Loss of Tankyrase-mediated destruction of 3BP2 is the underlying pathogenic mechanism of cherubism.

Cherubism is an autosomal-dominant syndrome characterized by inflammatory destructive bony lesions resulting in symmetrical deformities of the facial bones. Cherubism is caused by mutations in Sh3bp2, the gene that encodes the adaptor protein 3BP2. Most identified mutations in 3BP2 lie within the peptide sequence RSPPDG. A mouse model of cherubism develops hyperactive bone-remodeling osteoclasts and systemic inflammation characterized by expansion of the myelomonocytic lineage. The mechanism by which cherubism mutations alter 3BP2 function has remained obscure. Here we show that Tankyrase, a member of the poly(ADP-ribose)polymerase (PARP) family, regulates 3BP2 stability through ADP-ribosylation and subsequent ubiquitylation by the E3-ubiquitin ligase RNF146 in osteoclasts. Cherubism mutations uncouple 3BP2 from Tankyrase-mediated protein destruction, which results in its stabilization and subsequent hyperactivation of the SRC, SYK, and VAV signaling pathways.

Identification of ICAT as an APC Inhibitor, Revealing Wnt-Dependent Inhibition of APC-Axin Interaction.

Adenomatous polyposis coli (APC) and Axin are core components of the ß-catenin destruction complex. How APC's function is regulated and whether Wnt signaling influences the direct APC-Axin interaction to inhibit the ß-catenin destruction complex is not clear. Through a CRISPR screen of ß-catenin stability, we have identified ICAT, a polypeptide previously known to block ß-catenin-TCF interaction, as a natural inhibitor of APC. ICAT blocks ß-catenin-APC interaction and prevents ß-catenin-mediated APC-Axin interaction, enhancing stabilization of ß-catenin in cells harboring truncated APC or stimulated with Wnt, but not in cells deprived of a Wnt signal. Using ICAT as a tool to disengage ß-catenin-mediated APC-Axin interaction, we demonstrate that Wnt quickly inhibits the direct interaction between APC and Axin. Our study highlights an important scaffolding function of ß-catenin in the assembly of the destruction complex and suggests Wnt-inhibited APC-Axin interaction as a mechanism of Wnt-dependent inhibition of the destruction complex.

The SIAH E3 ubiquitin ligases promote Wnt/ß-catenin signaling through mediating Wnt-induced Axin degradation.

The Wnt/ß-catenin signaling pathway plays essential roles in embryonic development and adult tissue homeostasis. Axin is a concentration-limiting factor responsible for the formation of the ß-catenin destruction complex. Wnt signaling itself promotes the degradation of Axin. However, the underlying molecular mechanism and biological relevance of this targeting of Axin have not been elucidated. Here, we identify SIAH1/2 (SIAH) as the E3 ligase mediating Wnt-induced Axin degradation. SIAH proteins promote the ubiquitination and proteasomal degradation of Axin through interacting with a VxP motif in the GSK3-binding domain of Axin, and this function of SIAH is counteracted by GSK3 binding to Axin. Structural analysis reveals that the Axin segment responsible for SIAH binding is also involved in GSK3 binding but adopts distinct conformations in Axin/SIAH and Axin/GSK3 complexes. Knockout of SIAH1 blocks Wnt-induced Axin ubiquitination and attenuates Wnt-induced ß-catenin stabilization. Our data suggest that Wnt-induced dissociation of the Axin/GSK3 complex allows SIAH to interact with Axin not associated with GSK3 and promote its degradation and that SIAH-mediated Axin degradation represents an important feed-forward mechanism to achieve sustained Wnt/ß-catenin signaling.

Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin.

Cross-species transmission of viruses from wildlife animal reservoirs poses a marked threat to human and animal health 1 . Bats have been recognized as one of the most important reservoirs for emerging viruses and the transmission of a coronavirus that originated in bats to humans via intermediate hosts was responsible for the high-impact emerging zoonosis, severe acute respiratory syndrome (SARS) 2-10 . Here we provide virological, epidemiological, evolutionary and experimental evidence that a novel HKU2-related bat coronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), is the aetiological agent that was responsible for a large-scale outbreak of fatal disease in pigs in China that has caused the death of 24,693 piglets across four farms. Notably, the outbreak began in Guangdong province in the vicinity of the origin of the SARS pandemic. Furthermore, we identified SADS-related CoVs with 96-98% sequence identity in 9.8% (58 out of 591) of anal swabs collected from bats in Guangdong province during 2013-2016, predominantly in horseshoe bats (Rhinolophus spp.) that are known reservoirs of SARS-related CoVs. We found that there were striking similarities between the SADS and SARS outbreaks in geographical, temporal, ecological and aetiological settings. This study highlights the importance of identifying coronavirus diversity and distribution in bats to mitigate future outbreaks that could threaten livestock, public health and economic growth.

The establishment of a recombinase polymerase amplification technique for the detection of mouse poxvirus.

BACKGROUND: Ectromelia virus (ECTV) is the causative agent of mousepox in mice. In the past century, ECTV was a serious threat to laboratory mouse colonies worldwide. Recombinase polymerase amplification (RPA), which is widely used in virus detection, is an isothermal amplification method. RESULTS: In this study, a probe-based RPA detection method was established for rapid and sensitive detection of ECTV.Primers were designed for the highly conserved region of the crmD gene, the main core protein of recessive poxvirus, and standard plasmids were constructed. The lowest detection limit of the ECTV RT- RPA assay was 100 copies of DNA mol-ecules per reaction. In addition, the method showed high specificity and did not cross-react with other common mouse viruses.Therefore, the practicability of the RPA method in the field was confirmed by the detection of 135 clinical samples. The real-time RPA assay was very similar to the ECTV real-time PCR assay, with 100% agreement. CONCLUSIONS: In conclusion, this RPA assay offers a novel alternative for the simple, sensitive, and specific identification of ECTV, especially in low-resource settings.

Dishevelled promotes Wnt receptor degradation through recruitment of ZNRF3/RNF43 E3 ubiquitin ligases.

Tumor suppressors ZNRF3 and RNF43 inhibit Wnt signaling through promoting degradation of Wnt coreceptors Frizzled (FZD) and LRP6, and this activity is counteracted by stem cell growth factor R-spondin. The mechanism by which ZNRF3 and RNF43 recognize Wnt receptors remains unclear. Here we uncover an unexpected role of Dishevelled (DVL), a positive Wnt regulator, in promoting Wnt receptor degradation. DVL knockout cells have significantly increased cell surface levels of FZD and LRP6. DVL is required for ZNRF3/RNF43-mediated ubiquitination and degradation of FZD. Physical interaction with DVL is essential for the Wnt inhibitory activity of ZNRF3/RNF43. Binding of FZD through the DEP domain of DVL is required for DVL-mediated downregulation of FZD. Fusion of the DEP domain to ZNRF3/RNF43 overcomes their DVL dependency to downregulate FZD. Our study reveals DVL as a dual function adaptor to recruit negative regulators ZNRF3/RNF43 to Wnt receptors to ensure proper control of pathway activity.

Clinical outcomes and affecting factors of ipsilateral femoral neck and shaft fractures - Multination, multicenter analysis.

BACKGROUND: This study aimed to evaluate the clinical outcomes of ipsilateral femoral neck and shaft fractures and identify the risk factors associated with missed diagnosis of femoral neck fractures and clinical outcomes of this fracture. METHODS: The ipsilateral femoral neck and shaft fractures from seven centers were retrospectively reviewed. Data on injury mechanism, fracture pattern, and fracture classification; surgical factors including fixation method; and timing of detection of femoral neck fracture were analyzed. The clinical outcomes, complications, and the incidence of avascular necrosis of the femoral head (AVNFH) were reviewed. Risk factors for missed femoral neck fracture and complications were analyzed. RESULTS: In total, 74 patients with an average age of 43.6 years were included. Of the femoral shaft fractures, 56.8% were type A, 21.6% were type B, and 21.6% were type C. Sixteen patients had an open fracture of the femoral shaft. Femoral neck fracture was initially missed in 27% patients and the timing of delayed diagnosis was at an average of 11.1 days after injury. For detecting femoral neck fractures, minimal displacement of the femoral neck fracture was a risk factor, whereas computed tomography (CT) was a protective factor. The incidence of AVNFH was 6.8% at an average of 36.8 months after injury. The AVNFH group had more displaced femoral neck fractures at the time of surgery, but there was no difference in the timing of diagnosis compared to non-AVNFH group. The femoral shaft showed considerable healing problems, with an average union time of 29.7 weeks and a 20.2% nonunion rate. CONCLUSION: Ipsilateral femoral neck and shaft fractures had a high rate of missed diagnosis, especially in minimally displaced fractures; however, CT was a protective factor. AVNFH occurred in 6.8% and was related to femoral neck fracture displacement, but not delayed diagnosis. The femur nonunion rate was high, which warrants attention.

De novo variants in SIAH1, encoding an E3 ubiquitin ligase, are associated with developmental delay, hypotonia and dysmorphic features.

BACKGROUND: Ubiquitination has a central role in numerous biological processes, including cell development, stress responses and ageing. Perturbed ubiquitination has been implicated in human diseases ranging from cancer to neurodegenerative diseases. SIAH1 encodes a RING-type E3 ubiquitin ligase involved in protein ubiquitination. Among numerous other roles, SIAH1 regulates metabotropic glutamate receptor signalling and affects neural cell fate. Moreover, SIAH1 positively regulates Wnt signalling through ubiquitin-mediated degradation of Axin and accumulation of ß-catenin. METHODS: Trio exome sequencing followed by Sanger validation was undertaken in five individuals with syndromic developmental delay. Three-dimensional structural modelling was used to predict pathogenicity of affected residues. Wnt stimulatory activity was measured by luciferase reporter assays and Axin degradation assays in HEK293 cells transfected with wild-type and mutant SIAH1 expression plasmids. RESULTS: We report five unrelated individuals with shared features of developmental delay, infantile hypotonia, dysmorphic features and laryngomalacia, in whom exome sequencing identified de novo monoallelic variants in SIAH1. In silico protein modelling suggested alteration of conserved functional sites. In vitro experiments demonstrated loss of Wnt stimulatory activity with the SIAH1 mutants, suggesting variant pathogenicity. CONCLUSION: Our results lend support to SIAH1 as a candidate Mendelian disease gene for a recognisable syndrome, further strengthening the connection between SIAH1 and neurodevelopmental disorders. Furthermore, the results suggest that dysregulation of the Wnt/ß-catenin pathway may be involved in the pathogenesis.

Allosteric activation of the RNF146 ubiquitin ligase by a poly(ADP-ribosyl)ation signal.

Protein poly(ADP-ribosyl)ation (PARylation) has a role in diverse cellular processes such as DNA repair, transcription, Wnt signalling, and cell death. Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several crucial regulatory proteins, including Axin and 3BP2 (refs 7, 8, 9). The RING-type E3 ubiquitin ligase RNF146 (also known as Iduna) is responsible for PARylation-dependent ubiquitination (PARdU). Here we provide a structural basis for RNF146-catalysed PARdU and how PARdU specificity is achieved. First, we show that iso-ADP-ribose (iso-ADPr), the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to bind and activate a ubiquitin-conjugating enzyme (E2) efficiently. Binding of PAR or iso-ADPr induces a major conformational change that creates a functional RING structure. Thus, RNF146 represents a new mechanistic class of RING E3 ligases, the activities of which are regulated by non-covalent ligand binding, and that may provide a template for designing inducible protein-degradation systems. Second, we find that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146-TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction. We propose that the maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146-TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell.

Molecular characterization of the FCoV-like canine coronavirus HLJ-071 in China.

BACKGROUND: According to the differences of antigen and genetic composition, canine coronavirus (CCoV) consists of two genotypes, CCoV-I and CCoV-II. Since 2004, CCoVs with point mutations or deletions of NSPs are contributing to the changes in tropism and virulence in dogs. RESULTS: In this study, we isolated a CCoV, designated HLJ-071, from a dead 5-week-old female Welsh Corgi with severe diarrhea and vomit. Sequence analysis suggested that HLJ-071 bearing a complete ORF3abc compared with classic CCoV isolates (1-71, K378 and S378). In addition, a variable region was located between S gene and ORF 3a gene, in which a deletion with 104 nts for HLJ-071 when compared with classic CCoV strains 1-71, S378 and K378. Phylogenetic analysis based on the S gene and complete sequences showed that HLJ-071 was closely related to FCoV II. Recombination analysis suggested that HLJ-071 originated from the recombination of FCoV 79-1683, FCoV DF2 and CCoV A76. Finally, according to cell tropism experiments, it suggested that HLJ-071 could replicate in canine macrophages/monocytes cells. CONCLUSION: The present study involved the isolation and genetic characterization of a variant CCoV strain and spike protein and ORF3abc of CCoV might play a key role in viral tropism, which could affect the replication in monocyte/macrophage cells. It will provide essential information for further understanding the evolution in China.

mTORC1 signaling suppresses Wnt/ß-catenin signaling through DVL-dependent regulation of Wnt receptor FZD level.

Wnt/ß-catenin signaling plays pivotal roles in cell proliferation and tissue homeostasis by maintaining somatic stem cell functions. The mammalian target of rapamycin (mTOR) signaling functions as an integrative rheostat that orchestrates various cellular and metabolic activities that shape tissue homeostasis. Whether these two fundamental signaling pathways couple to exert physiological functions still remains mysterious. Using a genome-wide CRISPR-Cas9 screening, we discover that mTOR complex 1 (mTORC1) signaling suppresses canonical Wnt/ß-catenin signaling. Deficiency in tuberous sclerosis complex 1/2 (TSC1/2), core negative regulators of mTORC1 activity, represses Wnt/ß-catenin target gene expression, which can be rescued by RAD001. Mechanistically, mTORC1 signaling regulates the cell surface level of Wnt receptor Frizzled (FZD) in a Dishevelled (DVL)-dependent manner by influencing the association of DVL and clathrin AP-2 adaptor. Sustained mTORC1 activation impairs Wnt/ß-catenin signaling and causes loss of stemness in intestinal organoids ex vivo and primitive intestinal progenitors in vivo. Wnt/ß-catenin-dependent liver metabolic zonation gene expression program is also down-regulated by mTORC1 activation. Our study provides a paradigm that mTORC1 signaling cell autonomously regulates Wnt/ß-catenin pathway to influence stem cell maintenance.

ZNRF3 functions in mammalian sex determination by inhibiting canonical WNT signaling.

Mammalian sex determination is controlled by the antagonistic interactions of two genetic pathways: The SRY-SOX9-FGF9 network promotes testis determination partly by opposing proovarian pathways, while RSPO1/WNT-ß-catenin/FOXL2 signals control ovary development by inhibiting SRY-SOX9-FGF9. The molecular basis of this mutual antagonism is unclear. Here we show that ZNRF3, a WNT signaling antagonist and direct target of RSPO1-mediated inhibition, is required for sex determination in mice. XY mice lacking ZNRF3 exhibit complete or partial gonadal sex reversal, or related defects. These abnormalities are associated with ectopic WNT/ß-catenin activity and reduced Sox9 expression during fetal sex determination. Using exome sequencing of individuals with 46,XY disorders of sex development, we identified three human ZNRF3 variants in very rare cases of XY female presentation. We tested two missense variants and show that these disrupt ZNRF3 activity in both human cell lines and zebrafish embryo assays. Our data identify a testis-determining function for ZNRF3 and indicate a mechanism of direct molecular interaction between two mutually antagonistic organogenetic pathways.

Novel Regulation of Wnt Signaling at the Proximal Membrane Level.

Wnt pathways are crucial for embryonic development and adult tissue homeostasis in all multicellular animals. Our understanding of Wnt signaling networks has grown increasingly complex. Recent studies have revealed many regulatory proteins that function at the proximal membrane level to fine-tune signaling output and enhance signaling specificity. These proteins regulate crucial points in Wnt signaling, including post-translational modification of Wnt proteins, regulation of Wnt receptor degradation, internalization of Wnt receptor complex, and specific ligand-receptor complex formation. Such regulators not only provide us with molecular details of Wnt regulation but also serve as potential targets for therapeutic intervention. In this review we highlight new insights into Wnt regulation at the plasma membrane, especially newly identified feedback regulators.

Development of a recombinase polymerase amplification fluorescence assay to detect feline coronavirus.

Feline coronavirus (FCoV) is classified into two pathotypes: the avirulent feline enteric coronavirus (FECV), and the virulent feline infectious peritonitis virus (FIPV). Rapid pathogen detection, which is efficient and convenient, is the best approach for early confirmatory diagnosis. In this study, we first developed and evaluated a rapid recombinase polymerase amplification (RPA) detection method for FCoV that can detect FCoV within 15 min at 39 °C. The detection limit of that assay was 233 copies/µL DNA molecules per reaction. The specificity was high: it did not cross-react with canine distemper virus (CDV), canine coronavirus (CCoV), canine adenovirus (CAV), feline calicivirus (FCV), feline herpesvirus (FHV), or feline parvovirus (FPV). This assay was evaluated using 42 clinical samples (30 diarrhea samples and 12 ascites samples). The coincidence rate between FCoV-RPA and RT-qPCR for detection in clinical samples was 95.2%. In summary, FCoV-RPA analysis provides an efficient, rapid, and sensitive detection method for FCoV.

Application of recombinase polymerase amplification method for rapid detection of infectious laryngotracheitis virus.

Infectious laryngotracheitis is a significant respiratory disease of chickens that causes huge economic losses due to high morbidity and mortality and reduced egg production. A real-time recombinase polymerase amplification (RPA) assay was developed to accurately detect ILTV. The specific probe and primer sets were carefully designed and screened. The real-time RPA assay was carried out at 39 °C for 30 min, and results were obtained within 15 min. The results of the specificity assay showed no fluorescence signals with other avian-related viruses. The sensitivity of the assay was 1 × 102 copies/µL. The low CV value showed that the assay was reproducible. A total of 115 clinical samples were tested using the real-time RPA assay and the real-time PCR assay in parallel; the coincidence rates of the two detection methods were 100%. The results indicated that the real-time RPA assay is a specific, sensitive, rapid, and useful tool for epidemiological studies and clinical diagnosis, especially in the field and in resource-poor areas.

Development of a real-time reverse transcription recombinase polymerase amplification assay for rapid detection of spring viremia of carp virus.

Spring viremia of carp virus (SVCV) is a significant pathogenic agent that can cause large-scale outbreaks of spring viremia of carp (SVC) in many types of fish and bring huge economic losses to the aquaculture industry. A simple and convenient detection method is imperative for SVCV diagnosis. In this study, the real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and validated. Primers and probe targeting the conserved region of M gene were designed and applied to the real-time RT-RPA assay that performed at 39 °C for 20 min. The specificity analysis showed that no cross-reaction with other pathogenic viruses of fish was found, indicating appropriate specificity of the assay. In vitro transcribed RNA standards were used to estimate the sensitivity of the assay and the detection limit was 102copies/reaction. To further evaluate the assay, 65 clinical samples were tested using both real-time RT-RPA assay and real-time RT-PCR method. The same detection results were observed, suggesting the potential application of real-time RT-RPA assay in clinical sample detection. This is the first report on RPA assay for SVCV detection and this new developed assay would be useful in both laboratory and in the field for diagnosis of SVCV.

Recognition of the iso-ADP-ribose moiety in poly(ADP-ribose) by WWE domains suggests a general mechanism for poly(ADP-ribosyl)ation-dependent ubiquitination.

Protein poly(ADP-ribosyl)ation and ubiquitination are two key post-translational modifications regulating many biological processes. Through crystallographic and biochemical analysis, we show that the RNF146 WWE domain recognizes poly(ADP-ribose) (PAR) by interacting with iso-ADP-ribose (iso-ADPR), the smallest internal PAR structural unit containing the characteristic ribose-ribose glycosidic bond formed during poly(ADP-ribosyl)ation. The key iso-ADPR-binding residues we identified are highly conserved among WWE domains. Binding assays further demonstrate that PAR binding is a common function for the WWE domain family. Since many WWE domain-containing proteins are known E3 ubiquitin ligases, our results suggest that protein poly(ADP-ribosyl)ation may be a general mechanism to target proteins for ubiquitination.

Rapid and sensitive real-time recombinase polymerase amplification for detection of Marek's disease virus.

Marek's disease (MD) is one of the most devastating diseases of poultry. It's caused by the highly infectious alphaherpesvirus MD virus serotype 1 (MDV-1). In this study, a rapid and easy-to-use assay based on recombinase polymerase amplification (RPA) was developed for MDV detection. Primer-probe sets targeting the highly conserved region of Meq gene were designed and applied to the RPA assay. The assay was carried out on a real-time thermostatic fluorescence detector at 39 °C for 20 min. As revealed by the results, no cross-reactions were found with the Newcastle disease virus (NDV), chicken infectious anemia virus (CAV), infectious bursal disease virus (IBDV), avian infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avain influenza virus (AIV), avian leucosis virus (ALV), avian reovirus (ARV), Marek's disease virus serotype 2 (MDV-2) and turkey herpes virus (HVT), indicating appropriate specificity of the assay. Plasmid DNA standards were used to determine the sensitivity of the assay and the detection limit was 102copies/µL. To further evaluate the clinical performance, 94 clinical samples were subjected to the RPA assay and 28 samples were tested MDV positive, suggesting that the real-time RPA assay was sufficient enough for clinical sample detection. Thus, a highly specific and sensitive real-time RPA assay was established and validated as a candidate for MDV diagnosis. Additionally, the portability of real-time RPA assay makes it suitable to be potentially applied in clinical diagnosis in the field, especially in resource-limited settings.

High-throughput Luminex xMAP assay for simultaneous detection of antibodies against rabbit hemorrhagic disease virus, Sendai virus and rabbit rotavirus.

Rabbits are widely used as models in biological research, and the pathogen status of rabbits used in studies can directly affect the results of experiments. Serological surveillance is the common monitoring method used in laboratory animals. A rapid, sensitive, and cost-effective high-throughput Luminex xMAP assay could be an attractive alternative to labor-intensive enzyme-linked immunosorbent assay (ELISA) methods. In this study, recombinant proteins from rabbit hemorrhagic disease virus and rabbit rotavirus and whole viral lysates of Sendai virus were used as coating antigens in an xMAP assay for the simultaneous detection of antibodies against these pathogens. The xMAP assay showed high specificity, with no cross-reaction with other pathogens. The coefficient of variation for intra-assay and inter-assay comparisons was less than 3% and 4%, respectively, indicating good repeatability and stability of the assay. The xMAP assay exhibited similar limits of detection for rabbit hemorrhagic virus and Sendai virus and was less sensitive for the detection of rabbit rotavirus when compared with commercial ELISA kits. A total of 52 clinical samples were tested simultaneously using both the xMAP assay and ELISA kits. The results obtained using these two methods were 100% coincident. In summary, the novel xMAP assay offers an alternative choice for rapid and sensitive high-throughput detection of antibodies in rabbit serum and can be used as a daily monitoring tool for laboratory animals.

Development of a real-time loop-mediated isothermal amplification assay for detection of porcine circovirus 3.

BACKGROUND: Porcine circovirus type 3 (PCV3) is an emerging circovirus species, that has been reported in major pig-raising countries including the United States, China, South Korea, Brazil, Spain, and Poland. RESULTS: A real-time loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of porcine circovirus 3 (PCV3). The method had a detection limit of 1 × 101 copies/µL with no cross-reactions with classical swine fever virus (CSFV) C strain, foot-and-mouth disease virus (FMDV), porcine circovirus 2 (PCV2) LG vaccine strain, porcine epidemic diarrhoea virus (PEDV), porcine respiratory and reproductive syndrome virus (PRRSV), or pseudorabies virus (PRV). The PCV3 positive detection rate of 203 clinical samples for the real-time LAMP assay was 89.66% (182/203). CONCLUSIONS: The real-time LAMP assay is highly sensitive, and specific for use in epidemiological investigations of PCV3.