Hsien-Chi Wang

Phylogenetic analysis of canine parvovirus VP2 gene in Taiwan.

Canine parvovirus (CPV) is a non-enveloped virus with a single-stranded DNA genome and causes infectious enteritis in dog. In this study, 36 isolates of CPV infection were obtained in Taichung, Taiwan from 2003 to 2004. Using primers that can distinguish subtypes of CPV, we amplified part of viral VP2 gene by polymerase chain reaction (PCR) and the PCR product was sequenced; results demonstrated that two isolates could be classified as type 2a of CPV and the others were type 2b. The complete coding region of VP2 gene of type 2b was also sequenced, and phylogenetic analysis of these DNA sequences revealed that our Taichung isolate was close to the V-120, FPV-314, 97-008, Taiwan 9, LCPV-T1, and T4 isolates; however, because of the degeneracy of codons, the amino acid sequences of Taichung isolate was similar to that of the 97-008 isolate from Japan. It is known that two important amino acid residues (Asn-426 in type 2a and Asp-426 in type 2b; Ile-555 in type 2a and Val-555 in type 2b) are the determinants for the discrimination of type 2a or type 2b. After scrutinizing the complete VP2 gene of our Taichung isolate, we found the VP2 protein of the Taichung isolate did possess this molecular feature of type 2b virus. Previous studies reported that type 2a virus was the major type in Taiwan; our finding showed that CPV type 2b was the predominant type in the middle part of Taiwan. Moreover, a unique Ala-489 in VP2 of our Taichung isolate was found, contrasting to a Val-489 in the VP2 of other strains.

Phylogenetic analysis of parapoxviruses and the C-terminal heterogeneity of viral ATPase proteins.

Two outbreaks of orf virus (a parapoxvirus) infection in goats found in Nantou and Taiping of central Taiwan were investigated. The nucleotide and the amino acid sequences of viral B2L, E3L and A32L genes in these two outbreaks were analyzed, and each of their phylogenetic trees were also constructed. In the A32L gene, an unexpected deletion of 24 nucleotides was found in the Taiping strain. The A32L gene can encode an ATPase and is supposed to be involved in virion DNA packaging. The 24 nucleotides correspond to 8 amino acids residues of the viral ATPase, which are located near the C-terminal region of the enzyme. Moreover, two copies of the RGD sequence at C-terminal region of ATPase were found in the Nantou strain. The 24-nucleotide difference in the A32L gene indicated that the Nantou strain and the Taiping strain were two separate strains, and it can be used in differential molecular diagnosis. Moreover, the C-terminal heterogeneity was found to be a general feature of the viral ATPase. Lastly, similar functional motifs of the ATPase and the Ras proto-oncoprotein (a GTPase) are discussed.

Targeting L1 cell adhesion molecule using lentivirus-mediated short hairpin RNA interference reverses aggressiveness of oral squamous cell carcinoma

The L1 cell adhesion molecule (L1CAM) has been implicated in tumor progression of many types of cancers, but its role in oral squamous cell carcinoma (OSCC) has not been investigated. In the present study, we demonstrated overexpression of L1CAM in OSCC cells, but not in normal keratinocytes, using both clinical specimens and cell lines. This overexpression demonstrated a strong correlation with less differentiation and a higher invasion potential of cancer cells, supporting the significance of L1CAM in human OSCC tumor progression. Targeting L1CAM gene expression in SCC4 cells overexpressing L1CAM using a lentivirus-mediated small hairpin RNA (shRNA) led to a significant reduction in cell proliferation in vitro via retardation of cell cycle at the G1 phase. In addition, shRNA knockdown of L1CAM strongly attenuated the migration and invasion of SCC4 cells, and this was also observed to parallel increased E-cadherin levels and decreased levels of vimentin, fibronectin, and Snail-family transcription factors, indicating that L1CAM expression was related to the epithelial-mesenchymal transition. Furthermore, while mice receiving orthotopically placed control SCC4 cells died within 40 days due to invasive tumor growth and regional lymph node metastasis, prolonged animal survival and complete suppression of tumor progression was observed in mice implanted with L1CAM-deficent SCC4 cells, further substantiating the fundamental importance of L1CAM in OSCC pathophysiology. Our findings suggested that L1CAM is a critical mediator of tumor progression in OSCC, and targeting L1CAM using lentivirus-mediated shRNA may be a useful molecular pharmaceutical approach for the treatment of advanced OSCC.

Anesthesia with Isoflurane and Sevoflurane in the Crested Serpent Eagle (Spilornis cheela hoya): Minimum Anesthetic Concentration, Physiological Effects, Hematocrit, Plasma Chemistry and Behavioral Effects

ABSTRACT The initial goal of this study was to determine the minimum anesthetic concentration (MAC) for isoflurane (ISO) and sevoflurane (SEVO) for the crested serpent eagle. Next, we compared the anesthetic effects of each on the physiological effects, hematocrit, plasma chemistry values and behavior in spontaneously breathing captive adult crested serpent eagles. Sixteen eagles were randomly allocated to two groups for anesthesia with ISO (n=8) or SEVO (n=8). First, we measured the MAC values of ISO and SEVO, and four weeks later, we investigated the effect of each on the physiological effects, hematocrit (HCT) and plasma chemistry values. The MAC values of ISO and SEVO for crested serpent eagles were 1.46 ± 0.30 and 2.03 ± 0.32%, respectively. The results revealed no significant differences between the two anesthetics in induction time, while time of extubation to recovery was significantly shorter with SEVO. A time-related increase in end-tidal CO2 and decreases in body temperature and respiratory rates were observed during anesthesia with each anesthetic. There were no significant differences between the effect of the two anesthetics on heart rate, hematocrit, plasma chemistry values or respiration, although each caused minor respiration depression. We concluded that SEVO is a more effective inhalant agent than ISO for use in eagles, showing the most rapidest induction and recovery from anesthesia.

Functional expression of the recombinant ATPase of orf virus

Nucleotide sequence analysis has indicated that the A32L gene of orf virus can encode an ATPase (Chan et al. in Gene 432:44–53, 2009). In this work, we cloned the A32L gene into a prokaryotic expression vector, and the recombinant protein was expressed and purified. The antigenicity of recombinant ATPase was examined by immunoblotting, and its identity was confirmed by mass spectrometry. The ATP hydrolysis function of the purified recombinant protein was examined, and our results showed that it exhibited the ATPase activity. Similar to other viral ATPases, the ATPase of orf virus remained active in the presence of different divalent ions; nevertheless, unlike other viral ATPases, our recombinant ATPase exhibited similar enzymatic activity in reaction buffers of different pH.

Identification, expression and antigenic analysis of recombinant hemagglutinin proteins of canine distemper virus.

Canine distemper (CD) is a widely distributed disease of dogs, caused by the canine distemper virus (CDV). In the present study, the gene encoding the hemagglutinin (H) protein of a CDV isolate from central Taiwan was sequenced and compared with other strains. Sequence variations were noticed in the H gene from the field CDV strain that had previously been implicated in the increasing incidence of CD. To establish a serology-based diagnostic test, the full-length H protein, as well as five deletion mutants of a recombinant H protein of the local isolate, were produced using an E. coli expression system. Three truncated recombinant proteins with relatively high expression levels, designated HM3, HM4 and HM5, were used as antigens to examine their reactivity with canine sera. By using three negative sera and 17 CD-positive sera, the high specificity of recombinant H proteins was observed by ELISA. In addition, immunoblotting demonstrated that all three purified recombinant proteins exhibit an antigenic property recognized by the serum of a CD-suspected dog.

Lytic infection of pseudorabies virus in the presence of spermine, spermidine, or DFMO.

The effects of polyamines (spermine or spermidine) and DFMO (an ornithine decarboxylase inhibitor) on the infection of LM (tk-) cells by pseudorabies virus (PRV) were investigated. Results from radioactive methionine labeling showed that the synthesis of viral proteins was not affected; however, the expression of a distinctive cellular protein ( approximately 27 kDa) was induced after the treatment of spermine or spermidine. Using plaque assay, we found that the plaque formation of PRV was not affected by these three reagents either. Furthermore, the effects of these drugs on the transcription of PRV immediate-early gene (IE) promoter were examined by CAT assay, and results showed weak stimulation of transcription by these drugs. Taken together, our results demonstrated that lytic infection of PRV was not influenced by addition of exogenous polyamines or depletion of endogenous polyamines; this conclusion was similar to earlier studies by using herpes simplex virus.

EFFECTS OF INTRAVENOUS DEXMEDETOMIDINE ON CARDIAC CHARACTERISTICS MEASURED USING RADIOGRAPHY AND ECHOCARDIOGRAPHY IN SIX HEALTHY DOGS

Dexmedetomidine is a highly specific and selective α2-adrenergic receptor agonist widely used in dogs for sedation or analgesia. We hypothesized that dexmedetomidine may cause significant changes in radiographic and echocardiographic measurements. The objective of this prospective cross-sectional study was to test this hypothesis in a sample of six healthy dogs. Staff-owned dogs were recruited and received a single dose of dexmedetomidine 250 μg/m(2) intravenously. Thoracic radiography and echocardiography were performed 1 h before treatment, and repeated 10 and 30 min after treatment, respectively. One observer recorded cardiac measurements from radiographs and another observer recorded echocardiographic measurements. Vertebral heart score and cardiac size to thorax ratio on the ventrodorsal projection increased from 9.8 ± 0.6 v to 10.3 ± 0.7 v (P = 0.0007) and 0.61 ± 0.04 to 0.68 ± 0.03 (P = 0.0109), respectively. E point-to-septal separation and left ventricle internal diameter in diastole and systole increased from 2.4 ± 1.1 to 6.6 ± 1.9 mm, 32.3 ± 8.1 to 35.5 ± 8.8 mm, and 19.4 ± 6 to 27.0 ± 7.2 mm, respectively (P < 0.05). Fractional shortening and sphericity index decreased from 40.7 ± 5.8 to 24.4 ± 2.9%, and 1.81 ± 0.07 to 1.58 ± 0.04, respectively (P < 0.05). Moderate-to-severe mitral regurgitation and mild pulmonic regurgitation occurred in all dogs after dexmedetomidine administration. Findings indicated that dexmedetomidine could cause false-positive diagnoses of valvular regurgitation and cardiomegaly in dogs undergoing thoracic radiography and echocardiography.

Bovine lactoferrin and piroxicam as an adjunct treatment for lymphocytic-plasmacytic gingivitis stomatitis in cats.

Feline lymphocytic-plasmacytic gingivitis/stomatitis (LPGS) or caudal stomatitis is an inflammatory disease that causes painfully erosive lesions and proliferations of the oral mucosa. The disease is difficult to cure and can affect cats at an early age, resulting in lifetime therapy. In this study, a new treatment using a combination of bovine lactoferrin (bLf) oral spray and oral piroxicam was investigated using a randomized double-blinded clinical trial in 13 cats with caudal stomatitis. Oral lesion grading and scoring of clinical signs were conducted during and after the trial to assess treatment outcome. Oral mucosal biopsies were used to evaluate histological changes during and after treatment. Clinical signs were significantly improved in 77% of the cats. In a 4-week study, clinical signs were considerably ameliorated by oral piroxicam during the first 2 weeks. In a 12-week study, the combined bLf oral spray and piroxicam, when compared with piroxicam alone, exhibited an enhanced effect that reduced the severity of the oral lesions (P = 0.059), while also significantly improving clinical signs (P <0.05), quality of life (P <0.05), and weight gain (P <0.05). The remission of oral inflammation was closely correlated with the decreased number of macrophages (OR = 4.719, P < 0.05). There was no detectable influence on liver or kidney function during a 12-week assessment. It was concluded that combining oral bLf spray and piroxicam was safe and might be used to decrease the clinical signs of caudal stomatitis in cats.

Clinical diagnosis and surgical management of diaphragmatic retroperitoneal perirenal fat and kidney herniation in a pet rabbit

CASE DESCRIPTION A 6-year-old 2.08-kg (4.58-lb) neutered male Lionhead-mix pet rabbit (Oryctolagus cuniculus) was examined because of sneezing and increased respiratory effort. CLINICAL FINDINGS On the basis of the rabbits radiographic findings, a diagnosis of diaphragmatic retroperitoneal perirenal fat and kidney herniation was made. Nine months later, physical examination revealed increased respiratory rate and effort and slightly decreased body weight. Thoracic radiography revealed decreased lung aeration and further craniomedial displacement of the right kidney, compared with the initial evaluation findings, suggesting progressive herniation of the retroperitoneal perirenal fat. TREATMENT AND OUTCOME During exploratory celiotomy, a tear in the right dorsal tendinous portion of the diaphragm was noted. The right kidney and perirenal fat were found to be displaced into the thorax. Diaphragmatic herniorrhaphy was performed after replacement of the right kidney and the perirenal fat in the retroperitoneal space. The rabbit recovered uneventfully from anesthesia and surgery. Clinical signs did not recur during the following 16 months. CLINICAL RELEVANCE For rabbits with increased respiratory effort, diaphragmatic retroperitoneal perirenal fat and kidney herniation should be included as a differential diagnosis. As illustrated by the case described in this report, appropriate surgical management can provide a successful outcome for affected pet rabbits.