Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats.

While the spike proteins from severe acute respiratory syndrome coronaviruses-1 and 2 (SARS-CoV and SARS-CoV-2) bind to host angiotensin-converting enzyme 2 (ACE2) to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016A, as well as an ACE2-independent virus, RsHuB2019A. Although our stocks of RsHuB2019A rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed that ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light on the intricacies of coronavirus isolation and propagation in vitro. IMPORTANCE Several coronaviruses have been transmitted from animals to people, and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of the in vitro requirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for the exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.

Characterization of a mouse-adapted strain of bat severe acute respiratory syndrome-related coronavirus.

Bats carry genetically diverse severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs). Some of them utilize human angiotensin-converting enzyme 2 (hACE2) as a receptor and cannot efficiently replicate in wild-type mice. Our previous study demonstrated that the bat SARSr-CoV rRsSHC014S induces respiratory infection and lung damage in hACE2 transgenic mice but not wild-type mice. In this study, we generated a mouse-adapted strain of rRsSHC014S, which we named SMA1901, by serial passaging of wild-type virus in BALB/c mice. SMA1901 showed increased infectivity in mouse lungs and induced interstitial lung pneumonia in both young and aged mice after intranasal inoculation. Genome sequencing revealed mutations in not only the spike protein but the whole genome, which may be responsible for the enhanced pathogenicity of SMA1901 in wild-type BALB/c mice. SMA1901 induced age-related mortality similar to that observed in SARS and COVID-19. Drug testing using antibodies and antiviral molecules indicated that this mouse-adapted virus strain can be used to test prophylactic and therapeutic drug candidates against SARSr-CoVs. IMPORTANCE The genetic diversity of SARSr-CoVs in wildlife and their potential risk of cross-species infection highlights the importance of developing a powerful animal model to evaluate the antibodies and antiviral drugs. We acquired the mouse-adapted strain of a bat-origin coronavirus named SMA1901 by natural serial passaging of rRsSHC014S in BALB/c mice. The SMA1901 infection caused interstitial pneumonia and inflammatory immune responses in both young and aged BALB/c mice after intranasal inoculation. Our model exhibited age-related mortality similar to SARS and COVID-19. Therefore, our model will be of high value for investigating the pathogenesis of bat SARSr-CoVs and could serve as a prospective test platform for prophylactic and therapeutic candidates.

ACE2-Independent Bat Sarbecovirus Entry and Replication in Human and Bat Cells.

Hundreds of sarbecoviruses have been found in bats, but only a fraction of them have the ability to infect cells using angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV and -2. To date, only ACE2-dependent sarbecoviruses have been isolated from field samples or grown in the laboratory. ACE2-independent sarbecoviruses, comprising the majority of the subgenus, have not been propagated in any type of cell culture, as the factors and conditions needed for their replication are completely unknown. Given the significant zoonotic threat posed by sarbecoviruses, cell culture models and in vitro tools are urgently needed to study the rest of this subgenus. We previously showed that the exogenous protease trypsin could facilitate cell entry of viral-like particles pseudotyped with spike protein from some of the ACE2-independent sarbecoviruses. Here, we tested if these conditions were sufficient to support bona fide viral replication using recombinant bat sarbecoviruses. In the presence of trypsin, some of the spike proteins from clade 2 viruses were capable of supporting bat sarbecovirus infection and replication in human and bat cells. Protease experiments showed a specific viral dependence on high levels of trypsin, as TMPRSS2 and furin had no effect on clade 2 virus entry. These results shed light on how sarbecoviruses transmit and coexist in their natural hosts, provide key insights for future efforts to isolate and grow these viruses from field samples, and further underscore the need for broadly protective, universal coronavirus vaccines. IMPORTANCE Our studies demonstrate that some unexplored sarbecoviruses are capable of replicating in human and bat cells in an ACE2-independent way but need a high trypsin environment. We found that trypsin is not compensated by other known proteases involved in some coronavirus entry. This work provides important information that the trypsin-dependent entry may be a widely employed mechanism for coronaviruses and will help for further understanding the biological features of the less-studied viruses.

Efficacy and prognostic factors of neoadjuvant chemotherapy for triple-negative breast cancer.

BACKGROUND: Breast cancer mainly occurs in young and premenopausal women; its incidence is increasing annually. Patients with triple-negative breast cancer (TNBC) have relatively high recurrence and transfer rates during the operation and 3 years after postoperative adjuvant chemotherapy. Currently, the treatment for patients with TNBC is mainly based on a comprehensive combination of surgery and chemotherapy. Therefore, identifying additional effective treatments to improve patient prognosis is important. AIM: To explore and discuss the effects and prognostic factors of neoadjuvant chemotherapy in TNBC. METHODS: In total, 118 patients diagnosed with TNBC from January 2016 to January 2020 in our hospital were selected and divided into the observation (n = 60) and control (n = 58) groups according to therapeutic regimen. The control group received routine chemotherapy, and the observation group received neoadjuvant chemotherapy. The therapeutic effects of the two groups were observed, and the survival of patients was followed up. RESULTS: The karyopherin A2 (KPNA2)-positive and SRY-related HMG box-2 (SOX2)-positive expression rates of patients with TNBC with intravascular tumor thrombus and tumor-node-metastasis (TNM) stage IV were 92.00% and 91.67% and 96.00% and 95.83%, respectively, which were significantly higher than those of patients with no intravascular tumor thrombus and TNM stage III (P < 0.05). KPNA2 was positively associated with SOX2 expression (r s = 0.514, P < 0.50). The short-term curative effect of the observation group was better than that of the control group (P < 0.05), and the total effective rate was 58.33%. After treatment, carcinoembryonic antigen, cancer antigen (CA) 19-9, and CA125 Levels in the observation group were 11.40 ± 2.32 mg/L, 19.92 ± 3.42 kU/L, and 54.30 ± 12.28 kU/L, respectively, which were significantly lower than those in the control group (P < 0.05). The median survival time of the observation group was 33 mo (95%CI: 31.21-34.79), which was significantly longer than that of the control group (P < 0.05). TNM stage, degree of differentiation, lymph node metastasis, KPNA2 and SOX2 expressions, and treatment plan were prognostic factors of TNBC (relative risk = 1.575, 1.380, 1.366, 1.433, 1.411, and 0.581, respectively, P < 0.05). CONCLUSION: Neoadjuvant chemotherapy for TNBC treatment can achieve good curative effects. TNM stage, differentiation degree, lymph node metastasis, KPNA2 and SOX2 expressions, and treatment plan are prognostic factors of TNBC.