查看更多>>摘要：? 2021 Elsevier B.V.The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-CoV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus, targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose50(TCID50)/mL or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of ?107 plaque forming units/mL at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of ?106.5 TCID50/mL. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts.
查看更多>>摘要：? 2021Considering the global impact of the coronavirus disease 2019 (COVID-19) pandemic, generating suitable experimental models is imperative. For pre-clinical studies, researchers require animal models displaying pathological features similar to those observed in patients; therefore, establishing animal models for COVID-19 is crucial. The golden Syrian hamster model mimics conditions observed in humans with mild severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, a golden Syrian hamster model of severe infection has not been reported. J2N-k hamsters are utilized as a cardiomyopathy model; therefore, we used cardiomyopathic J2N-k hamsters showing conditions similar to those of severe COVID-19 complicated with cardiovascular diseases, as patients with cardiovascular diseases exhibit a higher risk of morbidity and mortality due to COVID-19 than patients without cardiovascular diseases. Unlike that in golden Syrian hamsters, SARS-CoV-2 infection was lethal in J2N-k hamsters, with a median lethal dose of 104.75 plaque-forming units for the S clade of SARS-CoV-2 (A, GenBank: MW466791.1). High viral titers and viral genomes were detected in the lungs of J2N-k and golden Syrian hamster models harvested 3 days after infection. Pathological features of SARS-CoV-2-associated lung injury were observed in both models. The J2N-k hamster model can aid in developing vaccines or therapeutics against COVID-19.
查看更多>>摘要：? 2021 Elsevier B.V.Introduction: Plaque assay (PA) is a gold standard for virus titration and neutralization of various cytopathic viruses, including avian nephritis virus (ANV), the etiological agent associated with kidney disorders in chickens. In this study, as an alternative to the labor-intensive PA, we developed a spectrophotometric microplate assay (MA) for ANV titration and neutralization based on the virus cytopathicity to primary chicken kidney (CK) cells. Methods: CK cells were infected with ANV in the presence or absence of chicken serum in a 96-well microplate, and the virus-induced cytolysis was quantified by measurement of neutral red uptake using a spectrophotometer. The absorbance values obtained were subjected to a sigmoidal four-parameter logistic regression analysis for the virus titer determination and serum neutralization assessment. Accuracy and reliability of the serum neutralization MA in comparison to the standard PA was statistically evaluated. Results: The ANV-MA was capable of quantifying infectious virus titers based on a virus dose-dependent cytolysis of CK cells, and serum neutralization could be assessed as an inhibition of the virus-induced cytolysis accordingly. Statistical evaluation using a 2 × 2 contingency table and receiver-operating characteristic analyses showed 82 % sensitivity, 99 % specificity and 0.97 area under the curve, supporting an overall diagnostic accuracy of the neutralization MA. Conclusion: The newly developed MA using simplified experimental procedures in the microplate format and direct spectophotometric data readout is readily applicable to general laboratories for high-throughput screening of serum neutralization of ANV.
de Camargo B.R.da Silva L.A.de Oliveira A.S.Ribeiro B.M....
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查看更多>>摘要：? 2021 Elsevier B.V.The COVID-19 pandemic has demanded a range of biotechnological products for detection of SARS-CoV-2 variants and evaluation of human seroconversion after infection or vaccination. In this work, we describe an easy pipeline for expression of SARS-CoV-2 nucleocapsid (N) protein in insect cells followed by its purification via affinity chromatography. The N gene was cloned into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via transposition and the resulting recombinant baculovirus was used for infection of lepidopteran Sf9 cells adapted to high-density suspension. Using Tris?HCl pH 8.0 buffer as mobile phase and eluting bound proteins with 175 mM imidazole as part of a three-step gradient, an average of 1 mg N protein could be purified from each 50 mg of total protein from clarified supernatant. Such protein amount allows the manufacturing of serological tests and the development of basic studies on cellular responses to SARS-CoV-2.
查看更多>>摘要：? 2021Mollicutes (Mycoplasma and Acholeplasma) are parasitic bacteria that adhere to cellular surfaces, naturally resistant to many antibiotics and extremely small. They are often found as contaminants in cultured cells, where they go unnoticed. They may be present in viral stocks because they are present in supernatants of cells where cultured viruses are released. The best way to keep laboratories free of Mycoplasma is to discard infected cultures, but, as judged by the very common finding of Mycoplasma-contaminated cultures in many laboratories, this is not done as often as it should be. A possible reason is that most procedures recommended take as long as performing a simple experiment and many laboratories delay testing to save money and time. Indeed, many methods exist to detect Mycoplasma infection of cell lines, but they take at least a couple of hours of hands-on work, if not more. Here we describe a procedure to screen viral stocks and tissue cultures for Mycoplasma presence. It relies on isolation of Mycoplasma on ordinary horse blood agar directly from exhausted tissue culture supernatants and does not require experienced personnel or expensive equipment. It only requires minutes of hands-on work, and, for this, it may be useful for weekly screening of cultures. It yields semiquantitative results in roughly 5 days, which is the time that usually passes between one subculture passage of cells in vitro to another. Because of its simplicity, it may be useful for detecting Mycoplasma in viral stocks and for frequent screening of cultures in research laboratories.
查看更多>>摘要：? 2021Human respiratory syncytial virus (HRSV) is a leading cause of acute respiratory illness in young children worldwide. Whole genome sequencing of HRSV offers enhanced resolution of strain variability for epidemiological surveillance and provides genomic information essential for antiviral and vaccine development. A 10-amplicon one-step RT-PCR assay and a 20-amplicon nested RT-PCR assay with enhanced sensitivity were developed to amplify whole HRSV genomes from samples containing high and low viral loads, respectively. Ninety-six HRSV-positive samples comprised of 58 clinical specimens and 38 virus isolates with Ct values ≤ 24 were amplified successfully using the 10-amplicon one-step RT-PCR method and multiplexed in a single MiSeq run. Genome coverage exceeded 99.3% for all 96 samples. The 20-amplicon nested RT-PCR NGS method was used to generate >99.6% HRSV full-length genome for 72 clinical specimens with Ct values ranging from 24 to 33. Phylogenetic analysis of the genome sequences obtained from the 130 clinical specimens revealed a wide diversity of HRSV genotypes demonstrating methodologic robustness.
查看更多>>摘要：? 2021Vaccination has greatly reduced global measles incidence, however measles remains endemic in many regions worldwide. Measles surveillance relies on high performance molecular detection of the virus. We have developed and validated a multiplex rRT-PCR assay for the detection of measles virus. The assay includes three independent probes with unique reporter dyes for the simultaneous detection of the measles hemagglutinin gene, nucleoprotein gene and endogenous RNaseP control. Using dilution series of synthetic RNAs the limits of detection were determined to be approximately 20 copies of measles RNA. The assay is extremely reproducible with very low intra-assay and inter-assay coefficients of varation for both the N and the H targets. After testing 68 confirmed measles positive and 86 measles negative archival clinical samples our data shows the multiplex assay has a sensitivity and specificity of 100 %, and a 100 % concordance with the expected results. No cross reactivity was identified with clinical specimens positive for six other viruses. According to the WHO, currently only the B3, D4, D8, H1 measles genotypes of the 24 recognized genotypes continue to circulate and this new multiplex assay successfully detected all four of those genotypes as well as six other genotypes.
查看更多>>摘要：? 2021In an era of decreasing genetic diversity of Measles Virus (MeV), effective surveillance requires a higher-resolution genotyping method or whole genome sequencing (WGS) to document elimination. Through optimization of MeV WGS protocol, we developed a MeV-specific probe enrichment method that allows next generation sequencing from clinical specimens. With the probe enrichment method, 70% of specimens can be sequenced at a read depth of greater than 10 reads with minimal off-target sequences.
查看更多>>摘要：? 2021The Middle East respiratory syndrome coronavirus (MERS-CoV), belonging to the family Coronaviridae and genus Betacoronavirus, has been recognized as a highly pathogenic virus. Due to the lack of therapeutic or preventive agents against MERS-CoV, developing an effective vaccine is essential for preventing a viral outbreak. To address this, we developed a recombinant S1 subunit of MERS-CoV spike protein fused with the human IgG4 Fc fragment (LV-MS1-Fc) in Chinese hamster ovary (CHO) cells. Thereafter, we identified the baculovirus gp64 signal peptide-directed secretion of LV-MS1-Fc protein in the extracellular fluid. To demonstrate the immunogenicity of the recombinant LV-MS1-Fc proteins, BALB/c mice were inoculated with 2.5 μg of LV-MS1-Fc. The inoculated mice demonstrated a significant humoral immune response, measured via total IgG and neutralizing antibodies. In addition, human dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with LV-MS1-Fc showed the protective capacity of LV-MS1-Fc against MERS-CoV with no inflammatory cell infiltration. These data showed that the S1 and Fc fusion protein induced potent humoral immunity and antigen-specific neutralizing antibodies in mice, and conferred protection against coronavirus viral challenge, indicating that LV-MS1-Fc is an effective vaccine candidate against MERS-CoV infection.
查看更多>>摘要：? 2021 Elsevier B.V.An apscaviroid, tentatively named citrus viroid VII (CVd-VII), was recently discovered in citrus in Australia. A diagnostic assay using real-time reverse transcription polymerase chain reaction was developed and validated to detect the viroid in citrus plants. The assay showed a high level of sensitivity, reliably detecting 2000 plasmid copies per reaction, while down to 20 plasmid copies per reaction were occasionally detected. The assay showed high specificity, producing no false positives or cross-reactivity with a range of other citrus graft-transmissible pathogens, including viroids, viruses and bacteria. The real-time assay was also found to be more sensitive than the available end-point reverse transcription polymerase chain reaction assay by a factor of 100,000 and could be a useful tool for the rapid detection of CVd-VII in diagnostic and research environments.
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