ABSTRACT
Turkey coronaviral enteritis caused by turkey coronavirus (TCoV) continues to infect turkey flocks, resulting in significant economic loss. Determining and understanding genetic relationships among different TCoV isolates or strains is important for controlling the disease. Using two-step RT-PCR assays that amplify the full length of TCoV spike (S) gene, TCoV isolates can be sequenced, analyzed, and genotyped. Described in this chapter is the protocol on PCR amplification and sequencing analysis of full-length TCoV S gene. Such protocol is useful in molecular epidemiology for establishing an effective strategy to control the transmission of TCoV among turkey flocks. Copyright © Springer Science+Business Media New York 2016
ABSTRACT
Turkey coronavirus (TCoV) infection causes acute atrophic enteritis in turkey poults, leading to significant economic loss in the turkey industry. Rapid detection, differentiation, and quantitation of TCoV are critical to the diagnosis and control of the disease. A specific one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using TCoV-specific primers and dual-labeled fluorescent probe for detection and quantitation of TCoV in feces and intestine tissues is described in this chapter. The fluorogenic probe labeled with a reporter dye (FAM, 6-carboxytetramethylrhodamine) and a quencher dye (Absolute QuencherTM) was designed to bind to a 186 base-pair fragment flanked by the two PCR primers targeting the 3' end of spike gene (S2) of TCoV. The assay is highly specific and sensitive and can quantitate between 102 and 1010 copies/mL of viral genome. It is useful in monitoring the progression of TCoV-induced atrophic enteritis in the turkey flocks. Copyright © 2016 Springer Science+Business Media New York.
ABSTRACT
A multiplex polymerase chain reaction (PCR) method for differential detection of turkey coronavirus (TCoV), infectious bronchitis virus (IBV), and bovine coronavirus (BCoV) is presented in this chapter. Primers are designed from the conserved or variable regions of nucleocapsid (N) or spike (S) protein genes of TCoV, IBV, and BCoV and used in the same PCR reaction. Reverse transcription followed by PCR reaction is used to amplify a portion of N or S gene of the corresponding coronaviruses. Two PCR products, a 356-bp band corresponding to N gene and a 727-bp band corresponding to S gene, are obtained for TCoV. In contrast, one PCR product of 356 bp corresponding to a fragment of N gene is obtained for IBV strains and one PCR product of 568 bp corresponding to a fragment of S gene is obtained for BCoV. Copyright © Springer Science+Business Media New York 2016.
ABSTRACT
Turkey coronavirus (TCoV) infection continues to threaten turkey industry. Because specific treatment and effective vaccination program are not available, rapid and cost-effective detection of antibodies to TCoV infection is an important control measure to monitor the disease status in the fields. Two antibody-capture enzyme-linked immunosorbent assay (ELISA) procedures for detection of antibodies to TCoV are outlined in this chapter. One ELISA method uses chicken infectious bronchitis coronavirus (IBV) as the coating antigen based on antigenic cross-reactivity between TCoV and IBV. The other method relies on a recombinant TCoV nucleocapsid protein. Both methods are useful for serological diagnosis of TCoV infection in the turkey flocks. Copyright © Springer Science+Business Media New York 2016.
ABSTRACT
Expression and purification of turkey coronavirus (TCoV) nucleocapsid (N) protein from a prokaryotic expression system as histidine-tagged fusion protein are presented in this chapter. Expression of histidine-tagged fusion N protein with a molecular mass of 57 kDa is induced with isopropyl beta-D-1-thiogalactopyranoside (IPTG). The expressed N protein inclusion body is extracted and purified by chromatography on nickel-agarose column to near homogeneity. The protein recovery can be 10 mg from 100 ml of bacterial culture. The purified N protein is a superior source of TCoV antigen for antibody-capture ELISA for detection of antibodies to TCoV. Copyright © Springer Science+Business Media New York 2016.
ABSTRACT
Turkey coronavirus (TCoV) infection induces the production of protective antibodies against the sequent exposure of TCoV. Serological tests to determine TCoV-specific antibodies are critical to evaluate previous exposure to TCoV in the turkey flocks and differentiate serotypes from different isolates or strains. A specific virus neutralization assay using embryonated turkey eggs and immunofluorescent antibody assay for determining TCoV-specific neutralizing antibodies is described in this chapter. Virus neutralization titer of turkey serum from turkeys infected with TCoV is the dilution of serum that can inhibit TCoV infection in 50 % of embryonated turkey eggs. Virus neutralization assay for TCoV is useful to monitor the immune status of turkey flocks infected with TCoV for the control of the disease. Copyright © Springer Science+Business Media New York 2016.
ABSTRACT
Background: Access to health care including clinical trials (CT) leading to paradigm-changing cancer treatments are critical for high quality cancer care and equity in society. In this report, we highlight methods in accruing to ETCTN wherein underrepresented rural, low-income, and racial minorities comprise >50% of enrollment. Methods: University of Kansas Cancer Center (KUCC) is one of eight National Cancer Institute (NCI) designated cancer centers awarded CATCH-UP.2020 (CATCH-UP), a congressionally mandated P30 supplement to enhance access for minority/underserved populations to ETCTN precision medicine CT. KUCC catchment area is 23% rural by Rural Urban Continuum Codes (RUCC);almost 90 % of counties are designated primary care HPSA's (Health Professional Shortage Areas). KUCC Early Phase and Masonic Cancer Alliance (rural outreach network) partnered to operationalize CATCH-UP. We engaged disease-focused champion investigators in disease working groups and MCA physicians who selected scientifically sound CT that fit catchment area needs. Patient and Investigator Voices Organizing Together, a patient research advocacy group provided practical feedback. MCA navigator coordinated recruitment. Telehealth was used for rural patients that would have a significant distance to travel just to be screened. Results: CATCH-UP was initiated in September 2020. Twenty-eight CT were activated, many in community sites. Average activation time was 81 days. Delays were mainly from CT amendments. KUCC enrolled the first patient in the CATCH-UP program. In 6 months, we met accrual requirements (24/year, 50% minorities). During first year, we enrolled 47 (>50% minorities), an increase of 680% from our average accrual of 6/year (>50% minorities) in ETCTN through Early Drug Development Opportunity Program (2016-2020). To date, we have enrolled 61, 54% from rural, HPSA, race and other minorities. Although the proportion of minorities did not change but remained high, this funding allowed us to substantially increase the number of patients from a catchment area with high proportion of geographically and socioeconomically underserved minorities given access to early phase CT through ETCTN. Conclusions: Amid COVID-19 pandemic, the NCI CATCH-UP program and methods we used allowed access to novel therapies for rural, medically underserved, and other minority groups.
ABSTRACT
Background The SARS-CoV-2 pandemic has assaulted all aspects of daily life. Medical professionals in oncology face additional challenges with balancing prompt cancer diagnosis and urgent treatment against potential COVID-19 exposure risk in these high-risk patients. We designed this prospective freewill study to offer testing for SAR2-CoV-2 viral RNA and/or anti-COVID-19, respectively in asymptomatic medical and research staff who work in direct contact with cancer patients. The overall goal was to evaluate the prevalence of infection in this group of asymptomatic healthcare providers to reduce exposure of cancer patients to asymptomatic staff. Methods Asymptomatic medical and research staff who work in direct contact with cancer patients were asked to voluntarily be tested for either SARS-CoV-2 viral RNA or antibodies or both. Either NP swabs and/or blood samples (EDTA tube) were collected. Tests are performed at Sinochips Kansas LLC, Sinochips Diagnostics (CLIA number:17D2176068, CAP number: 8709463). The PCR test is performed with FDA authorized 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel EUA. The Elecsys® Anti-SARS-CoV-2 (Roche Diagnostics) immunoassay was used to qualitative detection of antibodies to SARS-CoV-2 in human plasma. Results From 06/18/2020 to 12/18/2020, 861 participated in the study. 1095 tests were completed for SAR2-CoV-2 virus infection, and 918 were completed for antibody. Amount participants, 530 had both virus and antibody tested. 235 were tested more than once for viral infection and 166 were tested more than once for the antibody. Median age of participants was 39 years (IQR 32-51 years). Among these 84.7% were females, 84.4% white, 6.7% African American, 4.8% Asian and 84.7% non-Hispanic. The cumulative incidence of a positive test for the virus was 2.2% (16/712), and for the antibody test was 3.8% (26/679). 5 had both viral and antibody tests positive, with an average time of 4.1 weeks from viral testing positivity to detectable antibody among 3 cases and 2 cases with both viral infection and antibody detected at same time. There were 3 cases virus was detected more than once after turning positive. 2 remained positive at 16 and 22 days after initial test and one turned negative at 36 days as of last follow up. There were 7 cases where the antibody was tested more than once after turning positive and all 7 remained positive as of last follow up (range 7-103 days). Conclusion Prospective voluntary testing in asymptomatic medical and research staff who work in direct contact with cancer patients was feasible and resulted in identification of asymptomatic carriers who then placed in quarantine, thereby limiting exposure to cancer patients. Medical and research staff who work with cancer patients are general very cautious and the frequency of infections were significantly lower than general society. In addition, it seems that 1) virus and antibody may co-exist in the same person after exposure, and 2) the antibody may last for a relatively long time.