Development of a duplex real-time PCR assay for simultaneous detection and differentiation of Theileria equi and Babesia caballi.

Equine Piroplasmosis (EP) is a tick-borne disease caused by three apicomplexan protozoan parasites, Theileria equi (T. equi), Babesia caballi (B. caballi) and T. haneyi, which can cause similar clinical symptoms. There are five known 18S rRNA genotypes of T. equi group (including T. haneyi) and three of B. caballi. Real-time PCR methods for detecting EP based on 18S rRNA analysis have been developed, but these methods cannot detect all genotypes of EP in China, especially genotype A of T. equi. In this study, a duplex real-time PCR detection method was developed for the simultaneous detection and differentiation of T. equi and B. caballi. The primers and probes for this duplex real-time PCR assay were designed based on the conserved 18S rRNA gene sequences of all genotypes of T. equi and B. caballi including Chinese strain. Double-quenched probes were used in this method, which provide less background and more signal to decrease the number of false positives relative to single-quenched probes. The newly developed real-time PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. The real-time PCR assays were further validated by comparison with a nested PCR assay and a previous developed real-time PCR for EP and sequencing results in the analysis of 506 clinical samples collected from 2019 to 2020 in eleven provinces and regions of China. Based on clinical performance, the agreements between the duplex real-time PCR assay and the nPCR assay or the previous developed real-time PCR assay were 92.5% (T. equi) and 99.4% (B. caballi) or 87.4% (T. equi) and 97.2% (B. caballi). The detection results showed that the positivity rate of T. equi was 43.87% (222/506) (10 genotype A, 1 genotype B, 4 genotype C, 207 genotype E), while that of B. caballi was 5.10% (26/506) (26 genotype A), and the rate of T. equi and B. caballi co-infection was 2.40% (12/506). The established method could contribute to the accurate diagnosis, pathogenic surveillance and epidemiological investigation of T. equi and B. caballi infections in horses.

Olfactory Dysfunction in Patients With Coronavirus Disease 2019: A Review.

The coronavirus disease 2019 (COVID-19) pandemic is wreaking havoc on public-health and economic systems worldwide. Among the several neurological symptoms of patients with COVID-19 reported in clinical practice, olfactory dysfunction (OD) is the most common. OD occurs as the earliest or the only clinical manifestation in some patients. Increasing research attention has focused on OD, which is listed as one of the main diagnostic symptoms of severe acute respiratory syndrome-coronavirus-2 infection. Multiple clinical and basic-science studies on COVID-19-induced OD are underway to clarify the underlying mechanism of action. In this review, we summarize the clinical characteristics, mechanisms, evaluation methods, prognosis, and treatment options of COVID-19-induced OD. In this way, we hope to improve the understanding of COVID-19-induced OD to aid early identification and precise intervention.

Possible pharmaceutical applications can be developed from naturally occurring phenanthroindolizidine and phenanthroquinolizidine alkaloids.

Naturally occurring phenanthroindolizidine and phenanthroquinolizidine alkaloids (PIAs and PQAs) are two small groups of herbal metabolites sharing a similar pentacyclic structure with a highly oxygenated phenanthrene moiety fused with a saturated or an unsaturated N-heterocycle (indolizidine/quinolizidine moieties). Natural PIAs and PQAs only could be obtained from finite plant families (such as Asclepiadaceae, Lauraceae and Urticaceae families, etc.). Up to date, more than one hundred natural PIAs, while only nine natural PQAs had been described. PIA and PQA analogues have been applied to the development of potent anticancer agents all along because of their excellent cytotoxic activity. However, in the last two decades, other great biological properties, such as anti-inflammatory and antiviral activities were revealed successively by different pharmacological assays. Especially because of their potent antiviral activity against coronavirus (TGEV, SARS CoV and MHV) and tobacco mosaic virus, PIA and PQA analogues have attracted much pharmaceutical attention again, some of them have been used to present interesting targets for total or semi synthesis, and structure-activity relationship (SAR) study for the development of antiviral agents. In this review, natural PIA and PQA analogues obtained in the last two decades with their herbal origins, key spectroscopic characteristics for structural identification, biological activity with possible SARs and application prospects were systematically summarized. We hope this paper can stimulate further investigations on PIA and PQA analogues as an important source for potential drug discovery.

[Application of Reyanning Mixture in evaluating combining disease with syndrome of human coronavirus pneumonia with pestilence attacking lung syndrome].

In the previous research, our laboratory established a mouse model combining disease with syndrome of human coronavi-rus pneumonia with pestilence attacking the lung syndrome, based on the national traditional Chinese medicine clinical classification of Novel Coronavirus Infected Pneumonia Diagnosis and Treatment Plan. In this study, a mouse model combining disease with syndrome of human coronavirus pneumonia with pestilence attacking the lung syndrome was used to evaluate the effectiveness of Reyanning Mixture to provide animal experimental support for clinical application. Mice were divided into normal group, 229 E infection group, cold-dampness group, cold-dampness+229 E infection group(the model group), Reyanning high and low dose groups. The cold-dampness group, cold-dampness+229 E infection group, two Reyanning groups were given cold and damp stimulation for 7 days. On the 5 th day, the 229 E infection group, cold-dampness+229 E infection group, and two Reyanning groups were infected with HCoV-229 E virus. Reyanning was administered for 3 days, starting from the day of infection. Blood was collected on the 4 th day and the lung tissue was dissected to calculate the lung index and inhibition rate; flow cytometry was used to detect the percentage of T and B lymphocytes in peripheral blood; RT-PCR was used to detect the nucleic acid virus load in lung tissue; ELISA was used to detect motilin and gastrin in serum, and inflammatory factors TNF-α, IFN-γ, IL-6, IL-10 in lung tissue proteins. Reyanning Mixture could reduce the lung index(P<0.01) of coronavirus pneumonia mice with pestilence attacking the lung; it could significantly increase the percentage of CD8~+ T lymphocytes and CD4~+ T lymphocytes in peripheral blood of model mice(P<0.05, P<0.01). The low dose of Reyanning could effectively increase the percentage of total B lymphocytes(P<0.05), reduce virus load in lung tissue of model mice(P<0.01), reduce the levels of TNF-α, IFN-γ, IL-6, IL-10 in the lung tissue of model mice(P<0.01), reduce the content of motilin in the serum of model mice(P<0.01). Reyanning Mixture convey a better effect in treating coronavirus pneumonia mice with pestilence attacking the lung. It manifested obvious effects in improving lung lesions, enhancing the gastrointestinal function of mice, improving the autoimmune function of mice, and reducing the expression of inflammatory factors in vivo, which could provide evidences for clinical research.