Worldwide rapid spread of the novel rabbit haemorrhagic disease virus (GI.2/RHDV2/b)

We describe the extremely rapid worldwide spread of the Lagovirus europaeus/GI.2/RHDV2/b (henceforth GI.2), the causative infectious agent of the so‐called ‘novel’ rabbit haemorrhagic disease of the European rabbit (Oryctolagus cuniculus). We tracked down all novel confirmed detections of GI.2 between May 2010 and November 2018 by carrying out a two‐step in‐depth review. We suggest that such spread would not have been possible without anthropogenic involvement. Our results also point out the importance of reviewing and adapting the protocols of virus detection and management in order to control, mitigate and contain properly, not only GI.2, but also new viruses that may emerge in the future.     

Spillover Events of Infection of Brown Hares (Lepus europaeus) with Rabbit Haemorrhagic Disease Type 2 Virus (RHDV2) Caused Sporadic Cases of an European Brown Hare Syndrome‐Like Disease in Italy and Spain

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80–90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV‐related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS‐like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.     

Absence of Hepatitis E virus circulation in wild rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis) in Mediterranean ecosystems in Spain

In recent decades, cases of autochthonous hepatitis E (HE) have sharply increased in European countries where foodborne transmission is considered the main route of HE virus (HEV) transmission. Although rabbits are considered the main reservoir of the zoonotic HEV‐3ra subtype, information on the role of wild lagomorphs in the epidemiology of HEV remains scarce. The aim of this study therefore was to assess the circulation of HEV in European wild rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis), the most important lagomorph species in Spanish Mediterranean ecosystems. Liver samples from 372 wild rabbits and 78 Iberian hares were analysed using a broad‐spectrum RT‐PCR that detects HEV genotypes 1–8. None of the 450 lagomorphs tested were positive for HEV infection. To the best of our knowledge, this is the first study to assess HEV circulation in wild rabbits in Spain and the first to evaluate HEV infection in Iberian hares. Our results indicate absence of HEV circulation in wild rabbits and Iberian hares in southern Spain during the study period, which suggests that the risk of transmission of HEV from wild lagomorphs to other species, including humans, is low.     

Insights into the evolution of the new variant rabbit haemorrhagic disease virus (GI.2) and the identification of novel recombinant strains

Rabbit haemorrhagic disease (RHD ) is a viral disease that affects the European rabbit. RHD was detected in 1984 in China and rapidly disseminated worldwide causing a severe decline in wild rabbit populations. The aetiological agent, rabbit haemorrhagic disease virus (RHDV ), is an RNA virus of the family Caliciviridae , genus Lagovirus . Pathogenic (G1‐G6 or variants GI .1a‐GI .1d) and non‐pathogenic strains (GI .4) have been characterized. In 2010, a new variant of RHDV , RHDV 2/RHDV b/GI .2, was detected in France. GI .2 arrived to the Iberian Peninsula in 2011, and several recombination events were reported. Here, we sequenced full genomes of 19 samples collected in Portugal between 2014 and 2016. New GI .2 recombinant strains were detected, including triple recombinants. These recombinants possess a non‐structural protein p16 related to a non‐pathogenic strain. Evolutionary analyses were conducted on GI .2 VP 60 sequences. Estimated time to the most recent common ancestor (tMRCA ) suggests an emergence of GI .2 in July 2008, not distant from its first detection in 2010. This is the first study on GI .2 evolution and highlights the need of continued monitoring and characterization of complete genome sequences when studying lagoviruses’ evolution.     

Endemic Skunk amdoparvovirus in free‐ranging striped skunks (Mephitis mephitis) in California

The genus Amdoparvovirus includes the newly discovered skunk amdoparvovirus and the well‐characterized Aleutian disease virus which causes significant health impacts in farmed mink worldwide. In 2010–2013, an outbreak of fatal amdoparvovirus‐associated disease was documented in free‐ranging striped skunks (Mephitis mephitis) from the San Francisco Bay Area of California. To characterize the geographic distribution, earliest occurrence and abundance of this virus, as well as possible impacts on sympatric mustelids of conservation concern, we tested blood samples from skunks throughout California and fishers (Pekania pennanti) from northern California for amdoparvovirus DNA. Amdoparvovirus DNA was detected in 64.8% of sampled skunks (140/216), and test‐positive skunks were distributed widely throughout the state, from as far north as Humboldt County and south to San Diego County. The first test‐positive skunks were detected from 2004, prior to the 2010–2013 outbreak. No significant spatial or temporal clustering of infection was detected. Although healthy and clinically ill animals tested positive for amdoparvovirus DNA, histopathologic evaluation of a subset from clinically ill skunks indicated that positive PCR results were associated with pneumonia as well as there being more than one inflammatory type lesion. None of 38 fishers were PCR‐positive. Given the widespread geographic distribution and lack of a clear epizootic centre, our results suggest the presence of an endemic skunk‐associated amdoparvovirus strain or species. However, if the virus is not host‐specific, skunks’ ubiquitous presence across rural and urban habitats may pose a risk to susceptible domestic and wild species including mustelids of conservation concern such as fishers and Pacific martens (Martes caurina).     

Repair of large osteochondral defects in rabbits using porous hydroxyapatite/collagen (HAp/Col) and fibroblast growth factor‐2 (FGF‐2)

Articular cartilage has a limited capacity for self‐renewal. This article reports the development of a porous hydroxyapatite/collagen (HAp/Col) scaffold as a bone void filler and a vehicle for drug administration. The scaffold consists of HAp nanocrystals and type I atelocollagen. The purpose of this study was to investigate the efficacy of porous HAp/Col impregnated with FGF‐2 to repair large osteochondral defects in a rabbit model. Ninety‐six cylindrical osteochondral defects 5 mm in diameter and 5 mm in depth were created in the femoral trochlear groove of the right knee. Animals were assigned to one of four treatment groups: porous HAp/Col impregnated with 50 µl of FGF‐2 at a concentration of 10 or 100 µg/ml (FGF10 or FGF100 group); porous HAp/Col with 50 µl of PBS (HAp/Col group); and no implantation (defect group). The defect areas were examined grossly and histologically. Subchondral bone regeneration was quantified 3, 6, 12, and 24 weeks after surgery. Abundant bone formation was observed in the HAp/Col implanted groups as compared to the defect group. The FGF10 group displayed not only the most abundant bone regeneration but also the most satisfactory cartilage regeneration, with cartilage presenting a hyaline‐like appearance. These findings suggest that porous HAp/Col with FGF‐2 augments the cartilage repair process. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:677–686, 2010     

Arrival of rabbit haemorrhagic disease virus 2 to northern Europe: Emergence and outbreaks in wild and domestic rabbits (Oryctolagus cuniculus) in Sweden

Incursion of rabbit haemorrhagic disease virus (RHDV ) into Sweden was documented in 1990 and it is now considered endemic in wild rabbit (Oryctolagus cuniculus ) populations. Rabbit haemorrhagic disease virus 2 (RHDV 2), a new, related lagovirus was first detected in France in 2010, and has spread rapidly throughout Europe and beyond. However, knowledge of RHDV 2 in northern Europe is sporadic and incomplete, and in Sweden, routinely available diagnostic methods to detect rabbit haemorrhagic disease (RHD ) do not distinguish between types of virus causing disease. Using RHDV 2‐specific RT ‐qPCR , sequencing of the VP 60 gene and immunological virus typing of archived and prospective case material from the National Veterinary Institute's (SVA ) wildlife disease surveillance programme and diagnostic pathology service, we describe the emergence of RHDV 2 in Sweden in both wild and domestic rabbits. The earliest documented outbreak occurred on 22 May 2013, and from May 2013 to May 2016, 10 separate incidents of RHDV 2 were documented from six different municipalities in the southern half of Sweden. Phylogenetic analysis of the VP 60 gene shows clear clustering of Swedish isolates into three separate clusters within two different clades according to geographic location and time, suggesting viral evolution, multiple introduction events or both. Almost all cases of RHD examined by SVA from May 2013 to May 2016 were caused by RHDV 2, suggesting that RHDV 2 may be replacing RHDV as the predominant cause of RHD in Sweden.     

Fatal canine parvovirus‐2 (CPV‐2) infection in a rescued free‐ranging Taiwanese pangolin (Manis pentadactyla pentadactyla)

Carnivore protoparvovirus 1 includes feline parvovirus (FPV), variants of canine parvovirus‐2 (CPV‐2), mink enteritis virus, and raccoon parvovirus, important pathogens affecting both wild and domestic carnivores. In this report, we described a fatal CPV‐2 infection in a rescued Taiwanese pangolin, which provides the first evidence of CPV‐2 infection in a non‐carnivore. Post‐rescue, the Taiwanese pangolin died from complications resulting from a severe panleucocytopenia and bloody diarrhoea. A full autopsy was performed and microscopic examination of the tissues revealed ulcerative, necrotizing, and haemorrhagic glossitis, esophagitis and enteritis. The results of transmission electronic microscopy, polymerase chain reaction and in situ hybridization provided confirmatory evidence that the lesions in the tongue, oesophagus and intestine were associated with a protoparvovirus. Phylogenetic comparison of the whole VP2 gene from the current pangolin protoparvovirus strain showed close clustering with the CPV‐2c strains from domestic dogs in Taiwan, China and Singapore. The amino acid sequence of the pangolin protoparvovirus showed 100% identity to the CPV‐2c strains from domestic dogs in China, Italy, and Singapore. The current findings highlight that pangolins are susceptible to protoparvoviruses. The potential of cross‐species transmission of protoparvoviruses between Carnivora and Pholidota should be considered when housing pangolins in close proximity to carnivores and adopting strict biosecurity measures to avoid cross‐species transmission in rescue facilities and zoos.     

Bone morphogenetic protein‐2 promotes osteosarcoma growth by promoting epithelial‐mesenchymal transition (EMT) through the Wnt/β‐catenin signaling pathway

The correlation between BMP‐2 and osteosarcoma growth has gained increased interest in the recent years, however, there is still no consensus. In this study, we tested the effects of BMP‐2 on osteosarcoma cells through both in vitro and in vivo experiments. The effect of BMP‐2 on the proliferation, migration and invasion of osteosarcoma cells was tested in vitro. Subcutaneous and intratibial tumor models were used for the in vivo experiments in nude mice. The effects of BMP‐2 on EMT of osteosarcoma cells and the Wnt/β‐catenin signaling pathway were also tested using a variety of biochemical methods. In vitro tests did not show a significant effect of BMP‐2 on tumor cell proliferation. However, BMP‐2 increased the mobility of tumor cells and the invasion assay demonstrated that BMP‐2 promoted invasion of osteosarcoma cells in vitro. In vivo animal study showed that BMP‐2 dramatically enhanced tumor growth. We also found that BMP‐2 induced EMT of osteosarcoma cells. The expression levels of Axin2 and Dkk‐1 were both down regulated by BMP‐2 treatment, while β‐catenin, c‐myc and Cyclin‐D1 were all upregulated. The expression of Wnt3α and p‐GSK‐3β were also significantly upregulated indicating that the Wnt/β‐catenin signaling pathway was activated during the EMT of osteosarcoma driven by BMP‐2. From this study, we can conclude that BMP‐2 significantly promotes growth of osteosarcoma cells (143B, MG63), and enhances mobility and invasiveness of tumor cells as demonstrated in vitro. The underlying mechanism might be that BMP‐2 promotes EMT of osteosarcoma through the Wnt/β‐catenin signaling pathway. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1638–1648, 2019.     

Activated protein C (APC) can increase bone anabolism via a protease‐activated receptor (PAR)1/2 dependent mechanism

Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP‐2) induced ectopic bone formation model. Local co‐administration of 10 µg rhBMP‐2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (p < 0.01) compared to rhBMP‐2 alone. This was associated with a significant increase in CD31+ and TRAP+ cells in tissue sections of ectopic bone, consistent with enhanced vascularity and bone turnover. The actions of APC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease‐activated receptors (PARs). Cultured pre‐osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre‐osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP‐2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1549–1556, 2014.     

BMP‐2 stimulated non‐tenogenic differentiation and promoted proteoglycan deposition of tendon‐derived stem cells (TDSCs) in vitro

We hypothesized that BMP‐2 might induce non‐tenocyte differentiation and increase production of proteoglycans of tendon‐derived stem cells (TDSCs). This study investigated the effects of BMP‐2 on the differentiation and production of proteoglycans in TDSCs in vitro. Rat patellar TDSCs were treated without or with BMP‐2. The osteogenic, adipogenic, chondrogenic, and tenogenic differentiation of TDSCs were assessed by (1) Alizarin red‐S staining assay; (2) Oil Red‐O staining assay; (3) haematoxylin–eosin staining, Safranin‐O staining, immunohistochemical staining of Sox9, and collagen type II; and (4) qRT‐PCR analysis of lineage‐specific markers. The production of glycoaminoglycans (GAG) in the BMP‐2‐treated TDSCs was assessed by alcian blue staining. The mRNA expression of aggrecan (Acan), decorin (Dcn), biglycan (Bgn), and fibromodulin (Fmod) in TDSCs after BMP‐2 treatment was assessed by qRT‐PCR. BMP‐2 promoted the osteogenic, adipogenic, and chondrogenic differentiation but inhibited tenogenic marker expression of TDSCs. GAG production and Acan increased while Dcn, Bgn, and Fmod decreased in TDSCs after BMP‐2 stimulation. In conclusion, BMP‐2 promoted GAG deposition, aggrecan expression, and enhanced non‐tenocyte differentiation of TDSCs in vitro. The effect of BMP‐2 on TDSCs might provide insights into the histopathological changes of tendinopathy. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 746–753, 2013     

CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes osteoclastogenesis via induction of and interaction with dendritic cell–specific transmembrane protein (DC‐STAMP)

CCN family 2/connective tissue growth factor (CCN2/CTGF) promotes endochondral ossification. However, the role of CCN2 in the replacement of hypertrophic cartilage with bone is still unclear. The phenotype of Ccn2 null mice, having an expanded hypertrophic zone, indicates that the resorption of the cartilage extracellular matrix is impaired therein. Therefore, we analyzed the role of CCN2 in osteoclastogenesis because cartilage extracellular matrix is resorbed mainly by osteoclasts during endochondral ossification. Expression of the Ccn2 gene was upregulated in mouse macrophage cell line RAW264.7 on day 6 after treatment of glutathione S transferase (GST) fusion mouse receptor activator of NF‐κB ligand (GST‐RANKL), and a combination of recombinant CCN2 (rCCN2) and GST‐RANKL significantly enhanced tartrate‐resistant acid phosphatase (TRACP)–positive multinucleated cell formation compared with GST‐RANKL alone. Therefore, we suspected the involvement of CCN2 in cell‐cell fusion during osteoclastogenesis. To clarify the mechanism, we performed real‐time PCR analysis of gene expression, coimmunoprecipitation analysis, and solid‐phase binding assay of CCN2 and dendritic cell–specific transmembrane protein (DC‐STAMP), which is involved in cell‐cell fusion. The results showed that CCN2 induced and interacted with DC‐STAMP. Furthermore, GST‐RANKL–induced osteoclastogenesis was impaired in fetal liver cells from Ccn2 null mice, and the impaired osteoclast formation was rescued by the addition of exogenous rCCN2 or the forced expression of DC‐STAMP by a retroviral vector. These results suggest that CCN2 expressed during osteoclastogenesis promotes osteoclast formation via induction of and interaction with DC‐STAMP. © 2011 American Society for Bone and Mineral Research.     

Molecular detection and characterization of carnivore parvoviruses in free‐ranging Eurasian otters (Lutra lutra) in southern Italy

The most important Italian population of the Eurasian otter (Lutra lutra) occurs in the southern part of the peninsula with two isolated sub‐populations of about 250 adult individuals. The Eurasian otter is considered to be near threatened and it is a fully protected species. The aims of this study were to investigate for the first time the occurrence and characterize the parvoviruses included in the species Carnivore protoparvovirus 1 in seven carcasses of road‐killed Eurasian otters from the southern Italy. Carnivore protoparvovirus 1 are responsible for acute gastroenteritis and leukopenia in pets and free‐ranging carnivores. Initial screening of tissue samples by real‐time PCR revealed CPV/FPV DNA in tissue samples of five Eurasian otters; three of them, showed co‐infections by both CPV and FPV. Among the five positive Eurasian otters, we successfully obtained six DNA sequences from four individuals including two CPV‐2a, one CPV‐2b, one CPV‐2c, and two FPV sequences. Comparison of these sequences with 250 VP2 gene sequences deposited in the GenBank database, showed 10 nt differences resulting in two synonymous and eight non‐synonymous substitutions. On the basis of these results, two sequences here found were characterized as new CPV‐2a, one was characterized as new CPV‐2b variant, and one was characterized as FPV‐like mutant. The last two sequences belong to a FPV and CPV‐2c strain respectively. Carnivore protoparvovirus 1 is reported for the first time in the Eurasian otter showing high infection value in southern Italy. Occurrence of this infection should be studied further to understand its possible pathogenicity and virulence to the fragile and isolate Eurasian otter population which live in southern Italy.     

GFR ≤25 years postdonation in living kidney donors with (vs. without) a first‐degree relative with ESRD

An increased risk of ESRD has been reported for living kidney donors, and appears to be higher for those donating to a relative. The reasons for this are not clear. One possibility is that ESRD is due to the nephrectomy‐related reduction in GFR, followed by an age‐related decline that may be more rapid in related donors. Between 1/1/1990 and 12/31/2014, we did 2002 living donor nephrectomies. We compared long‐term postdonation eGFR trajectory for donors with (n = 1245) vs. without (n = 757) a first‐degree relative with ESRD. Linear mixed‐effects models were used to model the longitudinal trajectory of eGFR. With all other variables held constant, we noted a steady average increase in eGFR until donors reached age 70: 1.12 (95% CI: 0.92‐1.32) mL/min/1.73m^²/yr between 6 weeks and 5 years postdonation; 0.24 (0.00‐0.49) mL/min/1.73m^²/yr between 5 and 10 years; and 0.07 (?0.10 to +0.25) mL/min/1.73m^²/yr between 10 and 20 years for donors with attained age less than 70. After age 70, eGFR declined. After we adjusted for predonation factors, the difference in eGFR slopes between related and unrelated donors was 0.20 mL/min/1.753 m²/year (0.07‐0.33). Our data suggests that postdonation, kidney donor eGFR increases each year for a number of years and that eGFR trajectory does not explain any increase in ESRD after donation.     

FGF-2-containing dalteparin/protamine nanoparticles (FGF-2&D/P NPs) ameliorate UV-induced skin photoaging in hairless mice

Abstract

UVB exposure penetrates deeply into the dermis to alter skin barrier function, which is a primary factor in skin photoaging. We previously reported that dalteparin and protamine nanoparticles (D/P NPs) are effective carriers of FGF-2. This study aimed to examine the ability of FGF-2-containing D/P NPs (FGF-2&D/P NPs) to ameliorate UVB-induced skin photoaging in hairless mice. Dorsal skin of HR-1 hairless mice were exposed to UVB irradiation 5 days/week for 8 weeks (UV (+): final total, 2700?mJ/cm2). Mice were divided into four groups: Non-UVB (UV (?)) + saline, UV (+) + saline, UV (+) + FGF-2&D/P NPs, UV (+) + FGF-2, and UV (+) + D/P NPs, and following UVB irradiation, FGF-2&D/P NPs, FGF-2, and D/P NPs were applied to the groups of mice just after each UVB irradiation. Each group was subjected to evaluation of skin changes (elasticity), and histological examination using hematoxylin & eosin and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. UVB irradiation of mice significantly induced a decline in elasticity and acanthosis, which was alleviated by application of FGF-2&D/P NPs. Furthermore, TUNEL-staining showed the proportions of apoptotic dermal fibroblast cells (DFCs) and epidermal keratinocyte cells (EKCs) in the UV (+) + FGF-2&D/P NPs group were significantly lower than those in the UV (+) + saline, UV (+) + FGF-2, and UV (+) + D/P NPs groups. Thus, FGF-2&D/P NPs may be effective in preventing skin photoaging accelerated by UVB irradiation such as declining elasticity, acanthosis, and apoptosis of DFCs and EKCs.     

C-erb-B2 (HER2/neu) expression in synovial sarcoma of the head and neck

BACKGROUND: Synovial sarcoma is a malignant mesenchymal tumor composed of varying proportions of spindle and epithelial cell components. Because of the histologic and immunohistochemical similarity of synovial sarcoma to epithelial carcinomas, we hypothesized that the human epithelial growth factor receptor 2 (C-erb-B2, also termed HER2/neu) may contribute to the tumor phenotype and provide a new therapeutic target for this soft tissue tumor. METHODS: Three head and neck, one chest wall, and seven extremity synovial sarcomas were evaluated for C-erb-B2 (HER2/neu) expression by immunohistochemistry, Western immunoblotting, and fluorescence in situ hybridization (FISH). RESULTS: The head and neck cases demonstrated immunohistochemically strong positive staining, whereas tumors from other anatomic locations showed neither positive nor cytoplasmic restricted staining. Antigen-targeted antibody therapy (trastuzumab) was initiated in two patients. CONCLUSIONS: These results demonstrate that C-erb-B2 (HER2/neu) may play a role in the tumorigenesis of synovial sarcoma; and, therefore, antigrowth factor therapies may provide a previously unrecognized pharmaceutical approach to soft tissue tumors. The data also suggest that although synovial sarcoma of the head and neck and synovial sarcoma of the extremities have similar morphologic features, they may be clinically and mechanistically distinct entities.