Host proteins interact with viral elements and affect the life cycle of highly pathogenic avian influenza A virus H7N9.

Host-virus interactions can significantly impact the viral life cycle and pathogenesis; however, our understanding of the specific host factors involved in highly pathogenic avian influenza A virus H7N9 (HPAI H7N9) infection is currently restricted. Herein, we designed and synthesized 65 small interfering RNAs targeting host genes potentially associated with various aspects of RNA virus life cycles. Afterward, HPAI H7N9 viruses were isolated and RNA interference was used to screen for host factors likely to be involved in the life cycle of HPAI H7N9. Moreover, the research entailed assessing the associations between host proteins and HPAI H7N9 proteins. Twelve key host proteins were identified: Annexin A (ANXA)2, ANXA5, adaptor related protein complex 2 subunit sigma 1 (AP2S1), adaptor related protein complex 3 subunit sigma 1 (AP3S1), ATP synthase F1 subunit alpha (ATP5A1), COPI coat complex subunit alpha (COP)A, COPG1, heat shock protein family A (Hsp70) member 1A (HSPA)1A, HSPA8, heat shock protein 90 alpha family class A member 1 (HSP90AA1), RAB11B, and RAB18. Co-immunoprecipitation revealed intricate interactions between viral proteins (hemagglutinin, matrix 1 protein, neuraminidase, nucleoprotein, polymerase basic 1, and polymerase basic 2) and these host proteins, presumably playing a crucial role in modulating the life cycle of HPAI H7N9. Notably, ANXA5, AP2S1, AP3S1, ATP5A1, HSP90A1, and RAB18, were identified as novel interactors with HPAI H7N9 proteins rather than other influenza A viruses (IAVs). These findings underscore the significance of host-viral protein interactions in shaping the dynamics of HPAI H7N9 infection, while highlighting subtle variations compared with other IAVs. Deeper understanding of these interactions holds promise to advance disease treatment and prevention strategies.

Beyond the pandemic: The truth of life after COVID-19.

This study focused on how to deal with the psychological trauma from the perspective of a doctor on the front line of the fight against COVID-19. As the pandemic continues to ravage our world, post-pandemic psychological counseling urgently needs to be addressed. Based on the experience of fighting the epidemic, this study discusses the psychological changes since the COVID-19 outbreak in 2020. Taking a 19-year-old with breast cancer as an example, this study considered how to find spiritual comfort, and examined how to find meaning in today's complicated world and lives, as well as turning the crisis into an opportunity for spiritual renewal and adding meaning to our lives. It is hoped that this study will inspire readers to overcome the difficulties of the epidemic, find strength and see it as a life-changing opportunity.

Human brucellosis and fever of unknown origin.

BACKGROUND: Human brucellosis has become one of the major public health problems in China, and increases atypical manifestations, such as fever of unknown origin (FUO), and misdiagnosis rates has complicated the diagnosis of brucellosis. To date, no relevant study on the relationship between brucellosis and FUO has been conducted. METHODS: We retrospectively reviewed the medical charts of 35 patients with confirmed human brucellosis and prospectively recorded their outcomes by telephone interview. The patients were admitted to the Second Affiliated Hospital of Nanchang University between January 01, 2013 and October 31, 2019. Patient data were collected from hospital medical records. RESULTS: The percentage of males was significantly higher than that of female in FUO (78.95% vs. 21.05%, P < 0.05), and 80% of the patients had a clear history of exposure to cattle and sheep. Moreover, 19 (54%) cases were hospitalized with FUO, among which the patients with epidemiological histories were significantly more than those without (P < 0.05). The incidence of toxic hepatitis in FUO patients was higher than that in non-FUO patients (89% vs. 50%, P < 0.05). Meanwhile, the misdiagnosis rate was considerably higher in the FUO group than in the non-FUO group (100% vs. 63%; P < 0.05). CONCLUSION: Brucellosis is predominantly FUO admission in a non-endemic area of China, accompanied by irregular fever and toxic hepatitis. Careful examination of the epidemiological history and timely improvement of blood and bone marrow cultures can facilitate early diagnosis and prevent misdiagnosis.

Corrigendum: Chaperones, Membrane Trafficking and Signal Transduction Proteins Regulate Zaire Ebola Virus trVLPs and Interact With trVLP Elements.

[This corrects the article DOI: 10.3389/fmicb.2018.02724.].

Chaperones, Membrane Trafficking and Signal Transduction Proteins Regulate Zaire Ebola Virus trVLPs and Interact With trVLP Elements.

Ebolavirus (EBOV) life cycle involves interactions with numerous host factors, but it remains poorly understood, as does pathogenesis. Herein, we synthesized 65 siRNAs targeting host genes mostly connected with aspects of the negative-sense RNA virus life cycle (including viral entry, uncoating, fusion, replication, assembly, and budding). We produced EBOV transcription- and replication-competent virus-like particles (trVLPs) to mimic the EBOV life cycle. After screening host factors associated with the trVLP life cycle, we assessed interactions of host proteins with trVLP glycoprotein (GP), VP40, and RNA by co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP). The results demonstrate that RNAi silencing with 11 siRNAs (ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1, and YWHAZ) decreased the replication efficiency of trVLPs. Co-IP revealed nine candidate host proteins (FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1, and YWHAZ) potentially interacting with trVLP GP, and four (ANXA5, GRP78, HSPA1A, and HSP90AB1) potentially interacting with trVLP VP40. Ch-IP identified nine candidate host proteins (ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, MAPK11, MEK2, and NTRK1) interacting with trVLP RNA. This study was based on trVLP and could not replace live ebolavirus entirely; in particular, the interaction between trVLP RNA and host proteins cannot be assumed to be identical in live virus. However, the results provide valuable information for further studies and deepen our understanding of essential host factors involved in the EBOV life cycle.

Development and Characterization of Neutralizing Antibodies Against Zaire Ebolavirus Glycoprotein and Protein 40.

BACKGROUND/AIMS: Monoclonal antibodies (mAbs) are presently the most promising treatment against Ebola virus disease (EVD), and cocktail of two or more antibodies likely confers protection through complementary mechanisms. Zaire Ebolavirus (EBOV) glycoprotein (GP) and viral protein 40 (VP40) are targets for designing neutralizing antibodies. Currently, the antiviral therapeutics of mAb-cocktails are still limited solely to anti-GP antibodies，there is no Abs cocktail against Zaire EBOV GP and VP40, which both have important interactions with host cellular membrane. METHODS: We used hybridoma technology to produce anti-Zaire EBOV GP mAb against GP receptor binding domain, and anti-Zaire EBOV VP40 mAbs against the N-terminal domain, the C-terminal domain, respectively; synthesized Zaire EBOV transcription and replication competent virus like particles (trVLPs), which model even all aspects of the EBOV life cycles in order to evaluate the anti-viral effect of mAbs. Then, we characterized the anti- Zaire EBOV trVLPs effect of anti-GP and VP40 mAbs in vitro by real time-PCR, immunofluorescence assay and western blot analysis. RESULTS: Our results demonstrate that anti-GP or anti-VP40 mAbs effectively inhibit trVLPs replication. The cocktails of anti-GP and anti-VP40 mAbs, or between anti-VP40 mAbs, had synergistic anti-trVLPs effect. Meanwhile, the detailed DNA and amino acid sequences of the mAbs were checked. CONCLUSION: The study verifies neutralizing efficacy of anti-GP or anti-VP40 mAb, report promising cocktail of anti-GP and anti-VP40 mAb, or cocktail of two anti-VP40 mAbs. To our knowledge, this is the first account to report the important anti-viral effect of cocktails of anti-GP and anti-VP40 mAbs in vitro.

Process optimization for the rapid production of adenoviral vectors for clinical trials in a disposable bioreactor system.

Recombinant adenoviral (Ad) vectors are highly efficient gene transfer vectors widely used in vaccine development and immunotherapy. To promote the industrial application of Ad vectors, studies focusing on reducing the cost of manufacturing, shortening the preclinical research period, and improving the quality of products are needed. Here, we describe a highly efficient and economical process for producing Ad vector in a novel, single-use bioreactor system suitable for clinical trials. A mini-bioreactor was used for parameter optimization and development of medium replacement protocols for Ad5-GFP production before scale-up. HEK293 cell culture and virus infection were monitored in a disposable AmProtein Current Perfusion Bioreactor and Bioflo310 bioreactor using optimized parameters and medium replacement protocols. The total cell number increased from 2.0 × 109 to 3.2 × 1010 after 6 days of culture. The total number of viral particles obtained in a single batch was 1.2 × 1015. These results demonstrate the efficiency and suitability of this system for Ad vector production for research and GMP applications.

The Protective Effects of the A/ZJU01/ PR8/2013 Split H7N9 Avian Influenza Vaccine Against Highly Pathogenic H7N9 in BALB/c Mice.

BACKGROUND/AIMS: Since the first case of novel H7N9 infection was reported, China has experienced five epidemics of H7N9. During the fifth wave, a highly pathogenic H7N9 strain emerged. In order to assess whether the H7N9 vaccine based on A/Zhejiang/DTID-ZJU01/2013(H7N9) was effective in protecting against highly pathogenic H7N9, we conducted this study. METHODS: Groups of mice were immunized twice by intraperitoneal injection with 500 µl of either split vaccine alone or MF59-adjuvanted vaccine. Serum was collected 2 weeks after the second vaccine booster. The hemagglutinin inhibition test was conducted on vaccine seed and highly pathogenic H7N9 to evaluate the neutralization of highly pathogenic H7N9. We also immunized mice and challenged them with highly pathogenic H7N9. Mice were observed for illness, weight loss, and death at 1 week and 2 weeks post-infection. Then, the mice were sacrificed and lungs were removed. Antibody responses were assessed and pathological changes in the lung tissue were evaluated. RESULTS: The ability of serum to neutralize highly pathogenic H7N9 was reduced. In mice, highly pathogenic H7N9 was more virulent than A/Zhejiang/DTID-ZJU01/2013(H7N9). After challenge with highly pathogenic H7N9, all mice died while mice challenged with A/Zhejiang/DTID-ZJU01/2013(H7N9) all recovered. The A/ZJU01/PR8/2013 split H7N9 avian influenza vaccine was able to protect against infection with highly pathogenic H7N9 in mice, with or without MF59. Moreover, H7N9 vaccine adjuvanted with MF59 produced high antibody levels, which lead to better protection. CONCLUSIONS: The A/ZJU01/PR8/2013 split H7N9 avian influenza vaccine based on A/Zhejiang/DTID-ZJU01/2013(H7N9) is effective in protecting against highly pathogenic H7N9. H7N9 vaccine adjuvanted with MF59 offers better protection against infection with highly pathogenic H7N9. In order to make the H7N9 vaccine applicable to humans, further clinical trials are required to evaluate MF59 adjuvanted vaccine. Meanwhile, the vaccine strain should be updated based on the highly pathogenic H7N9 gene sequence.

The lifecycle of the Ebola virus in host cells.

Ebola haemorrhagic fever causes deadly disease in humans and non-human primates resulting from infection with the Ebola virus (EBOV) genus of the family Filoviridae. However, the mechanisms of EBOV lifecycle in host cells, including viral entry, membrane fusion, RNP formation, GP-tetherin interaction, and VP40-inner leaflet association remain poorly understood. This review describes the biological functions of EBOV proteins and their roles in the lifecycle, summarizes the factors related to EBOV proteins or RNA expression throughout the different phases, and reviews advances with regards to the molecular events and mechanisms of the EBOV lifecycle. Furthermore, the review outlines the aspects remain unclear that urgently need to be solved in future research.

The regulatory role of microRNA-1187 in TNF-α-mediated hepatocyte apoptosis in acute liver failure.

In the current study, we aimed at elucidating the regulatory mechanisms through which microR-1187 (miR-1187) participates in hepatocyte apoptosis in acute liver failure (ALF). An ALF model was induced with D-galactosamine (D-GalN) plus lipopolysaccharide (LPS) in BALB/c mice. The hepatic miRNA expression profile was detected by microarray analysis and verified by quantitative real-time PCR (qRT-PCR). The possible underlying mechanism was investigated in vitro using an embryonic murine hepatocyte cell line (BNLCL2) and miR-1187 mimic. Caspase-8 protein was detected by Western blotting and cell apoptosis was assayed by flow cytometry. Hepatic miR-1187 was down-regulated in ALF mice based on microarray data (P<0.001) and verified by qRT-PCR (P<0.01). Target scan revealed that caspase-8 was the putative target of miR-1187. In an in vitro study, miR-1187 showed the highest up-regulation in BNLCL2 cells transfected with the miR-1187 mimic at a 50 nM concentration for 12 h compared with cells transfected with the non-specific mimic (P<0.001). miR-1187 was down-regulated (P<0.01) but caspase-8 mRNA (P<0.01) as well as protein (P<0.05) were up-regulated in the BNLCL2 cells treated with D-GalN/TNF. Furthermore, overexpressed miR-1187 reduced caspase-8 expression at both the mRNA and protein levels significantly (P<0.01 and P<0.05 respectively), and significantly attenuated the apoptotic rate of BNLCL2 cells (P<0.05). We show that miR-1187 regulates hepatocyte apoptosis by targeting caspase-8. miR-1187 may serve as a potential therapeutic target for the treatment of ALF.

[The role of miRNA-122 expression during the acute liver failure in mice induced by D-GalN/LPS].

OBJECTIVE: To investigate the expression of miR-122 and its relationship with progression and development of acute liver failure in mice induced by D-GalN/LPS, and to explore new biomarker(s) for early diagnosis of acute liver failure. METHODS: BALB/C mice were randomly divided into four groups: the mice were given D-GalN (900 mg/kg body weight) and LPS (10 micog/kg body weight) intraperitoneally (i.p.) to construct the acute liver model; whereas the control groups were given D-GalN (900 mg/kg), LPS (10 microg/kg) and normal saline respectively. All biochemical and histological indexes were determined at 0, 1, 3, 5, 7 and 9 h respectively after administration. Real-time RT-PCR were used to detect the expression of miR-122 and pro-inflammatory cytokines, furthermore, the expression of miR-122 was verified by LNA (lock nucleic acid)-Northern-blot. ALT and AST levels were tested by biochemistry analyzer. Serum pro-inflammatory cytokine levels were tested by ELISA. RESULTS: The mortality rate was about 80% at 24h after D-GalN/LPS treatment, but no mortality was observed in the other three control groups. Liver special miRNA miR-122 was highly expressed in liver tissue of normal mice (ct is approximately equal to 14), it was up-regulated significantly (P = 0.013) at first hour after treatment then down-regulated according to the development of acute liver failure, the change was more obvious at 9 h (ct is approximately equal to 15, P = 0.002). ALT and AST levels increased obviously at 3h after treatment and reached peak at 7 hours then they were declined sharply. It was found that the expression of miR-122 was faster and more durable than ALT. Pro-inflammatory cytokines related to acute liver failure including TNFa and IL-6 were all up-regulated in serum as well as liver tissue (P less than 0.05). Correlation analysis showed that miR-122 had a negative correlation with ALT (correlation coefficients -0.505) and positive correlations with TNFa and IL-6 (correlation coefficients were 0.493 and 0.674 respectively). CONCLUSIONS: Liver-specific miR-122 supposed be a new marker molecule for early diagnosis of liver cells injury in the acute liver failure.