The porcine circovirus 3 humoral response: characterization of maternally derived antibodies and dynamic following experimental infection.

Since Porcine Circovirus 3 (PCV3) was first identified in 2016, our understanding of the humoral response is still relatively scarce. Current knowledge of the PCV3 humoral response is primarily based on field studies identifying the seroprevalence of PCV3 Cap-induced antibodies. Studies on the humoral response following experimental PCV3 infection have conflicting results where one study reports the development of the Cap IgG response 7 days postinfection with no concurrent Cap IgM response, while a second study shows a Cap IgM response at the same time point with no detection of Cap IgG. The dynamics of the PCV3 Cap and Rep IgG following maternal antibody transfer and experimental infection have not been well characterized. Additionally, the cross-reactivity of convalescent serum from PCV2 and PCV3 experimentally infected animals to serologic methods of the alternate PCV has limited evaluation. Here, we show that maternally derived antibodies were detectable in piglet serum 7-9 weeks postfarrowing for the Cap IgG and 5-weeks-post farrowing for the Rep IgG using Cap- and Rep-specific enzyme linked immunosorbent assays (ELISA) and immunofluorescent assays (IFA) methods. Following experimental inoculation, Cap IgG was detected at 2-weeks-post inoculation and Rep IgG detection was delayed until 4-weeks-post inoculation. Furthermore, convalescent serum from either PCV2 or PCV3 methods displayed no cross-reactivity by serological methods against the other PCV. The information gained in this study highlights the development of both the Cap- and Rep-specific antibodies following experimental infection and through the transfer of maternal antibodies. The increased understanding of the dynamics of maternal antibody transfer and development of the humoral response following infection gained in the present study may aid in the establishment of husbandry practices and potential application of prophylactics to control PCV3 clinical disease. IMPORTANCE: Research on Porcine Circovirus 3 (PCV3) immunology is vital for understanding and controlling this virus. Previous studies primarily relied on field observations, but they have shown conflicting results about the immunological response against PCV3. This study helps fill those gaps by looking at how antibodies develop in pigs, especially those maternal-derived, and their impact in neonatal pigs preventing PCV3-associated disease in piglets. In addition, we look at the dynamics of antibodies in experimental infections mimicking infection in pigs in the grower-phase condition. Understanding this process can help to develop better strategies to prevent PCV3 infection. Also, this research found that PCV2 and PCV3 do not cross-react, which is crucial for serological test development and results interpretation. Overall, this work is essential for improving swine health and farming practices in the face of PCV3 infections.

Rapid genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) using MinION nanopore sequencing.

The global distribution and constant evolution are challenges for the control of porcine reproductive and respiratory syndrome virus (PRRSV), one of the most important viruses affecting swine worldwide. Effective control of PRRSV benefits from genotyping, which currently relies on Sanger sequencing. Here we developed and optimized procedures for real-time genotyping and whole genome sequencing of PRRSV directly from clinical samples based on targeted amplicon- and long amplicon tiling sequencing using the MinION Oxford Nanopore platform. Procedures were developed and tested on 154 clinical samples (including lung, serum, oral fluid and processing fluid) with RT-PCR Ct values ranging from 15 to 35. The targeted amplicon sequencing (TAS) approach was developed to obtain sequences of the complete ORF5 (main target gene for PRRSV genotyping) and partial ORF4 and ORF6 sequences of both PRRSV-1 and PRRSV-2 species. After only 5 min of sequencing, PRRSV consensus sequences with identities to reference sequences above 99% were obtained, enabling rapid identification and genotyping of clinical PRRSV samples into lineages 1, 5 and 8. The long amplicon tiling sequencing (LATS) approach targets type 2 PRRSV, the most prevalent viral species in the U.S. and China. Complete PRRSV genomes were obtained within the first hour of sequencing for samples with Ct values below 24.9. Ninety-two whole genome sequences were obtained using the LATS procedure. Fifty out of 60 sera (83.3%) and 18 out of 20 lung samples (90%) had at least 80% of genome covered at a minimum of 20X sequence depth per position. The procedures developed and optimized in this study here are valuable tools with potential for field application during PRRSV elimination programs.

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.

IDENTIFICATION AND CORRELATION OF A NOVEL SIADENOVIRUS IN A FLOCK OF BUDGERIGARS (MELOPSITTACUS UNDULATES) INFECTED WITH SALMONELLA TYPHIMURIUM IN THE UNITED STATES.

A flock of budgerigars (Melopsittacus undulates) was purchased from a licensed breeder and quarantined at a zoologic facility within the United States in 2016. Following 82 deaths within the flock, the remaining 66 birds were depopulated because of ongoing clinical salmonellosis despite treatment. Gross necropsy was performed on all 66 birds. Histopathologic examination was performed on 10 birds identified with gross lesions and 10 birds without. Pathologic findings were most often observed in the liver, kidney, and spleen. Lesions noted in the livers and spleens were consistent with published reports of salmonellosis in psittacine species. Multisystemic changes associated with septicemia were not noted, most likely because of antibiotic intervention before euthanasia. Of the 20 budgerigars evaluated by histopathology, six had large basophilic intranuclear inclusion bodies within tubular epithelia in a portion of the kidneys. Electronic microscopy, next-generation sequencing, Sanger sequencing, and phylogenetic analyses were used to identify and categorize the identified virus as a novel siadenovirus strain BuAdV-1 USA-IA43444-2016. The strain was 99% similar to budgerigar adenovirus 1 (BuAdV-1), previously reported in Japan, and to a psittacine adenovirus 5 recently identified in a U.S. cockatiel. Salmonella typhimurium carriers were identified via polymerase chain reaction (PCR) and bacterial culture and compared with viral carriers identified via PCR. Inclusion bodies and Salmonella detection were significant in birds with gross lesions versus those without; however, there was no correlation between budgerigars positive with siadenovirus by PCR and concurrent Salmonella infection. Identifying subclinical siadenovirus strain BuAdV-1 USA-IA43444-2016 infection in this flock significantly differs from a previous report of clinical illness in five budgerigars resulting in death caused by BuAdV-1 in Japan. S. typhimurium remains a significant pathogen in budgerigars, and zoonotic concerns prompted depopulation to mitigate the public health risks of this flock.

Genetic diversity and evolution of the emerging picornavirus Senecavirus A.

Senecavirus A (SVA) is an emerging picornavirus that causes vesicular disease (VD) in swine. The virus has been circulating in swine in the United Stated (USA) since at least 1988, however, since 2014 a marked increase in the number of SVA outbreaks has been observed in swine worldwide. The factors that led to the emergence of SVA remain unknown. Evolutionary changes that accumulated in the SVA genome over the years may have contributed to the recent increase in disease incidence. Here we compared full-genome sequences of historical SVA strains (identified before 2010) from the USA and global contemporary SVA strains (identified after 2011). The results from the genetic analysis revealed 6.32 % genetic divergence between historical and contemporary SVA isolates. Selection pressure analysis revealed that the SVA polyprotein is undergoing selection, with four amino acid (aa) residues located in the VP1 (aa 735), 2A (aa 941), 3C (aa 1547) and 3D (aa 1850) coding regions being under positive/diversifying selection. Several aa substitutions were observed in the structural proteins (VP1, VP2 and VP3) of contemporary SVA isolates when compared to historical SVA strains. Some of these aa substitutions led to changes in the surface electrostatic potential of the structural proteins. This work provides important insights into the molecular evolution and epidemiology of SVA.

Reactivity of Porcine Epidemic Diarrhea Virus Structural Proteins to Antibodies against Porcine Enteric Coronaviruses: Diagnostic Implications.

The development of porcine epidemic diarrhea virus (PEDV) antibody-based assays is important for detecting infected animals, confirming previous virus exposure, and monitoring sow herd immunity. However, the potential cross-reactivity among porcine coronaviruses is a major concern for the development of pathogen-specific assays. In this study, we used serum samples (n = 792) from pigs of precisely known infection status and a multiplex fluorescent microbead-based immunoassay and/or enzyme-linked immunoassay platform to characterize the antibody response to PEDV whole-virus (WV) particles and recombinant polypeptides derived from the four PEDV structural proteins, i.e., spike (S), nucleocapsid (N), membrane (M), and envelope (E). Antibody assay cutoff values were selected to provide 100% diagnostic specificity for each target. The earliest IgG antibody response, mainly directed against S1 polypeptides, was observed at days 7 to 10 postinfection. With the exception of nonreactive protein E, we observed similar antibody ontogenies and patterns of seroconversion for S1, N, M, and WV antigens. Recombinant S1 provided the best diagnostic sensitivity, regardless of the PEDV strain, with no cross-reactivity detected against transmissible gastroenteritis virus (TGEV), porcine respiratory coronavirus (PRCV), or porcine deltacoronavirus (PDCoV) pig antisera. The WV particles showed some cross-reactivity to TGEV Miller and TGEV Purdue antisera, while N protein presented some cross-reactivity to TGEV Miller. The M protein was highly cross-reactive to TGEV and PRCV antisera. Differences in the antibody responses to specific PEDV structural proteins have important implications in the development and performance of antibody assays for the diagnosis of PEDV enteric disease.

A Novel Porcine Circovirus Distantly Related to Known Circoviruses Is Associated with Porcine Dermatitis and Nephropathy Syndrome and Reproductive Failure.

Porcine circovirus-associated disease (PCVAD) is clinically manifested by postweaning multisystemic wasting syndrome (PMWS), respiratory and enteric disease, reproductive failure, and porcine dermatitis and nephropathy syndrome (PDNS). Porcine circovirus 2 (PCV2) is an essential component of PCVAD, although an etiologic role in PDNS is not well established. Here, a novel circovirus, designated porcine circovirus 3 (PCV3), was identified in sows that died acutely with PDNS-like clinical signs. The capsid and replicase proteins of PCV3 are only 37% and 55% identical to PCV2 and bat circoviruses, respectively. Aborted fetuses from sows with PDNS contained high levels of PCV3 (7.57 × 107 genome copies/ml), and no other viruses were detected by PCR and metagenomic sequencing. Immunohistochemistry (IHC) analysis of sow tissue samples identified PCV3 antigen in skin, kidney, lung, and lymph node samples localized in typical PDNS lesions, including necrotizing vasculitis, glomerulonephritis, granulomatous lymphadenitis, and bronchointerstitial pneumonia. Further study of archived PDNS tissue samples that were negative for PCV2 by IHC analysis identified 45 of 48 that were PCV3 positive by quantitative PCR (qPCR), with 60% of a subset also testing positive for PCV3 by IHC analysis. Analysis by qPCR of 271 porcine respiratory disease diagnostic submission samples identified 34 PCV3-positive cases (12.5%), and enzyme-linked immunosorbent assay detection of anti-PCV3 capsid antibodies in serum samples found that 46 (55%) of 83 samples tested were positive. These results suggest that PCV3 commonly circulates within U.S. swine and may play an etiologic role in reproductive failure and PDNS. Because of the high economic impact of PCV2, this novel circovirus warrants further studies to elucidate its significance and role in PCVAD. IMPORTANCE: While porcine circovirus 2 (PCV2) was first identified in sporadic cases of postweaning multisystemic wasting syndrome in Canada in the early 1990s, an epidemic of severe systemic disease due to PCV2 spread worldwide in the ensuing decade. Despite being effectively controlled by commercial vaccines, PCV2 remains one of the most economically significant viruses of swine. Here, a novel porcine circovirus (PCV3) that is distantly related to known circoviruses was identified in sows with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2, which has previously been associated with these clinical presentations, was not identified. High levels of PCV3 nucleic acid were observed in aborted fetuses by quantitative PCR, and PCV3 antigen was localized in histologic lesions typical of PDNS in sows by immunohistochemistry (IHC) analysis. PCV3 was also identified in archival PDNS diagnostic samples that previously tested negative for PCV2 by IHC analysis. The emergence of PCV3 warrants further investigation.

Optic nerve astrocytoma in a dog.

Intracranial astrocytomas are relatively uncommon in dogs and optic nerve astrocytomas even more so. This neoplasm should be considered as differential in canine patients with vision loss, retinal detachment, ocular mass, and histopathologic findings of infiltrative fusiform to polygonal glial cells possibly associated with glomeruloid vascular proliferation.

Modulation of Proinflammatory Cytokines in Monocyte-Derived Dendritic Cells by Porcine Reproductive and Respiratory Syndrome Virus Through Interaction with the Porcine Intercellular-Adhesion-Molecule-3-Grabbing Nonintegrin.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important global swine pathogen. PRRSV infects porcine dendritic cells (DCs), but the effects of the interactions with DCs are largely unknown. Current research focuses on the production and regulation of interferons and selected inflammatory cytokines in DCs, which may play key roles in immune modulation. In addition, PRRSV also downregulates swine leukocyte antigen class I (SLA-I), SLA-II, and CD80/86 costimulatory molecules in DCs. In this study, we aim to evaluate the PRRSV immunomodulatory effects on monocyte-derived DCs (MoDCs) through interactions with porcine DC-SIGN (pDC-SIGN) receptor. We demonstrated that blocking the PRRSV and pDC-SIGN interactions in MoDCs with recombinant hICAM-3 did not affect the regulatory effects of PRRSV on SLA-I, SLA-II, or CD80/86 molecules. The hICAM-3 did not affect the morphological changes on MoDCs associated with their activation and maturation after PRRSV infection, and did not impair the virus infectivity in these cells either. The mRNA levels of tumor necrosis factor alpha (TNF-α), IL-12p35, IL-1ß, and IL-6 were upregulated after hICAM-3 treatment or PRRSV infection, but in the presence of the blockage of pDC-SIGN in MoDCs with hICAM-3, PRRSV did not modulate the expression of these genes. However, in the presence of an anti-pDC-SIGN monoclonal antibody (mAb), we showed that PRRSV infection significantly reduced the mRNA expression levels of TNF-α and IL-1α, but enhanced the expression of IL-12p35 in MoDCs. Both hICAM-3-Fc and pDC-SIGN mAb treatments did not modulate proinflammatory cytokine protein levels in the culture supernatants of PRRSV-infected MoDCs. The results indicate that blocking the PRRSV-pDC-SIGN interactions by recombinant hICAM-3-Fc did not significantly affect virus infectivity, DC maturation, and proinflammatory cytokine gene expression in infected MoDCs. However, blocking the PRRSV-pDC-SIGN interactions on MoDCs with an anti-pDC-SIGN mAb revealed differential regulatory effects on specific proinflammatory gene expressions in those cells.

Chronic inflammatory demyelinating polyradiculoneuropathy with cholesterol deposits in a dog.

Chronic inflammatory demyelinating polyradiculoneuropathy occurred in an 11-year-old Labrador Retriever dog. Spinal cord compression resulted from massive radiculitis with prominent cholesterol granulomas. Cholesterol deposition and associated granuloma formation is unique in chronic inflammatory demyelinating polyradiculoneuropathy, in both its human and canine expressions.

Chimeric porcine reproductive and respiratory syndrome virus containing shuffled multiple envelope genes confers cross-protection in pigs.

The extensive genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) strains is a major obstacle for vaccine development. We previously demonstrated that chimeric PRRSVs in which a single envelope gene (ORF3, ORF4, ORF5 or ORF6) was shuffled via DNA shuffling had an improved heterologous cross-neutralizing ability. In this study, we incorporate all of the individually-shuffled envelope genes together in different combinations into an infectious clone backbone of PRRSV MLV Fostera(®) PRRS. Five viable progeny chimeric viruses were rescued, and their growth characteristics were characterized in vitro. In a pilot pig study, two chimeric viruses (FV-SPDS-VR2,FV-SPDS-VR5) were found to induce cross-neutralizing antibodies against heterologous strains. A subsequent vaccination/challenge study in 72 pigs revealed that chimeric virus FV-SPDS-VR2 and parental virus conferred partial cross-protection when challenged with heterologous strains NADC20 or MN184B. The results have important implications for future development of an effective PRRSV vaccine that confers heterologous protection.

Histopathological and immunohistochemical findings of primary and metastatic medullary thyroid carcinoma in a young dog.

This report describes the gross, histological, and immunohistochemical features of medullary thyroid carcinoma (MTC) with pulmonary metastases in a young dog. Sheets of pleomorphic cells supported by fibrous stroma characterized the primary mass, while metastatic nodules had a neuroendocrine pattern. Despite differing histologic features, all masses showed marked immunoreactivity against calcitonin and multiple neuroendocrine markers consistent with MTC. Although MTC is a well-recognized entity, it may be difficult to distinguish this mass from other thyroid neoplasms, necessitating immunohistochemical characterization.

DNA shuffling of the GP3 genes of porcine reproductive and respiratory syndrome virus (PRRSV) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous PRRSV strain.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important swine pathogen. Here we applied the DNA shuffling approaches to molecularly breed the PRRSV GP3 gene, a neutralizing antibodies inducer, in an attempt to improve its heterologous cross-neutralizing ability. The GP3 genes of six different PRRSV strains were bred by traditional DNA shuffling. Additionally, synthetic DNA shuffling of the GP3 gene was also performed using degenerate oligonucleotides. The shuffled-GP3-libraries were cloned into the backbone of a DNA-launched PRRSV infectious clone pIR-VR2385-CA. Four traditional-shuffled chimeras each representing all 6 parental strains and four other synthetic-shuffled chimeras were successfully rescued. These chimeras displayed similar levels of replication both in vitro and in vivo, compared to the backbone parental virus, indicating that the GP3 shuffling did not impair the replication capability of the chimeras. One chimera GP3TS22 induced significantly higher levels of cross-neutralizing antibodies in pigs against a heterologous PRRSV strain FL-12.