Ferrets: A powerful model of SARS-CoV-2.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in recent years not only caused a global pandemic but resulted in enormous social, economic, and health burdens worldwide. Despite considerable efforts to combat coronavirus disease 2019 (COVID-19), various SARS-CoV-2 variants have emerged, and their underlying mechanisms of pathogenicity remain largely unknown. Furthermore, effective therapeutic drugs are still under development. Thus, an ideal animal model is crucial for studying the pathogenesis of COVID-19 and for the preclinical evaluation of vaccines and antivirals against SARS-CoV-2 and variant infections. Currently, several animal models, including mice, hamsters, ferrets, and non-human primates (NHPs), have been established to study COVID-19. Among them, ferrets are naturally susceptible to SARS-CoV-2 infection and are considered suitable for COVID-19 study. Here, we summarize recent developments and application of SARS-CoV-2 ferret models in studies on pathogenesis, therapeutic agents, and vaccines, and provide a perspective on the role of these models in preventing COVID-19 spread.

[Effect of electroacupuncture on postpartum uterine contraction pain and uterine involution: a randomized controlled trial].

OBJECTIVE: To observe the effect of electroacupuncture on postpartum uterine contraction pain and uterine involution. METHODS: A total of 80 patients with postpartum uterine contraction pain were randomly divided into an observation group and a control group, 40 cases in each group. The observation group was treated with electroacupuncture at Dahe (KI 12), Zhongzhu (KI 15), Hegu (LI 4), Xuehai (SP 10), etc. for 30 min, once a day, 3 days were as one course, and 2 courses with 1-day interval were required. The control group was treated with oral Xinshenghua tablet, 4 tablets each time, 3 times a day for 7 days. Before treatment, 24, 48, 72 h into treatment and after treatment, the visual analogue scale (VAS) score was observed; the maximum anteroposterior diameter of uterine hemorrhage, the area of hemorrhage, the space between uterus fundus and umbilical cord, and the summation of three diameters of uterus before and after treatment were compared, and the time of postpartum uterine contraction pain disappeared was recorded in the two groups. RESULTS: Compared before treatment, the VAS scores of 24, 48, 72 h into treatment and after treatment were decreased in both groups (P<0.05), and those in the observation group were lower than the control group (P<0.05). Compared before treatment, the maximum anteroposterior diameter of uterine hemorrhage, the area of hemorrhage and the summation of three diameters of uterus after treatment were decreased (P<0.05), the space between uterus fundus and umbilical cord was increased in both groups (P<0.05), and those in the observation group were superior to the control group (P<0.05). The number of days required to treat the uterine contraction pain in the observation group was shorter than the control group (P<0.05). CONCLUSION: Electroacupuncture can effectively relieve postpartum uterine contraction pain, accelerate the discharge of residual uterine hemorrhage in the uterine cavity, and promote uterine involution.

[Clinical observation on the therapeutic effect of warm acupuncture on endometrial cavity fluid from in vitro fertilization-embryo transfer].

OBJECTIVE: To observe the clinical effect of warm acupuncture on endometrial cavity fluid (ECF) from in vitro fertilization-embryo transfer (IVF-ET), and to explore the mechanism of warm acupuncture on ECF. METHODS: Twenty-nine patients who were prepared for IVF-ET from 2016 to 2019 and whose transplantation was cancelled due to ECF found by vaginal B-ultrasound examination were divided into an observation group (14 cases) and a control group (15 cases) according to random number table method. The warm acupuncture was applied at Zhongwan (CV 12), Qihai (CV 6), Guanyuan (CV 4), Zhongji (CV 3), Guilai (ST 29), Zigong (EX-CA 1), Zusanli (ST 36), Sanyinjiao (SP 6) after the end of menstruation in the observation group, the treatment lasted for 60 min, once a day, 5 times as a course, with 2 days interval between the courses and 3 consecutive courses of treatment were given, until the embryo transfer was performed in the IVF assisted pregnancy cycle. After the end of menstruation, fresh leonurus japonicus capsule was given in the control group, 3 times a day, 0.8 g each time, 7 days as a course, and 3 courses of continuous treatment were received, until the embryo transfer was performed in the IVF assisted pregnancy cycle. The changes of ECF before and after treatment, the time required to prepare for embryo transfer during IVF assisted pregnancy cycle, and the clinical outcome of embryo transfer were observed in the two groups. RESULTS: The decrease of ECF in the observation group was more significant than that in the control group (P<0.05). The time required for the embryo transfer in the IVF assisted pregnancy cycle in the observation group was shorter than that in the control group (P<0.05). The clinical pregnancy rate in the observation group was 42.9% (6/14), which was significantly higher than 26.7% (4/15) in the control group (P<0.05). CONCLUSION: Warm acupuncture may improve the clinical pregnancy rate by raising the local temperature of the lower abdomen, accelerating the blood circulation around the uterus and appendages, promoting the absorption of ECF, improving the uterine environment and endometrial receptivity.

[Study on combined immunization of rAd/MDC-VP1 and pcDNA3/MDC-VP1 against Coxsackievirus B3 challenge in mice].

AIM: To immunize the mice using the rAd/MDC-VP1 prime-pcDNA3/MDC-VP1 boost strategy and observe its immunological effect against Coxsackievirus B3(CVB3). METHODS: BALB/c mice were randomly divided into four groups: PBS group, rAd/MDC-VP1 group, pcDNA3/MDC-VP1 group and rAd/MDC-VP1 prime-pcDNA3/MDC-VP1 boost group. Mice in each group were immunized intramuscularly. The titers of serum IgG and neutralizing antibody were tested by ELISA and trace neutralization assays respectively. The Lymphocytes proliferation activity and specific CTL cytotoxic activity were tested by CCK-8 assay. The mice in each group were challenged with lethal dose of CVB3, and the serum virus titer was assayed and the protection efficacy against Coxsackievirus infection was observed. RESULTS: It was observed that the titers of CVB3 VP1 specific IgG and neutralizing antibody, non-specific lymphocytic proliferation activity and specific lymphocytic CTL cytotoxic activity of the rAd/MDC-VP1 prime-pcDNA3/MDC-VP1 boost group were much higher than those of the rest groups(P<0.05), what's more, after CVB3 challenged, the serum virus titer of this group was lower and the protection rate(41.67%) was higher (P<0.05). CONCLUSION: Both the cellular and humoral immune responses in mice could be significantly enhanced by the rAd/MDC-VP1 prime-pcDNA3/MDC-VP1 boost strategy and the protection rate after challenged by lethal dose of virus could be increased.

[The effects of inoculation route and adjuvant type on the immunizing potency of CVB3 VP1 protein].

AIM: To explore the effects of inoculation route and adjuvant type on the immunizing potency of coxsackievrus B type 3 (CVB3) VP1 protein. METHODS: The recombinant plasmid pET-His/VP1 expressed CVB3 VP1 was transformed into E.coli BL21(DE3) pLysS and induced to express VP1 protein by IPTG, and verified by Western blot analysis. The fusion VP1 protein was purified with Ni affinity chromatography. Firstly, BALB/c mice were administered via different inoculation route(subcutaneous, intraperitoneal, intramuscular), with twelves mice in each group. Secondly, combined with various adjuvants (Alum, Freund's adjuvant, Montanide ISA720), with eighteen mice in each group. The mice were immunized three times at a three week interval with 50 µg of VP1 protein. The titers of sera IgG and neutralizing antibody were detected by ELISA and neutralization assay. Cell mediated immune response was tested by the lymphocytes proliferation activity and specific CTL cytotoxic activity. The mice were challenged with lethal dose of CVB3, the titers of the sera virus were titrated. Furthermore, the survival rates of mice were observed. RESULTS: The VP1 protein was expressed in E.coli successfully and the fusion protein was purified. In different inoculation route, the titers of neutralizing antibody and specific IgG in intramuscular injection group was much higer than other groups (P<0.01). VP1 protein in combination with Montanide ISA720 and Freund's adjuvant elicit higher titer antibodies and cell mediated immune response, and the virus titers in blood were lower in comparison to Alum adjuvant group (P<0.05).The survival rate of Freund' adjuvant group was better than adjuvant AL(OH)(3); group (P<0.05). CONCLUSION: The VP1 protein combined with ISA720 and Freund's adjuvant given by intramuscular injection may induce an improved immune responses and the better survival rates of the mice after virus challenge.

[Immune effects of four coxsackievirus B3 VP1 DNA fusion vaccines in mice].

AIM: To compare the immunogenicity and protective effects on CVB3 infected mice of four DNA fusion vaccines coupling coxsackievirus B3 (CVB3) VP1 with macrophage-derived chemokine (MDC), C3d3, shiga toxin B subuit (STxB) and mouse beta-defensin-2 (mBD2), respectively. METHODS: BALB/c mice were divided into 6 groups randomly and inoculated in quadriceps at 3-week interval for 3 times with pcDNA3, pcDNA3/VP1, pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3, pcDNA3/STxB-VP1 and pcDNA3/mBD2 -VP1, respectively. Fourteen days after every inoculation, serum samples were collected and CVB3 specific neutralizing antibodies were determined. Three weeks after the last immunization, the mice were treated in three ways. First, the spleen cells were isolated from 3 mice of each group and specific CTL activities were tested. Second, 3 mice of each group were further challenged with 3LDLD(50); CVB3 and sacrificed 7 days later, and their blood viral titers were evaluated. Third, the rest mice of each group were subjected to intraperitoneal (i.p.) challenge with 5LDLD(50); CVB3 and their survival was observed. RESULTS: The neutralizing antibodies against CVB3 were induced in pcDNA3/VP1, pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3, pcDNA3/STxB-VP1 and pcDNA3/mBD2 -VP1 groups, and antibody titers correlated with the number of injections (P<0.01). After three immunizations, the antibody titers in pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3 and pcDNA3/mBD2 -VP1 groups were higher than the ones in pcDNA3/VP1and pcDNA3/STxB-VP1 groups (P<0.01). The specific CTL activities in both pcDNA3/STxB-VP1 and pcDNA3/mBD2-VP1 groups were significantly stronger than those in the other groups (P<0.01). After CVB3 challenge, the blood viral titers in the pcDNA3/MDC-VP1, pcDNA3/VP1-C3d3 and pcDNA3/mBD2-VP1 groups were lower than those in the other groups (P<0.01), and the pcDNA3/MDC-VP1 and pcDNA3/VP1-C3d3 mice survived longer than the others (P<0.05). CONCLUSION: Both pcDNA3/MDC-VP1 and pcDNA3/VP1-C3d3 vaccines could induce stronger immune responses, resulting in higher survival rates and better protective effects on CVB3 infection than pcDNA3/STxB-VP1, pcDNA3/mBD2-VP1 and pcDNA3/VP1 vaccines.