Dunaliella salina alga extract inhibits the production of interleukin-6, nitric oxide, and reactive oxygen species by regulating nuclear factor-κB/Janus kinase/signal transducer and activator of transcription in virus-infected RAW264.7 cells.

Recent investigations have demonstrated that carotenoid extract of Dunaliella salina alga (Alga) contains abundant ß-carotene and has good anti-inflammatory activities. Murine macrophage (RAW264.7 cells) was used to establish as an in vitro model of pseudorabies virus-induced reactive oxygen species (ROS) response. In this study, antioxidant activities of Alga were measured based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, trolox equivalent antioxidant capacity assays, reducing power, and virus-induced ROS formation in RAW264.7 cells. Anti-inflammatory activities of Alga were assessed by its ability to inhibit the production of interleukin-6 and nitric oxide (NO) using enzyme-linked immunosorbent assay, then the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway was investigated by measuring the inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-κB (p50 and p65), JAK, STAT-1/3, and suppressor of cytokine signaling 3 (SOCS3) by Western blotting. In addition, Alga inhibited virus replication by plaque assay. Our results showed that the Alga had high antioxidant activity, significantly reduced the virus-induced accumulation of ROS, and inhibited the levels of nitric oxide and interleukin-6. Further studies revealed that Alga also downregulated the gene and protein expressions of iNOS, COX-2, nuclear factor-κB (p50 and p65), and the JAK/STAT pathway. The inhibitory effects of Alga were similar to pretreatment with specific inhibitors of JAK and STAT-3 in pseudorabies virus -infected RAW264.7 cells. Alga enhanced the expression of SOCS3 to suppress the activity of the JAK/STAT signaling pathway in pseudorabies virus-infected RAW264.7 cells. In addition, Alga has decreased viral replication (p < 0.005) at an early stage. Therefore, our results demonstrate that Alga inhibits ROS, interleukin6, and nitric oxide production via suppression of the JAK/STAT pathways and enhanced the expression of SOCS3 in virus-infected RAW264.7 cells.

Luteolin inhibits viral-induced inflammatory response in RAW264.7 cells via suppression of STAT1/3 dependent NF-κB and activation of HO-1.

Luteolin is a common dietary flavonoid present in Chinese herbal medicines that has been reported to have important anti-inflammatory properties. Previous studies have shown that luteolin is an anti-inflammatory and anti-oxidative agent. In this study, the anti-virus inflammatory capacity of luteolin and its molecular mechanisms of action were analyzed. The cytotoxic effects of luteolin were assessed in the presence or absence of pseudorabies virus (PRV) via LDH and MTT assays. The results showed that luteolin (<10µM) had no toxic effects and there were tendencies toward higher cell survival. In PRV-infected RAW264.7 cells, luteolin potently inhibited the production of NO, iNOS, COX-2 and inflammatory cytokine production. Luteolin did not inhibit the phosphorylation of ERK 1/2, p38, and JNK 1/2 either. We found that PRV-induced NF-κB activation is regulated through inhibition of STAT1and STAT3 phosphorylation in response to luteolin. Additionally, luteolin caused the induction of HO-1 via upregulation of Nrf2, both of which are involved in the secretion of proinflammatory mediators. The blockade of HO-1 expression with SnPP, a HO-1 inhibitor, attenuated HO-1 induction by luteolin and thus mitigated its anti-inflammatory effects during PRV-infected RAW264.7 cells. Taken together, our data indicate that luteolin diminishes the proinflammatory mediators NO, inflammatory cytokines and the expression of their regulatory genes, iNOS and COX-2, in PRV-infected RAW264.7 cells by inhibiting STAT1/3 dependent NF-κB activation and inducing Nrf2mediated HO-1 expression.

Roles of nucleic acid substrates and cofactors in the vhs protein activity of pseudorabies virus.

Pseudorabies virus (PrV) belongs to the α-herpesvirinae of which human simplex virus (HSV) is the prototype virus. One of the hallmarks of HSV infection is shutoff of protein synthesis that is mediated by various viral proteins including vhs (virion host shutoff), which is encoded by the UL41 gene. However, the function of PrV vhs is poorly understood. Due to the low sequence similarity (39.3%) between the HSV and PrV UL41 proteins, vhs might not share the same biochemistry characteristics. The purpose of this study was to characterize the nuclease activity of the PrV vhs protein with respect to substrate specificity, its requirements in terms of cofactors, and the protein regions, as well as key amino acids, which contribute to vhs activity. Our results indicated that, similar to HSV vhs, PrV vhs is able to degrade ssRNA and mRNA. However, PrV vhs also targeted rRNA for degradation, which is novel compared to the HSV-1 vhs. Activity assays indicated that Mg(2+) alone enhances RNA degradation mediated by PrV vhs, while K(+) and ATP are not sufficient to induce activity. Finally, we demonstrated that each of the four highly conserved functional boxes of PrV vhs contributes to RNA degradation and that, in particular, residues 152, 169, 171, 172, 173 343, 345, 352 and 356, which are conserved among α-herpesviruses, are key amino acids needed for PrV vhs ribonuclease activity.

Regulation of virus-induced inflammatory response by Dunaliella salina alga extract in macrophages.

Previous reports have suggested that many constituents within various algal samples are able to attenuate LPS-induced inflammatory effects. To date no report has been published on the regulation of virus-induced inflammatory response of Dunaliella salina carotenoid extract. In the present study, the anti-inflammatory effect of D. salina carotenoid extract on pseudorabies virus (PRV)-infected RAW 264.7 macrophages was investigated. We evaluated the anti-inflammatory effect of D. salina carotenoid extract on PRV-infected RAW 264.7 cells by measuring cell viability, cytotoxicity, production of inflammatory mediators such as NO, iNOS, COX-2, pro-inflammatory cytokines and anti-virus replication by plaque assay. We found down-regulation of the expression of the iNOS, COX-2 and pro-inflammatory genes IL-1ß, IL-6, TNF-α, and MCP-1 in a dose-dependent manner. Although there was no effect on viral replication, there were tendencies toward lower virus titer and tendencies toward higher cell survival. Most importantly, we found that inhibition of TLR9, PI3K and Akt phosphorylation plays a crucial role in the extract-mediated NF-κB regulation by modulating IKK-IκB signaling in PRV-infected RAW264.7 cells. These results indicate that D. salina carotenoid extracts inhibited inflammation by inhibition of NF-κB activation by TLR9 dependent via PI3K/Akt inactivation.

Translational enhancing activity in 5' UTR of peste des petits ruminants virus fusion gene.

The fusion gene of peste des petits ruminants virus (PPRV-F), a paramyxovirus, contains an unusual long 5' untranslated region (5' UTR) with a high GC content that is capable of folding into secondary structure proximally to the 5' cap. Sequence analysis further suggested that the proximal end of this UTR contains a nine-nucleotide sequence which could perfectly complement the 18S rRNA and might affect translation through mRNA-rRNA interaction. Based on these features, we examined the functional role of the proximal PPRV-F 5' UTR on translational efficiency compared with two other morbilliviruses. From reporter gene assays, PPRV-F 5' UTR functioned as a strong enhancer of translational efficiency independent of cell and gene specificity. Northern blot analysis of the accumulative RNA levels and mRNA stability suggested that elevated gene expression driven by PPRV-F 5' UTR was accompanied by an increased mRNA level and enhanced mRNA stability. Deletion analysis identified the complementary sequence and distal nucleotides necessary for the enhancing activity, and results suggest RNA structural conformation is important. Taken together, we conclude that the proximal PPRV-F 5' UTR functions as a translational enhancer by promoting translation efficiency and mRNA stability.

Application of purified recombinant antigenic spike fragments to the diagnosis of avian infectious bronchitis virus infection.

The spike (S) protein, containing two subunits, S1 and S2, is the major immunity-eliciting antigen of avian infectious bronchitis virus (IBV), a highly contagious disease of chickens. Several immunogenic regions, mainly located within the S1 subunit, have been identified. Nonetheless, these immune-dominant regions were defined using selected monoclonal antibodies or using a short peptide approach that involves only certain limited regions of the S protein. In addition, some immune-dominant regions are located in hypervariable regions (HVRs) which are not present in all serotypes. Hence, the aim of this study was to determine a broader range of antigenic regions that have strong antibody eliciting ability; these could then be applied for development of an IBV-diagnostic tool. Initially, the S1 and part of the S2 subunit protein (24-567 amino acids) were expressed as five fragments in prokaryotic system. The antigenicity was confirmed using IBV immunized sera. Performance of the S subfragments was evaluated by ELISA using a panel of field chicken sera with known IBV titres determined by a commercial kit. This indicated that, among the five antigenic recombinant proteins, the region S-E showed the highest specificity and sensitivity, namely 95.38 % and 96.29 %, respectively. The κ value for the in-house ELISA using the S-E fragment compared to a commercial kit was 0.9172, indicating a high agreement between these two methods. As region S-E harbors strong immunogenicity within the spike protein, it has the potential to be exploited as an antigen when developing a cost-effective ELISA-based diagnosis tool.

Avian reovirus sigma C enhances the mucosal and systemic immune responses elicited by antigen-conjugated lactic acid bacteria.

Mucosal surfaces are common sites of pathogen colonization/entry. Effective mucosal immunity by vaccination should provide protection at this primary infection site. Our aim was to develop a new vaccination strategy that elicits a mucosal immune response. A new strain of Enterococcus faecium, a non pathogenic lactic acid bacteria (LAB) with strong cell adhesion ability, was identified and used as a vaccine vector to deliver two model antigens. Specifically, sigma (σ) C protein of avian reovirus (ARV), a functional homolog of mammalian reovirus σ1 protein and responsible for M-cell targeting, was administered together with a subfragment of the spike protein of infectious bronchitis virus (IBV). Next, the effect of immunization route on the immune response was assessed by delivering the antigens via the LAB strain. Intranasal (IN) immunization induced stronger humoral responses than intragastic (IG) immunization. IN immunization produced antigen specific IgA both systemically and in the lungs. A higher IgA titer was induced by the LAB with ARV σC protein attached. Moreover, the serum of mice immunized with LAB displaying divalent antigens had much stronger immune reactivity against ARV σC protein compared to IBV-S1. Our results indicate that ARV σC protein delivered by LAB via the IN route elicits strong mucosal immunity. A needle-free delivery approach is a convenient and cost effective method of vaccine administration, especially for respiratory infections in economic animals. Furthermore, ARV σC, a strong immunogen of ARV, may be able to serve as an immunoenhancer for other vaccines, especially avian vaccines.

Regulation of virus-induced inflammatory response by ß-carotene in RAW264.7 cells.

Carotenoids are effective antioxidants, which can quench singlet oxygen, suppress lipid peroxidation, and prevent oxidative damage. Both Pseudorabies virus (PRV) and human Herpes simplex virus (HSV) are DNA viruses, and their pathogenesis and immunobiology are similar. However, PRV does not infect humans. Therefore, PRV was used to infect murine macrophages (RAW264.7 cells), to mimic HSV-induced inflammation. Meanwhile, the influence of ß-carotene on PRV-induced inflammation was also investigated. Results indicated that ß-carotene inhibited (p<0.05) NO, IL-1ß, IL-6, and MCP-1 production in PRV-infected RAW264.7 cells. ß-Carotene also suppressed (p<0.05) NF-κB (p50 and p65), phosphorylation of extracellular-signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) expression. It could be concluded that the anti-inflammatory effect of ß-carotene is mainly through a suppression of cytokine expression in PRV-induced inflammation, which results from NF-κB inactivation. ß-Carotene can be considered a potential anti-inflammatory agent for DNA-virus infection.

Territorial behavior in Taiwanese kukrisnakes (Oligodon formosanus).

The independent evolutionary origin of a complex trait, within a lineage otherwise lacking it, provides a powerful opportunity to test hypotheses on selective forces. Territorial defense of an area containing resources (such as food or shelter) is widespread in lizards but not snakes. Our studies on an insular population of Taiwanese kukrisnakes (Oligodon formosanus) show that females of this species actively defend sea turtle nests by repelling conspecifics for long periods (weeks) until the turtle eggs hatch or are consumed. A clutch of turtle eggs comprises a large, long-lasting food resource, unlike the prey types exploited by other types of snakes. Snakes of this species have formidable weaponry (massively enlarged teeth that are used for slitting eggshells), and when threatened, these snakes wave their tails toward the aggressor (an apparent case of head-tail mimicry). Bites to the tail during intraspecific combat bouts thus can have high fitness costs for males (because the hemipenes are housed in the tail). In combination, unusual features of the species (ability to inflict severe damage to male conspecifics) and the local environment (a persistent prey resource, large relative to the snakes consuming it) render resource defense both feasible and advantageous for female kukrisnakes. The (apparently unique) evolution of territorial behavior in this snake species thus provides strong support for the hypothesis that resource defensibility is critical to the evolution of territoriality.

Attenuation of nitric oxide bioavailability in porcine aortic endothelial cells by classical swine fever virus.

Classical swine fever (CSF) causes severe disease in pigs, characterized by hemorrhage, fever, and leucopenia. A primary target of the virus is endothelial cells, where a pro-inflammatory and pro-coagulant response occurs with downregulation of gap junctional communication; these changes establish a basis for haemostatic imbalance. The aim of this study was to gain an understanding of the effect of classical swine fever virus (CSFV) on endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability. Porcine aortic endothelial cells (PAECs) were infected with CSFV at different multiplicity of infection (M.O.I.) for 48 h. Downregulation of the transcription and translation levels of eNOS was detected by semi-quantitative RT-PCR, immunoconfocal microscopy, and western blotting. This was accompanied by a reduction in NO bioavailability and attenuation of angiogenesis. Without influence from the progeny virus titer, the decrease in eNOS protein was reversed by an ERK inhibitor (PD98059) and two PI3/Akt inhibitors (LY294002 and wortmannin). In addition, we found that the transcription factors AP1, Sp1, and GATA1/2 may be involved in the downregulation of eNOS promoter activity. In conclusion, infection of PAECs with CSFV attenuated the expression of eNOS and reduced NO bioavailability through activation of the ERK and PI3/Akt pathways.

Surgical treatment of Kawasaki disease with intestinal pseudo-obstruction.

A 5-year-old boy suffering from abdominal pain accompanied by a fever of up to 39.5°C for 2 days was admitted to the hospital. Although Flomoxef was administered following admission, the boy's fever persisted and abdominal distension gradually worsened. On the 4th day, dry lips, red eyes and a strawberry tongue were noted. An echocardiogram revealed pericoronary enhancement with mild mitral valve regurgitation and a small degree of pericardial effusion, characteristics compatible with Kawasaki disease. Although intravenous immunoglobulin was administered, the fever and abdominal distension persisted. On the 8th day, a pediatric surgeon was consulted and an exploratory laparotomy was arranged. During the operation, intestinal pseudo-obstruction and fibrin coatings around the intestine near the splenic flexure were found. A colostomy was performed for decompression of the dilated bowel and a biopsy of the lymph node surrounding the splenic flexure was taken. The fever subsided dramatically after decompression of the bowel and the recovery course was uneventful. The pathologic report revealed necrotic lymphadenitis. We report this rare case and review the literature.

Comparison of two different screening methods for the KRAS mutation in colorectal cancer.

BACKGROUND: To compare high resolution melting (HRM) and primer extension (PE) for mutation screening of KRAS codons 12 and 13. METHODS: DNA samples were isolated from 60 colorectal cancer (CRC) patients. HRM and primer extension analyses were used for mutation screening of KRAS codons 12 and 13. RESULTS: Both methods can detect KRAS mutations in a DNA mixture containing as little as 5% mutant DNA. The concordant rate between the two methods was 100%. HRM analysis is able to distinguish mutant from wild type, however, it is unable to detect the actual base change. PE analysis needs more procedures, is time-consuming and slightly more expensive but it is able to detect the precise mutation. CONCLUSIONS: This study showed that HRM is a reliable screening method to identify mutations in the KRAS gene, and the PE can be used as a diagnostic method to accurately pinpoint the nature of mutations.

Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) for diagnosis of natural infection with canine distemper virus.

BACKGROUND: Canine distemper virus (CDV) is present worldwide and produces a lethal systemic infection of wild and domestic Canidae. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR) of the matrix (M) gene to the fusion (F) gene (designated M-F UTR) in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions. RESULTS: Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains. CONCLUSIONS: The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes.

Role of the UL41 protein of pseudorabies virus in host shutoff, pathogenesis and induction of TNF-α expression.

The vhs (virion host shutoff) is highly conserved in alphaherpesvirus, including pseudorabies virus (PRV). In an attempt to explore the function of vhs of PRV, we constructed and characterized a mutant virus (Δ41). In the absence of vhs activity, Δ41 mutant is highly attenuated in mice model and the lethality is correlated with the virus dissemination in neural tissues. As with herpes simplex virus type 1 (HSV-1), the prototype virus of alphaherpesvirus, the pronounced decrease in cellular protein synthesis triggered by wild type PRV was largely restored in cells infected with Δ41 virus. Furthermore, tumor necrosis factor-α (TNF-α) protein expression was elevated significantly in spleen of mice infected with vhs mutant virus. Since TNF-α has been indicated to be an important cytokine in the innate immune response against various infections, our results implicate vhs may contribute to the protection against PRV lethality via the action of TNF-α. Overall, we confirm the shutoff function of vhs protein in PRV, and demonstrate the role that vhs protein plays in virulence, and regulation of cytokine production.

Development of a high-resolution melting method for the detection of hemoglobin alpha variants.

OBJECTIVES: The present study was aimed at identifying hemoglobin (Hb) alpha variants. DESIGN AND METHODS: To identify Hb variants, a high-resolution melting (HRM) method was performed. RESULTS: The diagnostic strategy was found to be successful in identifying Hb alpha variants including HBA1:c.27G>T, (Hb Hekinan) HBA1:c.46G>C (Hb Ottawa), HBA2:c.31_33AG (Hb alpha2-globin gene codon del AG), HBA1:c.223G>C (Hb G-Taichung), HBA1:p.Phe118_Thr119insIle (Hb Phnom Penh), HBA2:c.369C>G (Hb Westmead), HBA2:c.364G>A (or HBA1) (Hb Owari), HBA2:c.377T>C (Hb Quong Sze), and HBA2:c.427T>C (Hb Constant Spring). Each Hb variant could be readily and easily identified through the difference in plotted curves. In addition, the Hb variants could be distinguished to be located at either HBA1 or HBA2 gene. CONCLUSIONS: The HRM analysis is found to be a good tool for identifying Hb variants in alpha globin genes.

Detection of N-, H-, and KRAS codons 12, 13, and 61 mutations with universal RAS primer multiplex PCR and N-, H-, and KRAS-specific primer extension.

OBJECTIVES: Mutations of all three RAS genes, N-, H-, and KRAS, are identified mainly in codons 12, 13, and 61 of exons 2 and 3 in human cancers. DESIGN AND METHODS: DNA samples were isolated from 58 oral cancer and 106 colorectal cancer patients. Multiplex amplification of codons 12 and 13 of exon 2 and codon 61 of exon 3 of three RAS genes using two pairs of universal primers for exons 2 and 3 was performed in a single tube. The products were cleaned and split in three tubes. Each was subjected for primer extension using seven different-sized RAS primers for different RAS gene separately to detect base changes in codons 12, 13, and 61 of each RAS gene. RESULTS: We compared the results with that from direct sequencing for detecting N-, H-, and KRAS mutations in 58 oral cancers and 106 colorectal cancers. The two methods yield identical results, but our method is superior to direct sequencing in terms the amount of work and time required. CONCLUSIONS: We presented a rapid method to detect codons 12, 13, and 61 mutations of N-, H-, and KRAS genes in human cancers.

Single nucleotide variation in exon 11 of canine BRCA2 in healthy and cancerous mammary tissue.

Germline mutations in the BRCA2 tumour suppressor gene are significant risk indicators of breast cancer in women, especially for hereditary breast cancer. The BRCA2 protein interacts via the BRC (breast cancer) domain with RAD51, an essential component of the cellular machinery for the maintenance of genome stability and double strand-breaks repair. Exon 11 is the largest exon of the BRCA2 gene and contains the region encoding eight repeats of the BRC domain. Little is known about the roles of BRCA2 exon 11 in canine mammary tumours. In present study, the entire BRCA2 exon 11 was sequenced in canine mammary tumours. Fifteen mammary gland samples were obtained from four normal mammary glands and 11 mammary tumours (10 malignant and one benign tumours). Comparing sequences of normal mammary glands with those in GenBank (AB043895 and Z75664), a single nucleotide polymorphism (SNP) at codon 2414 G>A (resulting in a lysine to an arginine substitution) was identified. When compared with the normal mammary gland, 19 sporadically distributed point mutations were found in mammary tumours, including 68% of missense and 32% of silent mutations. A high frequency of genetic variations in codon 511 A>C or 2414 A>G were identified in 6/11 cases, and two missense mutations (2414 A>G, 2383 A>C) were located at the fourth repeat of the BRC domains.

Rapid identification of HBB gene mutations by high-resolution melting analysis.

OBJECTIVE: This study was undertaken to identify HBB gene mutation. DESIGN AND METHODS: Herein we evaluated high-resolution melting analysis in the identification of HBB mutations. RESULTS: We have successfully established a diagnostic strategy for identifying HBB gene mutations including c.-78A>G, c.-79A>G, c.2T>G, c.79_80insT, c.84_85insC, c.123_124insT, c.125_128delTCTT, c.130 G>T, c.170G>A, c.216_217ins A and c.316-197 C>T from wild-type DNA using HRM analysis. The results of HRM analysis were confirmed by direct DNA sequencing. CONCLUSIONS: In summary, we report that HRM analysis is an appealing technique for the identification of HBB mutations. We also believe that HRM can be used as a method for prenatal diagnosis of beta-thalassemia.

Fast simultaneous detection of K-RAS mutations in colorectal cancer.

BACKGROUND: RAS genes acquire the most common somatic gain-of-function mutations in human cancer, and almost all of these mutations are located at codons 12, 13, 61, and 146. METHODS: We present a method for detecting these K-RAS hotspot mutations in 228 cases of colorectal cancer. The protocol is based on the multiplex amplification of exons 2, 3 and 4 in a single tube, followed by primer extension of the PCR products using various sizes of primers to detect base changes at codons 12, 13, 61 and 146. We compared the clinicopathological data of colorectal cancer patients with the K-RAS mutation status. RESULTS: K-RAS mutation occurred in 36% (83/228) of our colorectal cancer cases. Univariate analysis revealed a significant association between K-RAS mutation at codon 12 of exon 2 and poor 5-year survival (p = 0.023) and lymph node involvement (p = 0.048). Also, K-RAS mutation at codon 13 of exon 2 correlates with the size of the tumor (p = 0.03). Multivariate analysis adjusted for tumor size, histologic grade, and lymph node metastasis also indicated K-RAS mutations at codon 12 and 13 of exon 2 correlate significantly with overall survival (p = 0.002 and 0.025). No association was observed between codon 61 and 146 and clinicopathological features. CONCLUSION: We demonstrated a simple and fast way to identify K-RAS mutation.

Development of a loop-mediated isothermal amplification for rapid detection of orf virus.

A loop-mediated isothermal amplification (LAMP) assay using six primers targeting a highly conserved region of the B2L gene has been developed to diagnose orf virus. The assay produces a ladder-like pattern of products on an agarose gel that can be specifically digested with BsrGI enzyme. The sensitivity of the LAMP assay, which was determined to be a single copy of the standard plasmid, was 100 fold and 10 fold higher than PCR and nested PCR, respectively; furthermore, no cross-reactivity was founded with the other tested viruses. By staining the products directly in the tube with PicoGreen or ethidium bromide, the products can be visualized with a similar sensitivity as by gel electrophoresis. Clinical samples were tested using PCR, nested PCR and LAMP assay and the positive rates were 60%, 70% and 70%, respectively. The LAMP assay allows easy, rapid, accurate and sensitive detection of infection with orf virus and is especially applicable in a resource-limited situation.