The transmembrane replacement H7N9-VLP vaccine displays high levels of protection in mice.

The incidence of infections caused by the H7N9 subtype of the influenza virus has expanded rapidly in China in recent decades, generating massive economic loss and posing a significant threat to public health. In the absence of specialized antiviral treatments or long-term effective preventative vaccinations, it is critical to constantly enhance vaccines and create effective antiviral drugs to prevent the recurrence of pandemics. In the present study, a transmembrane-substituted (TM) virus-like particle (VLP)-based vaccine was created by replacing the transmembrane region of hemagglutinin (HA) protein with the transmembrane region of the H3 HA protein and then used to immunize BALB/c mice. Sera and T cells were collected from the immunized mice to evaluate the passive immune effects. Our results showed that naïve mice achieved 80-100% protection against homologous and heterologous H7N9 influenza strains after receiving passive serum immunization; the protective effect of the TM VLPs was more evident than that of the wild-type HA VLPs. In contrast, mice immunized with passive T cells achieved only 20 to 80% protection against homologous or heterologous strains. Our findings significantly contribute to understanding the control of the H7N9 virus and the development of a vaccine.

Overexpression of miR-125a-5p Inhibits Hepatocyte Proliferation through the STAT3 Regulation In Vivo and In Vitro.

microRNAs (miRNAs) are critically involved in liver regeneration (LR). miR-125a-5p (miR-125a) is a tumor-suppressing miRNA, but its role in LR has not been studied. Our previous studies have proved that miR-125a was related to LR at the initiation phase, while the mechanism hepatocyte proliferation triggered by miR-125a in LR has been rarely evaluated. Herein, we mainly studied the molecular mechanism of miR-125a in triggering hepatocyte proliferation and the proliferation stage of LR. Firstly, a striking reduction of miR-125a was found at 24 h as well as 30 h following partial hepatectomy (PH) in rat liver tissue by miRNAs expression profiles as well as qRT-PCR analysis. Furthermore, in vitro, upregulation of miR-125a decreased proliferation as well as G1/S conversion, which promoted hepatocytes apoptosis. STAT3 was the target of miR-125a. In vivo, upregulation of miR-125a by tail vein injection of agomir inhibited LR index. Upregulation of miR-125a inhibited LR index and hepatocytes proliferation by STAT3/p-STAT3/JUN/BCL2 axis. In summary, these current discoveries indicated that miR-125a inhibited hepatocytes proliferation as well as LR by targeting STAT3 and via acting on the STAT3/p-STAT3/JUN/BCL2 axis.

Mitochondrial Dysfunction and Chronic Liver Disease.

Mitochondria are generally considered the powerhouse of the cell, a small subcellular organelle that produces most of the cellular energy in the form of adenosine triphosphate (ATP). In addition, mitochondria are involved in various biological functions, such as biosynthesis, lipid metabolism, oxidative phosphorylation, cell signal transduction, and apoptosis. Mitochondrial dysfunction is manifested in different aspects, like increased mitochondrial reactive oxygen species (ROS), mitochondrial DNA (mtDNA) damage, adenosine triphosphate (ATP) synthesis disorder, abnormal mitophagy, as well as changes in mitochondrial morphology and structure. Mitochondrial dysfunction is related to the occurrence and development of various chronic liver diseases, including hepatocellular carcinoma (HCC), viral hepatitis, drug-induced liver injury (DILI), alcoholic fatty liver (AFL), and non-alcoholic fatty liver (NAFL). In this review, we summarize and discuss the role and mechanisms of mitochondrial dysfunction in chronic liver disease, focusing on and discussing some of the latest studies on mitochondria and chronic liver disease.

Combinational benefit of antihistamines and remdesivir for reducing SARS-CoV-2 replication and alleviating inflammation-induced lung injury in mice.

COVID-19 is an immune-mediated inflammatory disease caused by SARS-CoV-2 infection, the combination of anti-inflammatory and antiviral therapy is predicted to provide clinical benefits. We recently demonstrated that mast cells (MCs) are an essential mediator of SARS-CoV-2-initiated hyperinflammation. We also showed that spike protein-induced MC degranulation initiates alveolar epithelial inflammation for barrier disruption and suggested an off-label use of antihistamines as MC stabilizers to block degranulation and consequently suppress inflammation and prevent lung injury. In this study, we emphasized the essential role of MCs in SARS-CoV-2-induced lung lesions in vivo, and demonstrated the benefits of co-administration of antihistamines and antiviral drug remdesivir in SARS-CoV-2-infected mice. Specifically, SARS-CoV-2 spike protein-induced MC degranulation resulted in alveolar-capillary injury, while pretreatment of pulmonary microvascular endothelial cells with antihistamines prevented adhesion junction disruption; predictably, the combination of antiviral drug remdesivir with the antihistamine loratadine, a histamine receptor 1 (HR1) antagonist, dampened viral replication and inflammation, thereby greatly reducing lung injury. Our findings emphasize the crucial role of MCs in SARS-CoV-2-induced inflammation and lung injury and provide a feasible combination antiviral and anti-inflammatory therapy for COVID-19 treatment.

Evaluation of the Clinical Effectiveness of Oseltamivir for Influenza Treatment in Children.

Objective: To estimate the clinical effectiveness of oseltamivir in children with different subtypes of influenza virus infection. Methods: A total of 998 children with acute respiratory infection were enrolled from January to March 2018, and were divided into influenza A, influenza B, influenza A + B, and non-influenza infection (IV-negative) groups. Influenza-like symptoms and duration of fever were evaluated and compared between oseltamivir-treated and non-treated groups. Results: There were no significant differences in the reduction in total febrile period and duration of fever from the onset of therapy between the oseltamivir treated and non-treated children infected with influenza A (p = 0.6885 for total febrile period and 0.7904 for the duration of fever from the onset of treatment), influenza B (p = 0.1462 and 0.1966), influenza A + B (p = 0.5568 and 0.9320), and IV-negative (p = 0.7631 and 0.4655). The duration of fever in children received oseltamivir therapy within 48 h was not significantly shorter than that beyond 48 h (p > 0.05). Additionally, percentages and severities of influenza-like symptoms, including headache, myalgia, fatigue, bellyache, vomiting, diarrhea, sore throat, cough, and coryza were not decreased and alleviated after treatment of oseltamivir. Conclusion: Oseltamivir treatment does not significantly shorten the duration of fever, nor does it significantly relieve influenza-like symptoms in children with infection of influenza.

miR-541-3p Promoted Porcine Reproductive and Respiratory Syndrome Virus 2 (PRRSV-2) Replication by Targeting Interferon Regulatory Factor 7.

Porcine reproductive and respiratory syndrome (PRRS) is a disease caused by PRRS virus (PRRSV), which seriously harms the pig industry. Revealing the mechanism by which PRRSV inhibits immune response will help prevent and control PRRS. Here, we found that PRRSV-2 may hijack host miR-541-3p to inhibit host innate immune response. Firstly, this work showed that miR-541-3p mimics could facilitate the replication of PRRSV-2 and the results of the quantitative real time polymerase chain reaction (qRT-PCR) showed that PRRSV-2 could up-regulate the expression of miR-541-3p in MARC-145 cells. Since previous studies have shown that type I interferon could effectively inhibit the replication of PRRSV-2, the present work explored whether miR-541-3p regulated the expression of type I interferon and found that miR-541-3p could negatively regulate the transcription of type I interferon by targeting interferon regulatory factor 7 (IRF7). More importantly, PRRSV-2 infection could down-regulate the expression of IRF7 and over-expression of IRF7 could down-regulate the replication of PRRSV-2 in MARC-145 cells. In conclusion, PRRSV-2 infection up-regulated the expression of miR-541-3p to promote its replication in MARC-145 cells, since miR-541-3p can negatively regulate the transcription of type I interferon by targeting IRF7.

Corrigendum to "Trend of HIV-1 drug resistance in China: A systematic review and meta-analysis of data accumulated over 17 years (2001-2017)" [EClinicalMedicine 18 (2020) 100238].

[This corrects the article DOI: 10.1016/j.eclinm.2019.100238.].

Comparative evaluation of 19 reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has caused a global pandemics. To facilitate the detection of SARS-CoV-2 infection, various RT-LAMP assays using 19 sets of primers had been developed, but never been compared. We performed comparative evaluation of the 19 sets of primers using 4 RNA standards and 29 clinical samples from COVID-19 patients. Six of 15 sets of primers were firstly identified to have faster amplification when tested with four RNA standards, and were further subjected to parallel comparison with the remaining four primer sets using 29 clinical samples. Among these 10 primer sets, Set-4 had the highest positive detection rate of SARS-CoV-2 (82.8%), followed by Set-10, Set-11, and Set-13 and Set-17 (75.9%). Set-14 showed the fastest amplification speed (Tt value < 8.5 min), followed by Set-17 (Tt value < 12.5 min). Based on the overall detection performance, Set-4, Set-10, Set-11, Set-13, Set-14 and Set-17 that target Nsp3, S, S, E, N and N gene regions of SARS-CoV-2, respectively, were determined to be better than the other primer sets. Two RT-LAMP assays with the Set-4 primers in combination with any one of four other primer sets (Set-14, Set-10, Set-11, and Set-13) were recommended to be used in the COVID-19 surveillance.

Epidemiological and clinical characteristics of COVID-19 patients in Nantong, China.

INTRODUCTION: COVID-19 is a newly emerging life-threatening respiratory disease caused by a newly identified coronavirus SARS-CoV-2. METHODOLOGY: We included 28 COVID-19 patients admitted to Nantong Third Hospital from January 23 to February 26, 2020. SARS-CoV-2 infection was confirmed using real-time RT-PCR. The demographic, epidemiological, clinical, laboratory parameters were obtained from each patient. RESULTS: The vast majority (71.4%) of confirmed COVID-19 patients were brought in from outside of the city, and all others had contact history with these confirmed cases. The median age of patients was 50 years old and half had underlying diseases. The most common symptoms at the onset of illness were fever (96.4%), cough (67.9%), and chilly (28.6%), and 75.0% patients had two or more symptoms. Increased erythrocyte sedimentation rate, serum ferritin and C-reactive protein levels, and reduced absolute counts of total lymphocytes and T lymphocyte subsets were observed among the patients. The vast majority (85.7%) of patients showed bilateral or unilateral pneumonia, and three symptomatic patients and one asymptomatic case did not show abnormalities in their CT image. Among the 28 admitted patients, 24 were discharged as of February 26, 2020, with an average hospital stay of 14.96 (±4.27) days, which was not significantly associated with the interval between the onset of symptoms and admission. CONCLUSIONS: In the absence of specific antiviral drugs or a vaccine, quarantine or isolation is the most effective intervention strategy for preventing the spread of the virus. Adequate supportive medical care is crucial for good prognosis of COVID-19 patients.

The Phylodynamics of Seasonal Influenza A/H1N1pdm Virus in China Between 2009 and 2019.

Since its first introduction into China in 2009, influenza A/H1N1pdm virus has undergone a rapid expansion and replaced the classical seasonal A(H1N1) virus. To characterize the ongoing evolution and national transmission dynamics of this virus, we analyzed 335 complete genome, 1259 HA, and 1043 NA sequences of the A/H1N1pdm strains detected in China. We found that the dN/dS value and relative genetic diversity of the A/H1N1pdm virus experienced a decrease from 2009 to 2017, and then a rapid increase during 2018-2019. Importantly, elevated relative genetic diversity was observed in the A/H1N1pdm and the A/H3N2 viruses, as well as two lineages (Victoria and Yamagata) of influenza B virus during 2018-2019, suggesting the simultaneous changes of these viruses in terms of genetic diversity might be associated with the recent large outbreak of seasonal influenza epidemic in China during 2018-2019. Fifteen amino acid mutations were found to be fixed along the main trunks of both HA and NA phylogenetic trees, and some of them are located in the antigen binding site or the receptor binding site. A sequential accumulation of mutations relative to the 2009-vaccine strain was observed in the circulating A/H1N1pdm strains during 2009-2016, while a rapid accumulation of mutations relative to the 2015-vaccine strain appeared in the emerging variants in 2017 shortly after the release of the vaccine. Multiple introductions of the A/H1N1pdm lineages into China were observed during 2009-2019, and East China and South China were found to serve as two major epicenters responsible for the national migration of the virus. In summary, these data provide important insights into the understanding of the evolution, epidemiology and transmission of the A/H1N1pdm virus, and highlight the importance of strengthening influenza surveillance in East China and South China.

Correction to: Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2.

The corrected legend is given below.

Trend of HIV-1 drug resistance in China: A systematic review and meta-analysis of data accumulated over 17 years (2001-2017).

BACKGROUND: The emergence and spread of HIV-1 drug resistance may compromise HIV control globally. In response to HIV/AIDS epidemic, China launched national HIV/AIDS treatment program in 2003, and started to accumulate drug resistance data since 2001. In this study we aimed to assess the level, trend and distribution of HIV-1 drug resistance during a period of 17 years from 2001 to 2017, and to characterize crucial drug resistance mutations. METHODS: We systematically reviewed 4737 studies published between January 1, 2001 and March 31, 2019 in PubMed, Embase, China National Knowledge Infrastructure (CNKI), WanFang Database, Web of Science, conference abstracts from the Chinese Medical Association and the Chinese AIDS Academic Conferences, and selected 170 studies that met our study criteria. To assess the prevalence of drug resistance in whole country or a local region, we performed pooled analyses of raw data. The transformed proportions were pooled using the inverse variance fixed effects methods or the DerSimonian-Laired random effects methods. The temporal trend of transmitted drug resistance (TDR) was determined using generalized additive model implemented in the Mgcv version 1.8 package. HIV-1 genotypic resistance was analyzed using the Stanford HIVdb algorithm. FINDINGS: We assembled 218 datasets from 170 selected studies (129 in Chinese and 41 in English), covering 21,451 ART-naïve and 30,475 ART-treated individuals with HIV-1 infection. The pooled prevalence of TDR was 3.0% (95%CI: 2.8-3.2), including 0.7% (95%CI: 0.4-1.0), 1.4% (95%CI: 1.3-1.6) and 0.5% (95%CI: 0.4-0.6) for nucleoside reverse transcriptase inhibitor (NRTI), non-NRTI (NNRTI) and protease inhibitor (PI) resistance, respectively. The acquired drug resistance (ADR) prevalence was 44.7% (95%CI: 39.3-50.2), including 31.4% (95%CI: 28.2-34.6), 39.5% (95%CI: 35.6-43.5) and 1.0% (95%CI: 0.8-1.2) for NRTI, NNRTI and PI resistance, respectively. TDR and ADR prevalence had characteristic regional patterns. The worst prevalence of drug resistance occurred in Central China, and higher ADR prevalence occurred in South China than North China. TDR in whole country has risen since 2012, and this rise was driven mainly by NNRTI resistance. One NRTI-associated (M184V/I) and three NNRTI-associated (K103N/S, Y181C/I and G190A/S) mutations had high percentages in ART-naïve and ART-treated individuals, and these mutations conferred high-level resistance to 3TC, EFV and/or NVP. INTERPRETATION: These findings suggest that the current available first-line ART regimens containing 3TC and/or EFV or NVP need to be revised. In addition, scale-up of multiple viral load measurements per year and drug resistance testing prior to ART initiation are recommended. Furthermore, implementation of pre-treatment education and counseling to improve patient adherence to ART is encouraged. FUNDING: This work was supported by grants from the National Natural Science Foundation of China (81672033, U1302224, and 81271888) and Open Research Fund Program of the State Key Laboratory of Virology of China (2019IOV002).

Development of chimeric virus-like particles containing the E glycoprotein of duck Tembusu virus.

Duck Tembusu virus (DTMUV) has caused enormous economic losses to the poultry industry in China. In the current study, we generated chimeric virus-like particles (VLPs) containing E protein of the DTMUV and HA2 protein of the H3N2 avian influenza virus (AIV). The chimeric VLPs could induce specific antibody responses in both mice (n = 5/group) and ducks (n = 10/group). After immunizing ducklings with the chimeric VLPs, all immunized ducks (n = 10/group) were 100% (10/10) protected against homologous DTMUV strain and virus shedding was not detected on day 5 post-challenge, whereas 60% (6/10) of the ducklings immunized with PBS presented typical symptoms with a virus shedding rate of 90% (9/10). Furthermore, viral loads were significantly decreased in the birds of the chimeric VLPs immunized group, comparing to that of the PBS immunized group. Our data demonstrated that the chimeric VLPs used in the current study could be applied as a potential vaccine candidate to control DTMUV infections in young ducks.

Multiple amino acid substitutions involved in the adaption of three avian-origin H7N9 influenza viruses in mice.

BACKGROUND: Avian influenza A H7N9 virus has caused five outbreak waves of human infections in China since 2013 and posed a dual challenge to public health and poultry industry. The number of reported H7N9 virus human cases confirmed by laboratory has surpassed that of H5N1 virus. However, the mechanism for how H7N9 influenza virus overcomes host range barrier has not been clearly understood. METHODS: To generate mouse-adapted H7N9 influenza viruses, we passaged three avian-origin H7N9 viruses in mice by lung-to-lung passages independently. Then, the characteristics between the parental and mouse-adapted H7N9 viruses was compared in the following aspects, including virulence in mice, tropism of different tissues, replication in MDCK cells and molecular mutations. RESULTS: After ten passages in mice, MLD50 of the H7N9 viruses reduced >750-3,160,000 folds, and virus titers in MDCK cells increased 10-200 folds at 48 hours post-inoculation. Moreover, the mouse-adapted H7N9 viruses showed more expanded tissue tropism and more serious lung pathological lesions in mice. Further analysis of the amino acids changes revealed 10 amino acid substitutions located in PB2 (E627K), PB1 (W215R and D638G), PA (T97I), HA (H3 numbering: R220G, L226S, G279R and G493R) and NA (P3Q and R134I) proteins. Moreover, PB2 E627K substitution was shared by the three mouse-adapted viruses (two viruses belong to YRD lineage and one virus belongs to PRD lineage), and PA T97A substitution was shared by two mouse-adapted viruses (belong to YRD lineage). CONCLUSIONS: Our result indicated that the virulence in mice and virus titer in MDCK cells of H7N9 viruses significantly increased after adapted in mouse model. PB2 E627K and PA T97A substitutions are vital in mouse adaption and should be monitored during epidemiological study of H7N9 virus.

Isolation and characterization of a highly pathogenic strain of Porcine enteric alphacoronavirus causing watery diarrhoea and high mortality in newborn piglets.

Porcine enteric alphacoronavirus (PEAV) was first discovered in China in February 2017, and the origin and virulence of this novel porcine coronavirus were not fully characterized. Here, we isolated a strain of PEAV, named GDS04 that is identified by immunofluorescence and typical crown-shaped particles observed with electron microscopy. Genomic analysis reveals that PEAV GDS04 shares a close relationship with SADS-CoV and SeACoV. Furthermore, newborn piglets orally challenged with PEAV GDS04 developed typical clinical symptoms as watery diarrhoea in neonatal piglets. Viral RNA was detected in faeces and various tissues of the infected piglets. Moreover, macroscopic and microscopic lesions in whole intestinal tract were observed, and viral antigen could be detected in the small intestines by immunohistochemical staining and electron microscopy. Importantly, the mortality rate of inoculated-newborn piglets was 100% and half of the cohabiting piglets died. Collectively, we demonstrate that PEAV is highly pathogenic in newborn piglets.

Biological characteristics and immunological properties in Muscovy ducks of H5N6 virus-like particles composed of HA-TM/HA-TMH3 and M1.

Highly pathogenic avian influenza viruses (HPAIVs), including H5N6 strains, pose threats to the health of humans and poultry. Waterfowl play a crucial role as a reservoir of HPAIVs. Since current influenza vaccines induce poor antibody titres in waterfowl, there is an urgent need to develop an efficient vaccine against H5N6 infection. In this study, we constructed two H5N6 virus-like particles (VLPs) composed of matrix-1 (M1) and haemagglutinin of wildtype (HA-TM) or haemagglutinin with transmembrane domain replacement (HA-TMH3) (designated as H5N6 VLPs-TM and H5N6 VLPs-TMH3). Biological characteristics of the composed H5N6 VLPs were compared including localization, expression, contents of HA trimers, thermal stability, morphology and immunogenicity in Muscovy ducks. Our results indicate that the H5N6 VLPs-TMH3 contained more HA trimers and presented better thermal stability. Moreover, Muscovy ducks immunized with H5N6 VLPs-TMH3 produced higher titres of HI antibody and IFN-γ compared with those immunized with the same dose of H5N6 VLP-TM, thus providing a promising approach for the development of influenza virus vaccines for waterfowl. RESEARCH HIGHLIGHTS H5N6 VLPs-TMH3 had more HA trimers and resisted higher temperature than H5N6 VLPs-TM H5N6 VLPs-TMH3 induced higher titre of HI than H5N6 VLPs-TM in Muscovy ducks.

Neutralizing antibodies against porcine epidemic diarrhea virus block virus attachment and internalization.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) is emerging as a pathogenic coronavirus that causes a huge economic burden to the swine industry. Interaction of the viral spike (S) surface glycoprotein with the host cell receptor is recognized as the first step of infection and is the main determinant of virus tropism. The mechanisms by which neutralizing antibodies inhibit PEDV have not been defined. Isolating PEDV neutralizing antibodies are crucial to identifying the receptor-binding domains of the viral spike and elucidating the mechanism of protection against PEDV infection. METHODS: B cell hybridoma technique was used to generate hybridoma cells that secrete specific antibodies. E.coli prokaryotic expression system and Bac-to-Bac expression system were used to identify the target protein of each monoclonal antibody. qPCR was performed to analyze PEDV binding to Vero E6 cells with neutralizing antibody. RESULTS: We identified 10 monoclonal antibodies using hybridoma technology. Remarkably, 4 mAbs (designed 2G8, 2B11, 3D9, 1E3) neutralized virus infection potently, of which 2B11 and 1E3 targeted the conformational epitope of the PEDV S protein. qPCR results showed that both 2B11 and 2G8 blocked virus entry into Vero cells. CONCLUSION: The data suggested that PEDV neutralizing antibody inhibited virus infection by binding to infectious virions, which could work as a tool to find the receptor-binding domains.

H7 virus-like particles assembled by hemagglutinin containing H3N2 transmembrane domain and M1 induce broad homologous and heterologous protection in mice.

Influenza A H7N9 virus has caused five outbreak waves of human infections in China since 2013 and posed a dual challenge to public health and poultry industry. There is an urgent need to develop an effective vaccine to reduce its pandemic potential. In the present study, we evaluated the biochemical characteristics and immunogenicity of two H7 virus-like particles (VLPs) composed of the matrix 1 (M1) and hemagglutinin of wild-type (HA-WT) or hemagglutinin of whose transmembrane domain replaced by that from H3N2 subtype (HA-TM). H7 VLPs-WT and H7 VLPs-TM could assemble and release into the supernatant of Sf9 cells and they had similar morphological characteristics. However, compared to H7 VLPs-WT, H7 VLPs-TM had more trimeric HA proteins and could better resist thermal changes. In mice H7 VLPs-TM induced higher titers of HI, IgG, IgG2a and IFN-γ, and provided better protection against homologous and heterologous H7N9 viruses (no matter belonging to Yangtze River Delta or Pearl River Delta) challenge with less weight loss and higher survival rate. In summary, H7 VLPs-TM represents a potential strategy for the development of H7N9 vaccines.