[Advances in immunotherapy for pulmonary sarcomatoid carcinoma].

Pulmonary sarcomatoid carcinoma (PSC) is a rare, poorly differentiated non-small cell lung cancer (NSCLC) that contains sarcomatoid components or sarcomatoid differentiation, and accounts for less than 1% of all lung tumors. Compared to other types of NSCLC, PSC has more invasive biological behavior, is prone to metastasis, and has a higher recurrence rate after early surgery. Its greater resistance to traditional treatments leads to a poorer prognosis compared to other NSCLCs. Immunotherapy offers the possibility of long-term survival for PSC patients.

[Clinical analysis of lung adenocarcinoma with epidermal growth factor receptor mutation transformed into sarcoma].

Objective: To analyze the clinical data of a case of lung adenocarcinoma with Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) resistance transforming into sarcoma, and to conduct a literature review to improve the understanding of the resistance mechanism. Histological transformation is a unique form of acquired resistance of EGFR-TKIs in non-small cell lung cancer (NSCLC). Thereinto, the transformation of small cell carcinoma is more common, and the transformation of sarcoma is rarely reported. Methods: Clinicopathological data on the treatment process, pathological features, and clinical outcome of the patient with EGFR-TKIs-resistance lung adenocarcinoma transforming into sarcoma were collected. The literature was reviewed to analyze the pathogenetic mechanism for sarcomatoid carcinoma or sarcoma transformation after drug resistance of adenocarcinoma, as well as the clinical characteristics of the patients and the corresponding therapeutic schemes. Results: We reported a patient with lung adenocarcinoma who developed EGFR-T790M mutation after first-line treatment with icotinib and sarcoma transformation after second-line treatment with almonertinib. Chemotherapy, radioactive particle implantation, antiangiogenic therapy and immunotherapy were followed, but the results were unsatisfactory. There was no report of EGFR-TKIs-resistant lung adenocarcinoma transforming into sarcoma. Among the 14 reports of adenocarcinoma transforming into sarcomatoid carcinoma, 8 cases had EGFR mutation, 3 cases had ALK mutation, 2 cases had ROS1 mutation, and 1 case had no asscoiated sensitive mutation. The median survival of 14 patients with adenocarcinoma transforming to sarcomatoid carcinoma was only 3 months. Conclusions: Sarcoma transformation can be one of the forms of drug resistance in patients with lung adenocarcinoma with EGFR-TKIs. The prognosis of patients with adenocarcinoma after transformation into sarcoma is poor.

Cardiovascular capacity in adults with obstructive sleep apnoea.

BACKGROUND: Obstructive sleep apnoea (OSA) patients have a decreased exercise capacity. Potential cardiovascular capacity in patients with OSA with different severity without known cardiovascular disease has not been described.OBJECTIVE: To evaluate impaired potential exercise cardiovascular capacity during cardiopulmonary exercise testing (CPET) in OSA patients and to compare cardiovascular performance reaction with different severity during graded exercise stages.DESIGN: All participants were accompanied without cardiovascular disease, especially hypertension and arrhythmia. Parameters of different stages were compared between subjects with and without OSA, and among OSA patients with varied severity.RESULTS: Despite having significantly higher peak exercise diastolic blood pressure (DBP) and ventilatory equivalent for CO2 (EQCO2), patients with OSA had a lower peak oxygen uptake (VO2), heart rate (HR), heart rate recovery (HRR) and respiratory reserve (BR) than normal subjects. Furthermore, significant correlations were found between VO2, DBP, EQCO2, HRR, BR and the apnoea-hypopnea index. In severe OSA, there was a greater difference in HR and HRR during the anaerobic threshold stages.CONCLUSIONS: OSA patients demonstrate reduced potential cardiovascular capacity, even without documented cardiovascular disease. Patients with severe OSA develop impaired exercise capacity at early stage during exercise. These data point to exaggerated haemodynamic response to graded exercise and delayed post-exercise cardiovascular response recovery in OSA patients. CPET can be a supplement for assessment of OSA severity.

[Treatment of oligoprogression to immunotherapy resistance in advanced non-small cell lung cancer].

In recent years, the incidence of lung cancer has been increasing year by year. Traditional treatments have limited clinical effects in advanced, driver-gene-negative non-small cell lung cancer. Immune checkpoint inhibitors (ICI) have dramatically changed the treatment landscape of advanced non-small cell lung cancer. However, most patients are suffered from primary and acquired resistance inevitably. Oligoprogression is one of the main progression patterns of acquired resistance. Therefore, it is essential to further understand treatment of oligoprogression to immunotherapy resistance. This article aimed to conduct a systematic review of the treatment of oligoprogression to immunotherapy resistance.

[Clinical characteristics of epidermal growth factor receptor-mutated advanced adenocarcinoma transformed into small-cell lung cancer].

Objective: To explore the clinicopathological characteristics and genomic characteristics of four patients with epidermal growth factor receptor(EGFR)-mutated advanced adenocarcinoma transformed into small-cell lung cancer. Methods: Four cases of EGFR-mutated advanced adenocarcinoma of the lung transformed into small-cell lung cancer were studied by clinical data, pathological morphology, immunohistochemistry and gene detection. Result: EGFR-mutated adenocarcinoma of the lung was heterogeneous in clinical and genomic profiles, of ten characterized by RB1, TP53 and PIK3CA mutations. Its transformation into small-cell lung cancer was a particularly aggressive mechanism of drug resistance, but the machanisms were not clear NSE and other tumor indicators had low diagnostic value for transformation. Conclusions: EGFR-mutated adenocarcinoma of the lung transformed into small-cell lung cancer was one of the reasons for EGFR resistance with avery poor prognosis.

[Advances in the study of occult malignant tumor-related pyogenic liver abscesses in the digestive system].

Pyogenic liver abscess (PLA) accompanied by occult malignant tumors is a rare kind of life-threatening disease. Studies have shown that it can predict the occurrence of cancer, especially hepatobiliary and colorectal cancer. The risk of combined occult primary liver cancer, cholangiocarcinoma, and gastrointestinal cancer is high in PLA patients. Malignant tumor-related PLA lacks specific symptoms and signs. The iodine concentration ratio between the energy spectrum CT lesions and normal liver tissue is of certain value in the differentiation of liver cancer and liver abscess. Computed tomography colonography has a dual role. It can screen patients with PLA for occult colorectal cancer and determine the treatment response of abscess lesions. Klebsiella pneumoniae and Escherichia coli is the main microorganism of PLA related to colorectal cancer, hepatocellular carcinoma, and intrahepatic cholangiocarcinoma. PLA treatment related to hepatobiliary malignant tumor has high complications and mortality, and poor prognosis. Most occult colorectal cancers are in the early stage, and their early detection and prognosis are better than those of PLA patients combined with hepatobiliary malignancies.

[The efficacy and safety of tiotropium/olodaterol fixed-dose combination in Chinese patients with chronic obstructive pulmonary disease: a pooled subgroup analysis of TONADO 1+2].

Objective: To compare the efficacy and safety profiles of tiotropium/olodaterol with the mono-components in Chinese and total study population from TONADO trial. Methods: In the replicate, double-blind, parallel-group, active-controlled, randomized, 52-week, Phase Ⅲ TONADO studies (TONADO 1+2), patients received tiotropium/olodaterol, tiotropium, or olodaterol via the Respimat(®) Inhaler (Boehringer Ingelheim, Germany). Primary end points were forced expiratory volume in 1 second (FEV(1)) area under the curve from 0 to 3 hours (AUC(0-3h)) response and trough FEV(1) response, and St George's respiratory questionnaire (SGRQ) total score at 24 weeks. Adverse events were also collected. This subgroup analysis only focused on the efficacy and safety of the drug at the approved dose in China. Results: 548 Chinese patients were randomized, aged 41 to 82 years [mean age, (63±8) years] and most were male (526, 96%), 111 received tiotropium/olodaterol 5/5 µg, and 127 received tiotropium 5 µg and 95 received olodaterol 5 µg. The baseline characteristics of these groups were similar. After 24 weeks, treatment with tiotropium/olodaterol 5/5 µg, tiotropium 5 µg and olodaterol 5 µg resulted in an adjusted mean FEV(1) AUC(0-3h) response of 0.240, 0.157 and 0.079 L, and trough FEV(1) response of 0.117, 0.068 and-0.001 L, respectively. Tiotropium/olodaterol 5/5 µg significantly improved SGRQ scores in Chinese patients compared with olodaterol 5 µg (32.729 and 37.202, respectively). Generally, the safety profile of tiotropium/olodaterol was comparable with mono-components in 52 weeks. Conclusion: Compared with tiotropium or olodaterol, tiotropium/olodaterol in Chinese patients provided significant improvement in lung function and quality of life, and the safety profiles were similar.

Virus-neutralising antibody responses in horses following vaccination with Equivac® HeV: a field study.

OBJECTIVE: To determine the antibody responses to a commercial Hendra virus vaccine (Equivac® HeV) in a field environment. METHODS: A group of 61 horses received a primary vaccination course comprising two doses administered 3-6 weeks apart (V1, V2) and a 3rd dose (V3) given 6 months after the second. This was followed by booster vaccinations at 12 monthly intervals (V4, V5). Antibody titres were assessed using a virus-neutralisation test. RESULTS: Neutralising antibodies against HeV were not detected prior to vaccination. Antibodies were detected in 54/57 horses at 3 weeks after V1 and 51/51 had titres ≥ 32 at 8 weeks after V2. At 6 months after V2, antibody titres decreased in most (31/34) horses and were not detected in three horses. A rapid increase in antibody titres was recorded in 35/36 horses at 1 week following V3. By the first annual booster vaccination (V4), antibodies were still detectable in 29/29 horses, although titres had decreased; in 26/29 horses, titres remained ≥ 32. All horses showed an increase in antibody titres after V4. There was no statistically significant increase in mean antibody titre after V5, compared with after V4. CONCLUSION: Horses administered Equivac® HeV, using a primary vaccination course followed by annual booster vaccinations, mounted an effective secondary immune response and acquired antibody responses that were consistent with protective immunity against HeV in the form of virus-neutralising antibodies. No adverse events were observed after vaccine administration.

[Therapeutic effect and mechanism of carboxyamidotriazole on the pulmonary fibrosis of mice induced by bleomycin].

Objective: To investigate the treatment effect of carboxyamidotriazole (CAI) on bleomycin induced lung fibrosis in mice, and the potential mechanism involved. Methods: A total of 45 mice were divided into three groups randomly. Blank control group (blank group): after a one-time tail vein injection of saline solution 0.2 ml, mice were given polyethylene glycol 400 (PEG-400) 0.1 ml/10 g by gavage once daily for 14 days; the bleomycin group (BLM group): after a one-time tail vein injection of bleomycin 150 mg/kg, mice were given PEG-400 solution 0.1 ml/10 g by gavage once daily for 14 days; CAI group: after a one-time tail vein injection of bleomycin 150 mg/kg, mice were given CAI solution 40 mg/kg by gavage once daily for 14 days. All mice were sacrificed on day 28. Observation index: lung coefficient, survival analysis, pathological section and collagen staining of lung tissue, lung hydroxyproline, Transformation growth factor-ß(1)(TGF-ß(1)), γ-interferon(IFN-γ), matrix metalloproteinase 9(MMP-9) and tissue inhibitor of matrix metalloproteinasese 1(TIMP-1) content determination in lung homogenate. Results: On day 28 the lung coefficient of mice in BLM group and CAI group was significantly higher than the blank group, and the BLM group was with the highest (all P<0.05). Degree of pulmonary fibrosis in lung tissue pathological specimens (HE staining) was, from heavy to light, BLM group, CAI group, blank group. The content of hydroxyproline in mice lung homogenate was (0.406±0.020) µg/mg in blank group, (0.722±0.118) µg/mg in BLM group, (0.537±0.071) µg/mg CAI group, respectively (all P<0.05). The content of TGF-ß(1) in three groups was (15±5), (60±10), (41±10) ng/ml respectively (all P<0.05). The content of IFN-γ in three groups was (47±5), (126±24), (194±34) pg/ml respectively (all P<0.05). The content of TIMP-1 in three groups was (73±6), (369±58), (246±51) ng/ml respectively (all P<0.05). Comparisons of the content of MMP-9 between each group had no significant difference (P>0.05). Conclusions: CAI can reduce lung injury induced by bleomycin in mice. The mechanism of action is related to the effects of CAI on cytokines such as TGF-ß(1), IFN-γ, MMP-9 and TIMP-1.

Mechanism of action of KIAA1456 gene on the proliferation and apoptosis of alveolar epithelial cells.

OBJECTIVE: To explore the mechanism by which KIAA1456 acts on alveolar epithelial cells through lentiviral transfection. MATERIALS AND METHODS: After constructing a KIAA1456 gene vector, 293T cells were co-transfected with lentiviral vectors and after incubation cells were examined by fluorescence microscopy. CCL-149 cells were transfected with LV-KIAA1456 and were examined by fluorescence microscopy. The proliferation capacity of transfected CCL-149 cells was evaluated using flow cytometry. The effect of KIAA1456 overexpression on CCL-149 cells proliferation was studied using the CCK-8 method. RESULTS: The expression level of KIAA1456 in the LV- KIAA1456 group was significantly higher compared with the LV-Con group and the blank group. Compared with the LV-Con and the blank groups, the proportion of responding cells in G2/M phase showed statistically significant differences. Viable cells had adarker color and higher OD value measured by ELISA. Compared with the control and the blank groups, the growth and proliferation in the CCL-149 transfection group were significantly slower. CONCLUSIONS: KIAA1456 gene inhibited the proliferation of CCL-149 cells by negative regulation of the G2/M cell cycle. We suggest that it can be used as a specific target for the treatment of alveolar epithelium.

Inhibition of the integrin/FAK signaling axis and c-Myc synergistically disrupts ovarian cancer malignancy.

Integrins, a family of heterodimeric receptors for extracellular matrix, are promising therapeutic targets for ovarian cancer, particularly high-grade serous-type (HGSOC), as they drive tumor cell attachment, migration, proliferation and survival by activating focal adhesion kinase (FAK)-dependent signaling. Owing to the potential off-target effects of FAK inhibitors, disruption of the integrin signaling axis remains to be a challenge. Here, we tackled this barrier by screening for inhibitors being functionally cooperative with small-molecule VS-6063, a phase II FAK inhibitor. From this screening, JQ1, a potent inhibitor of Myc oncogenic network, emerged as the most robust collaborator. Treatment with a combination of VS-6063 and JQ1 synergistically caused an arrest of tumor cells at the G2/M phase and a decrease in the XIAP-linked cell survival. Our subsequent mechanistic analyses indicate that this functional cooperation was strongly associated with the concomitant disruption of activation or expression of FAK and c-Myc as well as their downstream signaling through the PI3K/Akt pathway. In line with these observations, we detected a strong co-amplification or upregulation at genomic or protein level for FAK and c-Myc in a large portion of primary tumors in the TCGA or a local HGSOC patient cohort. Taken together, our results suggest that the integrin-FAK signaling axis and c-Myc synergistically drive cell proliferation, survival and oncogenic potential in HGSOC. As such, our study provides key genetic, functional and signaling bases for the small-molecule-based co-targeting of these two distinct oncogenic drivers as a new line of targeted therapy against human ovarian cancer.

[Diagnostic value of endobronchial ultrasound-guided transbronchial needle aspiration in stage â and stage â ¡ of sarcoidosis].

OBJECTIVE: To evaluate the diagnostic value of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in stage Ⅰ and stage Ⅱ of sarcoidosis. METHODS: There were 55 patients of sarcoidosis selected from January 2012 to October 2014 in the First Affiliated Hospital of Soochow University. The diagnostic positive rate and the positive rate in stage Ⅰ and Ⅱ patients with sarcoidosis through EBUS-TBNA, conventional transbronchial needle aspiration (cTBNA), endobronchial biopsy (EBB) combined with transbronchial lung biopsy (TBLB) were calculated. The positive rate of single lymph node puncture and the positive rate of different size of lymph node were compared. The difference of the positive rate of lymph nodes in different stations was checked. By calculating the diagnostic yield of EBUS-TBNA in sarcoidosis patients, the practicality and safety of EBUS-TBNA in diagnosing stage Ⅰ or stage Ⅱ sarcoidosis was assessed. RESULTS: Among 55 patients, 46 patients obtained positive results through EBUS-TBNA or cTBNA. There were 18 patients who had only received EBUS-TBNA among a total of 55 patients diagnosed with sarcoidosis, positive results appeared in 17 cases, while cTBNA was 9/15. There were 20 cases diagnosed in which both had EBUS-TBNA and cTBNA. The diagnostic rate of cTBNA combined with EBB and TBLB was 25/26, while combined with EBUS-TBNA was 21/21. Totally 90 lymph nodes were punctured by EBUS-TBNA, in which 65 lymph nodes got the positive results (65/90) while 49 lymph nodes got the positive results by TBNA in 93 punctured lymph nodes (49/93). The diagnostic positive rate in the lymph nodes with a short diameter ≥2 cm was 33/37 by EBUS-TBNA, while it was 12/19 in those whose short diameter 1-<2 cm. By cTBNA, the diagnostic positive rate was 15/26 and 11/28. No serious adverse events occurred. CONCLUSIONS: The diagnosis of sarcoidosis in stage Ⅰ and stage Ⅱ by EBUS-TBNA is safe and effective, while choosing the most noticeable swelling lymph node to puncture is recommended. Combining the EBUS-TBNA with traditional bronchoscope technology can obtain a further higher diagnostic efficiency.

[The clinical characteristics analysis of Escherichia coli bloodstream infection].

OBJECTIVE: To explore the clinical features of Escherichia coli bloodstream infection. METHODS: The clinical data of underlying diseases, antimicrobial susceptibility, temperature at blood sampling, results of routine blood tests, venous catheterization, therapy and prognosis of Escherichia coli bloodstream infection in the First Affiliated Hospital of Soochow University from January 2007 to December 2014 were analyzed retrospectively. The pathogens were routinely isolated and identified. Susceptibilities against antimicrobial agents were determined by Kirby-Bauer methods. RESULTS: All patients had at least one underlying disease. Most of the basic diseases were hematological malignancies, malignant solid tumors, pneumonia and so on. Body temperature was normal in 40 patients (6.4%), fever in 587 patients (93.5%) and low temperature in 1 patient. There were 252 patients with leukopenia (40.1%), 237 patients with granulocytopenia (37.7%) and 216 patients with agranulocytosis. The resistance rate to imipenem was 3.3%, which was the lowest among the total antimicrobial susceptibilities of 628 Escherichia Coli. The extended-spectrum-ß-lactamase (ESBL)-producing strains accounted for 53.8% among the total patients. The resistance rates of ESBLs-producing-Escherichia coli for the Sulfamethoxazole, Ampicillin, Gentamicin, Cefazolin, Cefuroxime, Cefotaxime, Ceftriaxone, Cefepime, Ceftazidime, Cefoperazone, Piperacillin and Ciprofloxacin were 80.2%, 100.0%, 62.4%, 99.1%, 99.1%, 98.8%, 98.2%, 48.5%, 50.6%, 95.0%, 98.2%, 79.6%, respectively, which were higher than that of non-ESBLs-producing-Escherichia coli (67.9%, 79.7%, 47.6%, 50.0%, 47.2%, 41.0%, 40.3%, 27.2%, 24.1%, 40.0%, 56.2%, 58.3%, respectively), the differences were significant statistically (χ(2)=12.33, 75.90, 13.92, 209.00, 224.94, 259.25, 256.59, 27.79, 46.19, 222.85, 165.08, 33.59, all P<0.05). One hundred and seventy eight patients received venous catheterization when blood culture were performed. All the patients received antimicrobial treatment, mainly including carbapenem antibiotics and beta-lactamase inhibitors combinations. Of which 533 patients were improved, the improvement rate was 84.9%. CONCLUSIONS: There are many risk factors in relation to Escherichia coli bloodstream infection. The antimicrobial resistance rate of ESBLs-producing-Escherichia coli is higher than that of none-ESBLs-producing-Escherichia coli. Individualized strategies should be based on antimicrobial sensitivity.

Improved liquid crystal pretilt angles by patterned dual alignment coating structures.

The pretilt angles for the optically compensated bend (OCB) mode liquid crystals have been improved using novel patterned dual alignment coating structures in this study. The transition from the splay configuration to the bend configuration can thus be effectively reduced. The dual alignment coating structures consisted of a horizontal alignment polyimide (PI) and a patterned vertical alignment liquid crystal polymer (LCP). Three patterning masks were designed for the photolithography process. The pretilt angles were demonstrated to be increased to 34 degrees for the triangle lattice array-patterned cells. It became 31 degrees for the square lattice array-patterned cells, and 24 degrees for the honeycomb lattice array-patterned cells. The improved pretilt angles were illustrated by the force balance model that can be predicted by the LCP area ratio. The effective control over the pretilt angle could improve the response time to 2 ms when the voltage was ramped up to 5.5 V for the OCB mode liquid crystal devices.

Polymerase chain reaction tests for the identification of Ross River, Kunjin and Murray Valley encephalitis virus infections in horses.

OBJECTIVE: To develop and validate specific, sensitive and rapid diagnostic tests using RT-PCR for the detection of Ross River virus (RRV), Kunjin virus (KV) and Murray Valley encephalitis virus (MVEV) infections in horses. METHODS: Primer sets based on nucleotide sequence encoding the envelope glycoprotein E2 of RRV and on the nonstructural protein 5 (NS5) of KV and MVEV were designed and used in single round PCRs to test for the respective viruses in infected cell cultures and, in the case of RRV, in samples of horse blood and synovial fluid. RESULTS: The primer pairs designed for each of the three viruses amplified a product of expected size from prototype viruses that were grown in cell culture. The identity of each of the products was confirmed by nucleotide sequencing indicating that in the context used the RT-PCRs were specific. RRV was detected in serums from 8 horses for which there were clinical signs consistent with RRV infection such that an acute-phase serum sample was taken and submitted for RRV serology testing. The RRV RT-PCR was analytically sensitive in that it was estimated to detect as little as 50 TCID50 of RRV per mL of serum and was specific in that the primer pairs did not amplify other products from the 8 serum samples. The RRV primers also detected virus in three independent mosquito pools known to contain RRV by virus isolation in cell culture. Samples from horses suspected to be infected with KV and MVEV were not available. CONCLUSION: Despite much anecdotal and serological evidence for infection of horses with RRV actual infection and associated clinical disease are infrequently confirmed. The availability of a specific and analytically sensitive RT-PCR for the detection of RRV provides additional opportunities to confirm the presence of this virus in clinical samples. The RT-PCR primers for the diagnosis of KV and MVEV infections were shown to be specific for cell culture grown viruses but the further validation of these tests requires the availability of appropriate clinical samples from infected horses.

The effects of acetylsalicylic acid on proliferation, apoptosis, and invasion of cyclooxygenase-2 negative colon cancer cells.

BACKGROUND: Acetylsalicylic acid (ASA, aspirin), the most common nonsteroidal anti-inflammatory drug (NSAID), has been shown to have a protective effect against the incidence and mortality of colorectal cancer. However, the mechanism of its anticancer function remains unclear. The aim of this study was to determine the effects of acetylsalicylic acid on proliferation, apoptosis, and invasion in human cyclooxygenase-2 (COX-2) negative colorectal cancer cell lines. MATERIALS AND METHODS: After treatment with various concentrations of ASA, cell proliferation was measured in the human colon cancer cell line SW480. Apoptotic cells were identified by transmission electron microscopy, acridine orange staining, and flow cytometry. The invasive potential of SW480 cells was detected using an in vitro invasion assay. The production of carcinoembryonic antigen was measured by microparticle enzyme immunoassay. Expression of Bcl2, Bax, CD44v6, and nm23 were evaluated by immunocytochemistry. RESULTS: ASA significantly inhibited the proliferation of SW480 cells and stimulated apoptosis. Production of carcinoembryonic antigen and the invasive potential of SW480 cells were also inhibited by ASA. After treatment with ASA, down-regulation of Bcl2 and CD44v6 expression and up-regulation of nm23 expression were observed in SW480 cells. No obvious effect of ASA was found on Bax expression. CONCLUSION: Our findings reveal that ASA inhibits the proliferation and promotes apoptosis in the human colon cancer cell line SW480. Down-regulation of Bcl2 expression might represent a potential mechanism by which ASA induces apoptosis in this COX-2 negative colon cancer cell line. Our results also suggest that ASA decreases the invasive potential of these colon cancer cells. Decreased CEA content and CD44v6 expression and elevated nm23 expression may contribute to the effect of ASA on invasive potential of SW480 colon cancer cells.

Sequence conservation and antigenic variation of the structural proteins of equine rhinitis A virus.

The nucleotide and deduced amino acid sequences of the P1 region of the genomes of 10 independent equine rhinitis A virus (ERAV) isolates were determined and found to be very closely related. A panel of seven monoclonal antibodies to the prototype virus ERAV.393/76 that bound to nonneutralization epitopes conserved among all 10 isolates was raised. In serum neutralization assays, rabbit polyclonal sera and sera from naturally and experimentally infected horses reacted in a consistent and discriminating manner with the 10 isolates, which indicated the existence of variation in the neutralization epitopes of these viruses.

Selection of rotavirus VP4 cell receptor binding domains for MA104 cells using a phage display library.

Rotavirus infection of host cells, like other viruses, is a complex process that has not been fully elucidated, and much attention has been focused on the regions of the viral attachment protein, VP4, that are involved in binding to the cellular receptor. In this study, phage display technology was employed to generate a g3p VP4 gene-targeted phage display peptide library using the porcine rotavirus strain CRW8, and a method was optimised for panning this library on adherent MA104 cells to identify receptor binding domains. Recombinant phage that displayed expressed peptides from both the rotavirus VP4 trypsin cleavage products VP8* and VP5* were selected, and while some of the phage clones contained insert sequences from regions of VP4 implicated previously in cell binding and infection, new domains were also identified. In all, four regions within VP8* and six regions of VP5* were selected by panning. To our knowledge, this paper is the first description of using a gene-targeted phage display library to identify receptor binding domains on viral proteins.

Rotavirus VP7 epitope mapping using fragments of VP7 displayed on phages.

cDNA copies of the complete porcine rotavirus CRW-8 VP7 gene were randomly digested to fragments of about 30-60 or 30-500 base pairs by DNase1 in the presence of Mn(2+). The fragments were cloned and expressed in a filamentous phage fd-tet-derived vector to create specific-gene-related peptide libraries. Polyclonal antibodies were then used to pan the SGRP libraries for antibody-binding phages. Analysis of the phage isolates revealed that the majority (86%) of them only had a single insert. However, phages displaying composite inserts containing the VP7 antigenic regions A, B, and C, originally defined by neutralising monoclonal antibody escape mutants, were also isolated. Inserts containing A or C region peptide were found to contain extra sequences from the C region, while the B region epitope was linear and had additional sequence from either upstream or downstream. In addition a dominant and possibly non-neutralising VP7 epitope was identified around amino acids 263-270. One of the recreated antigenic epitopes has also been fused to the outer membrane protein A (OmpA) of Escherichia coli and shown to maintain its antigenicity. The results in this study may have significant implication for recreation of conformational epitopes and vaccine development.