DNAJC14-Independent Replication of the Atypical Porcine Pestivirus.

Atypical porcine pestiviruses (APPV; Pestivirus K) are a recently discovered, very divergent species of the genus Pestivirus within the family Flaviviridae. The presence of APPV in piglet-producing farms is associated with the occurrence of so-called "shaking piglets," suffering from mild to severe congenital tremor type A-II. Previous studies showed that the cellular protein DNAJC14 is an essential cofactor of the NS2 autoprotease of all classical pestiviruses. Consequently, genetically engineered DNAJC14 knockout cell lines were resistant to all tested noncytopathogenic (non-cp) pestiviruses. Surprisingly, we found that the non-cp APPV can replicate in these cells in the absence of DNAJC14, suggesting a divergent mechanism of polyprotein processing. A complete laboratory system for the study of APPV was established to learn more about the replication of this unusual virus. The inactivation of the APPV NS2 autoprotease using reverse genetics resulted in nonreplicative genomes. To further investigate whether a regulation of the NS2-3 cleavage is also existing in APPV, we constructed synthetic viral genomes with deletions and duplications leading to the NS2 independent release of mature NS3. As observed with other pestiviruses, the increase of mature NS3 resulted in elevated viral RNA replication levels and increased protein expression. Our data suggest that APPV exhibit a divergent mechanism for the regulation of the NS2 autoprotease activity most likely utilizing a different cellular protein for the adjustment of replication levels. IMPORTANCE DNAJC14 is an essential cofactor of the pestiviral NS2 autoprotease, limiting replication to tolerable levels as a prerequisite for the noncytopathogenic biotype of pestiviruses. Surprisingly, we found that the atypical porcine pestivirus (APPV) is able to replicate in the absence of DNAJC14. We further investigated the NS2-3 processing of APPV using a molecular clone, monoclonal antibodies, and DNAJC14 knockout cells. We identified two potential active site residues of the NS2 autoprotease and could demonstrate that the release of NS3 by the NS2 autoprotease is essential for APPV replication. Defective interfering genomes and viral genomes with duplicated NS3 sequences that produce mature NS3 independent of the NS2 autoprotease activity showed increased replication and antigen expression. It seems likely that an alternative cellular cofactor controls NS2-3 cleavage and thus replication of APPV. The replication-optimized synthetic APPV genomes might be suitable live vaccine candidates, whose establishment and testing warrant further research.

Influence of deoxynivalenol-contaminated feed on the immune response of pigs after PRRSV vaccination and infection.

The impact of the Fusarium mycotoxin deoxynivalenol (DON) on the immune response against porcine reproductive and respiratory syndrome virus (PRRSV) vaccination and infection was investigated. Forty-two weaned piglets were separated into seven groups and received three different diets: Low DON (1.09 ppm), High DON (2.81 ppm) or No DON. These three treatments were split further into either vaccinated (Ingelvac PRRSFLEX EU) and challenged with PRRSV 28 days post-vaccination, or only infected at day 28. A seventh group received no DON, no vaccination, and no infection. Two weeks after challenge infection, when pigs were euthanized, the number of IFN-γ producing lymphocytes in the blood of vaccinated animals was lower in pigs on High DON compared to animals on Low DON or No DON. Intracellular cytokine staining showed that vaccinated animals fed with the Low DON diet had higher frequencies of TNF-α/IFN-γ co-producing CD4+ T cells than the other two vaccinated groups, particularly in lung tissue. Vaccinated animals on High DON had similar viral loads in the lung as the non-vaccinated groups, but several animals of the Low DON or No DON group receiving vaccination had reduced titers. In these two groups, there was a negative correlation between lung virus titers and vaccine-specific TNF-α/IFN-γ co-producing CD4+ T cells located either in lung tissue or blood. These results indicate that after PRRSV vaccination and infection, high levels of DON negatively influence immune parameters and clearance of the virus, whereas low DON concentrations have immunomodulatory effects.

Discovery of a phylogenetically distinct poxvirus in diseased Crocodilurus amazonicus (family Teiidae).

A novel poxvirus was discovered in Crocodilurus amazonicus (Teiidae) presenting with a debilitating skin disease. The generated first genome sequence of a reptilian poxvirus revealed the closest phylogenetic relationship to avipoxviruses, highlighting potential virus exchanges between avian and reptilian species.

Progression of asexual to sexual stages of Cystoisospora suis in a host cell-free environment as a model for Coccidia.

Coccidia display a characteristic life cycle, where the parasites switch between asexual and sexual development, resulting in an environmental stage, the oocyst. The entero-pathogenic Cystoisospora suis, a coccidian parasite of swine and close relative to Toxoplasma gondii, undergoes development in one host-cycle. Despite the well-described intracellular development of Coccidia, the C. suis life cycle can progress in an in vitro, host cell-free system after initial intracellular development of merozoites. A novel host cell-free cultivation method was developed by transferring purified merozoites from cell culture supernatant (dpi 6) to culture medium and incubating them for 5 days to induce their progression to sexually differentiated stages. The development of sexual stages in the absence of host cells was verified by morphological studies, flow cytometry and the transcription analysis of three genes linked to sexual stages (HAP2, OWP and TyRP). The host cell-free culture permits the sexual development (and with this, the complete life cycle progression from sporozoites to oocysts) of C. suis in vitro and provides a new tool for detailed research on the development of C. suis and possibly other Coccidia. This will also be useful for the evaluation of novel drug or vaccine targets in these parasites.

Morphology and Molecular Composition of Purified Bovine Viral Diarrhea Virus Envelope.

The family Flaviviridae includes viruses that have different virion structures and morphogenesis mechanisms. Most cellular and molecular studies have been so far performed with viruses of the Hepacivirus and Flavivirus genera. Here, we studied bovine viral diarrhea virus (BVDV), a member of the Pestivirus genus. We set up a method to purify BVDV virions and analyzed their morphology by electron microscopy and their protein and lipid composition by mass spectrometry. Cryo-electron microscopy showed near spherical viral particles displaying an electron-dense capsid surrounded by a phospholipid bilayer with no visible spikes. Most particles had a diameter of 50 nm and about 2% were larger with a diameter of up to 65 nm, suggesting some size flexibility during BVDV morphogenesis. Morphological and biochemical data suggested a low envelope glycoprotein content of BVDV particles, E1 and E2 being apparently less abundant than Erns. Lipid content of BVDV particles displayed a ~2.3 to 3.5-fold enrichment in cholesterol, sphingomyelin and hexosyl-ceramide, concomitant with a 1.5 to 5-fold reduction of all glycerophospholipid classes, as compared to lipid content of MDBK cells. Although BVDV buds in the endoplasmic reticulum, its lipid content differs from a typical endoplasmic reticulum membrane composition. This suggests that BVDV morphogenesis includes a mechanism of lipid sorting. Functional analyses confirmed the importance of cholesterol and sphingomyelin for BVDV entry. Surprisingly, despite a high cholesterol and sphingolipid content of BVDV envelope, E2 was not found in detergent-resistant membranes. Our results indicate that there are differences between the structure and molecular composition of viral particles of Flaviviruses, Pestiviruses and Hepaciviruses within the Flaviviridae family.

Novel Pestivirus Species in Pigs, Austria, 2015.

A novel pestivirus species was discovered in a piglet-producing farm in Austria during virologic examinations of congenital tremor cases. The emergence of this novel pestivirus species, provisionally termed Linda virus, in domestic pigs may have implications for classical swine fever virus surveillance and porcine health management.

Congenital infection with atypical porcine pestivirus (APPV) is associated with disease and viral persistence.

In 2013, several Austrian piglet-producing farms recorded outbreaks of action-related repetitive myoclonia in newborn piglets ("shaking piglets"). Malnutrition was seen in numerous piglets as a complication of this tremor syndrome. Overall piglet mortality was increased and the number of weaned piglets per sow decreased by more than 10% due to this outbreak. Histological examination of the CNS of affected piglets revealed moderate hypomyelination of the white substance in cerebellum and spinal cord. We detected a recently discovered pestivirus, termed atypical porcine pestivirus (APPV) in all these cases by RT-PCR. A genomic sequence and seven partial sequences were determined and revealed a 90% identity to the US APPV sequences and 92% identity to German sequences. In confirmation with previous reports, APPV genomes were identified in different body fluids and tissues including the CNS of diseased piglets. APPV could be isolated from a "shaking piglet", which was incapable of consuming colostrum, and passaged on different porcine cells at very low titers. To assess the antibody response a blocking ELISA was developed targeting NS3. APPV specific antibodies were identified in sows and in PCR positive piglets affected by congenital tremor (CT). APPV genomes were detected continuously in piglets that gradually recovered from CT, while the antibody titers decreased over a 12-week interval, pointing towards maternally transmitted antibodies. High viral loads were detectable by qRT-PCR in saliva and semen of infected young adults indicating a persistent infection.

X-ray structure of the pestivirus NS3 helicase and its conformation in solution.

UNLABELLED: Pestiviruses form a genus in the Flaviviridae family of small enveloped viruses with a positive-sense single-stranded RNA genome. Viral replication in this family requires the activity of a superfamily 2 RNA helicase contained in the C-terminal domain of nonstructural protein 3 (NS3). NS3 features two conserved RecA-like domains (D1 and D2) with ATPase activity, plus a third domain (D3) that is important for unwinding nucleic acid duplexes. We report here the X-ray structure of the pestivirus NS3 helicase domain (pNS3h) at a 2.5-Å resolution. The structure deviates significantly from that of NS3 of other genera in the Flaviviridae family in D3, as it contains two important insertions that result in a narrower nucleic acid binding groove. We also show that mutations in pNS3h that rescue viruses from which the core protein is deleted map to D3, suggesting that this domain may be involved in interactions that facilitate particle assembly. Finally, structural comparisons of the enzyme in different crystalline environments, together with the findings of small-angle X-ray-scattering studies in solution, show that D2 is mobile with respect to the rest of the enzyme, oscillating between closed and open conformations. Binding of a nonhydrolyzable ATP analog locks pNS3h in a conformation that is more compact than the closest apo-form in our crystals. Together, our results provide new insight and bring up new questions about pNS3h function during pestivirus replication. IMPORTANCE: Although pestivirus infections impose an important toll on the livestock industry worldwide, little information is available about the nonstructural proteins essential for viral replication, such as the NS3 helicase. We provide here a comparative structural and functional analysis of pNS3h with respect to its orthologs in other viruses of the same family, the flaviviruses and hepatitis C virus. Our studies reveal differences in the nucleic acid binding groove that could have implications for understanding the unwinding specificity of pNS3h, which is active only on RNA duplexes. We also show that pNS3h has a highly dynamic behavior--a characteristic probably shared with NS3 helicases from all Flaviviridae members--that could be targeted for drug design by using recent algorithms to specifically block molecular motion. Compounds that lock the enzyme in a single conformation or limit its dynamic range of conformations are indeed likely to block its helicase function.

Characterization of two Austrian porcine reproductive and respiratory syndrome virus (PRRSV) field isolates reveals relationship to East Asian strains.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes major problems for the swine industry worldwide. Due to Austria's central location in Europe, a large number of animals are transported through the country. However, little is known about current PRRSV strains and epidemiology. We determined full-length genome sequences of two Austrian field isolates (AUT13-883 and AUT14-440) from recent PRRSV outbreaks and of a related German isolate (GER09-613). Phylogenetic analysis revealed that the strains belong to European genotype 1 subtype 1 and form a cluster together with a South Korean strain. Remarkably, AUT14-440 infected the simian cell line MARC-145 without prior adaptation. In addition, this isolate showed exceptional deletions in nonstructural protein 2, in the overlapping region of glycoprotein 3 and 4 and in the 3' untranslated region. Both Austrian isolates caused similar lung lesions but only pigs infected with AUT14-440 developed clear clinical signs of infection. Taken together, the genetic and biological characterization of two novel Austrian PRRSV field isolates revealed similarities to East Asian strains. This stresses the necessity for a more detailed analysis of current PRRSV strains in Europe beyond the determination of short ORF5 and ORF7 sequences.

PRRSV-infected monocyte-derived dendritic cells express high levels of SLA-DR and CD80/86 but do not stimulate PRRSV-naïve regulatory T cells to proliferate.

In vitro generated monocyte-derived dendritic cells (moDCs) have frequently been used to study the influence of porcine reproductive and respiratory syndrome virus (PRRSV) infection on antigen presenting cells. However, obtained results have often been conflicting in regard to expression of co-stimulatory molecules and interaction with T cells. In this study we performed a detailed phenotypic characterisation of PRRSV-infected moDCs and non-infected moDCs. For CD163 and CD169, which are involved in PRRSV-entry into host cells, our results show that prior to infection porcine moDCs express high levels of CD163 but only very low levels for CD169. Following infection with either PRRSV-1 or PRRSV-2 strains after 24 h, PRRSV-nucleoprotein (N-protein)(+) and N-protein(-) moDCs derived from the same microculture were analyzed for expression of swine leukocyte antigen-DR (SLA-DR) and CD80/86. N-protein(+) moDCs consistently expressed higher levels of SLA-DR and CD80/86 compared to N-protein(-) moDCs. We also investigated the influence of PRRSV-infected moDCs on proliferation and frequency of Foxp3(+) regulatory T cells present within CD4(+) T cells in in vitro co-cultures. Neither CD3-stimulated nor unstimulated CD4(+) T cells showed differences in regard to proliferation and frequency of Foxp3(+) T cells following co-cultivation with either PRRSV-1 or PRRSV-2 infected moDCs. Our results suggest that a more detailed characterisation of PRRSV-infected moDCs will lead to more consistent results across different laboratories and PRRSV strains as indicated by the major differences in SLA-DR and CD80/86 expression between PRRSV-infected and non-infected moDCs present in the same microculture.

Autocatalytic cleavage within classical swine fever virus NS3 leads to a functional separation of protease and helicase.

Classical swine fever virus (CSFV) is a positive-stranded RNA virus belonging to the genus Pestivirus within the Flaviviridae family. Pivotal for processing of a large portion of the viral polyprotein is a serine protease activity within nonstructural protein 3 (NS3) that also harbors helicase and NTPase activities essential for RNA replication. In CSFV-infected cells, NS3 appears as two forms, a fully processed NS3 of 80 kDa and the precursor molecule NS2-3 of 120 kDa. Here we report the identification and mapping of additional autocatalytic intramolecular cleavages. One cleavable peptide bond occurs between Leu1781 and Met1782, giving rise to a helicase subunit of 55 kDa and, depending on the substrate, a NS2-3 fragment of 78 kDa (NS2-3p) or a NS3 protease subunit of 26 kDa (NS3p). In trans-cleavage assays using NS4-5 as a substrate, NS3p acts as a fully functional protease that is able to process the polyprotein. NS3p comprises the minimal essential protease, as deletion of Leu1781 results in inactivation. A second intramolecular cleavage was mapped to the Leu1748/Lys1749 peptide bond that yields a proteolytically inactive NS3 fragment. Deletion of either of the cleavage site residues resulted in a loss of RNA infectivity, indicating the functional importance of amino acid identity at the respective positions. Our data suggest that internal cleavage within the NS3 moiety is a common process that further extends the functional repertoires of the multifunctional NS2-3 or NS3 and represents another level of the complex polyprotein processing of Flaviviridae.

Characterization of a Molecular Clone of Deformed Wing Virus B.

Honey bees (Apis mellifera) play a crucial role in agriculture through their pollination activities. However, they have faced significant health challenges over the past decades that can limit colony performance and even lead to collapse. A primary culprit is the parasitic mite Varroa destructor, known for transmitting harmful bee viruses. Among these viruses is deformed wing virus (DWV), which impacts bee pupae during their development, resulting in either pupal demise or in the emergence of crippled adult bees. In this study, we focused on DWV master variant B. DWV-B prevalence has risen sharply in recent decades and appears to be outcompeting variant A of DWV. We generated a molecular clone of a typical DWV-B strain to compare it with our established DWV-A clone, examining RNA replication, protein expression, and virulence. Initially, we analyzed the genome using RACE-PCR and RT-PCR techniques. Subsequently, we conducted full-genome RT-PCR and inserted the complete viral cDNA into a bacterial plasmid backbone. Phylogenetic comparisons with available full-length sequences were performed, followed by functional analyses using a live bee pupae model. Upon the transfection of in vitro-transcribed RNA, bee pupae exhibited symptoms of DWV infection, with detectable viral protein expression and stable RNA replication observed in subsequent virus passages. The DWV-B clone displayed a lower virulence compared to the DWV-A clone after the transfection of synthetic RNA, as evidenced by a reduced pupal mortality rate of only 20% compared to 80% in the case of DWV-A and a lack of malformations in 50% of the emerging bees. Comparable results were observed in experiments with low infection doses of the passaged virus clones. In these tests, 90% of bees infected with DWV-B showed no clinical symptoms, while 100% of pupae infected with DWV-A died. However, at high infection doses, both DWV-A and DWV-B caused mortality rates exceeding 90%. Taken together, we have generated an authentic virus clone of DWV-B and characterized it in animal experiments.

Pluck-pools as diagnostic samples for detecting porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in porcine abortion material and stillbirths.

Investigating infectious agents in porcine abortion material and stillborn piglets poses challenges for practitioners and diagnostic laboratories. In this study, pooled samples of individual reference organs (thymus and heart) from a total of 1000 aborted fetuses and stillborn piglets were investigated using quantitative PCR protocols for porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) and porcine circovirus type 2 (PCV2). Simultaneously, a pluck-pool containing equivalent portions of fetal thymus, heart, and lung tissue was collected, frozen at - 20 °C, and re-analyzed when a certain amount of either PRRSV-1 RNA or PCV2 DNA was detected in individual reference organs. Thirteen pluck-pools were assessed for PRRSV-1, all being PCR-positive. For PCV2, 11 of 15 pluck-pools investigated were PCR-positive. In all pluck-pools testing negative, viral loads in individual pools were low. This study indicates that pluck-pools can be valuable diagnostic material and the consolidation of multiple organs through a single RNA/DNA extraction optimizes the utilization of available laboratory resources. Additional research is required to assess the feasibility of follow-up investigations and to accurately define criteria for interpretation of viral loads in a clinical context.

Detection of PRRSV-1 in tongue fluids under experimental and field conditions and comparison of different sampling material for PRRSV sow herd monitoring.

BACKGROUND: Infection with porcine reproductive and respiratory syndrome virus (PRRSV) leads to significant economic losses worldwide. One of the initial measures following an outbreak is to stabilise the herd and to prevent vertical transmission of PRRSV. The objective of this study was to detect PRRSV in different sampling material, both in an experimental model and on a commercial piglet producing farm, with a focus on evaluating the suitability of tongue fluid samples. RESULTS: In the experimental model, PRRSV negative pregnant gilts were infected with PRRSV-1 AUT15-33 on gestation day 85 and necropsy of gilts and foetuses was performed three weeks later. 38.3% of individual foetal serum and 39.4% of individual foetal thymus samples were considered PRRSV RT-qPCR positive. Tongue fluids from individual foetuses showed a 33.0% positivity rate. PRRSV RNA was detected in all but one sample of litter-wise pooled processing fluids and tongue fluids. In the field study, the investigated farm remained PRRSV positive and unstable for five consecutive farrowing groups after the start of the sampling process. Tongue fluid samples pooled by litter in the first investigated farrowing group had a 54.5% positivity rate, with the overall highest viral load obtained in the field study. In this farrowing group, 33.3% of investigated litter-wise pooled processing fluid samples and all investigated serum samples (pools of 4-6 individuals, two piglets per litter) were considered positive. Across all investigated farrowing groups, tongue fluid samples consistently showed the highest viral load. Moreover, tongue fluid samples contained the virus in moderate amounts for the longest time compared to the other investigated sampling material. CONCLUSION: It can be concluded that the viral load in individual foetuses is higher in serum or thymus compared to tongue fluid samples. However, litter-wise pooled tongue fluid samples are well-suited for detecting vertical transmission within the herd, even when the suspected prevalence of vertical transmission events is low.

A Conserved Stem-Loop Structure within ORF5 Is a Frequent Recombination Hotspot for Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) with a Particular Modified Live Virus (MLV) Strain.

The emergence of recombinant PRRSV strains has been observed for more than a decade. These recombinant viruses are characterized by a genome that contains genetic material from at least two different parental strains. Due to the advanced sequencing techniques and a growing number of data bank entries, the role of PRRSV recombinants has become increasingly important since they are sometimes associated with clinical outbreaks. Chimeric viruses observed more recently are products of PRRSV wild-type and vaccine strains. Here, we report on three PRRSV-1 isolates from geographically distant farms with differing clinical manifestations. A sequencing and recombination analysis revealed that these strains are crossovers between different wild-type strains and the same modified live virus vaccine strain. Interestingly, the recombination breakpoint of all analyzed isolates appears at the beginning of open reading frame 5 (ORF5). RNA structure predictions indicate a conserved stem loop in close proximity to the recombination hotspot, which is a plausible cause of a polymerase template switch during RNA replication. Further research into the mechanisms of the stem loop is needed to help understand the PRRSV recombination process and the role of MLVs as parental strains.

Litters of Various-Sized Mummies (LVSM) and Stillborns after Porcine Reproductive and Respiratory Syndrome Virus Type 1 Infection-A Case Report.

Diverse origins and causes are described for papyraceous mummifications of porcine foetuses, but the porcine reproductive and respiratory syndrome virus (PRRSV) is not one of them. In contrast, PRRSV is unlikely to cause mid-term placental transmission but may cause late-term abortions and weakness of piglets. This case report describes a sudden occurrence of mummified foetuses of various sizes and stillborns and delayed birth (>115 days) in more than 50% of sows from one farrowing batch, while newborn piglets were mostly vital. Neither increased embryonic death nor infertility was reported. Three litters with mummies, autolysed piglets and stillborn piglets were investigated, and infections with porcine parvoviruses, porcine teschoviruses, porcine circoviruses, encephalomyocarditis virus, Leptospira spp. and Chlamydia spp. were excluded. Instead, high viral loads of PRRSV were detected in the thymus pools of piglets at all developmental stages, even in piglets with a crown-rump length between 80 and 150 mm, suggesting a potential mid-term in utero transmission of the virus. Genomic regions encoding structural proteins (ORF2-7) of the virus were sequenced and identified the virulent PRRSV-1 strain AUT15-33 as the closest relative. This case report confirms the diversity of PRRSV and its potential involvement in foetal death in mid-gestation.

Processing of the 3C/D Region of the Deformed Wing Virus (DWV).

The deformed wing virus (DWV) belongs to the genus Iflavirus and the family Iflaviridae within the order Picornavirales. It is an important pathogen of the Western honey bee, Apis mellifera, causing major losses among honey bee colonies in association with the ectoparasitic mite Varroa destructor. Although DWV is one of the best-studied insect viruses, the mechanisms of viral replication and polyprotein processing have been poorly studied in the past. We investigated the processing of the protease-polymerase region at the C-terminus of the polyprotein in more detail using recombinant expression, novel serological reagents, and virus clone mutagenesis. Edman degradation of purified maturated polypeptides uncovered the C- and N-termini of the mature 3C-like (3CL) protease and RNA-dependent RNA polymerase (3DL, RdRp), respectively. Autocatalytic processing of the recombinant DWV 3CL protease occurred at P1 Q2118 and P1' G2119 (KPQ/GST) as well as P1 Q2393 and P1' S2394 (HAQ/SPS) cleavage sites. New monoclonal antibodies (Mab) detected the mature 3CL protease with an apparent molecular mass of 32 kDa, mature 3DL with an apparent molecular mass of 55 kDa as well as a dominant 3CDL precursor of 90 kDa in DWV infected honey bee pupae. The observed pattern corresponds well to data obtained via recombinant expression and N-terminal sequencing. Finally, we were able to show that 3CL protease activity and availability of the specific protease cleavage sites are essential for viral replication, protein synthesis, and establishment of infection using our molecular clone of DWV-A.

Retrospective Analysis of the Detection of Pathogens Associated with the Porcine Respiratory Disease Complex in Routine Diagnostic Samples from Austrian Swine Stocks.

The diagnostic workup of respiratory disease in pigs is complex due to coinfections and non-infectious causes. The detection of pathogens associated with respiratory disease is a pivotal part of the diagnostic workup for respiratory disease. We aimed to report how frequently certain viruses and bacteria were detected in samples from pigs with respiratory symptoms in the course of routine diagnostic procedures. Altogether, 1975 routine diagnostic samples from pigs in Austrian swine stocks between 2016 and 2021 were analysed. PCR was performed to detect various pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) (n = 921), influenza A virus (n = 479), porcine circovirus type 2 (PCV2) (n = 518), Mycoplasma (M.) hyopneumoniae (n = 713), Actinobacillus pleuropneumoniae (n = 198), Glaesserella (G.) parasuis (n = 165) and M. hyorhinis (n = 180). M. hyorhinis (55.1%) had the highest detection rate, followed by PCV2 (38.0%) and Streptococcus (S.) suis (30.6%). PRRSV was detected most frequently in a pool of lung, tonsil and tracheobronchial lymph node (36.2%). G. parasuis was isolated more frequently from samples taken after euthanasia compared to field samples. PRRSV-positive samples were more likely to be positive for PCV2 (p = 0.001), M. hyopneumoniae (p = 0.032) and Pasteurella multocida (p < 0.001). M. hyopneumoniae-positive samples were more likely to be positive for P. multocida (p < 0.001) and S. suis (p = 0.046), but less likely for M. hyorhinis (p = 0.004). In conclusion, our data provide evidence that lung samples that were positive for a primary pathogenic agent were more likely to be positive for a secondary pathogenic agent.

Quantitative Analysis of Inflammatory Uterine Lesions of Pregnant Gilts with Digital Image Analysis Following Experimental PRRSV-1 Infection.

Reproductive disorders caused by porcine reproductive and respiratory syndrome virus-1 are not yet fully characterized. We report QuPath-based digital image analysis to count inflammatory cells in 141 routinely, and 35 CD163 immunohistochemically stained endometrial slides of vaccinated or unvaccinated pregnant gilts inoculated with a high or low virulent PRRSV-1 strain. To illustrate the superior statistical feasibility of the numerical data determined by digital cell counting, we defined the association between the number of these cells and endometrial, placental, and fetal features. There was strong concordance between the two manual scorers. Distributions of total cell counts and endometrial and placental qPCR results differed significantly between examiner1's endometritis grades. Total counts' distribution differed significantly between groups, except for the two unvaccinated. Higher vasculitis scores were associated with higher endometritis scores, and higher total cell counts were expected with high vasculitis/endometritis scores. Cell number thresholds of endometritis grades were determined. A significant correlation between fetal weights and total counts was shown in unvaccinated groups, and a significant positive correlation was found between these counts and endometrial qPCR results. We revealed significant negative correlations between CD163+ counts and qPCR results of the unvaccinated group infected with the highly virulent strain. Digital image analysis was efficiently applied to assess endometrial inflammation objectively.

Presence of Equine and Bovine Coronaviruses, Endoparasites, and Bacteria in Fecal Samples of Horses with Colic.

Acute abdominal pain (colic) is one of the major equine health threats worldwide and often necessitates intensive veterinary medical care and surgical intervention. Equine coronavirus (ECoV) infections can cause colic in horses but are rarely considered as a differential diagnosis. To determine the frequency of otherwise undetected ECoV infections in horses with acute colic, fresh fecal samples of 105 horses with acute colic and 36 healthy control horses were screened for viruses belonging to the Betacoronavirus 1 species by RT-PCR as well as for gastrointestinal helminths and bacteria commonly associated with colic. Horses with colic excreted significantly fewer strongyle eggs than horses without colic. The prevalence of anaerobic, spore-forming, gram-positive bacteria (Clostridium perfringens and Clostridioides difficile) was significantly higher in the feces of horses with colic. Six horses with colic (5.7%) and one horse from the control group (2.8%) tested positive for Betacoronaviruses. Coronavirus-positive samples were sequenced to classify the virus by molecular phylogeny (N gene). Interestingly, in three out of six coronavirus-positive horses with colic, sequences closely related to bovine coronaviruses (BCoV) were found. The pathogenic potential of BCoV in horses remains unclear and warrants further investigation.