Characterization of strain PTV-2 USA/IA65463/2014 and strain PTV-11 USA/IA09592/2013 of Teschovirus A: Experimental inoculation, distribution of nucleic acids and development of Teschovirus encephalomyelitis

Teschovirus A (previously Porcine teschovirus; PTV) is the etiologic agent of Teschovirus encephalomyelitis (TE). Historically, PTV-1 has been recognized as one of the most pathogenic serotypes, although other serotypes have been described to cause TE. Hence, most information about the pathogenicity, epidemiology and control of disease relies on data from studies involving PTV-1. In recent years, two cases submitted to the Iowa State University Veterinary Diagnostic Laboratory with a history of polioencephalitis and/or myelitis were identified and genomic sequencing characterized these isolates as (sero)types PTV-2 and PTV-11. Although multiple serotypes have been identified and genetically characterized, the neuropathogenicity of some of these serotypes has not been fully elucidated. To assess these isolates, we developed an experimental model to determine the neuropathogenicity of the PTV-2 and PTV-11 isolates and observed that both isolates caused histological lesions and clinical disease consistent with TE in cesarean-derived colostrum-deprived pigs. Furthermore, PTV RNA was detected in different tissues, serum and feces in all inoculated animals by RT-qPCR. The experimental approach used in this research permitted to develop a successful platform to induce clinical disease. This is the first description of a neuropathogenic PTV-11 strain in the U.S. and the first experimental inoculation using a PTV-2 autochthonous U.S. strain after the initial description of PTV-2 strain O3b made by Long at al. in 1966. This is also the first assessment of the viral shedding, viremia and distribution by real time RT-qPCR of nucleic acids of PTV-2 and PTV-11 in experimentally infected pigs with Teschovirus encephalomyelitis

Evaluation of a Microencapsulated Form of Zinc Oxide on Weanling Pig Growth Performance, Intestinal Morphology, and Zinc Excretion

A total of 350 pigs (DNA 200 × 400; initially 13.31 ± 0.04 lb BW) were used in a 42-d study with 5 pigs per pen and 12 pens per treatment. At weaning, pigs were randomly allocated to pens and pens were randomly allotted to dietary treatments. Dietary treatments were: 1) negative control (standard nursery diet containing 110 ppm Zn from trace mineral premix); 2) control diet with 3,000 ppm added Zn in the form of ZnO in phase 1 and 2,000 ppm added Zn in the form of ZnO in phase 2 (High-ZnO); 3) control diet with 400 ppm added Zn in the form of ZnO in phases 1 and 2 (Low- ZnO); 4) 3,000 ppm added Zn in the form of microencapsulated ZnO in phase 1 and 2,000 ppm added Zn in the form of microencapsulated ZnO in phase 2 (High-MZnO); and 5) 400 ppm added Zn in the form of microencapsulated ZnO in phases 1 and 2 (Low-MZnO). Pigs were weighed and feed disappearance was determined to evaluate ADG, ADFI, and F/G. On d 10 and d 28, fecal samples from 3 pigs per pen were collected for fecal Zn concentrations. On d 28, 30 pigs (6 pigs per treatment) were euthanized, and small intestinal tissue was collected to evaluate morphology. There was no evidence of differences in ADG, ADFI, or F/G for the entire treatment period (d 0 to d 28; P \u3e 0.05). During the common phase 3 (d 28 to 42) pigs fed the negative control, High-MZnO, or Low-MZnO had improved (P \u3c 0.0001) ADG compared to pigs fed High- or Low-ZnO, which was driven by an increase in ADFI (P \u3c 0.0001). For the entire experiment (d 0 to 42), pigs fed Low-ZnO or High-ZnO had reduced (P \u3c 0.0001) ADG compared those fed the negative control. There was no evidence that small intestinal morphology differed significantly between treatments (P \u3e 0.05). Finally, a significant treatment × day interaction (P = 0.04) was observed for fecal Zn concentrations, where pigs fed High-ZnO had greater fecal Zn levels on d 10 and d 28 compared to pigs fed all other treatments

Porcine astrovirus type 3 is an emerging cause of atypical neurologic disease: Diagnostic cases and infection dynamics on affected flows

Astroviruses (AstVs) are viral agents in the Family Astroviridae that are able to infect and cause disease in a wide variety of host species. Despite the broad host range and ability to cause disease, there are significant gaps in knowledge concerning the epidemiology, ecology, and pathophysiology of a majority of AstVs. Porcine astroviruses (PoAstVs) are distributed worldwide. Five PoAstV lineages (PoAstV1–PoAstV5) have been identified perhaps reflecting diverse origins, interspecies transmission, and recombination events, some presumably with human strains. In the past decade, different members of the genus Mamastrovirus have been associated with neurologic disease in humans, bovine, mink and most recently, porcine astrovirus 3 (PoAstV3) in swine

Ecology of Porcine Astrovirus Type 3 in a Herd with Associated Neurologic Disease

Astroviruses (AstVs) cause disease in a wide variety of species. Porcine AstVs are highly genetically diverse and conventionally assigned to five genetic lineages (PoAstV1-5). Due to the increasing evidence that porcine astrovirus type 3 (PoAstV3) is a cause of encephalomyelitis in swine and to elucidate important ecologic characteristics, the infection dynamics and environmental distribution of PoAstV3 were investigated in a herd with PoAstV3-associated neurologic disease. Over a 22 week period, the frequency of PoAstV3 fecal shedding varied by pig and age. The peak detection by RT-qPCR of PoAstV3 on fecal swabs (95%; 61 of 64) occurred at 3 weeks of age. The lowest frequency of detection was at 21 weeks of age (4%; 2 of 47); however, the frequency increased to 41% (19 of 46) at the final sampling time point (25 weeks of age). Viremia was rare (0.9%: 4 of 433). Detection in oral fluid was consistent with 75% to 100% of samples positive at each time point. Pens and feeders also had a high rate of detection with a majority of samples positive at a majority of sampling time points. Based on the data presented, PoAstV3 can be consistently detected in the environment with a majority of pigs being infected and a subset intermittently shedding the virus in feces out to 25 weeks of age. These findings suggest the importance of as-yet unidentified risk factors associated with the development of PoAstV3-associated polioencephalomyelitis

Comparative Analysis of Novel Strains of Porcine Astrovirus Type 3 in the USA

Porcine astrovirus type 3 (PoAstV3) has been previously identified as a cause of polioencephalomyelitis in swine and continues to cause disease in the US swine industry. Herein, we describe the characterization of both untranslated regions, frameshifting signal, putative genome-linked virus protein (VPg) and conserved antigenic epitopes of several novel PoAstV3 genomes. Twenty complete coding sequences (CDS) were obtained from 32 diagnostic cases originating from 11 individual farms/systems sharing a nucleotide (amino acid) percent identity of 89.74–100% (94.79–100%), 91.9–100% (96.3–100%) and 90.71–100% (93.51–100%) for ORF1a, ORF1ab and ORF2, respectively. Our results indicate that the 5′UTR of PoAstV3 is highly conserved highlighting the importance of this region in translation initiation while their 3′UTR is moderately conserved among strains, presenting alternative configurations including multiple putative protein binding sites and pseudoknots. Moreover, two predicted conserved antigenic epitopes were identified matching the 3′ termini of VP27 of PoAstV3 USA strains. These epitopes may aid in the design and development of vaccine components and diagnostic assays useful to control outbreaks of PoAstV3-associated CNS disease. In conclusion, this is the first analysis predicting the structure of important regulatory motifs of neurotropic mamastroviruses, which differ from those previously described in human astroviruses

Characterization of strain PTV-2 USA/IA65463/2014 and strain PTV-11 USA/IA09592/2013 of Teschovirus A: Experimental inoculation, distribution of nucleic acids and development of Teschovirus encephalomyelitis

Teschovirus A (previously Porcine teschovirus; PTV) is the etiologic agent of Teschovirus encephalomyelitis (TE). Historically, PTV-1 has been recognized as one of the most pathogenic serotypes, although other serotypes have been described to cause TE. Hence, most information about the pathogenicity, epidemiology and control of disease relies on data from studies involving PTV-1. In recent years, two cases submitted to the Iowa State University Veterinary Diagnostic Laboratory with a history of polioencephalitis and/or myelitis were identified and genomic sequencing characterized these isolates as (sero)types PTV-2 and PTV-11. Although multiple serotypes have been identified and genetically characterized, the neuropathogenicity of some of these serotypes has not been fully elucidated. To assess these isolates, we developed an experimental model to determine the neuropathogenicity of the PTV-2 and PTV-11 isolates and observed that both isolates caused histological lesions and clinical disease consistent with TE in cesarean-derived colostrum-deprived pigs. Furthermore, PTV RNA was detected in different tissues, serum and feces in all inoculated animals by RT-qPCR. The experimental approach used in this research permitted to develop a successful platform to induce clinical disease. This is the first description of a neuropathogenic PTV-11 strain in the U.S. and the first experimental inoculation using a PTV-2 autochthonous U.S. strain after the initial description of PTV-2 strain O3b made by Long at al. in 1966. This is also the first assessment of the viral shedding, viremia and distribution by real time RT-qPCR of nucleic acids of PTV-2 and PTV-11 in experimentally infected pigs with Teschovirus encephalomyelitis.</p

Porcine astrovirus type 3 as a cause of central nervous system disease in swine: Retrospective identification of diagnostic cases, molecular characterization and experimental reproduction of disease.

Porcine astrovirus type 3 (PoAstV3) is an emerging swine pathogen associated with central nervous system (CNS) disease. Its association with cases of CNS disease in swine was unrecognized until two simultaneous publications highlighted the involvement of this virus as a putative cause of polioencephalomyelitis in swine 2017. In order to elucidate multiple aspects of the pathogenesis, clinical presentation and course of disease of this virus, a retrospective study involving CNS diagnostic cases sent to the Iowa State University Veterinary Diagnostic Laboratory with histologic lesions consistent with a viral etiology was performed to investigate the plausible association of this virus as cause of CNS disease. PoAstV3 RNA was detected within histologic lesions in the CNS by RNA in situ hybridization in 13 out of 50 cases that originated from multiple farms and different production stages over a six year time period. PoAstV3 RNA tissue distribution was assessed in three cases by RT-qPCR. PoAstv3 was rarely detected outside of the CNS at time of clinical disease. The results of this study supports the role of PoAstV3 as a recurring putative cause of viral encephalomyelitis in swine. One of the most important obstacles in the research of PoAstV3-associated disease is the inability to replicate PoAstV3 in cell culture systems. This impediment is also observed with other neurotropic astroviruses where viral isolation and propagation are an infructuous task. As a result, we opted to perform whole genome and in silico characterization of several PoAstV3 strains. The results of this study, consisting of the largest collection of PoAstV3 genomes, describes previously uncharacterized secondary RNA structures intrinsic viral replication. In addition, we identified the secondary structure of the putative genome-linked viral protein (VPg) and several epitopes within its capsid protein. This study also identifies conserved antigens located at the VP27 capsid spike domain, one of the most immunogenic domains at the viral surface. The results from this study broaden our knowledge and understanding of this single stranded RNA virus, emphasizing previously unknown motifs and structures within its genome that could likely be utilized for the development of future immunogens and immunodiagnostic assays. Finally, to further investigate the pathogenesis of PoAstV3, CNS tissue homogenate containing PoAstV3 was administered to cesarean-derived, colostrum-deprived (CDCD) pigs. Clinical signs similar to those in cases of natural disease including tetraparesis and ataxia were observed. Histologic lesions consistent with a viral etiology (gliosis, neuronal necrosis and lymphocytic perivascular cuffing) were noted in all inoculated animals, and PoAstV3 was detected in affected tissues including dorsal root ganglion and nerve roots using in situ hybridization. An immune response to the coding region of the vp27 gene, part of the PoAstV3 ORF2 capsid, was detected by an indirect ELISA in all inoculated animals

Porcine astrovirus type 3 as a cause of central nervous system disease in swine: Retrospective identification of diagnostic cases, molecular characterization and experimental reproduction of disease.

Porcine astrovirus type 3 (PoAstV3) is an emerging swine pathogen associated with central nervous system (CNS) disease. Its association with cases of CNS disease in swine was unrecognized until two simultaneous publications highlighted the involvement of this virus as a putative cause of polioencephalomyelitis in swine 2017. In order to elucidate multiple aspects of the pathogenesis, clinical presentation and course of disease of this virus, a retrospective study involving CNS diagnostic cases sent to the Iowa State University Veterinary Diagnostic Laboratory with histologic lesions consistent with a viral etiology was performed to investigate the plausible association of this virus as cause of CNS disease. PoAstV3 RNA was detected within histologic lesions in the CNS by RNA in situ hybridization in 13 out of 50 cases that originated from multiple farms and different production stages over a six year time period. PoAstV3 RNA tissue distribution was assessed in three cases by RT-qPCR. PoAstv3 was rarely detected outside of the CNS at time of clinical disease. The results of this study supports the role of PoAstV3 as a recurring putative cause of viral encephalomyelitis in swine. One of the most important obstacles in the research of PoAstV3-associated disease is the inability to replicate PoAstV3 in cell culture systems. This impediment is also observed with other neurotropic astroviruses where viral isolation and propagation are an infructuous task. As a result, we opted to perform whole genome and in silico characterization of several PoAstV3 strains. The results of this study, consisting of the largest collection of PoAstV3 genomes, describes previously uncharacterized secondary RNA structures intrinsic viral replication. In addition, we identified the secondary structure of the putative genome-linked viral protein (VPg) and several epitopes within its capsid protein. This study also identifies conserved antigens located at the VP27 capsid spike domain, one of the most immunogenic domains at the viral surface. The results from this study broaden our knowledge and understanding of this single stranded RNA virus, emphasizing previously unknown motifs and structures within its genome that could likely be utilized for the development of future immunogens and immunodiagnostic assays. Finally, to further investigate the pathogenesis of PoAstV3, CNS tissue homogenate containing PoAstV3 was administered to cesarean-derived, colostrum-deprived (CDCD) pigs. Clinical signs similar to those in cases of natural disease including tetraparesis and ataxia were observed. Histologic lesions consistent with a viral etiology (gliosis, neuronal necrosis and lymphocytic perivascular cuffing) were noted in all inoculated animals, and PoAstV3 was detected in affected tissues including dorsal root ganglion and nerve roots using in situ hybridization. An immune response to the coding region of the vp27 gene, part of the PoAstV3 ORF2 capsid, was detected by an indirect ELISA in all inoculated animals

Development and Evaluation of Antigen-Specific Dual Matrix Pestivirus K ELISAs Using Longitudinal Known Infectious Status Samples

Pestivirus K, commonly known as atypical porcine pestivirus (APPV), is the most common cause of congenital tremor (CT) in pigs. Currently, there is limited information on the infection dynamics of and immune response against APPV and no robust serologic assay to assess the effectiveness of preventative measures. To that end, known infection status samples were generated using experimental inoculation of cesarean-derived, colostrum-deprived pigs. Pigs (2 per pen) were inoculated with minimum essential medium (n = 6; negative control) or APPV (n = 16). Serum, pen-based oral fluid samples, and nasal swabs were collected through 70 days postinoculation (dpi). The immune response to recombinant APPV Erns, E2, or NS3 antigens was evaluated using both serum and oral fluids via indirect enzyme-linked immunosorbent assays (ELISAs). APPV was detected by real-time reverse transcription-PCR (RT-qPCR) in all oral fluid and serum samples from APPV-inoculated animals by 24 and 35 dpi, respectively. All samples remained genome positive until 70 dpi. Detection of nasal shedding was less consistent, with APPV being detected by RT-qPCR in all inoculated animals at 42, 49, and 56 dpi. Antibodies were first detected in oral fluids at 14 dpi, 10 days before serum detection, and concurrently with the first oral fluids RT-qPCR detection. Across sample types and time points, the Erns ELISA outperformed the other targets. In conclusion, both oral fluid and serum APPV Erns ELISAs can be used to economically evaluate the individual and herd status prior to and following intervention strategies.This article is published as Arruda, Bailey L., Shollie Falkenberg, Juan-Carlos Mora-Díaz, Franco S. Matias Ferreyra, Ronaldo Magtoto, and Luis Giménez-Lirola. "Development and Evaluation of Antigen-Specific Dual Matrix Pestivirus K ELISAs Using Longitudinal Known Infectious Status Samples." Journal of Clinical Microbiology (2022): e00697-22. DOI: 10.1128/jcm.00697-22. Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted