SARS-CoV-2 related coronaviruses (SARS-CoV-2r) from Guangdong and Guangxi pangolins have been implicated in the emergence of SARS-CoV-2 and future pandemics. We previously reported the culture of a SARS-CoV-2r GX_P2V from Guangxi pangolins. Here we report the GX_P2V isolate rapidly adapted to Vero cells by acquiring two genomic mutations: an alanine to valine substitution in the nucleoprotein and a 104-nucleotide deletion in the hypervariable region (HVR) of the 3′-terminus untranslated region (3′-UTR). We further report the characterization of the GX_P2V variant (renamed GX_P2V(short_3UTR)) in in vitro and in vivo infection models. In cultured Vero, BGM and Calu-3 cells, GX_P2V(short_3UTR) had similar robust replication kinetics, and consistently produced minimum cell damage. GX_P2V(short_3UTR) infected golden hamsters and BALB/c mice but was highly attenuated. Golden hamsters infected intranasally had a short duration of productive infection in pulmonary, not extrapulmonary, tissues. These productive infections induced neutralizing antibodies against pseudoviruses of GX_P2V and SARS-CoV-2. Collectively, our data show that the GX_P2V(short_3UTR) is highly attenuated in in vitro and in vivo infection models. Attenuation of the variant is likely partially due to the 104-nt deletion in the HVR in the 3′-UTR. This study furthers our understanding of pangolin coronaviruses pathogenesis and provides novel insights for the design of live attenuated vaccines against SARS-CoV-2.
Lipid A is an essential basal component of lipopolysaccharide of most Gram-negative bacteria. Inhibitors targeting LpxC, a conserved enzyme in lipid A biosynthesis, are antibiotic candidates against Gram-negative pathogens. Here we report the characterization of the role of lipid A in Coxiella burnetii growth in axenic media, monkey kidney cells (BGMK and Vero), and macrophage-like THP-1 cells by using a potent LpxC inhibitor -LPC-011. We first determined the susceptibility of C. burnetii LpxC to LPC-011 in a surrogate E. coli model. In E. coli, the minimum inhibitory concentration (MIC) of LPC-011 against C. burnetii LpxC is < 0.05 μg/mL, a value lower than the inhibitor's MIC against E. coli LpxC. Considering the inhibitor's problematic pharmacokinetic properties in vivo and Coxiella's culturing time up to 7 days, the stability of LPC-011 in cell cultures was assessed. We found that regularly changing inhibitor-containing media was required for sustained inhibition of C. burnetii LpxC in cells. Under inhibitor treatment, Coxiella has reduced growth yields in axenic media and during replication in non-phagocytic cells, and has a reduced number of productive vacuoles in such cells. Inhibiting lipid A biosynthesis in C. burnetii by the inhibitor was shown in a phase II strain transformed with chlamydial kdtA. This exogenous KdtA enzyme modifies Coxiella lipid A with an α-Kdo-(2 → 8)-α-Kdo epitope that can be detected by anti-chlamydia genus antibodies. In inhibitor-treated THP-1 cells, Coxiella shows severe growth defects characterized by poor vacuole formation and low growth yields. Coxiella progenies prepared from inhibitor-treated cells retain the capability of normally infecting all tested cells in the absence of the inhibitor, which suggests a dispensable role of lipid A for infection and early vacuole development. In conclusion, our data suggest that lipid A has significance for optimal development of Coxiella-containing vacuoles, and for robust multiplication of C. burnetii in macrophage-like THP-1 cells. Unlike many bacteria, C. burnetii replication in axenic media and non-phagocytic cells was less dependent on normal lipid A biosynthesis.
23Coxiella burnetii carries a large conserved plasmid or plasmid-like chromosomally 24 integrated sequence of unknown function. Here we report the curing of QpH1 plasmid 25 from C. burnetii Nine Mile phase II, the characterization of QpH1-deficient C. 26 burnetii in in vitro and in vivo infection models, and the characterization of plasmid 27 biology. A shuttle vector pQGK, which is composed of the CBUA0036-0039a region 28 (predicted for QpH1 maintenance), an E. coli plasmid ori, eGFP and kanamycin 29 resistance genes was constructed. The pQGK vector can be stably transformed into 30 Nine Mile II and maintained at a similar low copy like QpH1. Importantly, 31 transformation with pQGK cured the endogenous QpH1 due to plasmid 32 incompatibility. Compared to a Nine Mile II transformant of a RSF1010-based vector, 33 the pQGK transformant shows an identical one-step growth curve in axenic media, a 34 similar growth curve in Buffalo green monkey kidney cells, an evident growth defect 35 in macrophage-like THP-1 cells, and dramatically reduced ability of colonizing bone 36 marrow-derived murine macrophages. In the SCID mouse infection model, the pQGK 37 transformants caused a lesser extent of splenomegaly. Moreover, the plasmid biology 38 was investigated by mutagenesis. We found CBUA0037-0039 are essential for 39 plasmid maintenance, and CBUA0037-0038 account for plasmid compatibility. Taken 40 together, our data suggest that QpH1 encodes factor(s) essential for colonizing murine 41 macrophages, and to a lesser extent for colonizing human macrophages. This study 42 highlights a critical role of QpH1 for C. burnetii persistence in rodents, and expands 43 the toolkit for genetic studies in C. burnetii. 45Author summary 46 It is postulated that C. burnetii recently evolved from an inherited symbiont of ticks 47 by the acquisition of novel virulence factors. All C. burnetii isolates carry a large 48 plasmid or have a chromosomally integrated plasmid-like sequence. The plasmid is a 49 candidate virulence factor that contributes to C. burnetii evolution. Here we describe 50 the construction of novel shuttle vectors that allow to make plasmid-deficient C. 51 burnetii mutants. With this plasmid-curing approach, we characterized the role of the 52 QpH1 plasmid in in vitro and in vivo C. burnetii infection models. We found that the 53 plasmid plays a critical role for C. burnetii growth in macrophages, especially in 54 murine macrophages, but not in axenic media and BGMK cells. Our work highlights 55 an essential role of the plasmid for the acquisition of colonizing capability in rodents 56 by C. burnetii. This study represents a major step toward unravelling the mystery of 57 the C. burnetii cryptic plasmids. 58 59 Introduction 60 C. burnetii is a Gram-negative intracellular bacterium that causes Q fever, a world 61 widely distributed zoonosis [1]. It is highly infectious and is classified as a potential 62 biowarfare agent [2]. Its infections in humans are mostly asymptomatic but may 63 manifest as an acut...
Breast milk has been found to inhibit coronavirus infection, while the key components and mechanisms are unknown. We aimed to determine the components that contribute to the antiviral effects of breastmilk and explore their potential mechanism. Lactoferrin (Lf) and milk fat globule membrane inhibit severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)‐related coronavirus GX_P2V and transcription‐ and replication‐competent SARS‐CoV‐2 virus‐like particles in vitro and block viral entry into cells. We confirmed that bovine Lf (bLf) blocked the binding between human angiotensin‐converting enzyme 2 and SARS‐CoV‐2 spike protein by combining receptor‐binding domain (RBD). Importantly, bLf inhibited RNA‐dependent RNA polymerase (RdRp) activity of both SARS‐CoV‐2 and SARS‐CoV in vitro in the nanomolar range. So far, no biological macromolecules have been reported to inhibit coronavirus RdRp. Our result indicated that bLf plays a major role in inhibiting viral replication. bLf treatment reduced viral load in lungs and tracheae and alleviated pathological damage. Our study provides evidence that bLf prevents SARS‐CoV‐2 infection by combining SARS‐CoV‐2 spike protein RBD and inhibiting coronaviruses' RdRp activity, and may be a promising candidate for the treatment of coronavirus disease 2019.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.