Foundation and Clinical Evaluation of a New Method for Detecting SARS-CoV-2 Antigen by Fluorescent Microsphere Immunochromatography.

Purpose: To develop a rapid detection reagent for SARS-CoV-2 antigen for the auxiliary diagnosis of new coronary pneumonia (COVID-19), and perform the methodological evaluation and clinical evaluation of the reagent. Method: SARS-CoV-2 N-protein test strip was created by combining fluorescent microsphere labeling technology and immunochromatographic technology, based on the principle of double antibody sandwich. Then we evaluated the analytical capability and clinical application of the strips. Result: The limit of detection of the strips for recombinant N protein was 100 ng/ml and for activated SARS -CoV-2 virus was 1 × 103 TCID50/ml. The strips also have high analytical specificity and anti-interference capability. According to the predetermined cut-off value, the specificity of the test strip in healthy controls and patients with other respiratory disease was 100.00 and 97.29%, the sensitivity in COVID-19 cases at progress stage and cured stage was 67.15 and 7.02%. The positive percentage agreement and negative percentage agreement of antigen strip to RNA test were 83.16 and 94.45%. Conclusion: SARS-CoV-2 fluorescence immunochromatographic test strip can achieve fast, sensitive and accurate detection, which can meet the clinical requirements for rapid detection of viruses on the spot.

Identification of Putative UL54 (ICP27) Transcription Regulatory Sequences Binding to Oct-1, v-Myb, Pax-6 and Hairy in Herpes Simplex Viruses.

An oncolytic herpes simplex virus (oHSV) has proven amenable in oncolytic virotherapy and was approved to treat melanoma. The immediate-early (IE) protein ICP27 encoded by gene UL54 is essential for HSV infection. Post-transcriptional modification of UL54 would increase tumor targeting of oHSVs. However, UL54 gene transcription regulatory sequences and factors were not reported yet. Here we isolated a new strain LXMW of type 1 HSV (HSV-1-LXMW) in China and found it's closely related to HSV-1 strains Patton and H129 in the US by the first and next generation DNA sequencing viral DNA phylogenetic analysis. Using a weight matrix-based program Match, we found the UL54 transcription regulatory sequences binding to the transcription factors Oct-1, v-Myb and Pax-6 in HSV-1-LXMW, while the sequences binding to Oct-1 and Hairy in a HSV-2 strain. Further validation showed that HSV-1 and HSV-2 shared the common sequence binding to Oct-1, but had unique sequences to bind v-Myb and Pax-6, or Hairy, respectively, by DNA sequence alignment of total 11 HSV strains. The published results howed that the expression of transcription factors is consistent with the tissue tropism of HSV-1 and HSV-2. In the current article a new HSV-1 strain LXMW was isolated and its putative HSV UL54 transcription regulatory sequences and factors were identified for the first time. Our findings highlight the new understanding of the principles of transcriptional regulation in HSV biology and oncolytic virotherapy.

CRISPR/Cas9 genome editing technology significantly accelerated herpes simplex virus research.

Herpes simplex viruses (HSVs) are important pathogens and ideal for gene therapy due to its large genome size. Previous researches on HSVs were hampered because the technology to construct recombinant HSVs were based on DNA homology-dependent repair (HDR) and plaque assay, which are inefficient, laborious, and time-consuming. Fortunately, clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) recently provided the possibility to precisely, efficiently, and rapidly edit genomes and indeed is successfully being used in HSVs. Importantly, CRISPR/Cas9 technology increased HSV HDR efficiency exponentially by a 10,000-1,000,000 times when making recombinant HSVs, and its combination with flow cytometric technology made HSV recombination practically automatic. These may have a significant impact on virus and gene therapy researches. This review will summarize the latest development and molecular mechanisms of CRISPR/Cas9 genome editing technology and its recent application in HSVs.

Oncolytic herpes simplex virus tumor targeting and neutralization escape by engineering viral envelope glycoproteins.

Oncolytic herpes simplex viruses (oHSVs) have been approved for clinical usage and become more and more popular for tumor virotherapy. However, there are still many issues for the oHSVs used in clinics and clinical trials. The main issues are the limited anti-tumor effects, intratumor injection, and some side effects. To overcome such challenges, here we review the genetic engineering of the envelope glycoproteins for oHSVs to target tumors specifically, and at the same time we summarize the many neutralization antibodies against the envelope glycoproteins and align the neutralization epitopes with functional domains of the respective glycoproteins for future identification of new functions of the glycoproteins and future engineering of the epitopes to escape from host neutralization.

Detection of yellow fever virus genomes from four imported cases in China.

Yellow fever virus (YFV), as the first proven human-pathogenic virus, is still a major public health problem with a dramatic upsurge in recent years. This is a report on four imported cases of yellow fever virus into China identified by whole genome sequencing. Phylogenetic analysis was performed and the results showed that these four viruses were highly homologous with Angola 71 strains (AY968064). In addition, effective mutations of amino acids were not observed in the E protein domain of four viruses, thus confirming the effectiveness of the YFV-17D vaccine (X03700). Although there is low risk of local transmission in most part of China, the increasing public health risk of YF caused by international exchange should not be ignored.

Seroprevalence of Lyme disease and associated risk factors in rural population of Beijing.

A seroepidemiological survey of 801 local residents from 28 villages was conducted to assess the seroprevalence of Lyme disease and to identify the risk factors of becoming seropositive for Lyme disease in the northern suburb of Beijing. Forty-one serum samples were positive for IgG against B burgdorferi and the seroprevalence was 5.1% (41/801), indicating that Lyme disease is endemic in the rural population. In the multivariable analysis, sowing and harvesting in summer (OR, 2.377, 95% CI, 1.233-4.583), weed in the yard (OR, 1.914, 95% CI, 1.003-3.655) were positively associated with Lyme disease, while wearing protective clothes (OR, 0.173, 95% CI, 0.041-0.732) was negatively associated with Lyme disease. People living in the area are easily infected just near the house or in the cropland. They were barely diagnosed and cured. Without clear tick knowledge, the people are at high risk of exposure to tick bite and Lyme disease.

Autoprocessing: an essential step for expression and purification of enterovirus 71 3C(pro) in Escherichia coli.

A gene encoding the 3BC of human enterovirus 71 (EV71) was cloned and inserted into a derivative of plasmid pET-32a(+) driven by T7 promoter. The expressed 3C protease (3C(pro)) autocatalytically cleaved itself from the recombinant protein Trx-3BC and the mature 3C(pro) partitioned in the soluble fraction of bacterial lysate. The 13-amino-acid peptide substrates with the junction of 3B/3C were used to verify the proteolysis activity of the purified 3C(pro). The EV71 3C(pro) had a Km value of 63 µM (measured by a continuous fluorescence assay). The other solid-phase activity assay of the EV71 3C(pro) was developed using HPLC to analyze the proteolytic products. The combination of two activity assays contributes to promote the identification of the specific inhibitors targeted to the EV71 3C(pro).

A case-control study of risk factors associated with scrub typhus infection in Beijing, China.

To investigate the risk factors of scrub typhus infection in Beijing, China, a case-control study was carried out. Cases (n = 56) were defined as persons who were diagnosed by PCR and serological method within three years. Three neighborhood control subjects were selected by matching for age and occupation. Living at the edge of the village, living in the houses near grassland, vegetable field or ditch, house yard without cement floor, piling weeds in the house or yard, all of these were risk factors for scrub typhus infection. Working in vegetable fields and hilly areas, and harvesting in autumn posed the highest risks, with odds ratios (ORs) and 95% confidence intervals (CIs) of 3.7 (1.1-11.9), 8.2 (1.4-49.5), and 17.2 (5.1-57.9), respectively. These results would be useful for the establishment of a detail control strategy for scrub typhus infection in Beijing, China.

Laboratory diagnosis and genotype identification of scrub typhus from Pinggu district, Beijing, 2008 and 2010.

This study was conducted to determine the diagnosis and genotype of Orientia tsutsugamushi in Pinggu district, Beijing. Indirect immunofluorescence assay (IFA) was performed to detect O. tsutsugamushi-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies. Nested polymerase chain reaction (PCR) and DNA sequencing analysis targeting the O. tsutsugamushi-specific groEL gene and 56 kDa protein gene were performed on whole-blood samples from scrub typhus patients. We confirmed that 47 patients were infected with scrub typhus in Pinggu district, Beijing. Representative sequences amplified by primers according to the groEL gene (BJ-PG-2008; GenBank accession No. JQ894502) and the 56 kDa protein gene (PG-56kDa; GenBank accession No. JX843795) both clustered with Kawasaki. PG-56kDa had sequence homology of 100% with TADY12-0308, shandong-XDM2, Neimeng-90, and sdu-1 and sequence homology of 96% with Kawasaki, Taguchi, Oishi, and Kanda. We confirmed the genotype of O. tsutsugamushi in Pinggu district, Beijing, as Kawasaki, and the patient in 2008 confirmed in this study was the first patient with confirmed scrub typhus in Beijing.