Transmembrane domain-driven PD-1 dimers mediate T cell inhibition.

Programmed cell death-1 (PD-1) is a potent immune checkpoint receptor on T lymphocytes. Upon engagement by its ligands, PD-L1 or PD-L2, PD-1 inhibits T cell activation and can promote immune tolerance. Antagonism of PD-1 signaling has proven effective in cancer immunotherapy, and conversely, agonists of the receptor may have a role in treating autoimmune disease. Some immune receptors function as dimers, but PD-1 has been considered monomeric. Here, we show that PD-1 and its ligands form dimers as a consequence of transmembrane domain interactions and that propensity for dimerization correlates with the ability of PD-1 to inhibit immune responses, antitumor immunity, cytotoxic T cell function, and autoimmune tissue destruction. These observations contribute to our understanding of the PD-1 axis and how it can potentially be manipulated for improved treatment of cancer and autoimmune diseases.

Saikosaponin B2, Punicalin, and Punicalagin in Vitro Block Cellular Entry of Feline Herpesvirus-1.

In the realm of clinical practice, nucleoside analogs are the prevailing antiviral drugs employed to combat feline herpesvirus-1 (FHV-1) infections. However, these drugs, initially formulated for herpes simplex virus (HSV) infections, operate through a singular mechanism and are susceptible to the emergence of drug resistance. These challenges underscore the imperative to innovate and develop alternative antiviral medications featuring unique mechanisms of action, such as viral entry inhibitors. This research endeavors to address this pressing need. Utilizing Bio-layer interferometry (BLI), we meticulously screened drugs to identify natural compounds exhibiting high binding affinity for the herpesvirus functional protein envelope glycoprotein B (gB). The selected drugs underwent a rigorous assessment to gauge their antiviral activity against feline herpesvirus-1 (FHV-1) and to elucidate their mode of action. Our findings unequivocally demonstrated that Saikosaponin B2, Punicalin, and Punicalagin displayed robust antiviral efficacy against FHV-1 at concentrations devoid of cytotoxicity. Specifically, these compounds, Saikosaponin B2, Punicalin, and Punicalagin, are effective in exerting their antiviral effects in the early stages of viral infection without compromising the integrity of the viral particle. Considering the potency and efficacy exhibited by Saikosaponin B2, Punicalin, and Punicalagin in impeding the early entry of FHV-1, it is foreseeable that their chemical structures will be further explored and developed as promising antiviral agents against FHV-1 infection.

Exploring PLGA-OH-CATH30 Microspheres for Oral Therapy of Escherichia coli-Induced Enteritis.

Antibiotic therapy effectively addresses Escherichia coli-induced enteric diseases, but its excessive utilization results in microbial imbalance and heightened resistance. This study evaluates the therapeutic efficacy of orally administered poly (lactic-co-glycolic acid) (PLGA)-loaded antimicrobial peptide OH-CATH30 microspheres in murine bacterial enteritis. Mice were categorized into the healthy control group (CG), untreated model group (MG), OH-CATH30 treatment group (OC), PLGA-OH-CATH30 treatment group (POC), and gentamicin sulfate treatment group (GS). Except for the control group, all other experimental groups underwent Escherichia coli-induced enteritis, followed by a 5-day treatment period. The evaluation encompassed clinical symptoms, intestinal morphology, blood parameters, inflammatory response, and gut microbiota. PLGA-OH-CATH30 microspheres significantly alleviated weight loss and intestinal damage while also reducing the infection-induced increase in spleen index. Furthermore, these microspheres normalized white blood cell count and neutrophil ratio, suppressed inflammatory factors (IL-1ß, IL-6, and TNF-α), and elevated the anti-inflammatory factor IL-10. Analysis of 16S rRNA sequencing results demonstrated that microsphere treatment increased the abundance of beneficial bacteria, including Phocaeicola vulgatus, in the intestinal tract while concurrently decreasing the abundance of pathogenic bacteria, such as Escherichia. In conclusion, PLGA-OH-CATH30 microspheres have the potential to ameliorate intestinal damage and modulate the intestinal microbiota, making them a promising alternative to antibiotics for treating enteric diseases induced by Escherichia coli.

Assessing the Efficacy of PLGA-Loaded Antimicrobial Peptide OH-CATH30 Microspheres for the Treatment of Bacterial Keratitis: A Promising Approach.

Bacterial keratitis in animals presents challenges due to ocular structural barriers, hindering effective drug delivery. In this study, we used biocompatible and biodegradable poly(lactic-co-glycolic acid) (PLGA) to encapsulate the naturally occurring antimicrobial peptide OH-CATH30, an alternative to conventional antibiotics, for the treatment of bacterial keratitis in animals. Microspheres (MS) were prepared using a modified water-in-oil-in-water (W/O/W) double-emulsion method with optimized osmotic pressure. We conducted comprehensive evaluations, including in vitro characterization, encapsulation efficiency determination, in vitro release kinetics, and in vivo/vitro assessments of irritation and bacterial inhibition. The optimized method yielded microspheres with impressive encapsulation efficiency of 75.2 ± 3.62% and a loading capacity of 18.25 ± 5.73%, exhibiting a well-defined particle size distribution (200-1000 nm) and a ζ-potential of -17.3 ± 1.91 mV. The microspheres demonstrated initial burst release followed by sustained and controlled release in vitro. Both in vitro and in vivo tolerance tests confirmed the biocompatibility of the drug-loaded microspheres, as they did not elicit significant irritation in ocular tissues. Remarkable antibacterial effects were observed in both in vitro and in vivo experiments. Our developed PLGA microspheres show promise as an alternative therapeutic option for topical administration in managing keratitis, offering exceptional drug delivery capabilities, improved bioavailability, and potent antibacterial efficacy.

High expression of antimicrobial peptides cathelicidin-BF in Pichia pastoris and verification of its activity.

Antibacterial peptides are endogenous polypeptides produced by multicellular organisms to protect the host against pathogenic microbes, they show broad spectrum antimicrobial activities against various microorganisms and possess low propensity for developing resistance. The purpose of this study is to develop recombinant antibacterial peptide cathelicidin-BF by genetic engineering and protein engineering technology, and study its antibacterial activity in vitro and in vivo, so as to provide reference for the production and application of recombinant antibacterial peptide cathelicidin-BF. In this study, on account of Pichia pastoris eukaryotic expression system, we expressed and prepared antibacterial peptide cathelicidin-BF. Then, the minimum inhibitory concentration of antibacterial peptide cathelicidin-BF and the comparison with the antibacterial activity of antibiotics were determined through the antibacterial experiment in vitro. Chickens as infection model were used to verify the antibacterial peptide activity in vivo. The results show that the bacteriostatic ability of antibacterial peptide cathelicidin-BF is similar to that of antibiotics in certain concentration, and can reach the treatment level of antibiotics. Although the mode of administration of antibacterial peptide is still limited, this study can provide reference for the future research of antibacterial peptide.

Vaccines for African swine fever: an update.

African swine fever (ASF) is a fatal infectious disease of swine caused by the African swine fever virus (ASFV). Currently, the disease is listed as a legally notifiable disease that must be reported to the World Organization for Animal Health (WOAH). The economic losses to the global pig industry have been insurmountable since the outbreak of ASF. Control and eradication of ASF are very critical during the current pandemic. Vaccination is the optimal strategy to prevent and control the ASF epidemic, but since inactivated ASFV vaccines have poor immune protection and there aren't enough cell lines for efficient in vitro ASFV replication, an ASF vaccine with high immunoprotective potential still remains to be explored. Knowledge of the course of disease evolution, the way of virus transmission, and the breakthrough point of vaccine design will facilitate the development of an ASF vaccine. In this review, the paper aims to highlight the recent advances and breakthroughs in the epidemic and transmission of ASF, virus mutation, and the development of vaccines in recent years, focusing on future directions and trends.

Changyanning regulates gut microbiota and metabolism to ameliorate intestinal injury induced by ETEC K88.

Enterotoxigenic Escherichia coli (ETEC) is a common pathogen of swine colibacillosis, which can causing a variety of diseases initiate serious economic losses to the animal husbandry industry. The traditional Chinese medicine Changyanning (CYN) often used for diarrhea caused by the accumulation of damp heat in the gastrointestinal tract, has anti-bacterial, anti-inflammatory and anti-oxidation effects. This study investigated the effect of CYN on gut microbiota and metabolism in mice infected with ETEC K88. A total of 60 Kunming mices were divided into Control group, ETEC K88 group, CYN.L group (2.5 g/kg), CYN.M group (5 g/kg), CYN.H group (10 g/kg) and BTW group (10 g/kg), determined clinical symptoms, intestinal morphology, inflammatory responses, gut microbiota as well as serum metabolites. CYN administration elevated ETEC K88-induced body weight loss, ameliorated duodenum, ilem, colon pathological injury, and reduced the increase of spleen index caused by ETEC. CYN also reduced the levels of pro-inflammatory cytokines (IL-6, TNE-α) in the serum. 16s rRNA gene sequencing results showed that CYN increased the abundance of beneficial bacteria Lactobacillus but decreased the abundance of pathogenic bacteria Escherichia in the feces of mice. Moreover, CYN participates in amino acid biosynthesis and metabolism in the process of serum metabolism to regulates ameliorate intestinal injury induced by ETEC K88. In conclusion, CYN regulates gut microbiota and metabolism to ameliorate intestinal injury induced by ETEC K88.

A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies.

Monoclonal and polyclonal antibodies are mostly used for the development of traditional enzyme-linked immunosorbent assays (ELISAs), but the use of certain conventional antibodies may be limited by their low yield, the difficulty of their isolation, and their high cost. Heavy-chain antibodies derived from camelids with naturally missing light chains can overcome these deficiencies and are an excellent alternative to conventional antibodies. In this study, a nanobody (Nb)-AviTag fusion protein was constructed, and the feasibility of its use as a high-sensitivity probe in a blocking ELISA (bELISA) for classical swine fever virus (CSFV) was investigated. The CSFV E2 recombinant protein expressed by the CHO expression system exhibited good reactogenicity and immunogenicity and induced the production of high CSFV antibody levels in rabbits. Three different clones of Nbs were successfully isolated using a phage display system in alpaca, and an Nb1-AviTag fusion protein was successfully expressed using an Escherichia coli expression system. The purified Nb1-AviTag fusion protein was then biotinylated in vitro to obtain Nb1-biotin. A novel bELISA was developed for the detection of CSFV antibodies in clinical serum using Nb1-biotin as a probe. The cutoff value of bELISA was 32.18%, the sensitivity of bELISA was higher than that of the bELISA kit with IDEXX antibody, and the coincidence rate was 94.7%. A rapid, low-cost, highly sensitive and highly specific CSFV E2 antibody-based bELISA method was successfully established and can be used for the serological evaluation of CSFV E2 subunit vaccines and the ELISA-based diagnosis of CSFV infection. IMPORTANCE Currently, the epidemic situation of classical swine fever (CSF) is sporadic, and cases of atypical swine fever are on the rise in China. Therefore, it is necessary to accurately eliminate suspected cases by using highly sensitive and specific diagnostic techniques. In our study, a rapid, low-cost, highly sensitivity, highly reliable and reproducible, and highly specific classical swine fever virus (CSFV) E2 antibody-based blocking ELISA method was successfully established by using the phage display system and the Nb1-AviTag fusion expression platform. It provides a new technique for serological evaluation of CSFV vaccines and ELISA-based diagnosis of CSFV infection.

Efficacy of a gB + gD-based subunit vaccine and the adjuvant granulocyte-macrophage colony stimulating factor for pseudorabies virus in rabbits.

Pseudorabies (PR), which is caused by the pseudorabies virus (PRV), is a severe infectious disease that causes abortions in adult sows and fatal encephalitis in piglets; the disease can occur in pigs of all ages and other mammals, which can lead to significant economic loss around the worldwide. The new PRV variant invalidated the available commercial attenuated and inactivated vaccines. Consequently, subunit vaccines have been suggested as novel strategies for PR control, while they are usually formulated with adjuvants due to their lower immunogenicity. We aimed to select a safe and efficient adjuvant for subunit vaccines for PR. In our study, glycoprotein B (gB) and glycoprotein D (gD) were expressed based on a baculovirus expression system, and granulocyte-macrophage colony-stimulating factor (GM-CSF) was expressed using an Escherichia coli (E. coli) expression system; subsequently, a gB + gD subunit vaccine adjuvanted by GM-CSF was constructed. A rabbit model infected with a PRV SD-2017 strain was established, the TCID50 and LD50 were measured, and the typical clinical symptoms were observed. After a lethal challenge of 5 LD50 with a PRV SD-2017 strain, the rabbits exhibited typical clinical symptoms, including itching and high temperature, and histopathology revealed severe inflammation in the brain, which is the dominant target organ of PRV. Rabbits immunized with the gB + gD + GM-CSF subunit vaccines produced higher levels of antibodies than those immunized with gB + gD + ISA 201, which was adjuvanted with a frequently used oil adjuvant. The survival rate of rabbits vaccinated with gB + gD + GM-CSF was 100%, which was superior to that of rabbits vaccinated with gB + gD + ISA 201 (80%), inactivated PRV + GM-CSF (60%) and commercial inactivated vaccine (60%) after challenge with PRV SD-2017. These data suggested that the gB + gD + GM-CSF-based subunit vaccine had good protective efficacy against the PRV SD-2017 strain in rabbits and that GM-CSF could be developed as a candidate adjuvant for use in a vaccine regimen to prevent and even eradicate PR.

JNJ0966 inhibits PDGF-BB-induced airway smooth muscle cell proliferation and extracellular matrix production by regulating MMP-9.

The increased proliferation and extracellular matrix (ECM) production of airway smooth muscle cells (ASMCs) are crucial factors in asthma progression. JNJ0966, one of the metalloproteinase-9 (MMP-9)-specific inhibitors, has been demonstrated to be involved in the progression and development of diversified diseases. Nevertheless, the function of JNJ0966 in ASMCs remains unclear. This study aimed at investigating the effects of JNJ0966 on asthma progression. In our study, the platelet-derived growth factor BB (PDGF-BB) was first utilized to stimulate the cell model for asthma. Results demonstrated that the cell viability of ASMCs was increased by PDGF-BB (0, 10, 20, and 30 ng/mL) in a dose-dependent manner. Further investigation revealed that JNJ0966 inhibited the cell activity and migration ability of PDGF-BB-induced ASMCs. In addition, JNJ0966 relieved ECM deposition in PDGF-BB-induced ASMCs. Finally, through rescue assays, the results showed that overexpression of MMP-9 reversed the inhibitory effects of JNJ0966 on cell viability and ECM deposition in ASMCs. In conclusion, our findings suggested that JNJ0966 inhibited PDGF-BB-induced ASMC proliferation and ECM production by modulating MMP-9. These findings might provide novel insight for the treatment of asthma.

Molecular markers of tuberculosis and their clinical relevance: a systematic review and meta-analysis.

BACKGROUND: Since research on disease biomarkers of tuberculosis (TB) and latent tuberculosis infection (LTBI) provides hope for simple point-of-care testing, we aim to summarize and analyze the evidence for the clinical relevance of IFN-γ-inducible protein 10 (IP-10) and IFN-γ/interleukin 2 (IL-2) as diagnostic biomarkers for TB. METHODS: The search terms tuberculosis, tuberculous pleurisy, pulmonary tuberculosis, latent tuberculosis infection, biomarkers, markers, IFN-γ-inducible protein 10, IP-10, interleukin 2, and IL-2 were used to search the PubMed, Cochrane Central Register of Controlled Trials, EMBASE, Web of Science, China National Knowledge Infrastructure (CNKI), Wanfang, and Weipu databases. The retrieval time was from the establishment of the database to September 2021. The Cochrane risk of bias tool was used to evaluate the quality of the included studies, and the meta-analysis was performed using RevMan 5.20. RESULTS: A total of 9 articles were included for meta-analysis. The quality assessment showed that the overall quality of the included articles was met the requirements. The results showed that the overall sensitivity and specificity of IP-10 were 0.77 (95% CI, 0.71-0.82) and 0.84 (95% CI, 0.80-0.88), respectively. The overall sensitivity and specificity of IL-2 were 0.82 (95% CI, 0.74-0.89) and 0.95 (95% CI, 0.88-0.98), respectively. The areas under the curves (AUCs) of the IP-10 and IL-2 summary receiver operating characteristic (SROC) curves were 0.8592 and 0.9666, respectively. DISCUSSION: The results of this systematic review and meta-analysis showed that IP-10 and IL-2 as biomarkers have good clinical relevance to TB and can be used for the clinical screening of high-risk TB populations. However, a prospective cohort study across multiple regions using a large sample size should also be conducted.

The dNTPase activity of SAMHD1 is important for its suppression of innate immune responses in differentiated monocytic cells.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphohydrolase (dNTPase) with a nuclear localization signal (NLS). SAMHD1 suppresses innate immune responses to viral infection and inflammatory stimuli by inhibiting the NF-κB and type I interferon (IFN-I) pathways. However, whether the dNTPase activity and nuclear localization of SAMHD1 are required for its suppression of innate immunity remains unknown. Here, we report that the dNTPase activity, but not nuclear localization of SAMHD1, is important for its suppression of innate immune responses in differentiated monocytic cells. We generated monocytic U937 cell lines stably expressing WT SAMHD1 or mutated variants defective in dNTPase activity (HD/RN) or nuclear localization (mNLS). WT SAMHD1 in differentiated U937 cells significantly inhibited lipopolysaccharide-induced expression of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) mRNAs, as well as IFN-α, IFN-ß, and TNF-α mRNA levels induced by Sendai virus infection. In contrast, the HD/RN mutant did not exhibit this inhibition in either U937 or THP-1 cells, indicating that the dNTPase activity of SAMHD1 is important for suppressing NF-κB activation. Of note, in lipopolysaccharide-treated or Sendai virus-infected U937 or THP-1 cells, the mNLS variant reduced TNF-α or IFN-ß mRNA expression to a similar extent as did WT SAMHD1, suggesting that SAMHD1-mediated inhibition of innate immune responses is independent of SAMHD1's nuclear localization. Moreover, WT and mutant SAMHD1 similarly interacted with key proteins in NF-κB and IFN-I pathways in cells. This study further defines the role and mechanisms of SAMHD1 in suppressing innate immunity.

Expression and localization study of pIgR in the late stage of embryo development in turbot (Scophthalmus maximus).

The receptor responsible for maternofetal transmission of immunoglobulin (Igs) in the teleosts is not clear. Polymeric immunoglobulin receptor (pIgR) specifically binds with IgA and IgM and mediates the transcytosis of intracellular polymeric immunoglobulins (pIgs) at the mucosal surface to protect against pathogens. Hence there is a possibility that it may be involved in the transmission of maternal Igs. The aim of the present study was to detect the expression and localization of pIgR during embryonal development in turbot (Scophthalmus maximus). pIgR gene was first cloned from eggs and embryos of turbot with or without parent immunization. The expression and distribution of pIgR in unfertilized egg and in embryos ranging from day 1 to day 5 after fertilization were analyzed using reverse transcriptase quantitative polymerase chain reaction and in situ hybridization. pIgR gene was detected in all eggs and embryos at different stages of development, with the highest level detected on the 5th day. pIgR mRNA was observed to be first located in the whole blastoderm and enveloped the yolk sac. Later, it was located around entoderm including primary digestive tract and pronephric tubule tract, and finally it was located at the joint of abdomen and vitelline membrane. Then, Eukaryotic expression plasmid carrying pIgR gene was constructed and transfected into HEK293T cells. Results showed mature pIgR protein located on the cellular membrane, and could bound IgM in vitro. Our findings provide information for studying the involvement of pIgR in maternal Igs transportation in turbot.

SAMHD1 Suppression of Antiviral Immune Responses.

SAMHD1 is a host triphosphohydrolase that degrades intracellular deoxynucleoside triphosphates (dNTPs) to a lower level that restricts viral DNA synthesis, and thus prevents replication of diverse viruses in nondividing cells. Recent progress indicates that SAMHD1 negatively regulates antiviral innate immune responses and inflammation through interacting with various key proteins in immune signaling and DNA damage-repair pathways. SAMHD1 can also modulate antibody production in adaptive immune responses. In this review, we summarize how SAMHD1 regulates antiviral immune responses through distinct mechanisms, and discuss the implications of these new functions of SAMHD1. Furthermore, we propose important new questions and future directions that can advance functional and mechanistic studies of SAMHD1-mediated immune regulation during viral infections.

Isolation and antiproliferation of tumor cells by a novel peptide (TC22) from the beetle Tribolium castaneum.

Anticancer peptides (ACPs) are biologically anticancer active molecules that are produced by mammals, plants, insects and microorganisms. Here, a new peptide (TC22) with the amino acid sequence MTVVLLLIVLPLLGGVHSSGIL was identified and characterized from the beetle Tribolium castaneum. We found it inhibited the growth and viability of HeLa and MCF-7 cells. Flow cytometry analysis demonstrated the TC22 induced HeLa cell apoptosis, and activated caspase-9 and caspase-3. Furthermore, TC22 led to ROS generation, and triggered p53 transcription and expression. Taken together, our results indicated that TC22 exhibited high anticancer capacity via activating p53, inducing ROS generation and through a mitochondrial pathway. This research provided a novel natural source peptide with strong anticancer capacity. These findings provide some novel insights on the potential candidate reagent in cancer treatment.

SAMHD1 suppresses innate immune responses to viral infections and inflammatory stimuli by inhibiting the NF-κB and interferon pathways.

Sterile alpha motif and HD-domain-containing protein 1 (SAMHD1) blocks replication of retroviruses and certain DNA viruses by reducing the intracellular dNTP pool. SAMHD1 has been suggested to down-regulate IFN and inflammatory responses to viral infections, although the functions and mechanisms of SAMHD1 in modulating innate immunity remain unclear. Here, we show that SAMHD1 suppresses the innate immune responses to viral infections and inflammatory stimuli by inhibiting nuclear factor-κB (NF-κB) activation and type I interferon (IFN-I) induction. Compared with control cells, infection of SAMHD1-silenced human monocytic cells or primary macrophages with Sendai virus (SeV) or HIV-1, or treatment with inflammatory stimuli, induces significantly higher levels of NF-κB activation and IFN-I induction. Exogenous SAMHD1 expression in cells or SAMHD1 reconstitution in knockout cells suppresses NF-κB activation and IFN-I induction by SeV infection or inflammatory stimuli. Mechanistically, SAMHD1 inhibits NF-κB activation by interacting with NF-κB1/2 and reducing phosphorylation of the NF-κB inhibitory protein IκBα. SAMHD1 also interacts with the inhibitor-κB kinase ε (IKKε) and IFN regulatory factor 7 (IRF7), leading to the suppression of the IFN-I induction pathway by reducing IKKε-mediated IRF7 phosphorylation. Interactions of endogenous SAMHD1 with NF-κB and IFN-I pathway proteins were validated in human monocytic cells and primary macrophages. Comparing splenocytes from SAMHD1 knockout and heterozygous mice, we further confirmed SAMHD1-mediated suppression of NF-κB activation, suggesting an evolutionarily conserved property of SAMHD1. Our findings reveal functions of SAMHD1 in down-regulating innate immune responses to viral infections and inflammatory stimuli, highlighting the importance of SAMHD1 in modulating antiviral immunity.

Clofazimine for Treatment of Extensively Drug-Resistant Pulmonary Tuberculosis in China.

We performed a multicenter, prospective, randomized study to investigate the efficacy and safety of clofazimine (CLO) for treatment of extensively drug-resistant tuberculosis (XDR-TB) in China. Forty-nine patients infected with XDR-TB were randomly assigned to either the control group or the CLO group, both of which received 36 months of individually customized treatment. The primary endpoint was the time to sputum culture conversion on solid medium. Clinical outcomes of patients were evaluated at the time of treatment completion. Of the 22 patients in the experimental group, 7 (31.8%) met the treatment criterion of "cure" and 1 (4.5%) "complete treatment," for a total of 8 (36.4%) exhibiting successful treatment outcomes without relapse. In the control group, 6 patients (22.2%) were cured and 6 (22.2%) completed treatment by the end of the study. Statistical analysis revealed no significant difference in successful outcome rates between the CLO group and the control group. The average sputum culture conversion time for the experimental group was 19.7 months, which was not statistically different from that for the control group (20.3 months; P = 0.57). Of the 22 patients in the CLO group, 12 (54.5%) experienced adverse events after starting CLO treatment. The most frequently observed adverse event was liver damage, with 31.8% of patients (7/22 patients) in the CLO group versus 11.1% (3/27 patients) in the control group exhibiting this adverse event. Our study demonstrates that inclusion of CLO in background treatment regimens for XDR-TB is of limited benefit, especially since hepatic disorders arise as major adverse events with CLO treatment. (This study is registered with the Chinese Clinical Trial Registry [ChiCTR, www.chictr.org.cn] under identifier ChiCTR1800014800.).

Transmission and pathogenicity of novel reassortants derived from Eurasian avian-like and 2009 pandemic H1N1 influenza viruses in mice and guinea pigs.

Given the present extensive co-circulation in pigs of Eurasian avian-like (EA) swine H1N1 and 2009 pandemic (pdm/09) H1N1 viruses, reassortment between them is highly plausible but largely uncharacterized. Here, experimentally co-infected pigs with a representative EA virus and a pdm/09 virus yielded 55 novel reassortant viruses that could be categorized into 17 genotypes from Gt1 to Gt17 based on segment segregation. Majority of novel reassortants were isolated from the lower respiratory tract. Most of reassortant viruses were more pathogenic and contagious than the parental EA viruses in mice and guinea pigs. The most transmissible reassortant genotypes demonstrated in guinea pigs (Gt2, Gt3, Gt7, Gt10 and Gt13) were also the most lethal in mice. Notably, nearly all these highly virulent reassortants (all except Gt13) were characterized with possession of EA H1 and full complement of pdm/09 ribonucleoprotein genes. Compositionally, we demonstrated that EA H1-222G contributed to virulence by its ability to bind avian-type sialic acid receptors, and that pdm/09 RNP conferred the most robust polymerase activity to reassortants. The present study revealed high reassortment compatibility between EA and pdm/09 viruses in pigs, which could give rise to progeny reassortant viruses with enhanced virulence and transmissibility in mice and guinea pig models.

C-terminal elongation of NS1 of H9N2 influenza virus induces a high level of inflammatory cytokines and increases transmission.

H9N2 avian influenza viruses are enzootic around the world and can infect many different avian and mammalian hosts, including humans. Unlike the H9N2 viruses, which mainly originated in other countries and possess a non-structural protein 1 (NS1) of 230 aa, 98 % of the H9N2 viruses isolated in China lack the 13 aa at the C terminus of NS1 (217 aa in total). The biological significance of NS1 elongation remains elusive. To examine the effect of NS1 C-terminal elongation in the influenza virus, we used reverse genetics to generate a wt avian influenza H9N2 virus containing a 217 aa NS1 (H9N2NS1217) and two mutant viruses with elongated NS1s of 230 and 237 aa (H9N2NS1230 and H9N2NS1237). C-terminal elongation of NS1 did not have a significant impact on virus replication in Madin-Darby canine kidney cells or DF-1 cells. The three variants exhibited similar replicability in mice; however, the H9N2NS1230 and H9N2NS1237 variants exhibited an upregulation in the level of inflammatory cytokines. In addition, both the H9N2NS1230 and H9N2NS1237 viruses increased replication and induced a high level of inflammatory cytokines and transmission in chickens, compared with the wt virus. These findings suggest that the NS1 extension conferred a gain of fitness to some extent.