Emergence of a virulent porcine reproductive and respiratory syndrome virus (PRRSV) 1 strain in Lower Austria

Abstract Background In spring 2015, an outbreak of porcine reproductive and respiratory syndrome (PRRS) struck Lower Austria caused by a PRRS virus (PRRSV) strain spreading rapidly among both previously PRRSV negative and vaccinated pig herds. This case report describes the first well-documented emergence of the PRRSV strain responsible for this outbreak. Case presentation A PRRSV seronegative piglet-producing farm in Lower Austria encountered losses in foetuses and suckling piglets of up to 90 %; clinical signs in sows and nursery piglets included fever and reduced feed intake. Additionally, high percentages of repeat breeders and losses of up to 40 % in nursery piglets occurred. An infection with PRRSV was suggested by the detection of antibodies by enzyme linked immunosorbent assay and confirmed by quantitative real time PCR. The underlying PRRSV strain, termed AUT15-33, was isolated by passage on porcine alveolar macrophages, partially sequenced (ORF2-7) and grouped as PRRSV-1, subtype 1. In phylogenetic analysis of the genome region coding for the structural proteins, ORF2-7, AUT15-33 clustered with Belgian strains but identities were as low as 88 %. In contrast, analysis of ORF7 sequences revealed a close relationship to Croatian strains from 2012 with an identity of 94 – 95 %. Conclusions In the year following the outbreak, the same PRRSV strain was identified repeatedly in different regions of Austria. It can be speculated that the new strain has novel advantageous properties

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

Use of Three Air Samplers for the Detection of PRRSV-1 under Experimental and Field Conditions

Airborne transmission of porcine reproductive and respiratory syndrome virus (PRRSV) has been known for a long time. Most experiments were performed using PRRSV-2 strains and fairly little information is available on the airborne spread of PRRSV-1. The aim of this study was to assess three different air samplers for their ability to detect PRRSV-1 under experimental and field conditions. All three devices were able to detect PRRSV-1 by quantitative reverse trascription polymerase chain reaction (qRT-PCR) under experimental conditions. However, the detection of PRRSV-1 in a PRRSV-positive farm with active virus circulation was not successful

Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV)

<div><p>European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of <i>Deformed wing virus</i> (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete <i>in vitro</i> model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch’s postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.</p></div

Circulating Cancer Associated Macrophage-like Cells as a Potential New Prognostic Marker in Pancreatic Ductal Adenocarcinoma

Background: Circulating Cancer Associated Macrophage-like cells (CAMLs) have been described as novel liquid biopsy analytes and unfavorable prognostic markers in some tumor entities, with scarce data for Pancreatic Ductal Adenocarcinomas (PDAC). Methods: Baseline and follow-up blood was drawn from resected curative (n = 36) and palliative (n = 19) PDAC patients. A microfluidic size-based cell enrichment approach (ParsortixTM) was used for CAML detection, followed by immunofluorescence staining using pan-keratin, CD14, and CD45 antibodies to differentiate between CAMLs, circulating tumor cells (CTCs), and leukocytes. Results: CAMLs were detectable at baseline in 36.1% of resected patients and 47.4% of palliative PDAC patients. CAML detection was tumor stage independent. Follow-up data indicated that detection of CAMLs (in 45.5% of curative patients) was an independent prognostic factor for shorter recurrence-free survival (RFS) (HR: 4.3, p = 0.023). Furthermore, a combined analysis with CTCs showed the detectability of at least one of these cell populations in 68.2% of resected patients at follow-up. The combined detection of CAMLs and CTCs was also significantly associated with short RFS (HR: 8.7, p = 0.003). Conclusions: This pilot study shows that detection of CAMLs in PDAC patients can provide prognostic information, either alone or even more pronounced in combination with CTCs, which indicates the power of liquid biopsy marker analyses

DWV infection model.

<p>(A) Bee pupae at the day p.i.. A black arrow at one bee pupa marks the injury caused by injection. (B) Typical outcome of infections with rDWV-A 1414 and wtDWV-A 1414: (B1) Healthy bee, which emerged at day 21 of development after mock infection. (B2) Bee pupa, which died three days after infection with wtDWV-A 1414. (B3) Nonviable adult bee, which emerged at day 21 after infection with rDWV-A 1414 showing typical wing and limb deformities.</p

Sequence analyses of DWV-A 1414.

<p>(A) DWV 5’-NTR sequences were obtained from tailing RACE-PCRs. Numbers of clones containing the respective 5’-terminal nucleotides are indicated below the sequence. (B) Sequence comparison of the 5’-terminus of DWV-A 1414 and related Iflaviruses. Genbank entries are provided behind the strain designations. (C) Neighbor joining analysis using genomic sequences of different Iflaviruses. The phylogenetic analysis documents a close relationship of DWV-A 1414 to other DWV-A isolates from Europe and America. The number of substitutions per site is given as a scale and bootstrap values for 1,000 replicates were indicated above all nodes.</p

Western blot analysis of the rDWV rescue.

<p>(A) Bee pupae infected with a DWV-A field isolate (wtDWV, lane 2 and 3) or transfected with synthetic RNA of the DWV-A clone (rDWV, lane 4 and 5) were probed two days and four days after inoculation using mAb DVWVP1A1. Bands with an apparent molecular mass of 46 kDa (VP1) and 19 kDa (unknown) were visible in wtDWV infected and rDWV transfected pupae but absent in the mock control (lane 1). (B) Bee pupae infected with passage three of wtDWV (lane 7) and rDWV (lane 8) were tested for VP1 expression using mAb DVWVP1B1 two days after infection. Interestingly, the 19 kDa protein was not detectable using mAb DVWVP1B1.</p

Recombinant DWV VP1 (rVP1) and VP1 specific antibodies.

<p>(A) rVP1 was expressed in <i>E</i>. <i>coli</i> via plasmid pL443. Total protein from non-induced (NI) and induced (I) cultures was separated by SDS-PAGE and either stained with Coomassie blue or probed with an anti His-tag antibody. (B) IMAC purified rVP1 revealed by Coomassie stain and western blot analysis. (C) Reactivity and specificity of two mouse monoclonal antibodies is shown using <i>E</i>. <i>coli</i> expressing rVP1. Arrows indicate the protein bands of rVP1 and rVP1 dimer.</p