Vaccine Candidate Protects Against Heterologous Senecavirus A Challenge.

Senecavirus A (SVA) is an emerging picornavirus causing vesicular disease (VD) clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Currently there are no vaccines currently available for SVA. Here we developed a recombinant SVA strain (rSVAm SacII) using reverse genetics and assessed its immunogenicity and protective efficacy in pigs. In vivo characterization of the rSVAm SacII strain demonstrated that the virus is attenuated, as evidenced by absence of lesions, decreased viremia and virus shedding in inoculated animals. Notably, while attenuated, rSVA mSacII virus retained its immunogenicity as high neutralizing antibody (NA) responses were detected in inoculated animals. To assess the immunogenicity and protective efficacy of rSVA mSacII, 4-week-old piglets were sham-immunized or immunized with inactivated or live rSVA mSacII virus-based formulations. A single immunization with live rSVA mSacII virus via the intramuscular (IM) and intranasal (IN) routes resulted in robust NA responses with antibodies being detected between days 3-7 pi. Neutralizing antibody responses in animals immunized with the inactivated virus via the IM route were delayed and only detected after a booster on day 21 pi. Immunization with live virus resulted in recall T cell proliferation (CD4+, CD8+, and CD4+/CD8+ T cells), demonstrating efficient stimulation of cellular immunity. Notably, a single dose of the live attenuated vaccine candidate resulted in protection against heterologous SVA challenge, as demonstrated by absence of overt disease and reduced viremia, virus shedding and viral load in tissues. The live attenuated vaccine candidate developed here represents a promising alternative to prevent and control SVA in swine.

Evolutionary and Structural Overview of Human Picornavirus Capsid Antibody Evasion.

Picornaviruses constitute one of the most relevant viral groups according to their impact on human and animal health. Etiologic agents of a broad spectrum of illnesses with a clinical presentation that ranges from asymptomatic to fatal disease, they have been the cause of uncountable epidemics throughout history. Picornaviruses are small naked RNA-positive single-stranded viruses that include some of the most important pillars in the development of virology, comprising poliovirus, rhinovirus, and hepatitis A virus. Picornavirus infectious particles use the fecal-oral or respiratory routes as primary modes of transmission. In this regard, successful viral spread relies on the capability of viral capsids to (i) shelter the viral genome, (ii) display molecular determinants for cell receptor recognition, (iii) facilitate efficient genome delivery, and (iv) escape from the immune system. Importantly, picornaviruses display a substantial amount of genetic variability driven by both mutation and recombination. Therefore, the outcome of their replication results in the emergence of a genetically diverse cloud of individuals presenting phenotypic variance. The host humoral response against the capsid protein represents the most active immune pressure and primary weapon to control the infection. Since the preservation of the capsid function is deeply rooted in the virus evolutionary dynamics, here we review the current structural evidence focused on capsid antibody evasion mechanisms from that perspective.

Seneca Valley virus 2C and 3C inhibit type I interferon production by inducing the degradation of RIG-I.

Seneca Valley virus (SVV) is a member of the Picornaviridae family, which has been used to treat neuroendocrine cancer. The innate immune system plays an important role in SVV infection. However, few studies have elucidated the relationship between SVV infection and the host's antiviral response. In this study, SVV replication could induce the degradation of RIG-I in HEK-293T, SW620 and SK6 cells. And overexpressing retinoic acid-inducible gene I (RIG-I) could significantly inhibit SVV propagation. The viral protein 2C and 3C were essential for the degradation of RIG-I. Furthermore, 2C and 3C significantly reduced Sev or RIG-I-induced IFN-ß production. Mechanistically, 2C and 3C induced RIG-I degradation through the caspase signaling pathway. Taken together, we demonstrate the antiviral role of RIG-I against SVV and the mechanism by which SVV 2C and 3C weaken the host innate immune system.

Seneca Valley Virus 3C protease negatively regulates the type I interferon pathway by acting as a viral deubiquitinase.

The mechanisms that enable Seneca Valley Virus (SVV) to escape the host innate immune response are not well known. Previous studies demonstrated that SVV 3Cpro suppresses innate immune responses by cleavage of host proteins and degradation of IRF3 and IRF7 protein expression. Here, we showed that SVV 3C protease (3Cpro) has deubiquitinating activity. Overexpressed 3Cpro inhibits the ubiquitination of cellular substrates, acting on both lysine-48- and lysine-63-linked polyubiquitin chains. SVV infection also possessed deubiquitinating activity. The ubiquitin-proteasome system was significantly involved in SVV replication. Furthermore, 3Cpro inhibited the ubiquitination of retinoic acid-inducible gene I (RIG-I), TANK-binding kinase 1 (TBK1), and TNF receptor-associated factor 3 (TRAF3), thereby blocking the expression of interferon (IFN)-ß and IFN stimulated gene 54 (ISG54) mRNAs. A detailed analysis revealed that mutations (H48A, C160A, or H48A/C160A) that ablate the Cys and His residues of 3Cpro abrogated its deubiquitinating activity and the ability of 3Cpro to block IFN-ß induction. Together, our results demonstrate a novel mechanism developed by SVV 3Cpro to promote viral replication, and may also provide a novel strategy for improving ubiquitination-based therapy.

RIG-I is responsible for activation of type I interferon pathway in Seneca Valley virus-infected porcine cells to suppress viral replication.

BACKGROUND: Retinoic acid-inducible gene I (RIG-I) is a key cytosolic receptor of the innate immune system. Seneca valley virus (SVV) is a newly emerging RNA virus that infects pigs causing significant economic losses in pig industry. RIG-I plays different roles during different viruses infections. The role of RIG-I in SVV-infected cells remains unknown. Understanding of the role of RIG-I during SVV infection will help to clarify the infection process of SVV in the infected cells. METHODS: In this study, we generated a RIG-I knockout (KO) porcine kidney PK-15 cell line using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing tool. The RIG-I gene sequence of RIG-I KO cells were determined by Sanger sequencing method, and the expression of RIG-I protein in the RIG-I KO cells were detected by Western bloting. The activation status of type I interferon pathway in Sendai virus (SeV)- or SVV-infected RIG-I KO cells was investigated by measuring the mRNA expression levels of interferon (IFN)-ß and IFN-stimulated genes (ISGs). The replicative state of SVV in the RIG-I KO cells was evaluated by qPCR, Western bloting, TCID50 assay and indirect immunofluorescence assay. RESULTS: Gene editing of RIG-I in PK-15 cells successfully resulted in the destruction of RIG-I expression. RIG-I KO PK-15 cells had a lower expression of IFN-ß and ISGs compared with wildtype (WT) PK-15 cells when stimulated by the model RNA virus SeV. The amounts of viral RNA and viral protein as well as viral yields in SVV-infected RIG-I WT and KO cells were determined and compared, which showed that knockout of RIG-I significantly increased SVV replication and propagation. Meanwhile, the expression of IFN-ß and ISGs were considerably decreased in RIG-I KO cells compared with that in RIG-I WT cells during SVV infection. CONCLUSION: Altogether, this study indicated that RIG-I showed an antiviral role against SVV and was essential for activation of type I IFN signaling during SVV infection. In addition, this study suggested that the CRISPR/Cas9 system can be used as an effective tool to modify cell lines to increase viral yields during SVV vaccine development.

Pathogenicity and cross-reactive immune responses of a historical and a contemporary Senecavirus A strains in pigs.

The goals of this study were to compare the pathogenicity and infection dynamics of a historical and a contemporary SVA strains (SVV 001 and SD15-26) and to assess cross-neutralizing and cross-reactive T cell responses following experimental infection in pigs. Both SVA strains successfully infected all inoculated animals, resulting in viremia and robust antibody and cellular immune responses. SVA SD15-26 infection resulted in characteristic clinical signs and vesicular lesions, however, SVA SVV 001 did not cause overt clinical disease with inoculated animals remaining clinically normal during the experiment. Notably, neutralization- and -recall IFN-γ expression-assays revealed marked cross-neutralizing antibody and cross-reactive T cell responses between the two viral strains. Together these results demonstrate that the historical SVA SVV 001 strain presents low virulence in pigs when compared to the contemporary SVA SD15-26 strain. Additionally, immunological assays indicate that SVA SVV 001 and SD15-26 are antigenically related and share conserved antigenic determinants.

The full-length genome characterization, genetic diversity and evolutionary analyses of Senecavirus A isolated in Thailand in 2016.

Senecavirus A (SVA) is a novel picornavirus that causes porcine idiopathic vesicular disease characterized by lameness, coronary band hyperemia, and vesicles on the snout and coronary bands. An increase in the detection rate of SVA in several countries suggests that the disease has become a widespread problem. Herein, we report the detection of SVA in Thailand and the characterization of full-length genomic sequences of six Thai SVA isolates. Phylogenetic, genetic, recombination, and evolutionary analyses were performed. The full-length genome, excluding the poly (A) tail of the Thai SVA isolates, was 7282 nucleotides long, with the genomic organization resembling other previously reported SVA isolates. Phylogenetic and genetic analyses based on full-length genome demonstrated that the Thai SVA isolates were grouped in a novel cluster, separated from SVA isolates from other countries. Although the Thai SVA isolates were closely related to 11-55910-3, the first SVA isolate from Canada, with 97.9-98.2%, but they are different. Evolutionary and recombinant analyses suggested that the Thai SVA isolates shared a common ancestor with the 11-55910-3 isolate. The positive selection in the VP4 and 3D genes suggests that the virus was not externally introduced, but rather continuously evolved in the population prior to the first detection. Addition, the presence of SVA could have been ignored due to the presence of other pathogens causing similar clinical diseases. This study warrants further investigations into molecular epidemiology and genetic evolution of the SVA in Thailand.

Protective efficacy of a bivalent live attenuated vaccine against duck hepatitis A virus types 1 and 3 in ducklings.

Duck hepatitis A virus (DHAV) infection is characterized by an acute, rapidly spreading that affects young ducklings. DHAV-1 or DHAV-3 infection is prevalent, and simultaneous co-infection with both viruses has recently become increasingly frequent in the domestic duck farms. In this study, we developed a bivalent live attenuated vaccine (DHV-HSBP100 and AP-04203P100) for DHAV-1 and DHAV-3 and reported the protective efficacy and safety of the vaccine. At 1-day-old, the ducklings received a bivalent vaccine via intramuscular injection. The immunized ducklings showed effective and rapid protection against virulent DHAV-1 and DHAV-3 at 2 or 3 days post vaccination. Moreover, the ducklings showed a potent humoral immune response that peaked at 3 weeks and were maintained at 6 weeks after vaccination. The bivalent vaccine was safe; ducklings administered 10 doses of bivalent vaccines showed no clinical signs, mortality, gross lesions, and body weight changes compared with those observed in the negative controls. Ducklings vaccinated with a bivalent vaccine were evaluated for tissue tropism and viral replication of vaccine strains. Both bivalent vaccine strains were detected in various organs, and the highest virus replication was detected in the kidneys, among the tested organs. No interference occurred during the replication of both vaccine strains. Thus, these experiments suggest that bivalent vaccines would be useful as a promising and practical strategy for control DHAV outbreaks caused by DHAV-1 and DHAV-3 in duck farms.

Immunogenicity and protective efficacy of an inactivated cell culture-derived Seneca Valley virus vaccine in pigs.

Seneca Valley virus (SVV) infection in pigs is associated with porcine idiopathic vesicular disease (PIVD). Outbreaks of SVV infection in pig herds have been reported in several Asia and Americas countries. Recently, a series of outbreaks of SVV infection occurred in China, Canada, Thailand and the United States. However, no available vaccines have been developed to limit the transmission of SVV. The SVV CH-FJ-2017 from Fujian province in China is a representative of the epidemic strains, and shows 98.5-99.9% capsid protein amino acid identity with the recent SVV strains. In the present study, we developed a SVV CH-FJ-2017 inactivated vaccine. The SVV was produced by cultivation of BHK-21 cells in roller bottles, inactivated with binary ethylenimine, and mixed with oil adjuvant (Montanide ISA). The immunogenicity of the inactivated vaccine in pigs was evaluated by neutralizing test, and the immunized pigs were challenged with SVV CH-FJ-2017. The results showed that animals receiving one dose of the inactivated vaccine (2 µg/dose) with oil adjuvant developed high neutralizing antibody titers and showed no clinical signs after virus challenge comparing with the non-vaccinated animals, indicating a good protective efficacy of the produced vaccine against SVV infection. This is the first reported SVV vaccine that can be used for control of SVV infection in pigs.

Molecular epidemiology of duck hepatitis a virus types 1 and 3 in China, 2010-2015.

Duck hepatitis A virus (DHAV) is the most common aetiologic agent of duck virus hepatitis (DVH), causing substantial economic losses in the duck industry worldwide. In China, officially approved DHAV-1 live-attenuated vaccines have been used widely to vaccinate breeder ducks since 2013. However, following the reports of DVH outbreaks, it has become necessary to assess the epidemiological situation of this virus in China. We conducted molecular epidemiological analyses of 32 DHAV field isolates while analysing the samples from ducks suspected of having hepatitis collected from commercial duck farms in China between May 2010 and December 2015. Considerable changes were observed in the epidemiology of DHAV-1 and DHAV-3 in China over time. A higher number of DHAV-1 strains were isolated during 2010-2012, coinciding with the widespread use of officially approved DHAV-1 live vaccine strains beginning in 2013. In contrast, a higher rate of DHAV-3 causing DHAV infections was observed between 2013 and 2015. Phylogenetic analyses based on the full-length VP1 gene were performed on these field isolates and using reference strains available in GenBank. DHAV-1 field isolates were evaluated in two groups: one group closely related to prototype strains and circulating in China between 2010 and 2012 and another group exhibiting genetic and serological differences from prototype strains. All DHAV-3 strains isolated in this study were grouped as monophyletic, which has become the predominant viral type, particularly in Shandong and Sichuan provinces, since 2013. In conclusion, these data provide updated information on the genetic and serological diversity of DHAV-1 and DHAV-3, and our findings may serve as a foundation for the prevention of, and vaccine development for, DHAV in China.

Clearance of a persistent picornavirus infection is associated with enhanced pro-apoptotic and cellular immune responses.

Long-term persistent viral infections cause substantial morbidity and associated economic losses in human and veterinary contexts. Yet, the mechanisms associated with establishment of persistent infections are poorly elucidated. We investigated immunomodulatory mechanisms associated with clearance versus persistence of foot-and-mouth disease virus (FMDV) in micro-dissected compartments of the bovine nasopharynx by microarray. The use of laser-capture microdissection allowed elucidation of differential gene regulation within distinct anatomic compartments critical to FMDV infection. Analysis of samples from transitional and persistent phases of infection demonstrated significant differences in transcriptome profiles of animals that cleared infection versus those that became persistently infected carriers. Specifically, it was demonstrated that clearance of FMDV from the nasopharyngeal mucosa was associated with upregulation of targets associated with activation of T cell-mediated immunity. Contrastingly, gene regulation in FMDV carriers suggested inhibition of T cell activation and promotion of Th2 polarization. These findings were corroborated by immunofluorescence microscopy which demonstrated relative abundance of CD8+ T cells in the nasopharyngeal mucosa in association with clearance of FMDV. The findings presented herein emphasize that a critical balance between Th1 and Th2 -mediated immunity is essential for successful clearance of FMDV infection and should be considered for development of next-generation vaccines and antiviral products.

Histopathological, immunohistochemical, and ultrastructural evidence of spontaneous Senecavirus A-induced lesions at the choroid plexus of newborn piglets.

Epidemic Transient Neonatal Losses (ETNL) is a disease of piglets caused by Senecavirus A (SVA) in which the method of dissemination and associated lesions are not well-defined. This study investigated the possible SVA-induced lesions by examining spontaneous infections in newborn piglets. Histopathology revealed ballooning degeneration of transitional epithelium, nonsuppurative meningoencephalitis, plexus choroiditis, and atrophic enteritis. RT-PCR identified SVA in all tissues evaluated and sequencing confirmed these results. Positive immunoreactivity to SVA was observed in endothelial and epithelial tissues of all organs evaluated. Semithin analysis revealed vacuolization of apical enterocytes of the small intestine, balloon degeneration and necrosis of endothelial cells of the choroid plexus (CP) and nonsuppurative choroid plexitis. Ultrathin evaluation demonstrated hydropic degeneration of apical enterocytes, degeneration and necrosis of endothelium of CP fenestrated capillaries, degeneration of ependymocytes associated with intralesional viral particles. It is proposed that SVA initially infects apical enterocytes of newborn piglets and probably enters the circulatory system with entry to the brain via the CP, by first producing an initial inflammatory reaction, with subsequent encephalitic dissemination. Consequently, SVA probably uses an enteric-neurological method of dissemination.