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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.

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 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.

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, Set-13 and Set-14 (75.9%), and Set-14 showed the fastest amplification speed (< 8.5 minutes), followed by Set-17 (< 12.5 minutes). 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, are determined to be better than the other primer sets. Two RT-LAMP assays with the Set-14 primers in combination with any one of four other primer sets (Set-4, Set-10, Set-11, and Set-13) are recommended to be used in the COVID-19 surveillance.

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.

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). Keywords: HIV-1, Drug resistance, China, Antiretroviral therapy (ART), Meta-analysis, Drug resistance mutation

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.

In order to optimize the planning and investment construction of multi-station integration (MSI), this paper proposes a comprehensive indices system for safety and benefit evaluation of MSI. According to the characteristics of safety and benefit of MSI, the method divides the indicators into normative safety indicators, general safety indicators and benefit indicators, while the whole index system is divided into target layer, category layer, indicator layer and sub-indicator layer. Based on the fuzzy analytic hierarchy process (AHP), a fuzzy evaluation operator model is proposed to calculate the index. Finally, a hierarchical dominant fuzzy evaluation model based on multiplication operator is built. Case study results show that the method can accurately evaluate the comprehensive attributes of MSI.

Table S1. GeneBank accession numbers corresponding to the three H7N9 viruses. (DOCX 15 kb)

Subgroup J avian leukosis virus (ALV-J) is an oncogenic retrovirus known to induce tumor formation and immunosuppression in infected chickens. One of the organs susceptible to ALV-J is the bone marrow, from which specialized antigen-presenting cells named dendritic cells (BM-DCs) are derived. Notably, these cells possess the unique ability to induce primary immune responses. In the present study, a method of cultivating and purifying DCs from chicken bone marrow in vitro was established to investigate the effects of ALV-J infection on BM-DC differentiation or generation. The results indicated that ALV-J not only infects the chicken bone marrow mononuclear cells but also appears to inhibit the differentiation and maturation of BM-DCs and to trigger apoptosis. Moreover, substantial reductions in the mRNA expression of TLR1, TLR2, TLR3, MHCI, and MHCII and in cytokine production were detected in the surviving BM-DCs following ALV-J infection. These findings indicate that ALV-J infection disrupts the process of bone marrow mononuclear cell differentiation into BM-DCs likely via altered antigen presentation, resulting in a downstream immune response in affected chickens.

The corrected legend is given below.

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-TM

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. Electronic supplementary material The online version of this article (10.1186/s12985-018-1042-3) contains supplementary material, which is available to authorized users.

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. Electronic supplementary material The online version of this article (10.1186/s12985-018-1109-1) contains supplementary material, which is available to authorized users.

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.

Highlights • H7 VLPs-WT and H7 VLPs-TM have similar morphological and cleavage characteristics. • H7 VLPs-TM has more HA trimers and better resists thermal changes than H7 VLPs-WT. • H7 VLPs-TM induces stronger Th1 immune response than H7 VLPs-WT. • H7 VLPs-TM induces 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.

Since February 2013, H7N9 influenza virus, causing human infections with high mortality in China, has been a potential pandemic threat. The H7N9 viruses are found to diverge into distinct genotypes as other influenza viruses; thus a vaccine that can provide sufficient cross-protection against different genotypes of H7N9 viruses is urgently needed. Our previous studies demonstrated that the HA-based structural design approach by introducing a CFLLC minidomain into transmembrane domain (TM) of H1, H5 or H9 hemagglutinin (HA) proteins by replacing with H3 subtype HA TM could enhance their cross-protection. In this study, we used Sf9 insect cell expression system to express recombinant H7 HA proteins H7-53WT, in which HA gene was derived from H7N9-53 strain, and H7-53TM containing CFLLC minidomian by replacing its TM domain with H3 HA TM. We investigated whether introduction of CFLLC minidomain into H7 HA (H7-53TM) could increase its cross-reactivity and cross-protection against different genotypes of H7N9 viruses. The results showed that the H7-53TM either with or without squalene adjuvant induced increased HI antibodies, serum IgG antibodies, and IFN-γ production to a panel of 7 H7N9 viruses in mice. Vaccinated animals with H7-53TM alone showed complete protection against challenge with heterologous H7N9-MCX strain, while H7-53WT alone showed incomplete protection (80%). Furthermore, mice vaccinated with H7-53TM HA showed less body weight loss and less pulmonary lesions and inflammation after challenge with homologous or heterologous H7N9 viruses, comparing to H7-53WT. In summary, this study presents a better subunit vaccine candidate (H7-53TM) against potential H7N9 pandemic.

Suspected cases of hemorrhagic enteritis associated with hemorrhagic enteritis virus (HEV) are becoming more frequent among yellow chickens in the Guangdong Province of China. In this study, we have developed a one-step, ecumenical, real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay for the rapid diagnosis of HEV. The RealAmp assay was performed at 63°C and reduced the assay time to 15min, using a simple and portable device, the ESE-Quant Tube Scanner. The detection limit of DNA was 1fg/μl, and the detection was specific only to HEV. We also used nested PCR to evaluate the application of the RealAmp assay. The coincidence rate of the two methods was 100%. Our data indicated that the RealAmp assay provides a sensitive, specific, and user-friendly diagnostic tool for the identification and quantification of HEV for field diagnosis and in laboratory research.

The integration of retroviruses into the host genome following nonrandom genome-wide patterns may lead to the deregulation of gene expression and oncogene activation near the integration sites. Slow-transforming retroviruses have been widely used to perform genetic screens for the identification of genes involved in cancer. To investigate the involvement of avian leukosis virus subgroup J (ALV-J) integration in myeloid leukosis (ML) in chickens, we utilized an ALV-J insertional identification platform based on hybrid capture target enrichment and next-generation sequencing (NGS). Using high-definition mapping of the viral integration sites in the chicken genome, 241 unique insertion sites were obtained from six different ALV-J-induced ML samples. On the basis of previous statistical definitions, MYC , TERT , and ZIC1 genes were identified as common insertion sites (CIS) of provirus integration in tumor cells; these three genes have previously been shown to be involved in the malignant transformation of different human cell types. Compared to control samples, the expression levels of all three CIS genes were significantly upregulated in chicken ML samples. Furthermore, they were frequently, but not in all field ML cases, deregulated at the mRNA level as a result of ALV-J infection. Our findings contribute to the understanding of the relationship between multipathotypes associated with ALV-J infection and the molecular background of tumorigenesis. IMPORTANCE ALV-Js have been successfully eradicated from chicken breeding flocks in the poultry industries of developed countries, and the control and eradication of ALV-J in China are now progressing steadily. To further study the pathogenesis of ALV-J infections, it will be necessary to elucidate the in vivo viral integration and tumorigenesis mechanism. In this study, 241 unique insertion sites were obtained from six different ALV-J-induced ML samples. In addition, MYC , TERT , and ZIC1 genes were identified as the CIS of ALV-J in tumor cells, which might be a putative “driver” for the activation of the oncogene. In addition, the CIS genes showed deregulated expression compared to nontumor samples. These results have potentially important implications for the mechanism of viral carcinogenesis.

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.

Influenza A H7N9 virus is the latest emerging pandemic threat, and has rapidly diverged into three clades, demanding a H7N9 virus vaccine with broadened protection against unmatched strains. Hemagglutinin (HA)-based structural design approaches for stabilizing HA proteins have provided excitingly promising results. However, none of the HA-based structural design approaches has been applied to a recombinant replicative influenza virus. Here we report that our HA-based structural design approach is a first in the field to generate a recombinant replicative H7N9 virus (H7N9-53TM) showing broadened protection. The H7N9-53TM contains a replaced H3 HA transmembrane domain (TM) in its HA protein. In mice, the inactivated H7N9-53TM vaccine induced significantly higher HI titers, HA-specific IgG titers, and IFN-γ production than the corresponding H7N9-53WT inactivated virus vaccine containing wild-type HA. More excitingly, mice immunized with the H7N9-53TM showed full protection against homologous (H7N9-53) and interclade (H7N9-MCX) challenges with minimal weight loss, no detectable lung viral loads, and no apparent pulmonary lesions and inflammation, while mice immunized with the H7N9-53WT showed partial protection (only 60% against H7N9-MCX) with severe weight loss, detectable lung viral loads, and severe pulmonary lesions and inflammation. In summary, this study presents a better vaccine candidate (H7N9-53TM) against H7N9 pandemics. Furthermore, our HA-based structural design approach would be conceivably applicable to other subtype influenza viruses, especially the viruses from emerging pandemic and epidemic influenza viruses such as H5N1 and H1N1.