ABSTRACT
Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/µl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains.
Subject(s)
DNA Virus Infections/veterinary , Fish Diseases/diagnosis , Fish Diseases/virology , Iridoviridae/isolation & purification , Molecular Diagnostic Techniques/methods , Real-Time Polymerase Chain Reaction/methods , Veterinary Medicine/methods , Animals , DNA Primers/genetics , DNA Virus Infections/diagnosis , DNA Virus Infections/virology , DNA, Viral/chemistry , DNA, Viral/genetics , Fishes , Genotype , Molecular Sequence Data , Reproducibility of Results , Sensitivity and Specificity , Sequence Analysis, DNAABSTRACT
We describe two non-small-cell lung cancer (NSCLC) patients in which treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKIs) gefitinib produced a prolonged control of bone disease. In the first patient, a 48-year-old male with adenocarcinoma (ADC) of the lung and multiple bone metastases, the bone scan became completely negative following treatment with gefitinib for 9 months. The patient remained alive and with no evidence of bone metastases for 20 months, despite two local recurrences that were surgically removed. Similarly, the bone scan of the second patient, a 49-year-old male with ADC of the lung and bone metastases, became negative after 6 months on gefitinib. The molecular mechanisms potentially involved in this phenomenon are discussed.
