Characterization of a pangolin SARS-CoV-2-related virus isolate that uses the human ACE2 receptor.

Various SARS-CoV-2-related coronaviruses have been increasingly identified in pangolins, showing a potential threat to humans. Here we report the infectivity and pathogenicity of the SARS-CoV-2-related virus, PCoV-GX/P2V, which was isolated from a Malayan pangolin (Manis javanica). PCoV-GX/P2V could grow in human hepatoma, colorectal adenocarcinoma cells, and human primary nasal epithelial cells. It replicated more efficiently in cells expressing human angiotensin-converting enzyme 2 (hACE2) as SARS-CoV-2 did. After intranasal inoculation to the hACE2-transgenic mice, PCoV-GX/P2V not only replicated in nasal turbinate and lungs, but also caused interstitial pneumonia, characterized by infiltration of mixed inflammatory cells and multifocal alveolar hemorrhage. Existing population immunity established by SARS-CoV-2 infection and vaccination may not protect people from PCoV-GX/P2V infection. These findings further verify the hACE2 utility of PCoV-GX/P2V by in vivo experiments using authentic viruses and highlight the importance for intensive surveillance to prevent possible cross-species transmission.

Isolation and characterization of a pangolin-borne HKU4-related coronavirus that potentially infects human-DPP4-transgenic mice.

We recently detected a HKU4-related coronavirus in subgenus Merbecovirus (named pangolin-CoV-HKU4-P251T) from a Malayan pangolin1. Here we report isolation and characterization of pangolin-CoV-HKU4-P251T, the genome sequence of which is closest to that of a coronavirus from the greater bamboo bat (Tylonycteris robustula) in Yunnan Province, China, with a 94.3% nucleotide identity. Pangolin-CoV-HKU4-P251T is able to infect human cell lines, and replicates more efficiently in cells that express human-dipeptidyl-peptidase-4 (hDPP4)-expressing and pangolin-DPP4-expressing cells than in bat-DPP4-expressing cells. After intranasal inoculation with pangolin-CoV-HKU4-P251, hDPP4-transgenic female mice are likely infected, showing persistent viral RNA copy numbers in the lungs. Progressive interstitial pneumonia developed in the infected mice, characterized by the accumulation of macrophages, and increase of antiviral cytokines, proinflammatory cytokines, and chemokines in lung tissues. These findings suggest that the pangolin-borne HKU4-related coronavirus has a potential for emerging as a human pathogen by using hDPP4.

Treatment of a patient with severe lactic acidosis and multiple organ failure due to mitochondrial myopathy: A case report.

BACKGROUND: Mitochondrial myopathy is a rare genetic disease with maternal inheritance that may involve multiple organ systems. Due to the lack of typical characteristics, its clinical diagnosis is difficult, and it is often misdiagnosed or even missed. CASE SUMMARY: The patient was a young college student. When he presented at the hospital, he had severe lactic acidosis, respiratory failure, and shock with multiple organ dysfunction syndrome (MODS). He was treated by mechanical ventilation, veno-arterial extracorporeal membrane oxygenation, and other organ support. However, his condition continued to worsen. After a thorough and detailed medical and family history was taken, a mitochondrial crisis was suspected. A muscle biopsy was taken. Further genetic testing confirmed a mitochondrial gene mutation (TRNL1 3243A>G). The final diagnosis of mitochondrial myopathy was made. Although there is no known specific treatment, intravenous methylprednisone and intravenous immunoglobulin were started. The patient's shock eventually improved. The further course was complicated by severe infection in multiple sites, severe muscle weakness, and recurrent MODS. After 2 mo of multidisciplinary management and intensive rehabilitation, the patient could walk with assistance 4 mo after admission and walk independently 6 mo after admission. CONCLUSION: More attention should be paid to mitochondrial myopathy to avoid missed diagnosis and misdiagnosis.

Extended Kalman filter algorithm for non-roughness and moving damage identification.

It is a promising method to identify structural damage using bridge dynamic response under moving vehicle excitation, but the lack of accurate information about road roughness and vehicle parameters will lead to the failure of this method. The paper proposed a step-by-step EKF damage identification method, which transforms the inversion problem of unknown structural parameters under unknown loads (vehicle and road roughness) into two separate inversion problems: moving contact force identification and damage parameters identification. Firstly, the VBI model is converted into bridge vibration model under a moving contact force, and the moving contact force covering the information of road roughness and vehicle parameters can be calculated by EKF iteration. Secondly, the moving contact force identified in the first step is loaded on the bridge as a known condition, and the bridge damage problem is also solved by the EKF method. Numerical analyses of a simply-supported bridge under the moving vehicle are conducted to investigate the accuracy and efficiency of the proposed method. Effects of the vehicle speed, the damage cases, the measurement noise, and the roughness levels on the accuracy of the identification results are investigated. The results demonstrate the proposed algorithm is efficient and robust, and the algorithm can be developed into an effective tool for structural health monitoring of bridges.

Lipid nanoparticle-encapsulated mRNA antibody provides long-term protection against SARS-CoV-2 in mice and hamsters.

Monoclonal antibodies represent important weapons in our arsenal to against the COVID-19 pandemic. However, this potential is severely limited by the time-consuming process of developing effective antibodies and the relative high cost of manufacturing. Herein, we present a rapid and cost-effective lipid nanoparticle (LNP) encapsulated-mRNA platform for in vivo delivery of SARS-CoV-2 neutralization antibodies. Two mRNAs encoding the light and heavy chains of a potent SARS-CoV-2 neutralizing antibody HB27, which is currently being evaluated in clinical trials, were encapsulated into clinical grade LNP formulations (named as mRNA-HB27-LNP). In vivo characterization demonstrated that intravenous administration of mRNA-HB27-LNP in mice resulted in a longer circulating half-life compared with the original HB27 antibody in protein format. More importantly, a single prophylactic administration of mRNA-HB27-LNP provided protection against SARS-CoV-2 challenge in mice at 1, 7 and even 63 days post administration. In a close contact transmission model, prophylactic administration of mRNA-HB27-LNP prevented SARS-CoV-2 infection between hamsters in a dose-dependent manner. Overall, our results demonstrate a superior long-term protection against SARS-CoV-2 conferred by a single administration of this unique mRNA antibody, highlighting the potential of this universal platform for antibody-based disease prevention and therapy against COVID-19 as well as a variety of other infectious diseases.

Parabrachial nucleus astrocytes regulate wakefulness and isoflurane anesthesia in mice.

Background: The parabrachial nucleus (PBN) is an important structure regulating the sleep-wake behavior and general anesthesia. Astrocytes in the central nervous system modulate neuronal activity and consequential behavior. However, the specific role of the parabrachial nucleus astrocytes in regulating the sleep-wake behavior and general anesthesia remains unclear. Methods: We used chemogenetic approach to activate or inhibit the activity of PBN astrocytes by injecting AAV-GFAabc1d-hM3Dq-eGFP or AAV-GFAabc1d-hM4Di-eGFP into the PBN. We investigated the effects of intraperitoneal injection of CNO or vehicle on the amount of wakefulness, NREM sleep and REM sleep in sleep-wake behavior, and on the time of loss of righting reflex, time of recovery of righting reflex, sensitivity to isoflurane, electroencephalogram (EEG) power spectrum and burst suppression ratio (BSR) in isoflurane anesthesia. Results: The activation of PBN astrocytes increased wakefulness amount for 4 h, while the inhibition of PBN astrocytes decreased total amount of wakefulness during the 3-hour post-injection period. Chemogenetic activation of PBN astrocytes decreased isoflurane sensitivity and shortened the emergence time from isoflurane-induced general anesthesia. Cortical EEG recordings revealed that PBN astrocyte activation decreased the EEG delta power and BSR during isoflurane anesthesia. Chemogenetic Inhibition of PBN astrocytes increased the EEG delta power and BSR during isoflurane anesthesia. Conclusion: PBN astrocytes are a key neural substrate regulating wakefulness and emergence from isoflurane anesthesia.

Role of heparinase in the gastrointestinal dysfunction of sepsis (Review).

Heparinase (HPA) is a ß-D glucuronidase that belongs to the endoglycosidase enzyme family, and plays an important role in numerous pathological and physiological processes, including inflammation, angiogenesis and tumor metastasis. When the expression of HPA is abnormally high, the side chain of heparin sulfate proteoglycans degrades, destroying the cell barrier and leading to the occurrence and development of inflammation, with systemic inflammation occurring in severe cases. Sepsis is a major cause of mortality in critically ill patients. In sepsis, the gastrointestinal tract is the first and most frequently involved target organ, which often leads to gastrointestinal dysfunction. HPA overexpression has been determined to accelerate sepsis progression and gastrointestinal dysfunction; thus, it was hypothesized that HPA may play an important role and may serve as an index for the diagnosis of gastrointestinal dysfunction in sepsis. HPA inhibitors may therefore become applicable as targeted drugs for the treatment of gastrointestinal dysfunction in patients with sepsis. The present review mainly discussed the role of HPA in gastrointestinal dysfunction of sepsis.

Long-term stability and protection efficacy of the RBD-targeting COVID-19 mRNA vaccine in nonhuman primates.

Messenger RNA (mRNA) vaccine technology has shown its power in preventing the ongoing COVID-19 pandemic. Two mRNA vaccines targeting the full-length S protein of SARS-CoV-2 have been authorized for emergency use. Recently, we have developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor-binding domain (RBD) of SARS-CoV-2 (termed ARCoV), which confers complete protection in mouse model. Herein, we further characterized the protection efficacy of ARCoV in nonhuman primates and the long-term stability under normal refrigerator temperature. Intramuscular immunization of two doses of ARCoV elicited robust neutralizing antibodies as well as cellular response against SARS-CoV-2 in cynomolgus macaques. More importantly, ARCoV vaccination in macaques significantly protected animals from acute lung lesions caused by SARS-CoV-2, and viral replication in lungs and secretion in nasal swabs were completely cleared in all animals immunized with low or high doses of ARCoV. No evidence of antibody-dependent enhancement of infection was observed throughout the study. Finally, extensive stability assays showed that ARCoV can be stored at 2-8 °C for at least 6 months without decrease of immunogenicity. All these promising results strongly support the ongoing clinical trial.

Lasting antibody and T cell responses to SARS-CoV-2 in COVID-19 patients three months after infection.

The dynamics, duration, and nature of immunity produced during SARS-CoV-2 infection are still unclear. Here, we longitudinally measured virus-neutralising antibody, specific antibodies against the spike (S) protein, receptor-binding domain (RBD), and the nucleoprotein (N) of SARS-CoV-2, as well as T cell responses, in 25 SARS-CoV-2-infected patients up to 121 days post-symptom onset (PSO). All patients seroconvert for IgG against N, S, or RBD, as well as IgM against RBD, and produce neutralising antibodies (NAb) by 14 days PSO, with the peak levels attained by 15-30 days PSO. Anti-SARS-CoV-2 IgG and NAb remain detectable and relatively stable 3-4 months PSO, whereas IgM antibody rapidly decay. Approximately 65% of patients have detectable SARS-CoV-2-specific CD4+ or CD8+ T cell responses 3-4 months PSO. Our results thus provide critical evidence that IgG, NAb, and T cell responses persist in the majority of patients for at least 3-4 months after infection.

Aerosol and Surface Distribution of Severe Acute Respiratory Syndrome Coronavirus 2 in Hospital Wards, Wuhan, China, 2020.

To determine distribution of severe acute respiratory syndrome coronavirus 2 in hospital wards in Wuhan, China, we tested air and surface samples. Contamination was greater in intensive care units than general wards. Virus was widely distributed on floors, computer mice, trash cans, and sickbed handrails and was detected in air ≈4 m from patients.

Development and evaluation of serotype-specific recombinase polymerase amplification combined with lateral flow dipstick assays for the diagnosis of foot-and-mouth disease virus serotype A, O and Asia1.

BACKGROUND: Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV) is one of the most highly infectious diseases in livestock, and leads to huge economic losses. Early diagnosis and rapid differentiation of FMDV serotype is therefore integral to the prevention and control of FMD. In this study, a series of serotype-specific reverse transcription recombinase polymerase amplification assays combined with lateral flow dipstick (RPA-LFD) were establish to differentiate FMDV serotypes A, O or Asia 1, respectively. RESULTS: The serotype-specific primers and probes of RPA-LFD were designed to target conserved regions of the FMDV VP1 gene sequence, and three primer and probe sets of serotype-specific RPA-LFD were selected for amplification of FMDV serotypes A, O or Asia 1, respectively. Following incubation at 38 °C for 20 min, the RPA amplification products could be visualized by LFD. Analytical sensitivity of the RPA assay was then determined with ten-fold serial dilutions of RNA of VP1 gene and the recombinant vector respectively containing VP1 gene from FMDV serotypes A, O or Asia1, the detection limits of these assays were 3 copies of plasmid DNA or 50 copies of viral RNA per reaction. Moreover, the specificity of the assay was assessed, and there was no cross reactions with other viruses leading to bovine vesicular lesions. Furthermore, 126 clinical samples were respectively detected with RPA-LFD and real-time PCR (rPCR), there was 98.41% concordance between the two assays, and two samples were positive by RPA-LFD but negative in rPCR, these were confirmed as FMDV-positive through viral isolation in BHK-21 cells. It showed that RPA-LFD assay was more sensitive than the rPCR method in this study. CONCLUSION: The development of serotype-specific RPA-LFD assay provides a rapid, sensitive, and specific method for differentiation of FMDV serotype A, O or Asia1, respectively. It is possible that the serotype-specific RPA-LFD assay may be used as a integral protocol for field detection of FMDV.

Serological survey of avian influenza virus infection in non-avian wildlife in Xinjiang, China.

We conducted a serological survey to detect antibodies against avian influenza virus (AIV) in Gazella subgutturosa, Canis lupus, Capreolus pygargus, Sus scrofa, Cervus elaphus, Capra ibex, Ovis ammon, Bos grunniens and Pseudois nayaur in Xinjiang, China. Two hundred forty-six sera collected from 2009 to 2013 were assayed for antibodies against H5, H7 and H9 AIVs using hemagglutination inhibition (HI) tests and a pan-influenza competitive ELISA. Across all tested wildlife species, 4.47 % harbored anti-AIV antibodies that were detected by the HI assay. The seroprevalence for each AIV subtype across all species evaluated was 0 % for H5 AIV, 0.81 % for H7 AIV, and 3.66 % for H9 AIV. H7-reactive antibodies were found in Canis lupus (9.09 %) and Ovis ammon (4.55 %). H9-reactive antibodies were found in Gazella subgutturosa (4.55 %), Canis lupus (27.27 %), Pseudois nayaur (23.08 %), and Ovis ammon (4.55 %). The pan-influenza competitive ELISA results closely corresponded to the cumulative prevalence of AIV exposure as measured by subtype-specific HI assays, suggesting that H7 and H9 AIV subtypes predominate in the wildlife species evaluated. These data provide evidence of prior infection with H7 and H9 AIVs in non-avian wildlife in Xinjiang, China.

Mechanisms of hypercoagulability in nephrotic syndrome associated with membranous nephropathy as assessed by thromboelastography.

INTRODUCTION: Thromboelastography (TEG) was performed to assess potential hypercoagulability in Nephrotic syndrome (NS) patients with membranous nephropathy (MN) and to explore correlated factors contributing to hypercoagulable status MATERIALS AND METHODS: 101 MN patients, 61 minimal change disease (MCD) patients and 20 healthy controls met the inclusion criteria. The MN and MCD patients were stratified into two layers according to serum albumin (SALB) levels (<20g/l or 20-30g/l). Primary outcome measures included reaction time (R), α-angle, maximum amplitude (MA) and coagulation index (CI). TEG parameters of four patient subgroups were analyzed in factorial designed ANOVA with factors disease and SALB. RESULTS: By linear regression analysis, TEG parameters in MN patients correlated with SALB (P<0.01) and the ANOVA for factorial designed data confirmed that the main effects of factors SALB and disease were both statistically significant. Besides, comparison between control group and patient subgroups showed that R value in normal controls was significantly higher than that in MN subgroups, but was not statistically different from that in MCD subgroups. NS patients (MCD, MN) had significantly higherα-angle, MA and CI values than healthy controls (p<0.05). CONCLUSIONS: MN patients tend to be more hypercoagulable than normal and MCD patients. Hypercoagulability in MN patients involves the whole thrombotic processes acceleration (activated intrinsic pathway, fibrinogen, platelet function and fibrin-platelet interaction), whereas hypercoagulable state in MCD patients may be that the coagulation factors are not fully activated. Greater efforts should be made to prevent hypercoagulability especially for MN patients with severe hypoalbuminemia.

Protective effect of salidroside on contrast-induced nephropathy in comparison with N-acetylcysteine and its underlying mechanism.

OBJECTIVE: To study the prevention effect of salidroside on contrast-induced-nephropathy (CIN) and its underlying mechanism. METHODS: A total of 24 Wistar rats were randomly divided into 4 groups with 6 in each group. Rats were firstly administrated with normal saline (control and model groups), N-acetylcysteine (NAC, NAC group) and salidroside (salidroside group) for 7 days before model establishment in each group, respectively. Histopathological analysis was performed by periodic acid-Schiff (PAS) staining. Oxidative stress related parameters including superoxide dismutase (SOD) and methane dicarboxylic aldehyde (MDA), nitric oxide (NO), angiotensin II (Ang II), 8-hydroxy-2'-deoxyguanosine (8-OHdG), mRNA and protein levels of endothelial nitric oxide synthase (eNOS), and nitric oxide synthase (NOS) activity were measured. RESULTS: Compared with the control group, the levels of MDA, Ang II and 8-OHdG were all significantly increased and levels of SOD, NO, and eNOS mRNA and protein were decreased significantly in the model group (P<0.05). Meanwhile, the NOS activity was also significantly decreased in the model group (P<0.05). In addition, the levels of these parameters were all improved in the NAC (P<0.05) and salidroside groups and no significant different was found between these two groups (P>0.05). CONCLUSION: Salidroside can be the potential substitute of NAC to prevent CIN. The underlying mechanism may be associated with oxidative stress damage caused by contrast agents.

A meta-analysis of the effects of angiotensin converting enzyme inhibitors and angiotensin II receptor blockers on insulin sensitivity in hypertensive patients without diabetes.

AIMS: This study sought to compare the effects of angiotensin converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB) on insulin sensitivity (IS) in hypertensive patients without diabetes. METHODS: Studies on the observation of IS in hypertensive patients without diabetes who received ACEI and ARB prior to December 2013 was collected using computer-based retrieval of the PUBMED, EMBASE, and COCHRANE databases. The primary indicators included IS, fasting plasma glucose (FPG), and fasting plasma insulin (FPI). The secondary indicators included systolic blood pressure (SBP) and diastolic blood pressure (DBP). A meta-analysis was performed using the STATA and Review Manager 5.2 software. The effects of these two drugs on IS in hypertensive patients without diabetes were analyzed using the fixed effect model and the random effect model. RESULTS: A total of 203 cases of patients involved in 4 clinical studies were included. As compared to ARB, ACEI treatment resulted in more effective improvement of IS in hypertensive patients without diabetes (SMD: 0.45, 95% CI 0.17-0.73), although these two drugs did not show significant differences with regards to FPG (WMD: 0.00, 95% CI -0.19-0.20), FPI (WMD: -0.34, 95% CI -1.31-0.63), SBP (WMD: 2.85, 95% CI -1.55-7.24), and DBP (WMD: 0.81, 95% CI -1.12-2.75). CONCLUSION: In patients showing no significant difference in blood pressure control, the comparison between ACEI and ARB showed that the former type of drug more effectively relieved IS in hypertensive patients without diabetes.

Characterization of a novel reassortant influenza A virus (H2N2) from a domestic duck in Eastern China.

While H2N2 viruses have been sporadically isolated from wild and domestic birds, H2N2 viruses have not been detected among human populations since 1968. Should H2N2 viruses adapt to domestic poultry they may pose a risk of infection to people, as most anyone born after 1968 would likely be susceptible to their infection. We report the isolation of a novel influenza A virus (H2N2) cultured in 2013 from a healthy domestic duck at a live poultry market in Wuxi City, China. Sequence data revealed that the novel H2N2 virus was similar to Eurasian avian lineage avian influenza viruses, the virus had been circulating for ≥ two years among poultry, had an increase in α2,6 binding affinity, and was not highly pathogenic. Approximately 9% of 100 healthy chickens sampled from the same area had elevated antibodies against the H2 antigen. Fortunately, there was sparse serological evidence that the virus was infecting poultry workers or had adapted to infect other mammals. These findings suggest that a novel H2N2 virus has been circulating among domestic poultry in Wuxi City, China and has some has increased human receptor affinity. It seems wise to conduct better surveillance for novel influenza viruses at Chinese live bird markets.

Production and characterization of a fusion peptide derived from the rabies virus glycoprotein (RVG29).

Gene therapy targeting the brain holds great promise in curing nervous system degenerative diseases in clinical applications. With this in mind, in a previous study a 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) with a nonamer stretch of arginine residues (RVG29-9R) at its carboxy-terminus was exploited as a ligand for brain-targeting gene delivery. Importantly, the report demonstrated that the RVG29-9R vector was able to cross the blood-brain barrier. RVG29-9R is currently synthesized by commercial companies with high associated costs. In this study, in order to reduce the costs of producing RVG29-9R, we have expressed and purified 6mg of a recombinant peptide (RVG29-9R-6His) from 0.4g of cultured Escherichia coli. We assessed the physiochemical properties of RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro 2a cells (which express the acetylcholine receptor). Our results reveal that the RVG29-9R-6His peptide recognized Neuro 2a cells in a dose-dependent manner and it was also able to bind plasmid DNA and deliver it into the Neuro 2a cells effectively. Therefore, our study has demonstrated that the recombinant RVG29-9R-6His peptide retains the functions of RVG29-9R and so may provide an economically viable and alternative production method for the manufacture of RVG29-9R.

Aptamers targeting rabies virus-infected cells inhibit street rabies virus in vivo.

Rabies is a viral infection of the CNS that is almost always fatal once symptoms occur. No effective treatment of the disease is available and novel antiviral strategies are urgently required. Street rabies viruses are field isolates known to be highly neurotropic. Aptamers are single-stranded oligonucleotides that bind their targets with high affinity and specificity and thus have potential for use in diagnostic and therapeutic applications. In this study, we demonstrate that the aptamers FO24 and FO21, which target RABV-infected cells, can significantly protect mice from a lethal dose of the street rabies virus FJ strain in vivo. Groups receiving preexposure prophylaxis had higher survival rates than the groups receiving postexposure prophylaxis. When mice were inoculated with aptamers (4 nmol) for 24h by intracranial or intramuscular injection prior to intramuscular inoculation with the FJ strain, approximately 60% of the mice survived. These results indicate that the FO21 and FO24 aptamers may be used to develop preventative antiviral therapy against rabies disease.

Selection of an aptamer against rabies virus: a new class of molecules with antiviral activity.

Rabies is a fatal central nervous system (CNS) disease caused by the neurotropic rabies virus (RABV). The therapeutic management of RABV infections is still problematic, and novel antiviral strategies are urgently required. We established the RVG-BHK-21 cell line, which expresses RABV glycoprotein on the cell surface, to select aptamers. Through 28 iterative rounds of selection, single-stranded DNA (ssDNA) aptamers were generated by exponential enrichment (SELEX). A virus titer assay and a real-time quantitative reverse transcription PCR (qRT-PCR) assay revealed that four aptamers could inhibit the replication of RABV in cultured baby hamster kidney (BHK)-21 cells. However, the aptamers did not inhibit the replication of other virus, e.g., canine distemper virus (CDV) and canine parvovirus (CPV). In addition, the GE54 aptamer was found to effectively protect mice against lethal RABV challenge. After inoculation with aptamers for 24h or 48h, followed by inoculation with CVS-11, approximately 25-33% of the mice survived. In summary, we selected aptamers that could significantly protect from a lethal dose of RABV in vitro and in vivo.