Isolation of a Bluetongue virus group-specific monoclonal antibody and application to a diagnostic competitive ELISA.

The Bluetongue virus (BTV) VP7 protein represents an important group-specific antigen that can serve as a basis for diagnostic tests. Here, we report the generation of a novel BTV group-specific monoclonal antibody (Mab; herein named 4H7) that recognizes a conformational epitope in the VP7 protein. We used a phage-displayed peptide screen and site-directed mutagenesis to define the VP7 amino acid residues that most strongly contribute to the conformational epitope recognized by Mab 4H7. Amino acid residues at positions 175, 185, 186, and 278 of the BTV VP7 protein strongly contributed to Mab 4H7 binding. These key amino acid residues are conserved among all BTV serotypes, whereas related Orbiviruses possess at least one amino acid substitution at these positions. We developed a competitive enzyme-linked immunosorbent assay (c-ELISA) using Mab 4H7 and recombinant BTV VP7 protein to detect serum antibodies against this BTV group-specific VP7 epitope. The c-ELISA was used to screen 833 clinical samples collected from animals in three provinces of China. BTV seroprevalence in the three provinces ranged from 25.42 to 47.45 %. This work provides the foundation for a new diagnostic c-ELISA that can be further applied to BTV surveillance activities and informs our understanding of the structure of the BTV VP7 protein.

In silico prediction and in vitro identification of bluetongue virus 4 VP5 protein B-cell epitopes.

VP5, the outer capsid protein of bluetongue virus (BTV), plays an important role in viral penetration and antibody-mediated viral neutralization. Therefore, VP5 represents an important target for development of vaccines and diagnostic tests. In this study, we use bioinformatic tools to predict nine antigenic B cell epitopes in the VP5 protein of a BTV serotype 4 (BTV4) isolate from China. Further, we generate five BTV4 VP5-specific monoclonal antibodies (MAbs) and define their corresponding epitopes using a set of VP5-derived peptides expressed as maltose-binding protein (MBP) fusion proteins. The five identified epitopes map to amino acids 119-134, 257-272, 286-301, 322-337, and 481-496 of the VP5 protein. Importantly, the epitopes identified using VP5-derived peptides do not correlate with our bioinformatic prediction of antibody epitopes. Identification and characterization of BTV4 VP5 protein epitopes may aid the development of diagnostic tools and provide information with which to study the structure of the BTV VP5 protein.

Identification of a novel bluetongue virus 1-specific B-cell epitope using a monoclonal antibody against the VP2 protein.

Bluetongue virus (BTV) VP2 is an important antigenic protein that can be used for the differential diagnosis of different BTV serotypes. Here, we generated a serotype-specific monoclonal antibody (mab) against BTV1. A series of peptides synthesized based on the amino acid sequence of BTV1 VP2 were screened to define (115)AQPLKVGL(122) as the minimal linear peptide epitope recognized by mab 4B6. Using an immunofluorescence assay (IFA), we found that mab 4B6 reacted strongly with BTV1, but did not react with other BTV serotypes (BTV2-24). The 4B6 will serve as a novel reagent in the development of diagnostic tests for BTV1 infection.

Hospitals with briefer than average lengths of stays for common surgical procedures do not have greater odds of either re-admission or use of short-term care facilities.

We considered whether senior hospital managers and department chairs need to be concerned that small reductions in average hospital length of stay (LOS) may be associated with greater rates of re-admission, use of home health care, and/or transfers to short-term care facilities. The 2013 United States Nationwide Readmissions Database was used to study surgical Diagnosis Related Groups (DRG) with 1) national median LOS ≥3 days and 2) ≥10 hospitals in the database that each had ≥100 discharges for the DRG. Dependent variables were considered individually: 1) re-admission within 30 days of discharge, 2) discharge disposition to home health care, and/or 3) discharge disposition of transfer to short-term care facility (i.e., inpatient rehabilitation hospital or skilled nursing facility). While controlling for DRG, each one-day decrease in hospital median LOS was associated with an odds of re-admission nationwide of 0.95 (95% confidence interval [CI] 0.92-0.99; P=0.012), odds of disposition upon discharge being home care of 0.95 (95% CI 0.83-1.10; P=0.64), and odds of transfer to short-term care facility of 0.68 (95% CI 0.54-0.85; P=0.0008). Results were insensitive to the addition of patient-specific data. In the USA, patients at hospitals with briefer median LOS across multiple common surgical procedures did not have a greater risk for either hospital re-admission within 30 days of discharge or transfer to an inpatient rehabilitation hospital or a skilled nursing facility. The generalisable implication is that, across many surgical procedures, DRG-based financial incentives to shorten hospital stays seem not to influence post-acute care decisions.

Development of a novel protein chip for the detection of bluetongue virus in China.

Bluetongue (BT), which is caused by the BT virus (BTV), is an important disease in ruminants that leads to significant economic losses in the husbandry industry. To detect BTV-specific antibodies in serum, a protein chip detection method based on a novel solid supporting material known as polymer-coated initiator-integrated poly (dimethyl siloxane) (iPDMS) was developed. With a threshold of 25% (signal-to-noise percentage), the sensitivity and specificity of the protein chip were 98.6% and 94.8%, respectively. Furthermore, spot serum samples obtained from six provinces of China were tested with the protein chip and a commercially available BTV enzyme-linked immunosorbent assay (ELISA) kit (IDEXX). Of 615 samples, BTV-specific antibodies were detected in 200 (32.52%) by the protein chip and in 176 (28.62%) by the IDEXX BTV ELISA kit. Comparison of the protein chip with the commercial IDEXX BTV ELISA kit yielded the following spot serum detection results: a total coincidence, a negative coincidence and a positive coincidence of 95.12%, 99.28% and 86.5%, respectively. With the protein chip, the BTV-specific serum antibody was detected in samples from all six provinces, and the positive rates ranged from 4.12 to 74.4%. These results indicate that this protein chip detection method based on iPDMS is useful for the serological diagnosis of BTV infection and for epidemiological investigation.

Emergence of a Novel Bluetongue Virus Serotype, China 2014.

One hundred and twenty-six blood samples were collected from healthy sheep and goats in Xinjiang, China, during July 2014. Seventy-three samples (57.93%) were bluetongue virus (BTV) serology-positive, and 39 samples (30.95%) were BTV NS1 gene-positive. BTV strain XJ1407 was isolated from the blood of BTV NS1 gene-positive animals and sequenced. Analysis of its genome sequence suggests that XJ1407 is a novel BTV serotype.

Epidemiology of squamous cell conjunctival cancer.

The etiology of squamous cell carcinoma of the conjunctiva (SCCC) is not well known. A possible role of UVB radiation is suggested by an excess of SCCC in tropical countries and by the association between squamous cell skin cancer and exposure to UVB. Human papillomavirus type 16 also may be involved, given that it has been detected in benign and malignant conjunctival lesions and is the primary etiological agent involved in carcinoma of the anogenital tract. To examine the relationship between UVB exposure and SCCC, population-based age-adjusted incidence rates of SCCC and of conjunctival melanoma and squamous cell cancer of the eyelid were plotted against the UVB insolation of each registry site. Incidence data were examined further for patterns of second primary cancers among people with SCCC. SCCC was rare in the United States, with an incidence rate of 0.03 per 100,000 persons, although the rate was approximately 5-fold higher among males and whites. Regression analysis suggested a link between UVB exposure and SCCC rates (beta = 2.25; r = 0.58) that was as strong as that for squamous cell carcinoma of the eyelid (beta = 2.73; r = 0.62) and much stronger than for conjunctival melanoma (beta = 0.28; r = 0.02). Risk of a second malignancy after SCCC was not increased overall (20 observed and 14.1 expected), although a significant excess of salivary gland cancer (4 observed and 0.03 expected) and a borderline excess of lung cancer (6 observed and 2.4 expected) were noted. These observations suggest that UV radiation likely contributes to SCCC development. Additional research is needed to define the other exposures and host susceptibility that likely interact with UV-related genetic damage in the multifactorial development of this rare neoplasm.

Salivary gland cancer in the United States.

The risk of salivary gland cancer (SGC) is increased in atomic bomb survivors and after radiotherapy, but other risk factors are not well established. Some studies have suggested an association of SGC with breast cancer and with exposure to various viruses or UVB radiation. Corroborating evidence of these associations was sought by using population-based registries to examine the demographic distribution of SGC, patterns of secondary primary cancers after SGC, and risk of SGC with AIDS. SGC incidence per 100,000 persons did not change between 1973 and 1992, averaging 1.2 in males and 0.8 in females, with a steep age gradient. To examine the relationship between UVB exposure and SGC, population-based, age-adjusted incidence rates of SGC were plotted against the UVB insolation of each registry site. Regression analysis suggested no correlation between SGC incidence and increasing UVB insolation (beta = 0.10, R2 = 0.08). SGC also did not appear to be associated with second cancers that have been linked to herpes or papilloma viruses or with AIDS [observed/expected (O/E) ratio, <2.8], but all of these conditions are so uncommon that only very large relative risks would have been statistically significant. Women with SGC before age 35 had a statistically nonsignificant elevation in breast cancer risk [O/E, 3.30; 95% confidence interval (CI), 0.66-9.65], and older women had no increased risk of breast cancer. SGC patients were at increased risk for nonsalivary, second-primary oropharyngeal cancers (O/E, 3.27; 95% CI, 2.00-5.05), thyroid cancer (O/E, 3.31; 95% CI, 1.07-7.73), and lung cancer (O/E, 1.86; 95% CI, 1.45-2.35), particularly in patients whose SGC was treated with radiotherapy (O/E, 2.83; 95% CI, 2.06-3.80). In summary, SGC remains rare and does not appear to be associated with AIDS, virally related malignancies, or UVB. Patients who have had SGC, however, should be monitored for subsequent oropharyngeal, thyroid, and lung cancers.

Phage display identifies an Eastern equine encephalitis virus glycoprotein E2-specific B cell epitope.

The present study identified a linear B-cell epitope in the Eastern equine encephalitis virus (EEEV) E2 glycoprotein by screening a phage-displayed random 12-mer peptide library using an EEEV E2 specific monoclonal antibody (mAb) 7C11 and defined L/F-E/R-Y-T-W-G/R-N-H/W-P as the consensus binding motif. A sequence ((321)EGLEYTWGNHPP(332)) encompassing this consensus motif was found in the EEEV E2 glycoprotein and synthesized for further epitope confirmation. Meanwhile, the corresponding epitope peptides in E2 protein of associated alphaviruses were synthesized for specificity identification. Results showed the mAb 7C11 and murine antisera all reacted strongly against the synthesized polypeptide of EEEV antigen complex, but no reaction with Western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) was detected. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against EEEV.

Proteasome complex as a potential cellular target of hepatitis B virus X protein.

Although the biological importance of hepatitis B virus X protein (HBX) in the life cycle of hepatitis B virus has been well established, the cellular and molecular basis of its function remains largely undefined. Despite the association of multiple activities with HBX, none of them appear to provide a unifying hypothesis regarding the true biological function of HBX. Identification and characterization of cellular targets of HBX remain an essential goal in the elucidation of the molecular mechanisms of HBX. Using the Saccharomyces cerevisiae two-hybrid system, we have identified and characterized a novel subunit of the proteasome complex (XAPC7) that interacts specifically with HBX. We also showed that HBX binds specifically to XAPC7 in vitro. Mutagenesis studies have defined the domains of interaction to be critical for the function of HBX. Furthermore, overexpression of XAPC7 appeared to activate transcription by itself and antisense expression of XAPC7 was able to block transactivation by HBX. Therefore, the proteasome complex is possibly a functional target of HBX in cells.

Patellar metastasis from a squamous carcinoma of the lung: a case report.

Bone is a common site of metastasis from lung cancer. Metastasis to the patella, however, is rare. A 76-year-old man presented with knee pain caused by an isolated patellar metastasis from squamous cell carcinoma of the lung. Treatment was delayed secondary to delay in diagnosis. In cases of bone pain that are unexplained or out of proportion to a traumatic event, more extensive diagnostic studies should be done.