Huangkui capsule attenuates diabetic kidney disease through the induction of mitophagy mediated by STING1/PINK1 signaling in tubular cells.

BACKGROUND: Mitochondria is critic to tubulopathy, especially in diabetic kidney disease (DKD). Huangkui capsule (HKC; a new ethanol extract from the dried corolla of Abelmoschus manihot) has significant clinical effect on DKD. Previous studies have shown that HKC protects kidney by regulating mitochondrial function, but its mechanism is still unclear. The latest research found that the stimulator of interferon genes (STING1) signal pathway is closely related to mitophagy. However, whether HKC induces mitophagy through targeting STING1/PTEN-Induced putative kinase (PINK1) in renal tubular remains elusive. OBJECTIVE: This study aims to clarify the therapeutic effect of HKC on renal tubular mitophagy in DKD and its potential mechanism in vivo and in vitro. METHODS: Forty male C57BL/6 mice were randomly divided into 5 groups: CON group, DKD group, HKC-L (1.0 g/kg/day, by gavage), HKC-H (2.0 g/kg/day), and LST group. Diabetes model was induced by high-fat diet (HFD) combined with intraperitoneal injection of Streptozotocin (STZ). LST (losartan) is used as a positive control drug. Then, the glomeruli, renal tubular lesions, mitochondrial morphology and function of renal tubular cells and mitophagy levels were detected in mice. In addition, a high glucose injury model was established using HK2 human renal tubular cells. Pretreate HK2 cells with HKC or LST and detect mitochondrial function, mitophagy level, and autophagic flux. In addition, small interfering RNAs (siRNAs) of STING1 and PINK1 and overexpressing pcDNA3.1 plasmids were transfected into HK-2 cells to validate the mitophagy mechanism regulated by STING1/PINK1 signaling. RESULTS: The ratio of urinary albumin to creatinine (ACR), fasting blood glucose, body weight in the early DKD mice model was increased, with damage to the glomerulus and renal tubules, mitochondrial structure and dysfunction in the renal tubules, and inhibition of STING1/PINK1 mediated mitophagy. Although the fasting blood glucose, body weight and serum creatinine levels were hardly ameliated, high dose HKC (2.0 g/kg/day) treatment significantly reduced ACR in the DKD mice to some extent, improved renal tubular injury, accurately upregulated STING1/PINK1 signaling mediated mitophagy levels, improved autophagic flux, and restored healthy mitochondrial pools. In vitro, an increase in mitochondrial fragments, fusion to fission, ROS and apoptosis, and a decrease in respiratory function, mtDNA, and membrane potential were observed in HK2 cells exposed to high glucose. HKC treatment significantly protected mitochondrial dynamics and function, which is consistent with in vivo results. Further research has shown that HKC can increase the level of mitophagy mediated by STING1/PINK1 in HK2 cells. CONCLUSIONS: Our results suggest that HKC ameliorates renal tubulopathy in DKD and induces mitophagy partly through the up-regulation of the STING1/PINK1 pathway. These findings may provide an innovative therapeutic basis for DKD treatment.

RBF Protein with MA103 Adjuvant Elicited Protective Immunity against Human Respiratory Syncytial Virus in BALB/c Mice.

The development of a vaccine against human respiratory syncytial virus (HRSV) has been hampered by enhanced respiratory disease due to the Th2-biased immune response. In the present study, MA103 and aluminum phosphate (Adju-Phos) adjuvants were used to verify the immunogenicity of the recombinant fusion (RBF) protein (F protein expressed by Escherichia coli). Both adjuvants significantly increased the neutralizing antibody titer and number of interferon gamma (IFN-γ)-secreting CD4+ T cells in mice. Based on the immunoglobulin G1 (IgG1)/IgG2a and IFN-γ/interleukin 4-secreting CD4+ T cell ratio, however, MA103 significantly enhanced the Th1-biased immune response. The pathological damage to the lung in the RBF/MA103 group was less than what was seen in the RBF/Adju-Phos group. Additionally, the number of HRSV copies in the lungs of the RBF/MA103 group decreased by approximately 3 × log10. These results suggested that MA103 provides better protection against HRSV in mice.

Comparison of the efficacy and safety of different immunization routes induced by human respiratory syncytial virus F protein with CpG adjuvant in mice.

Human respiratory syncytial virus (HRSV) is a leading cause worldwide of severe respiratory illness in infants and the elderly. The ideal HRSV vaccine should induce a systemic immune response, especially mucosal immunity. In this study, mice were immunized twice with F protein combined with CpG adjuvant to compare the safety and efficacy of 4 immunization routes, including intranasal primed/intramuscular boosted immunization (CpG + F/in+im), intramuscular primed/intranasal boosted immunization (CpG + F/im+in), intramuscular primed/intramuscular boosted immunization (CpG + F/im + im) and intranasal primed/intranasal boosted immunization (CpG + F/in+in). Compared with the control group (CpG/in+im, CpG/im+in, CpG/im + im and CpG/in+in), all 4 immunization routes induced a high titer of neutralizing antibodies and a strong cellular immune response. Mice in the CpG + F/in+in group induced the highest antibody neutralization titer, and IgA antibody in bronchoalveolar lavage fluid (BALF) was the highest. The copy of HRSVs in the lung decreased by approximately 3 log10. As seen from the IgG1/IgG2a and IFN-γ/IL-4-secreting lymphocyte ratios, compared with the mice in the CpG + F/im + im group, mice in the CpG + F/in+in group induced Th1-baised humoral and cellular immune responses and significantly reduced lung pathological injury. In conclusion, among the 4 immunization routes, the safety and efficacy induced by the mice in the CpG + F/in+in group were the best. We can conclude that intranasal immunization is superior to intramuscular immunization using F protein with CpG adjuvant as vaccine candidates.

Substitution of I222L-E119V in neuraminidase from highly pathogenic avian influenza H7N9 virus exhibited synergistic resistance effect to oseltamivir in mice.

That the high frequency and good replication capacity of strains with reduced susceptibility to neuraminidase inhibitors (NAIs) in highly pathogenic avian influenza H7N9 (HPAI H7N9) virus made it a significance to further study its drug resistance. HPAI H7N9 viruses bearing NA I222L or E119V substitution and two mutations of I222L-E119V as well as their NAIs-sensitive counterpart were generated by reverse genetics for NA inhibition test and replication capability evaluation in vitro. The attenuated H7N9/PR8 recombinant viruses were developed to study the pathogenicity and drug resistance brought by the above substitutions to mice. The IC50 fold change of oseltamivir to HPAI H7N9 with NA222L-119V is 306.34 times than that of its susceptible strain, and 3.5 times than the E119V mutant virus. HPAI H7N9 bearing NA222L-119V had good replication ability with peak value of more than 6log10 TCID50/ml in MDCK cells. H7N9/PR8 virus bearing NA222L-119V substitutions leaded to diffuse pneumonia, significant weight loss and fatality in mice. NA E119V made H7N9/PR8 virus resistant to oseltamivir, and I222L-E119V had synergistic resistance to oseltamivir in mice. Due to the good fitness of drug resistant strains of HPAI H7N9 virus, it is necessary to strengthen drug resistance surveillance and new drug research.

Impact of RNA degradation on influenza diagnosis in the surveillance system.

BACKGROUND: The continuous evolution of influenza viruses is monitored by the World Health Organization Global Influenza Surveillance and Response System. Sample quality is essential for surveillance quality. METHODS: To evaluate the RNA degradation of clinical samples, influenza-like illness samples were collected from four sentinel hospitals, and seasonal influenza was tested by real-time reverse transcription polymerase chain reaction and quantified by digital reverse transcription polymerase chain reaction at different time points. RESULTS: RNA degradation was observed in the majority of samples eight days after sample collection. A significant and faster rate of RNA content reduction was observed in low viral load samples (<10 copies/µl) than in high viral load samples (>10 copies/µl), stored at 2 to 8°C for up to eight days. RNase P (RNP) RNA, which is a key indicator to evaluate sample collection quality, was detected. Sample collection quality was uneven in different hospitals. CONCLUSION: Low viral load samples increase the risk of false negatives due to RNA degradation to undetectable levels.

Phase 2a, open-label, dose-escalating, multi-center pharmacokinetic study of favipiravir (T-705) in combination with oseltamivir in patients with severe influenza.

BACKGROUND: The pharmacokinetics and appropriate dose regimens of favipiravir are unknown in hospitalized influenza patients; such data are also needed to determine dosage selection for favipiravir trials in COVID-19. METHODS: In this dose-escalating study, favipiravir pharmacokinetics and tolerability were assessed in critically ill influenza patients. Participants received one of two dosing regimens; Japan licensed dose (1600 mg BID on day 1 and 600 mg BID on the following days) and the higher dose (1800 mg/800 mg BID) trialed in uncomplicated influenza. The primary pharmacokinetic endpoint was the proportion of patients with a minimum observed plasma trough concentration (Ctrough) ≥20 mg/L at all measured time points after the second dose. RESULTS: Sixteen patients were enrolled into the low dose group and 19 patients into the high dose group of the study. Favipiravir Ctrough decreased significantly over time in both groups (p <0.01). Relative to day 2 (48 hrs), concentrations were 91.7% and 90.3% lower in the 1600/600 mg group and 79.3% and 89.5% lower in the 1800/800 mg group at day 7 and 10, respectively. In contrast, oseltamivir concentrations did not change significantly over time. A 2-compartment disposition model with first-order absorption and elimination described the observed favipiravir concentration-time data well. Modeling demonstrated that less than 50% of patients achieved Ctrough ≥20 mg/L for >80% of the duration of treatment of the two dose regimens evaluated (18.8% and 42.1% of patients for low and high dose regimen, respectively). Increasing the favipravir dosage predicted a higher proportion of patients reaching this threshold of 20 mg/L, suggesting that dosing regimens of ≥3600/2600 mg might be required for adequate concentrations. The two dosing regimens were well-tolerated in critical ill patients with influenza. CONCLUSION: The two dosing regimens proposed for uncomplicated influenza did not achieve our pre-defined treatment threshold.

Effectiveness of favipiravir (T-705) against wild-type and oseltamivir-resistant influenza B virus in mice.

The emergence of resistant mutants to the wildly used neuraminidase inhibitors (NAIs) makes the development of novel drugs necessary. Favipiravir (T-705) is one of the RNA-dependent RNA polymerase (RdRp) inhibitors developed in recent years. To examine the efficacy of T-705 against influenza B virus infections in vivo, C57BL/6 mice infected with wild-type or oseltamivir-resistant influenza B/Memphis/20/96 viruses were treated with T-705. Starting 2 h post inoculation (hpi), T-705 was orally administered to mice BID at dosages of 50, 150, or 300 mg/kg/day for 5 days. Oseltamivir was used as control. Here, we showed that T-705 protected mice from lethal infection in a dose-dependent manner. T-705 administration also significantly reduced viral loads and suppressed pulmonary pathology. In addition, phenotypic assays demonstrated that no T-705-resistant viruses emerged after T-705 treatment. In conclusion, T-705 can be effective to protect mice from lethal infection with both wild-type and oseltamivir-resistant influenza B viruses.

The Nsp12-coding region of type 2 PRRSV is required for viral subgenomic mRNA synthesis.

As one of many nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV), nonstructural protein 12 (Nsp12) has received relatively little attention, and its role in virus replication, if any, is essentially unknown. By the application of reverse genetic manipulation of an infectious PRRSV clone, the current study is the first to demonstrate that Nsp12 is a key component of PRRSV replication. In addition, the biochemical properties of Nsp12 were evaluated, revealing that Nsp12 forms dimers when exposed to oxidative conditions. Furthermore, we systemically analyzed the function of Nsp12 in PRRSV RNA synthesis using a strand-specific PCR method. To our surprise, Nsp12 was not found to be involved in minus-strand genomic RNA (-gRNA) synthesis; importantly, our results indicate that Nsp12 is involved in the synthesis of both plus- and minus-strand subgenomic mRNAs (+sgmRNA and -sgmRNA). Finally, we found that the combination of cysteine 35 and cysteine 79 in Nsp12 is required for sgmRNA synthesis. To our knowledge, we are the first to report the biological role of Nsp12 in the PRRSV lifecycle, and we conclude that Nsp12 is involved in the synthesis of both + sgRNA and -sgRNA.

Live attenuated pseudorabies virus developed using the CRISPR/Cas9 system.

Currently, pseudorabies virus (PRV) variant strains are outbreaking in China; these variants belong to genotype II PRV. The traditional Bartha-K61 vaccine has failed to provide complete protection against the emergent variant strains. Therefore, rapid attenuation of current epidemic strains is needed for effective PRV control. In this study, we report a rapid method for editing the PRV genome using the CRISPR-Cas9 system. We developed a triple gE/gI/TK gene-inactivated HeN1 PRV strain, because mice were more susceptible to PRV infection, we then evaluated the attenuation of PRV in the mice and demonstrated that modified PRV was fully attenuated. Furthermore, the attenuated strain also induced immune protection in response to a parental PRV challenge. Overall, we showed that PRVs can be rapidly attenuated using CRISPR-Cas9 technology, which will be critical for PRV control, especially when new variant PRV strains emerge.

Open reading frames 1a and 1b of the porcine reproductive and respiratory syndrome virus (PRRSV) collaboratively initiate viral minus-strand RNA synthesis.

The porcine reproductive and respiratory syndrome virus (PRRSV) causes a persistent threat to the swine industry, especially when highly pathogenic PRRSV (HP-PRRSV) emerges. Previous studies have indicated that PRRSV RNA synthesis was correlated with HP-PRRSV virulence. PRRSV RNA synthesis includes genomic RNA and sub-genomic mRNA, and these processes require minus-strand RNA as a template. However, the mechanisms involved in PRRSV minus-strand RNA synthesis are not fully understood. A mini-genome system can be used to assess viral replication mechanisms and to evaluate the effects of potential antiviral drugs on viral replicase activities. In this study, we developed a mini-genome system that uses firefly luciferase as a reporter. Based on this system, we found that PRRSV RNA-dependent RNA polymerase nsp9 alone failed to activate virus minus-strand RNA synthesis. We also demonstrated that combinations of open reading frames 1a (ORF1a) and ORF1b are necessary for viral minus-strand RNA synthesis.

Recombinant Pseudorabies Virus (PRV) Expressing Firefly Luciferase Effectively Screened for CRISPR/Cas9 Single Guide RNAs and Antiviral Compounds.

A Pseudorabies virus (PRV) variant has emerged in China since 2011 that is not protected by commercial vaccines, and has not been well studied. The PRV genome is large and difficult to manipulate, but it is feasible to use clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. However, identification of single guide RNA (sgRNA) through screening is critical to the CRISPR/Cas9 system, and is traditionally time and labor intensive, and not suitable for rapid and high throughput screening of effective PRV sgRNAs. In this study, we developed a recombinant PRV strain expressing firefly luciferase and enhanced green fluorescent protein (EGFP) as a reporter virus for PRV-specific sgRNA screens and rapid evaluation of antiviral compounds. Luciferase activity was apparent as soon as 4 h after infection and was stably expressed through 10 passages. In a proof of the principle screen, we were able to identify several PRV specific sgRNAs and confirmed that they inhibited PRV replication using traditional methods. Using the reporter virus, we also identified PRV variants lacking US3, US2, and US9 gene function, and showed anti-PRV activity for chloroquine. Our results suggest that the reporter PRV strain will be a useful tool for basic virology studies, and for developing PRV control and prevention measures.