Detection of porcine circovirus‐like virus P1 in Hebei,China

Porcine circovirus‐like virus P1 is a novel unclassified circovirus that was first detected in China and may be associated with post‐weaning multisystemic wasting syndrome (PMWS ) and congenital tremor. In this study, we detected P1 infection in pigs in Hebei Province, China, in 2017. One hundred and forty of 500 (28.0%) serum samples from 25 pig farms with different PMWS status in seven cities were P1 positive on PCR . Twelve P1 strains were sequenced, and the complete genomes of 11 P1 strains were 648 nucleotides (nt) in length, whereas that of strain ZJK 02 was 647 nt, with a G deletion at position of 183 in its genome. The complete genomic and capsid protein sequences of the 12 P1 strains analysed in this study shared 98.8%–100.0% and 86.5%–100.0% identity, respectively. A phylogenetic analysis based on the complete genomic and capsid sequences of 26 P1 strains showed that the 12 P1 sequences from Hebei Province clustered on two small branches. Further studies of the evolution and pathogenesis of P1 are required.     

Evidence of porcine circovirus‐like virus P1 in piglets with an unusual congenital tremor

Outbreaks of trembling and shaking were reported among pigs at two pig farms in Jiangsu Province, China. Serum and tissue samples tested positive for porcine circovirus‐like virus P1 and negative for classical swine fever virus, porcine circovirus type 2, astrovirus and porcine pestivirus using PCR/RT‐PCR and immunohistochemical techniques. High P1 viral genome loads were identified in sera, brain and lymph node tissue samples by qPCR. In addition, one of the most notable pathological changes was dissolution of the nucleus in Purkinje cells. The results of this study provide molecular evidence of an association between congenital tremor in pigs and P1 virus.     

Detection of a novel porcine circovirus‐like agent in aborted pig foetuses

A large outbreak of swine abortion, which began in January 2017, is ongoing in Jiangsu and Anhui provinces, China. We identified a new porcine circovirus‐like agent, designated P4, in the aborted foetuses from several of these cases of swine reproductive failure. PCR and qRT ‐PCR analyses confirmed that other common abortogenic agents were not present in these animals. P4 contains a 710‐nucleotide circular viral genome, and all strains examined in this study were closely related to the dominant genotype of porcine circovirus type 2. Further studies, such as in vivo analyses, are needed to confirm P4 as the aetiological agent.     

Serological Survey of Porcine circovirus‐2 in Captive Wild Boars (Sus scrofa) from Registered Farms of South and South‐east Regions of Brazil

This study aimed to survey captive wild boars for antibodies against Porcine circovirus‐2 (PCV‐2) in registered farms. Serum samples (n = 1305) were collected from 90‐day‐old wild boars from 118 farms of the Brazilian South‐east region, including the states of Minas Gerais and São Paulo, and South region, including the states of Paraná, Rio Grande do Sul and Santa Catarina. All herds (100%) presented reactive animals, in varying numbers and from low‐to‐high antibody titres, with the occurrence ranging from 82 to 89%. Considering farms, the average prevalence was of 84.9% (P < 0.05) and ranged from 54.1 to 94.95%. Regarding the geographic regions studied, the prevalence was of 100%, with PCV2 antibodies detected in wild boars of all regions. This study provides the first evidence of PCV2 antibodies in captive wild boars in Brazil.     

Identification of Natural Infections in Sheep/Goats with HoBi‐like Pestiviruses in China

The natural infections of HoBi‐like pestiviruses in cattle have been reported in South America, Europe and Asia. In China, although the detections of HoBi‐like pestivirus have been reported, the epidemiological investigation was limited. From January 2014 to October 2015, several flocks of sheep/goats in Henan province in central China suffered respiratory diseases which were recovered slowly after antibiotics treatment. To test whether it is the HoBi‐like pestivirus caused this symptom, 49 serum samples and 22 nasal swabs were then collected for analysis by serology and RT‐PCR. Serological result revealed that prevalence of pestivirus in small ruminants was 12.2% (6/49) in central China. Sequence analysis of partial 5′‐UTR nucleotides of pestivirus‐positive samples suggested that HoBi‐like pestivirus might have circulated in sheep/goats of China for a period and have evolved into new genotype clusters. It is apparent that the study provides the molecular evidence of natural infections in goat/sheep species with HoBi‐like pestiviruses in China.     

Pathogenicity of porcine reproductive and respiratory syndrome virus (ORF5 RFLP 1‐7‐4 viruses) in China

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that causes reproductive failure in sows and respiratory problems in piglets. PRRSV infection leads to substantial pig mortality and causing huge economic losses so that disease outbreaks caused by the new PRRSV strain from other regions have caused great concern in China. In this study, we analysed the pathogenicity of the novel ORF5 RFLP 1‐7‐4‐like PRRSV strain, named PRRSV‐ZDXYL‐China‐2018‐1 in pigs. The viral challenge test showed that PRRSV‐ZDXYL‐China‐2018‐1 infection can cause persistent fever, moderate dyspnoea, serum viraemia and interstitial pneumonia in piglets. The levels of viral loads in serum and PRRSV‐specific antigen were also detected in lung tissues were used one‐step Taq‐Man RT‐qPCR and Immunohistochemistry, respectively. At 28dpi, the level of specific antibodies was increased among infected piglets. Importantly, the new virus appeared be a moderately virulent isolate with pathogenicity compared to HP‐PRRSV strain LQ (JXA1‐like strain). Histological examination revealed severe monocyte haemorrhage and interstitial pneumonia associated with monocyte infiltration in the lung tissue of pigs infected with PRRSV‐ZDXYL‐China‐2018‐1 and LQ‐JXA1 strains. Immunohistochemistry (IHC) results showed positive brown‐red epithelial cells and macrophages in pig lungs. Therefore, it is critical to establish an effective strategy to control the spread of PRRSV in China.     

Porcine reproductive and respiratory syndrome virus NADC30‐like strain accelerates Streptococcus suis serotype 2 infection in vivo and in vitro

Porcine reproductive and respiratory syndrome (PRRS), an economically significant pandemic disease, commonly results in increased impact of bacterial infections, including those by Streptococcus suis (S. suis). In recent years, PRRS virus (PRRSV) NADC30‐like strain has emerged in different regions of China, and coinfected with S. suis and PRRSV has also gradually increased in clinical performance. However, the mechanisms involved in host innate responses towards S. suis and their implications of coinfection with NADC30‐like strain remain unknown. Therefore, the pathogenicity of NADC30‐like strain and S. suis serotype 2 (SS2) coinfection in vivo and in vitro was investigated in this study. The results showed that NADC30‐like increased the invasion and proliferation of SS2 in blood and tissues, resulting in more severe pneumonia, myocarditis, and peritonitisas well as higher mortality rate in pigs. In vitro, NADC30‐like strain increased the invasion and survival of SS2 in porcine alveolar macrophages (PAM) cells, causing more drastic expression of inflammatory cytokines and activation of NF‐ĸB signalling. These results pave the way for understanding the interaction of S. suis with the swine immune system and their modulation in a viral coinfection.     

Foot‐and‐mouth disease virus serotype SAT1 in cattle,Nigeria

The knowledge of foot‐and‐mouth disease virus (FMDV) dynamics and epidemiology in Nigeria and the West Africa subregion is important to support local and regional control plans and international risk assessment. Foot‐and‐mouth disease virus serotype South African territories (SAT)1 was isolated, identified and characterized from an FMD outbreak in cattle in Nigeria in 2015, 35 years after the last report of FMDV SAT1 in West Africa. The VP1 coding sequence of the Nigerian 2015 SAT1 isolates diverges from reported SAT1 topotypes resulting in a separate topotype. The reporting of a novel FMDV SAT1 strain in the virus pool 5 (West and Central Africa) highlights the dynamic and complex nature of FMDV in this region of Africa. Sustained surveillance is needed to understand the origin, the extent and distribution of this novel SAT1 topotype in the region as well as to detect and monitor the occurrence of (re‐)emerging FMDV strains.     

Improved transplantation outcome through delivery of DNA encoding secretion signal peptide‐linked glucagon‐like peptide‐1 into mouse islets

Glucagon‐like peptide‐1 (GLP‐1) stimulates cell proliferation and has anti‐apoptotic effects on pancreatic islet β cells. In our previous study, the transduction of mouse islets with a recombinant adenovirus containing GLP‐1 cDNA enhanced islet graft survival. In this study, we sought to deliver the GLP‐1 gene using a nonviral vector, which raises fewer safety issues in clinical application. We constructed a plasmid, pβ‐SP‐GLP‐1, in which a secretion signal peptide (SP) was inserted to increase GLP‐1 secretion, and transfected mouse islets using the nonviral carrier Effectene. Transfection of pβ‐SP‐GLP‐1 induced a significant increase in bioactive GLP‐1 levels in islet cultures. Islets transfected with pβ‐SP‐GLP‐1 were protected from H₂O₂‐induced cell damage in vitro. In addition, glucose‐stimulated insulin secretion was significantly increased in pβ‐SP‐GLP‐1‐transfected islets. Diabetic syngeneic mice transplanted under the kidney capsule with a marginal mass of pβ‐SP‐GLP‐1‐transfected islets rapidly became normoglycemic, with 88% of recipients being normoglycemic at 30 days post‐transplantation compared with 52% of mice that received pβ‐transfected islet grafts (P < 0.05). Islet grafts retrieved 7 days after transplantation revealed that the pβ‐SP‐GLP‐1‐transfected group had significantly more Ki67‐positive cells as compared with the pβ‐transfected group. In conclusion, delivery of a plasmid containing a secretion SP and GLP‐1 cDNA using a nonviral carrier leads to efficient secretion of GLP‐1 in mouse islet cells, enhances islet cell survival during the early post‐transplant period, and improves islet transplantation outcome.     

A natural recombinant PRRSV between HP‐PRRSV JXA1‐like and NADC30‐like strains

Porcine reproductive and respiratory syndrome virus (PRRSV ) is a major economically significant pathogen that has adversely affected China's swine industry. Currently, a novel type 2 PRRSV , called the NADC 30‐like strain, is epidemic in numerous provinces of China, and commercial vaccines provide limited protection for infected animals. The extensive recombination phenomenon among NADC 30‐like PRRSV s is identified as a unique molecular characteristic of the virus. However, our understanding of how recombination influences NADC 30‐like PRRSV s is largely inadequate. In this study, we analysed the genetic characteristics of a recombinant NADC 30‐like PRRSV (SC ‐d) and examined its pathogenicity compared with a non‐recombinant NADC 30‐like PRRSV (SD ‐A19) and a highly pathogenic PRRSV (HuN4). SC ‐d has three discontinuous deletions in nsp2, consistent with NADC 30 isolated from the United States in 2008. Furthermore, we identified four recombination breakpoints in the SC ‐d genome, which separated the SC ‐d genome into four regions (regions A, B, C and D). Regions A and C are closely related to the JXA 1‐like strain, one of the earliest Chinese HP ‐PRRSV strains, and regions B and D are closely related to the NADC 30 strain. Moreover, SC ‐d inoculated piglets exhibited a persistent fever, moderate weight loss, mild thymus atrophy and obvious microscopic lung lesions. In summary, the recombinant NADC 30‐like PRRSV SC ‐d strain displayed a higher pathogenicity than the non‐recombinant NADC 30‐like PRRSV SD ‐A19 strain; however, the pathogenicity of the NADC 30‐like PRRSV SC ‐d was lower compared with the HP ‐PRRSV HuN4 strain in piglets. Our findings demonstrate that recombination is responsible for the enormous genetic diversity and pathogenicity variance of the NADC 30‐like PRRSV in China. This study provides a theoretical basis for developing a more reasonable PRRSV control and prevention strategy.     

Full‐Genome Sequence of Porcine Circovirus type 3 recovered from serum of sows with stillbirths in Brazil

Two full‐genome sequences of porcine circovirus type 3 (PCV 3) are reported. The genomes were recovered from pooled serum samples from sows who had just delivered litters with variable numbers of stillbirths. The two circular genomes (PCV 3‐BR /RS /6 and PCV 3‐BR /RS /8) are 2,000 nucleotides long and contain two open reading frames (ORFs) oriented in opposite directions that encode the putative capsid (Cap) and replicase (Rep) proteins. The intergenic region contains a stem‐loop motif, as reported for other circoviruses. Rolling circle replication motifs and putative helicase domains were identified in the Rep coding region. The degree of overall nucleotide similarity between the genomes reported here and those available at GenBank was higher than 97%. No PCV 3 sequence was detected in pooled serum samples from sows which had no stillbirths on the same farms. However, further studies are necessary to confirm the association between PCV 3 and the occurrence of stillbirths.     

Development and Evaluation of Single‐tube Nested PCR (STNPCR) for the Detection of Porcine Circovirus type 2 (PCV2)

Porcine circovirus 2 (PCV2) is a common virus in pig population and is associated with the postweaning multisystemic wasting disease (PMWS). In this study, it was developed and evaluated the single‐tube nested PCR (STNPCR) method for the detection of PCV2 DNA. PCV2 reference controls and swine tissue samples were used, and primers were selected for targeting specific regions of the viral genome. In comparison of the methods, STNPCR was 10 times more sensitive than conventional PCR and showed the same sensitivity to nested PCR (NPCR), but with reduction in the risk of cross‐contamination. In clinical application, 55 tissue samples were analysed by conventional PCR and resulted in 67% (37/55) of positive reactions, while the NPCR and STNPCR were able to identify the presence of viral DNA in 100% (55/55) of the samples. The high sensitivity combined with the elimination of cross‐contamination makes the STNPCR method suitable for the epidemiological studies of PCV2 and can aid in the diagnosis of PMWS.     

Decrease of the insulin‐like growth factor‐1 bioavailability in spontaneously hypertensive rats with erectile dysfunction

We investigated the role of insulin‐like growth factor‐1 (IGF‐1) in spontaneously hypertensive rats with erectile dysfunction. Firstly, we evaluated intracavernous pressure. The bioavailability of IGF‐1 at both mRNA and protein levels were measured by quantitative real‐time PCR and Western blot respectively. Then, cavernous cyclic guanosine monophosphate concentrations were detected by enzyme‐linked immunosorbent assay. The cavernosal pressure was significantly decreased in the hypertensive and the propranolol treatment groups compared to the normal control group (P < 0.01). Cavernous IGF‐1 bioavailability and the concentrations of cavernous cyclic guanosine monophosphate were both significantly decreased in the hypertensive and the propranolol treatment groups compared to the normal control group (P < 0.01). This study suggests that an obvious decrease in cavernous IGF‐1 levels might play an important role in spontaneously hypertensive rats with erectile dysfunction.     

Sarcoidosis‐like reaction in metastatic triple negative breast cancer treated by anti‐PD‐L1

We report the first case of sarcoidosis‐like reaction in a patient treated by anti‐PD‐L1 for a breast cancer. A 69‐year‐old woman presented with a histologically confirmed lung metastasis of a triple negative breast cancer. She was treated by nab‐paclitaxel plus anti‐PD‐L1 in first line. After 2 months, a dramatic lung response was noticed but an involvement of mediastinal lymph nodes appeared. Endoscopic ultrasound‐guided fine‐needle aspiration of these lymph nodes revealed multiple epitheloid granulomas without caseating necrosis in favour of a sarcoidosis‐like reaction. The patient remained free of symptom and in complete lung response on anti‐PD‐L1 treatment as a maintenance therapy.     

Retrospective detection of Porcine circovirus 3 (PCV‐3) in pig serum samples from Spain

Porcine circovirus 3 (PCV ‐3) is an emerging circovirus species that has recently been reported in different countries around the world, suggesting a widespread circulation. In this study, sera samples originating from 654 pigs of different production phases and clinical/pathological conditions, submitted for diagnostic purposes between 1996 and 2017, were randomly selected. Detection of PCV ‐3 genome in such samples was attempted with a previously described PCR method, and the partial genome sequence was obtained from selected PCV ‐3‐positive samples from different years. Compiled data confirmed that PCV ‐3 has been circulating in the Spanish pig population since 1996. The overall frequency of PCV ‐3 PCR ‐positive samples in the study period was 11.47% (75 of 654). Phylogenetic analysis of twelve PCV ‐3 partial sequences obtained showed a high nucleotide identity with the already known PCV ‐3 sequences, with minor variations among years. No significant correlation was found between the detection of PCV ‐3 and any production phase nor clinical/pathological condition. These results confirm PCV ‐3 circulation at least since 1996 in the Spanish pig population with a low/moderate frequency. Although the information obtained was limited, PCV ‐3 did not appear to be linked to any specific pathological condition or age group.     

Association of Insulin‐like Growth Factor‐1, Bone Mass and Inflammation to Low‐energy Distal Radius Fractures and Fracture Healing in Elderly Women Attending Emergency Care

Objective

Elderly patients suffer fractures through low‐energy mechanisms. The distal radius is the most frequent fracture localization. Insulin‐like growth factor‐1 (IGF1) plays an important role in the maintenance of bone mass and its levels decline with advancing age and in states of malnutrition. Our aim was to investigate the association of IGF1 levels, bone mass, nutritional status, and inflammation to low‐energy distal radius fractures and also study if fracture healing is influenced by IGF1, nutritional status, and inflammation.

Methods

Postmenopausal women, 55 years or older, with low‐energy distal radius fractures occurring due to falling on slippery ground, indoors or outdoors, were recruited in the emergency department (ED) and followed 1 and 5 weeks after the initial trauma with biomarkers for nutritional status and inflammation. Fractures were diagnosed according to standard procedure by physical examination and X‐ray. All patients were conservatively treated with plaster casts in the ED. Patients who needed interventions were excluded from our study. Fracture healing was evaluated from radiographs. Fracture healing assessment was made with a five‐point scale where the radiological assessment included callus formation, fracture line, and stage of union. Blood samples were taken within 24 h after fracture and analyzed in the routine laboratory. Bone mineral density (BMD) was measured by dual‐energy X‐ray absorptiometry (DXA).

Results

Thirty‐eight Caucasian women, aged 70.5 ± 8.9 years (mean ± SD) old, were recruited. Nutritional status, as evaluated by albumin (40.3 ± 3.1 g/L), IGF1 (125.3 ± 39.9 μg/L), body mass index (26.9 ± 3.6 kg/m²), arm diameter (28.9 ± 8.9 cm), and arm skinfold (2.5 ± 0.7 cm), was normal. A positive correlation was found between IGF1 at visit 1 and the lowest BMD for hip, spine, or radius (r = 0.39, P = 0.04). High sensitive C‐reactive protein (hsCRP) and leukocytes were higher at the fracture event compared to 5 weeks later (P = 0.07 and P < 0.001, respectively). Fracture healing parameters (i.e. callus formation, fracture line, and stage of union) were positively correlated with the initial leukocyte count and to difference in thrombocyte count between visit 1 and 3.

Conclusions

In elderly women with low‐energy distal radius fractures, an association between IGF1 and lowest measures of BMD was found, indicating that low IGF1 could be an indirect risk factor for fractures. Fracture healing was associated with initial leukocytosis and a lower thrombocyte count, suggesting that inflammation and thrombocytes are important components in fracture healing.

     

Suppression of osteoblast toll‐like receptor 2 signaling by endothelin‐1

Peripheral endothelin‐1 (ET‐1) levels are increased in chronic systemic disorders such as congestive cardiac failure, diabetes and chronic renal failure. Bone infections are also associated with poor prognoses in these conditions. In the present study, we examined the alterations in Toll‐like receptor 2 (TLR2) signaling induced by ET‐1 in an in vitro osteoblast cell model. The TLR2‐positive murine osteoblast cell line MC3T3‐E1 was treated with heat‐killed Listeria monocytogenes (HKLM), a TLR2 ligand, in the presence or absence of ET‐1. We examined TLR2 expression, intranuclear NF‐κB phosphorylation and interleukin 6 (IL‐6) production. ET‐1 suppressed cell surface expression of TLR2, NF‐κB phosphorylation and IL‐6 production. As TLR2 represents an important mechanism by which osteoblasts recognize bacterial pathogens, a continuously elevated ET‐1 status may impair pathogenic recognition by osteoblasts and consequently affect bone metabolism during infections. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:910–914, 2014.     

HoBi‐like is the most prevalent ruminant pestivirus in Northeastern Brazil

The ruminant pestiviral species BVDV ‐1, BVDV ‐2 and BDV , along with the putative species HoBi‐like, may cause substantial economic losses in cattle, sheep and goats. Brazil's large size, variable biomes and wide range of ruminant animal production within different geographic regions suggest that the presence and prevalence of ruminant pestivirus may differ by regions within Brazil. This study investigated the genetic diversity of ruminant pestiviruses and determined the frequency of active infections within two states of the Northeast Region of Brazil, Maranhão and Rio Grande do Norte. Serum samples from 16,621 cattle and 2,672 small ruminants from 569 different herds residing in this region were tested by RT ‐PCR followed by DNA sequencing. Seventeen positive cattle were detected (0.1%) from fifteen different herds (2.64%). All isolates were classified as HoBi‐like pestiviruses based on phylogenetic analysis. All small ruminant samples tested negative. The findings presented herein suggest that the Northeast Region of Brazil has a uniquely high prevalence of HoBi‐like viruses. The increasing reports of HoBi‐like viruses detected in cattle in the field suggest that natural infection with these viruses may be more widespread than previously thought. The identification of HoBi‐like viruses as the most prevalent type of ruminant pestivirus circulating in the Northeast Region of Brazil indicates the need for both continued monitoring and determination of the extent of economic losses associated with HoBi‐like virus infections. In addition, it must be taken into account in the choice of diagnostic tests and in vaccine formulations.     

Bluetongue virus serotype 27: Experimental infection of goats,sheep and cattle with three BTV‐27 variants reveal atypical characteristics and likely direct contact transmission BTV‐27 between goats

Bluetongue virus (BTV) hitherto consisted of 26 recognized serotypes, of which all except BTV‐26 are primarily transmitted by certain species of Culicoides biting midges. Three variants of an additional 27th bluetongue virus serotype (BTV‐27v01‐v03) were recently detected in asymptomatic goats in Corsica, France, 2014–2015. Molecular characterization revealed genetic differences between the three variants. Therefore, in vivo characteristics were investigated by experimental infection of a total of 15 goats, 11 sheep and 4 cattle with any one of the three variants in separated animal trials. In goat trials, BTV‐naïve animals of the same species were kept in a facility where direct contact was unhindered. Of the 15 inoculated goats, 13 and 14 animals were found positive for BTV‐RNA and antibodies (Ab), respectively, until the end of the experiments. Surprisingly, BTV‐Ab levels as measured with ELISA and neutralization test (SNT) were remarkably low in all seropositive goats. Virus isolation from whole‐blood was possible at the peak of viremia until 49 dpi. Moreover, detection of BTV‐27v02‐RNA and Ab in one contact goat indicated that—similar to BTV‐26—at least one of three BTV‐27 variants may be transmitted by contact between goats. In the field, BTV‐27 RNA can be detected up to 6 months in the whole‐blood of BTV‐27‐infected Corsican goats. In contrast, BTV RNA was not detected in the blood of cattle or sheep. In addition, BTV‐27 Abs were not detected in cattle and only a transient increase in Ab levels was observed in some sheep. None of the 30 animals showed obvious BT‐like clinical signs. In summary, the phenotypes observed for BTV‐27v01‐v03 phenotypes correspond to a mixture of characteristics known for BTV‐25 and 26.