Examination on the Occurrence of Coinfections in Diagnostic Transmittals in Cases of Stillbirth, Mummification, Embryonic Death, and Infertility (SMEDI) Syndrome in Germany.

The stillbirth, mummification, embryonic death, and infertility (SMEDI) syndrome is most commonly associated with porcine parvovirus 1 (PPV1) infections. Little is known about the occurrence of coinfections with SMEDI-associated pathogens and the associations among these pathogens. In our study, we included 40 SMEDI-affected litters from 18 different farms. In total, 158 out of 358 available fetuses from diagnostic transmittals were selected by systematic random sampling and examined for PCV2, PCV3, PPV1, and Leptospira spp. by q-PCR. Results from diagnostic materials showed the following results: in eleven farms, PCV2 was present; in nine farms, PPV1 was present; in five farms, PCV3 was present; and in two farms, Leptospira spp. was present. The detection of Leptospira spp. was significantly associated with a PCV2 coinfection (OR: 26.3; p < 0.001). PCV3 positivity resulted in a reduced probability of detecting PCV2 in the corresponding fetus (OR: 0.078; p = 0.008). Fetal maceration was associated with Leptospira spp. detection (OR: 8.6; p = 0.003), whereas mummification (p = 0.047), reduced crown-rump length (p < 0.001), and bodyweight (p = 0.001) of fetuses were significantly associated with PPV1 and PCV2 coinfection and thus, presumably, a shorter time to death after infection, indicating an enhanced negative effect on the development of fetuses with PCV2 + PPV1 coinfection.

Newly Designed Poxviral Promoters to Improve Immunogenicity and Efficacy of MVA-NP Candidate Vaccines against Lethal Influenza Virus Infection in Mice.

Influenza, a respiratory disease mainly caused by influenza A and B, viruses of the Orthomyxoviridae, is still a burden on our society's health and economic system. Influenza A viruses (IAV) circulate in mammalian and avian populations, causing seasonal outbreaks with high numbers of cases. Due to the high variability in seasonal IAV triggered by antigenic drift, annual vaccination is necessary, highlighting the need for a more broadly protective vaccine against IAV. The safety tested Modified Vaccinia virus Ankara (MVA) is licensed as a third-generation vaccine against smallpox and serves as a potent vector system for the development of new candidate vaccines against different pathogens. Here, we generated and characterized recombinant MVA candidate vaccines that deliver the highly conserved internal nucleoprotein (NP) of IAV under the transcriptional control of five newly designed chimeric poxviral promoters to further increase the immunogenic properties of the recombinant viruses (MVA-NP). Infections of avian cell cultures with the recombinant MVA-NPs demonstrated efficient synthesis of the IAV-NP which was expressed under the control of the five new promoters. Prime-boost or single shot immunizations in C57BL/6 mice readily induced circulating serum antibodies' binding to recombinant IAV-NP and the robust activation of IAV-NP-specific CD8+ T cell responses. Moreover, the MVA-NP candidate vaccines protected C57BL/6 mice against lethal respiratory infection with mouse-adapted IAV (A/Puerto Rico/8/1934/H1N1). Thus, further studies are warranted to evaluate the immunogenicity and efficacy of these recombinant MVA-NP vaccines in other IAV challenge models in more detail.

Bacterial and Viral Pathogens with One Health Relevance in Invasive Raccoons (Procyon lotor, Linné 1758) in Southwest Germany.

In Europe, raccoons are invasive neozoons with their largest population in Germany. Globally, this mesocarnivore acts as a wildlife reservoir for many (non-)zoonotic (re-)emerging pathogens, but very little epidemiological data is available for southwest Germany. This exploratory study aimed to screen free-ranging raccoons in Baden-Wuerttemberg (BW, Germany) for the occurrence of selected pathogens with One Health relevance. Organ tissue and blood samples collected from 102 animals, obtained by hunters in 2019 and 2020, were subsequently analysed for two bacterial and four viral pathogens using a qPCR approach. Single samples were positive for the carnivore protoparvovirus-1 (7.8%, n = 8), canine distemper virus (6.9%, n = 7), pathogenic Leptospira spp. (3.9%, n = 4) and Anaplasma phagocytophilum (15.7%, n = 16). West Nile virus and influenza A virus were not detected. Due to their invasive behaviour and synanthropic habit, raccoons may increase the risk of infections for wildlife, domestic animals, zoo animals and humans by acting as a link between them. Therefore, further studies should be initiated to evaluate these risks.

Increased neutralization and IgG epitope identification after MVA-MERS-S booster vaccination against Middle East respiratory syndrome.

Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate the safety (primary objective) and immunogenicity (secondary and exploratory objectives: magnitude and characterization of vaccine-induced humoral responses) of a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. MVA-MERS-S booster vaccination is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n = 1) and the S2 subunit (n = 3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials. Trial registration: Clinicaltrials.gov NCT03615911.

Reproductive failure in an Austrian piglet-producing farm due to porcine circovirus genotype 2d.

Infections of pigs with porcine circovirus type 2 (PCV2) can lead to various clinical conditions including reproductive disorders (PCV2-RD). In general, a transplacental infection of fetuses leads to mummification and stillbirth. So far, PCV2-RD has mainly been described in specific-pathogen-free (SPF) herds or farms with a high proportion of gilts. From December 2018 to February 2019, a high abundance of mummified fetuses (15.5%) was observed in two farrowing groups in an Austrian piglet-producing farm. PCV2 DNA was detected using qPCR in organs of all six investigated fetuses (2.07 × 108-1.09 × 1012 PCV2) genome equivalents/g tissue and via in situ hybridisation in organs from five fetuses, while histologic lesions were not observed in a single fetal heart. All isolates were sequenced and identified as PCV2d. After the implementation of a regular vaccination of all sows against PCV2, the abundance of mummified fetuses dropped to 3.5% in May 2019. In contrast to previous reports about PCV2-RD, this farm was neither an SPF herd nor a start-up herd with a high proportion of gilts. The implementation of regular PCV2 vaccination helped to reduce the abundance of mummified fetuses substantially.

Cross-Sectional Study on the Prevalence of PCV Types 2 and 3 DNA in Suckling Piglets Compared to Grow-Finish Pigs in Downstream Production.

Vertical transmission is a consistently discussed pathway of porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3) transmission in pigs. To evaluate the presence of PCV2 and PCV3 in piglets, we collected tissue samples from 185 piglets that were crushed within the first week of life from 16 farms located in Germany and Austria. Pooled samples consisting of thymus, inguinal lymph node, myocardium, lung and spleen were examined for PCV2 and PCV3 by qPCR. Furthermore, oral fluid samples (OFS) from grow−finish pigs were collected and examined the same way. In piglets, PCV2 was highly prevalent (litters: 69.4%; piglets: 61.6%), whereas PCV3 prevalence was low (litters: 13.4%; piglets: 13.0%). In total, 72.6% and 67.2% of all collected OFS were PCV2 or PCV3 positive, respectively. Sow vaccination against PCV2 was identified as a protective factor concerning PCV2 in piglets (OR: 0.279; CI: 0.134−0.578; p < 0.001), whereas the porcine reproductive and respiratory syndrome virus (PRRSV) vaccination of sows was identified as a protective factor concerning PCV3 in piglets (OR: 0.252 CI: 0.104−0.610; p = 0.002). Our results show that PCV2, but not PCV3, is ubiquitous in suckling piglets and that early PCV3 infections might be modulated by PRRSV−PCV3 interaction. However, the ubiquitous nature of both viruses in older pigs could be confirmed.

Macrophages in dermal disease progression of phospholipase D4-deficient Fleckvieh calves.

A new gene defect in Fleckvieh calves leads to a syndrome with partial phenotype overlap with bovine hereditary zinc deficiency. A mutation in a gene encoding phospholipase D4 (PLD4), an endosomal exonuclease, causes the disorder. In mice, PLD4 activity indirectly regulates the Toll-like receptor 9 (TLR9) pathway via degradation of microbial DNA. PLD4 absence thus results in visceral macrophage activation comparable to human macrophage activation syndrome. In this study, disease progression and the role of macrophages in affected calves were monitored clinically, clinicopathologically, and histologically over time. Breeding data identified 73 risk matings of heterozygous carriers resulting in 54 potentially PLD4-deficient calves born on farms. PLD4 status was examined via 5'-exonuclease assay, detecting 6 calves carrying the defect. These were purchased and monitored daily until final necropsy. The calves developed progressive skin lesions starting with small scaling areas terminating in severe crusting dermatitis, especially in areas with mechanical exposure. Histological and immunohistochemical analyses indicated that macrophages with cytoplasmic vacuolation increased considerably in skin sections obtained weekly during the disease course. Macrophage increase correlated with increased dermal lesion severity. Macrophage activation was confirmed by prominent phagocytic activity in the superficial dermis using electron microscopy. Dermal mRNA abundance of CCL2 and CCL3 measured by quantitative polymerase chain reaction verified macrophage activation. Further increase in mRNA of downstream molecule MyD88 and cytokine IL12b connected bovine PLD4 deficiency to increased TLR9 pathway activation. In contrast to human macrophage activation syndrome, the main feature of bovine PLD4 deficiency was local disease in organs with contact to microbial DNA (skin, intestine, lungs).

Detection of porcine circovirus type 3 DNA in serum and semen samples of boars from a German boar stud.

Porcine circovirus type 3 (PCV3) is regularly reported in association with various clinical presentations, including porcine dermatitis and nephropathy syndrome (PDNS)-like lesions, respiratory signs, congenital tremor, and reproductive disorders. To investigate the epidemiology of PCV3 in a boar stud, we analysed fresh boar semen and matching sera from 181 boars from a German stud supplying semen for artificial insemination (AI) to approximately 740 breeder farms for PCV3 DNA. PCV3 DNA was detected in 1.7% semen samples and 24.3% sera. Spearman rho correlation demonstrated a significant positive correlation between boar age and quantitative DNA (by PCR quantification cycles [Cq] values) in serum samples (r = 0.636; P < 0.001). Sera from boars up to 12 months of age had higher viral loads (P < 0.001) and were PCV3-positive significantly more often (P < 0.01) than older boars. Detection of PCV3 DNA was not associated with breed (P> 0.05). PCV3 DNA was detected sporadically in fresh boar semen. Based on the assumption that processing fresh semen reduces viral load in semen used for AI, it is likely that the risk of sexual transmission of PCV3 during AI in is low. However, young boars may contribute to the maintenance of PCV3 infection in boar studs.

Identification and Characterization of Circular Single-Stranded DNA Genomes in Sheep and Goat Milk.

In recent years, a variety of circular replicase-encoding single-stranded (CRESS) DNA viruses and unclassified virus-like DNA elements have been discovered in a broad range of animal species and environmental samples. Key questions to be answered concern their presence in the human diet and their potential impact on disease emergence. Especially DNA elements termed bovine meat and milk factors (BMMF) are suspected to act as co-factors in the development of colon and breast cancer. To expand our knowledge on the occurrence of these potential pathogens in human nutrition, a total of 73 sheep and 40 goat milk samples were assayed by combining rolling circle amplification (RCA), PCR and Sanger sequencing. The present study further includes retail milk from the aforementioned species. We recovered 15 single stranded (ss) circular genomes. Of those, nine belong to the family Genomoviridae and six are members of the unclassified group of BMMF. Thus, dairy sheep and goats add to dispersal of CRESS viruses and circular ssDNA elements, which enter the food chain via milk. The presence of these entities is therefore more widespread in Bovidae than initially assumed and seems to be part of the common human nutrition.

Activation of interferon regulatory factor 3 by replication-competent vaccinia viruses improves antitumor efficacy mediated by T cell responses.

Recently, oncolytic vaccinia viruses (VACVs) have shown their potential to provide for clinically effective cancer treatments. The reason for this clinical usefulness is not only the direct destruction of infected cancer cells but also activation of immune responses directed against tumor antigens. For eliciting a robust antitumor immunity, a dominant T helper 1 (Th1) cell differentiation of the response is preferred, and such polarization can be achieved by activating the Toll-like receptor 3 (TLR3)-interferon regulatory factor 3 (IRF3) signaling pathway. However, current VACVs used as oncolytic viruses to date still encode several immune evasion proteins involved in the inhibition of this signaling pathway. By inactivating genes of selected regulatory virus proteins, we aimed for a candidate virus with increased potency to activate cellular antitumor immunity but at the same time with a fully maintained replicative capacity in cancer cells. The removal of up to three key genes (C10L, N2L, and C6L) from VACV did not reduce the strength of viral replication, both in vitro and in vivo, but resulted in the rescue of IRF3 phosphorylation upon infection of cancer cells. In syngeneic mouse tumor models, this activation translated to enhanced cytotoxic T lymphocyte (CTL) responses directed against tumor-associated antigens and neo-epitopes and improved antitumor activity.

Circular Rep-Encoding Single-Stranded DNA Sequences in Milk from Water Buffaloes (Bubalus arnee f. bubalis).

Isolation and characterization of circular replicase-encoding single-stranded (ss) DNA from animal, plant and environmental samples are rapidly evolving in virology. We detected 21 circular DNA elements, including one genomoviral sequence, in individual milk samples from domesticated Asian water buffaloes (Bubalus arnee f. bubalis). Most of the obtained genomes are related to Sphinx 1.76 and Sphinx 2.36 sequences and share a high degree of similarity to recently published circular DNAs-named BMMF (bovine meat and milk factors)-that have been isolated from commercial milk, as well as from bovine serum. Characteristic features such as rep genes, tandem repeats and inverted repeats were detected. These BMMF have recently been found to be present in taurine-type dairy cattle breeds descending from the aurochs (Bos primigenius). Importantly, the occurrence of BMMF has been linked to the higher incidence of colorectal and breast cancer in North America and Western Europe compared with Asia. This is the first report of circular ssDNA detected in milk from the domesticated form of the wild Asian water buffalo (B. arnee) belonging to the subfamily Bovinae. This novelty should be taken into account in view of the above-mentioned cancer hypothesis.

Equid herpesvirus-1 Distribution in Equine Lymphoid and Neural Tissues 70 Days Post Infection.

Equid herpesvirus-1 (EHV-1) causes respiratory disease, abortion and myeloencephalopathy in horses worldwide. As member of the Alphaherpesvirinae, latency is key to EHV-1 epidemiology. EHV-1 latent infection has been detected in the trigeminal ganglion (TG), respiratory associated lymphoid tissue (RALT) and peripheral blood mononuclear cells (PBMC) but additional locations are likely. The aim of this study was to investigate the distribution of viral DNA throughout the equine body. Twenty-five horses divided into three groups were experimentally infected via intranasal instillation with one of three EHV-1 viruses and euthanized on Day 70, post infection. During necropsy, TG, various sympathetic/parasympathetic ganglia of head, neck, thorax and abdomen, spinal cord dorsal root ganglia, RALT, mesenteric lymph nodes, spleen and PBMC of each horse were collected. Genomic viral loads and L-(late) gene transcriptional activity in each tissue and PBMC were measured using qPCR. In addition, immunohistochemistry (IHC) was applied on neural parenchyma tissue sections. EHV-1 DNA was detected in many neural and lymphoid tissue sections, but not in PBMC. L-gene transcriptional activity was not detected in any sample, and translational activity was not apparent on IHC. Tissue tropism differed between the Ab4 wild type and the two mutant viruses.

Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR.

BACKGROUND: The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. METHODS: Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. RESULTS: All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. CONCLUSION: Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

Viral Load and Cell Tropism During Early Latent Equid Herpesvirus 1 Infection Differ Over Time in Lymphoid and Neural Tissue Samples From Experimentally Infected Horses.

Upper respiratory tract infections with Equid Herpesvirus 1 (EHV-1) typically result in a peripheral blood mononuclear cell-associated viremia, which can lead to vasculopathy in the central nervous system. Primary EHV-1 infection also likely establishes latency in trigeminal ganglia (TG) via retrograde axonal transport and in respiratory tract-associated lymphatic tissue. However, latency establishment and reactivation are poorly understood. To characterize the pathogenesis of EHV-1 latency establishment and maintenance, two separate groups of yearling horses were experimentally infected intranasally with EHV-1, strain Ab4, and euthanized 30 days post infection (dpi), (n = 9) and 70 dpi (n = 6). During necropsy, TG, sympathetic trunk (ST), retropharyngeal and mesenteric lymph nodes (RLn, MesLn) and kidney samples were collected. Viral DNA was detected by quantitative PCR (qPCR) in TG, ST, RLn, and MesLn samples in horses 30 and 70 dpi. The number of positive TG, RLn and MesLn samples was reduced when comparing horses 30 and 70 dpi and the viral copy number in TG and RLn significantly declined from 30 to 70 dpi. EHV-1 late gene glycoprotein B reverse transcriptase PCR and IHC results for viral protein were consistently negative, thus lytic replication was excluded in the present study. Mild inflammation could be detected in all neural tissue samples and inflammatory infiltrates mainly consisted of CD3+ T-lymphocytes (T-cells), frequently localized in close proximity to neuronal cell bodies. To identify latently infected cell types, in situ hybridization (ISH, RNAScope®) detecting viral DNA was used on selected qPCR- positive neural tissue sections. In ganglia 30 dpi, EHV-1 ISH signal was located in the neurons of TG and ST, but also in non-neuronal support or interstitial cells surrounding the neuron. In contrast, distinct EHV-1 signal could only be observed in neurons of TG 70 dpi. Overall, detection of latent EHV-1 in abdominal tissue samples and non-neuronal cell localization suggests, that EHV-1 uses T-cells during viremia as alternative route toward latency locations in addition to retrograde neuronal transport. We therefore hypothesize that EHV-1 follows the same latency pathways as its close relative human pathogen Varicella Zoster Virus.

Safety and immunogenicity of a modified vaccinia virus Ankara vector vaccine candidate for Middle East respiratory syndrome: an open-label, phase 1 trial.

BACKGROUND: The Middle East respiratory syndrome coronavirus (MERS-CoV) causes a respiratory disease with a case fatality rate of up to 35%. Given its potential to cause a public health emergency and the absence of efficacious drugs or vaccines, MERS is one of the WHO priority diseases warranting urgent research and development of countermeasures. We aimed to assess safety and tolerability of an anti-MERS-CoV modified vaccinia virus Ankara (MVA)-based vaccine candidate that expresses the MERS-CoV spike glycoprotein, MVA-MERS-S, in healthy adults. METHODS: This open-label, phase 1 trial was done at the University Medical Center Hamburg-Eppendorf (Hamburg, Germany). Participants were healthy men and women aged 18-55 years with no clinically significant health problems as determined during medical history and physical examination, a body-mass index of 18·5-30·0 kg/m2 and weight of more than 50 kg at screening, and a negative pregnancy test for women. A key exclusion criterion was a previous MVA vaccination. For the prime immunisation, participants received doses of 1 × 107 plaque-forming unit (PFU; low-dose group) or 1 × 108 PFU (high-dose group) MVA-MERS-S intramuscularly. A second identical dose was administered intramuscularly as a booster immunisation 28 days after first injection. As a control group for immunogenicity analyses, blood samples were drawn at identical study timepoints from six healthy adults, who did not receive any injections. The primary objectives of the study were safety and tolerability of the two dosage levels and reactogenicity after administration. Immunogenicity was assessed as a secondary endpoint by ELISA and neutralisation tests. T-cell immunity was evaluated by interferon-γ-linked enzyme-linked immune absorbent spot assay. All participants who were vaccinated at least once were included in the safety analysis. Immunogenicity was analysed in the participants who completed 6 months of follow-up. This trial is registered with ClinicalTrials.gov, NCT03615911, and EudraCT, 2014-003195-23 FINDINGS: From Dec 17, 2017, to June 5, 2018, 26 participants (14 in the low-dose group and 12 in the high-dose group) were enrolled and received the first dose of the vaccine according to their group allocation. Of these, 23 participants (12 in the low-dose group and 11 in the high-dose group) received a second dose of MVA-MERS-S according to their group allocation after a 28-day interval and completed follow-up. Homologous prime-boost immunisation with MVA-MERS-S revealed a benign safety profile with only transient mild-to-moderate reactogenicity. Participants had no severe or serious adverse events. 67 vaccine-related adverse events were reported in ten (71%) of 14 participants in the low-dose group, and 111 were reported in ten (83%) of 12 participants in the high-dose group. Solicited local reactions were the most common adverse events: pain was observed in 17 (65%; seven in the low-dose group vs ten in the high-dose group) participants, swelling in ten (38%; two vs eight) participants, and induration in ten (38%; one vs nine) participants. Headaches (observed in seven participants in the low-dose group vs nine in the high-dose group) and fatigue or malaise (ten vs seven participants) were the most common solicited systemic adverse events. All adverse events resolved swiftly (within 1-3 days) and without sequelae. Following booster immunisation, nine (75%) of 12 participants in the low-dose group and 11 (100%) participants in the high-dose group showed seroconversion using a MERS-CoV S1 ELISA at any timepoint during the study. Binding antibody titres correlated with MERS-CoV-specific neutralising antibodies (Spearman's correlation r=0·86 [95% CI 0·6960-0·9427], p=0·0001). MERS-CoV spike-specific T-cell responses were detected in ten (83%) of 12 immunised participants in the low-dose group and ten (91%) of 11 immunised participants in the high-dose group. INTERPRETATION: Vaccination with MVA-MERS-S had a favourable safety profile without serious or severe adverse events. Homologous prime-boost immunisation induced humoral and cell-mediated responses against MERS-CoV. A dose-effect relationship was demonstrated for reactogenicity, but not for vaccine-induced immune responses. The data presented here support further clinical testing of MVA-MERS-S in larger cohorts to advance MERS vaccine development. FUNDING: German Center for Infection Research.

Genotyping Porcine Circovirus 3 (PCV-3) Nowadays: Does It Make Sense?

The discovery of a globally distributed porcine circovirus (Porcine circovirus 3; PCV-3) has led to intense research activity and the production of a large amount of molecular data. Different research groups have proposed several, not always concordant, genotypes for this virus. While such categories could aid an easier interpretation of PCV-3 molecular epidemiology, any classification, to be useful in practical settings, must be univocal and of help in the understanding of underlying biological features and epidemiology. Based on these premises, the possibility of defining PCV-3 genotypes was evaluated on the broadest available dataset of PCV-3 complete genome (n = 357) and open reading frame 2 (ORF2, n = 653) sequences. Genetic distance and phylogenetic clustering were selected as the main objective criteria. Additional factors, including the number of within-cluster sequences, host and geographic clustering, concordance between different genomic regions, and analysis method were also taken in account to generate a classification that could be effectively applied in research and diagnostic settings. A maximum within-genotype genetic distance of 3% at the complete genome and 6% at the ORF2 levels, bootstrap support higher than 90%, and concordance between analysis methods allowed us to clearly define two clades which could be potentially defined as genotypes. Further subdivision was not suggested due to the absence of a meaningful association between PCV-3 and its biological/epidemiological features. Nevertheless, since one of the clades included two strains only, thus far we formally propose the definition of only one PCV-3 genotype (PCV-3a). The established criteria will allow us to automatically recognize other genotypes when more strain sequences are characterized.

Proliferative Kidney Disease and Proliferative Darkening Syndrome are Linked with Brown Trout (Salmo trutta fario) Mortalities in the Pre-Alpine Isar River.

For many years, brown trout (Salmo trutta fario) mortalities within the pre-alpine Isar River in Germany were reported by the Bavarian Fisheries Association (Landesfischereiverband Bayern e.V.) and local recreational anglers during August and September. Moribund fish seemed to be affected by proliferative darkening syndrome (PDS). In addition, proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae was discussed. To investigate this phenomenon, the present field study monitored brown trout mortalities by daily river inspection in 2017 and 2018. Moribund brown trout (n = 31) were collected and examined using histology, immunohistochemistry, qPCR, and quantitative stereology. Our investigations identified 29 (93.5%) brown trout affected by PKD. Four brown trout (12.9%) displayed combined hepatic and splenic lesions fitting the pathology of PDS. The piscine orthoreovirus 3, suspected as causative agent of PDS, was not detectable in any of the samples. Quantitative stereological analysis of the kidneys revealed a significant increase of the renal tissue volumes with interstitial inflammation and hematopoietic hyperplasia in PKD-affected fish as compared to healthy brown trout. The identified T. bryosalmonae strain was classified as part of the North American clade by phylogenetical analysis. This study highlights PKD and PDS as contributing factors to recurrent autumnal brown trout mortalities.

Piscine Orthoreovirus 3 Is Not the Causative Pathogen of Proliferative Darkening Syndrome (PDS) of Brown Trout (Salmo trutta fario).

The proliferative darkening syndrome (PDS) is a lethal disease of brown trout (Salmo trutta fario) which occurs in several alpine Bavarian limestone rivers. Because mortality can reach 100%, PDS is a serious threat for affected fish populations. Recently, Kuehn and colleagues reported that a high throughput RNA sequencing approach identified a piscine orthoreovirus (PRV) as a causative agent of PDS. We investigated samples from PDS-affected fish obtained from two exposure experiments performed at the river Iller in 2008 and 2009. Using a RT-qPCR and a well-established next-generation RNA sequencing pipeline for pathogen detection, PRV-specific RNA was not detectable in PDS fish from 2009. In contrast, PRV RNA was readily detectable in several organs from diseased fish in 2008. However, similar virus loads were detectable in the control fish which were not exposed to Iller water and did not show any signs of the disease. Therefore, we conclude that PRV is not the causative agent of PDS of brown trout in the rhithral region of alpine Bavarian limestone rivers. The abovementioned study by Kuehn used only samples from the exposure experiment from 2008 and detected a subclinical PRV bystander infection. Work is ongoing to identify the causative agent of PDS.

Cross-sectional study on viraemia and shedding of porcine circovirus type 2 in a subclinically infected multiplier sow herd.

Vertical and horizontal transmission of porcine circovirus type 2 (PCV2) plays an important role for the spread of PCV2 within piglet-producing farms and following production steps. Further information is crucial to learn about the principles of PCV2 circulation among sows in piglet-producing farms to improve preventive healthcare concerning porcine circovirus diseases (PCVD) in downstream production steps. The present study was conducted as a cross-sectional study in a 400 sow multiplier herd in Germany with no PCV2 vaccination. Blood, faeces and saliva of the sows in all stages of production were tested for PCV2-DNA by real-time PCR. Results were analysed under respect of the parity and stage of production of the sows. PCV2-DNA in faeces or saliva was observed especially in young sows. Highest rates of viraemia in productive sows were found in the early stages of pregnancy. The results revealed that particularly gilts from the quarantine and rearing area and sows up to the second parity play a major role for the spread of PCV2 and thus for the maintenance of PCV2 infection in sow herds. Furthermore, the stage of production had a significant influence on the detection rate of PCV2-DNA in serum, saliva or faeces of the sows.

CD8+ T Cells Responding to the Middle East Respiratory Syndrome Coronavirus Nucleocapsid Protein Delivered by Vaccinia Virus MVA in Mice.

Middle East respiratory syndrome coronavirus (MERS-CoV), a novel infectious agent causing severe respiratory disease and death in humans, was first described in 2012. Antibodies directed against the MERS-CoV spike (S) protein are thought to play a major role in controlling MERS-CoV infection and in mediating vaccine-induced protective immunity. In contrast, relatively little is known about the role of T cell responses and the antigenic targets of MERS-CoV that are recognized by CD8+ T cells. In this study, the highly conserved MERS-CoV nucleocapsid (N) protein served as a target immunogen to elicit MERS-CoV-specific cellular immune responses. Modified Vaccinia virus Ankara (MVA), a safety-tested strain of vaccinia virus for preclinical and clinical vaccine research, was used for generating MVA-MERS-N expressing recombinant N protein. Overlapping peptides spanning the whole MERS-CoV N polypeptide were used to identify major histocompatibility complex class I/II-restricted T cell responses in BALB/c mice immunized with MVA-MERS-N. We have identified a H2-d restricted decamer peptide epitope in the MERS-N protein with CD8+ T cell antigenicity. The identification of this epitope, and the availability of the MVA-MERS-N candidate vaccine, will help to evaluate MERS-N-specific immune responses and the potential immune correlates of vaccine-mediated protection in the appropriate murine models of MERS-CoV infection.