Guangxiang Wang

Comparison of a loop-mediated isothermal amplification for orf virus with quantitative real-time PCR

BackgroundOrf virus (ORFV) causes orf (also known as contagious ecthyma or contagiouspapular dermatitis), a severe infectious skin disease in goats, sheep andother ruminants. Therefore, a rapid, highly specific and accurate method forthe diagnosis of ORFV infections is essential to ensure that the appropriatetreatments are administered and to reduce economic losses.MethodsA loop-mediated isothermal amplification (LAMP) assay based on theidentification of the F1L gene was developed for the specific detection ofORFV infections. The sensitivity and specificity of the LAMP assay wereevaluated, and the effectiveness of this method was compared with that ofreal-time PCR.ResultsThe sensitivity of this assay was determined to be 10 copies of a standardplasmid. Furthermore, no cross-reactivity was found with either capripoxvirus or FMDV. The LAMP and real-time PCR assays were both able to detectintracutaneous- and cohabitation-infection samples, with a concordance of97.83%. LAMP demonstrated a sensitivity of 89.13%.ConclusionThe LAMP assay is a highly efficient and practical method for detecting ORFVinfection. This LAMP method shows great potential for monitoring theprevalence of orf, and it could prove to be a powerful supplemental tool forcurrent diagnostic methods.

Pathogenic characteristics of three genotype II porcine reproductive and respiratory syndrome viruses isolated from China

BackgroundWe examined differences in pathogenicity in pigs from China that had been experimentally infected with porcine reproductive and respiratory syndrome virus (PRRSV).MethodsWe compared pathogenic characteristics of a field isolate (GX-1/2008F), two PRRSV isolates (HN-1/2008, YN-1/2008) propagated in cells, and GX-1/2008F that had been propagated in cells (GX-1/2008). The clinical courses, along with humoral and cell-mediated responses, were monitored for 21 days post-infection (DPI). Animals were sacrificed and tissue samples used for gross pathological, histopathological and ultrastructure examination.ResultsAt 2–3 DPI, animals infected with cell-propagated viruses exhibited signs of coughing, anorexia and fever. However their rectal temperature did not exceed 40.5°C. Viremia was detectable as early as 3 DPI in animals infected with HN-1/2008 and YN-1/2008. Animals inoculated with GX-1/2008F displayed clinical signs at 6 DPI; the rectal temperature of two animals in this group exceeded 41.0°C, with viremia first detected at 7 DPI. Seroconversion for all challenged pigs, except those infected with GX-1/2008, was seen as early as 7 DPI. All of these pigs had fully seroconverted by 11 DPI. All animals challenged with GX-1/2008 remained seronegative until the end of the experiment. Innate immunity was inhibited, with levels of IFN-α and IL-1 not significantly different between control and infected animals. The cytokines IFN-γ and IL-6 transiently increased during acute infection. All virus strains caused gross lesions including multifocal interstitial pneumonia and hyperplasia of lymph nodes. Inflammation of the stomach and small intestine was also observed. Lesions in the group infected with GX-1/2008F were more serious than in other groups. Transmission electron microscopy revealed that alveolar macrophages, plasmacytes and lymphocytes had fractured cytomembranes, and hepatocytes had disrupted organelles and swollen mitochondria.ConclusionsThe pathogenicity of the PRRSV field isolate became attenuated when propagated in MARC-145 cells. Tissue tropism of highly pathogenic strains prevailing in China was altered compared with classical PRRSV strains. The observed damage to immune cells and modulation of cytokine production could be mechanisms that PRRSV employs to evade host immune responses.

How foot-and-mouth disease virus receptor mediates foot-and-mouth disease virus infection

This study reviews the FMDV receptor-binding domain, integrin receptors, and heparan sulfate receptors to provide references for studies regarding the mechanisms underlying FMDV infection.

Erratum to: Diagnosis and phylogenetic analysis of Orf virus from goats in China: a case report

Background: Orf virus (ORFV) is the etiological agent of contagious pustular dermatitis and is the prototype of the genus Parapoxvirus (PPV). It causes a severe exanthematous dermatitis that afflicts domestic and wild small ruminants. Case presentation: In the present study, an outbreak of proliferative dermatitis in farmed goats. The presence of ORFV in tissue scrapings from the lips was confirmed by B2L gene polymerase chain reaction (PCR) amplification. The molecular characterization of the ORFV was performed using PCR amplification, DNA sequencing and phylogenetic analysis of the B2L gene. Conclusion: The results of this investigation indicated that the outbreak was caused by infection with an ORFV that was closely related genetically to Nantou (DQ934351), which was isolated from the Tai wan province of China and Hoping (EU935106), which originated from South Korea in 2008. This is the first report of the phylogenetic analysis of ORFV from goats in China. Background The ORFV is the prototype member of the genus Parapoxvirus, which also includes pseudocowpox virus (PCPV) in cattle, bovine papular stomatitis virus (BPSV) in cattle, squirrel parapoxvirus (SPPV) and parapoxvirus of red deer in New Zealand (PVNZ) [1,2]. Contagious pustular dermatitis is a common viral skin disease that occurs in a range of species, not only in wild ruminants [3] but also in humans [4-6]. Humans with immunodefi-ciency diseases, in particular, can develop serious infections [7]. The diseases caused by ORFV have worldwide distribution and have been reported from many countries [1]. The disease not only has an economic impact on farmers worldwide but also has a considerable negative effect on animal welfare. Infected animals are sickly, fail to thrive, and are more susceptible to adventitious bacterial infections [8]. The typical progress of orf in goats and sheep moves from erythema, via vesicle formation, to pustules and then to scabs. Characteristic of the disease

Comparative analysis of cloned cDNAs encoding Chinese yellow cattle and Gansu black swine integrin receptors for foot-and-mouth disease virus.

To analyze foot-and-mouth disease virus tropism and host range with respect to the integrin receptor, we cloned cDNAs encoding the integrin αν, β1, β3, β6 and β8 subunits from Chinese yellow cattle and Gansu black swine and carried out comparative analysis of their molecular characteristics. The lengths of the mature proteins and the functional domains of the four integrin β subunits were the same between bovine and swine; however, the number of putative N-linked glycosylation sites and cysteine residues and their arrangement varied. Homology analysis of the nucleotide and amino acid sequences showed that FMDV integrin receptors of Chinese yellow cattle and Gansu black swine are highly conserved. Phylogenetic analysis showed that all FMDV integrin receptor subunits of cattle and swine are clustered into the Artiodactyla group; however, Chinese yellow cattle are phylogenetically closer to sheep than to Gansu black swine. We postulate that the host tropism of FMDV may, in part, be related to the divergence of integrin subunits among different species.

Validated hydrophilic interaction LC–MS/MS method for determination of N-methyl-2-pyrrolidinone residue: Applied to a depletion study of N-methyl-2-pyrrolidinone in swine liver following intramuscular administration of drug N-methyl-2-pyrrolidinone formulation

A hydrophilic interaction high-performance liquid chromatography coupled with tandem mass spectrometry method for determination of N-methyl-2-pyrrolidinone in swine liver was developed and validated. After the fortification of N-methyl-d(3)-2-pyrrolidinone-d(6) as the deuterium-labeled internal standard, N-methyl-2-pyrrolidinone in swine liver was extracted by acetonitrile and the supernatant was led through a C18+WAX mixed-mode SPE cartridge for removal of the matrix interferences. The final eluate was acidified by formic acid and then injected onto a 3μm 15cm×2.1mm TX column for hydrophilic interaction chromatographic analysis. Mass spectrometry detection was carried on a PE Sciex API 4000 triple quadrupole mass spectrometer using positive turbo-ion spray ionization mode. The MRM transitions were 100→58 for N-methyl-2-pyrrolidinone and 109→62 for N-methyl-d(3)-2-pyrrolidinone-d(6). Solvent calibration standards could be readily used for quantitative analysis of N-methyl-2-pyrrolidinone with excellent precision and accuracy, although there are endogenous levels of N-methyl-2-pyrrolidinone in many blank matrices. The true recovery was nearly 100% and the MRM signal of N-methyl-2-pyrrolidinone was suppressed about 30% because of the matrix effect. Nevertheless, N-methyl-d(3)-2-pyrrolidinone-d(6) completely compensated the ion-suppression effect and the injection-to-injection variation. The detection limit was 5ngg(-1) swine liver. The validated method was applied to a depletion study of N-methyl-2-pyrrolidinone in swine liver following intramuscular administration of a drug N-methyl-2-pyrrolidinone formulation.