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.

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...

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.

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.

Coxiella burnetii strains carry one of four large, conserved, autonomously replicating plasmids (QpH1, QpRS, QpDV, and QpDG) or a QpRS-like chromosomally integrated sequence of unknown function. Here we report the characterization of the QpH1 plasmid of C. burnetii Nine Mile phase II by making QpH1-deficient strains. A shuttle vector pQGK containing the CBUA0036-0039a region (predicted as being required for the QpH1 maintenance) was constructed. The pQGK vector can be stably transformed into the Nine Mile II and maintained at a similar low copy like QpH1. Importantly, transformation with pQGK cured the endogenous QpH1 due to plasmid incompatibility. Compared to a Nine Mile II transformant of a RSF1010-ori based vector, the pQGK transformant shows a similar growth curve in both axenic media and Buffalo green monkey kidney cells, a variable growth defect in macrophage-like THP-1 cells depending on the origin of inoculum, and dramatically reduced ability of colonizing wild-type bone marrow-derived murine macrophages. Furthermore, we found CBUA0037-0039 ORFs are essential for plasmid maintenance, and CBUA0037-0038 ORFs account for plasmid compatibility. And plasmid-deficient C. burnetii can be isolated by using CBUA0037 or -0038 deletion vectors. Furthermore, QpH1-deficient C. burnetii strains caused a lesser extent of splenomegaly in SCID mice but, intriguingly, they had significant growth in SCID mouse-sourced macrophages. Taken together, our data suggest that QpH1 encodes factor(s) essential for colonizing murine, not human, macrophages. This study suggests a critical role of QpH1 for C. burnetii persistence in rodents and expands the toolkit for the genetic studies in C. burnetii. Author summary All C. burnetii isolates carry one of four large, conserved, autonomously replicating plasmids or a plasmid-like chromosomally integrated sequence. The plasmid is a candidate virulence factor of unknown function. Here we describe the construction of novel shuttle vectors that allow making plasmid-deficient C. burnetii mutants. With this plasmid-curing approach, we characterized the role of the QpH1 plasmid in in vitro and in vivo C. burnetii infection models. We found that the plasmid plays a critical role for C. burnetii growth in murine macrophages. Our work suggests an essential role of the QpH1 plasmid for the acquisition of colonizing capability in rodents by C. burnetii. This study represents a major step toward unravelling the mystery of the C. burnetii cryptic plasmids.

Background Hematological comparison of coronavirus disease (COVID-19) and other viral pneumonias can provide insights into COVID-19 treatment. Methods In this retrospective case–control single-center study, we compared the data of 126 patients with viral pneumonia during different outbreaks [severe acute respiratory syndrome (SARS) in 2003, influenza A (H1N1) in 2009, human adenovirus type 7 in 2018, and COVID-19 in 2020]. Results One of the COVID-19 characteristics was a continuous decline in the hemoglobin level. The neutrophil count was related to the aggravation of COVID-19 and SARS. Thrombocytopenia occurred in patients with SARS and severe COVID-19 even at the recovery stage. Lymphocytes were related to the entire course of adenovirus infection, recovery of COVID-19, and disease development of SARS. Conclusions Dynamic changes in hematological counts could provide a reference for the pathogenesis and prognosis of pneumonia caused by respiratory viruses in clinics.

Trachoma, the leading infectious cause of blindness worldwide, is an ancient human disease. Its existence in China can be traced back to as early as the twenty-seventh century BC. In modern China, the overall prevalence of trachoma has dramatically reduced, but trachoma is still endemic in many areas of the country. Here, we report that 26 (8%) of 322 students from two rural boarding schools of Qinghai province, west China, were identified as having ocular C. trachomatis infection; and 15 ocular C. trachomatis strains were isolated from these trachoma patients. Chlamydiae in 37 clinical samples were genotyped as type B based on ompA gene analyses. Three ompA variants with one or two in-between SNP differences in the second or fourth variable domain were found. C. trachomatis strains QH111L and QH111R were from the same patient's left and right conjunctival swabs, respectively, but their ompA genes have a non-synonymous base difference in the second variable domain. Moreover, this SNP only exists in this single sample, suggesting QH111L is a newly emerged ompA variant. Interestingly, chromosomal phylogeny analysis found QH111L clusters between a branch of two type B strains and a branch of both A and C strains, but is significantly divergent from both branches. Comparative chromosome analysis found that compared to sequences of reference B/TZ1A828/OT strain, 12 of 22 QH111L's chromosomal genes exhibiting more than nine SNPs have the best homology with reciprocal genes of UGT strains while 9 of 22 genes are closest to those of type C strains. Consistent with findings of UGT-type genetic features in the chromosome, the QH111L plasmid appears to be intermediate between UGT and classical ocular plasmids due to the existence of UGT-type SNPs in the QH111L plasmid. Moreover, the QH111L strain has a unique evolutionarily older cytotoxin region compared to cytotoxin regions of other C. trachomatis strains. The genome analyses suggest that the QH111L strain is derived from recombinations between UGT and classical ocular ancestors. This is the first study of culture and characterization of ocular C. trachomatis in Qinghai Tibetan areas.

20Coxiella burnetii is a Gram-negative, facultative intracellular microorganism that can 21 cause acute or chronic Q fever in human. It was recognized as an obligate intracellular 22 organism until the revolutionary design of an axenic cystine culture medium (ACCM). 23Present axenic culture of C. burnetii strictly requires a hypoxic condition (<10% 24 oxygen). Here we investigated the normoxic growth of C. burnetii strains in ACCM-2 25 with or without tryptophan supplementation. Three C. burnetii strains -Henzerling 26 phase I, Nine Mile phase II and a Nine Mile phase II transformant, were included. The 27 transformant contains a pMMGK plasmid that is composed of a RSF1010 ori, a 28 repABC operon, an eGFP gene and a kanamycin resistance cassette. We found that, 29 under normoxia if staring from an appropriate concentration of fresh age inocula, 30 Nine Mile phase II can grow significantly in ACCM-2 with tryptophan, while the 31 transformant can grow robustly in ACCM-2 with or without tryptophan. In contrast, 32 long-term frozen stocks of phase II and its transformant, and Henzerling phase I of 33 different ages had no growth capability under normoxia under any circumstances. 34 Furthermore, frozen stocks of the transformant consistently caused large 35 splenomegaly in SCID mice, while wild type Nine Mile phase II induced a lesser 36 extent of splenomegaly. Taken together, our data show that normoxic cultivation of 37 phase II C. burnetii can be achieved under certain conditions. Our data suggests that 38 tryptophan and an unknown temperature sensitive signal are involved in the 39 expression of genes for normoxic growth regulated by quorum sensing in C. burnetii. 40 129 Mile phase II and NMIIpMMGK were used. In the second experiment, 9 five weeks 130 old mice were averaged into three infection groups: one month old Nine Mile phase II, 131 one-month old NMIIpMMGK and 19 months old NMIIpMMGK. At indicated days 132 post-infection, mice were weighted and sacrificed to harvest spleens to determine 133 splenomegaly (spleen weight/body weight). Each spleen was homogenized in 2 mL 134 PBS. Total DNAs from 20 μL of each tissue homogenate were purified with DNeasy 135