p53-dependent elimination of aneuploid mitotic offspring by entosis.

Entosis was proposed to promote aneuploidy and genome instability by cell-in-cell mediated engulfment in tumor cells. We reported here, in epithelial cells, that entosis coupled with mitotic arrest functions to counteract genome instability by targeting aneuploid mitotic progenies for engulfment and elimination. We found that the formation of cell-in-cell structures associated with prolonged mitosis, which was sufficient to induce entosis. This process was controlled by the tumor suppressor p53 (wild-type) that upregulates Rnd3 expression in response to DNA damages associated with prolonged metaphase. Rnd3-compartmentalized RhoA activities accumulated during prolonged metaphase to drive cell-in-cell formation. Remarkably, this prolonged mitosis-induced entosis selectively targets non-diploid progenies for internalization, blockade of which increased aneuploidy. Thus, our work uncovered a heretofore unrecognized mechanism of mitotic surveillance for entosis, which eliminates newly born abnormal daughter cells in a p53-dependent way, implicating in the maintenance of genome integrity.

Mechanical Ring Interfaces between Adherens Junction and Contractile Actomyosin to Coordinate Entotic Cell-in-Cell Formation.

Entosis is a cell-in-cell (CIC)-mediated death program. Contractile actomyosin (CA) and the adherens junction (AJ) are two core elements essential for entotic CIC formation, but the molecular structures interfacing them remain poorly understood. Here, we report the characterization of a ring-like structure interfacing between the peripheries of invading and engulfing cells. The ring-like structure is a multi-molecular complex consisting of adhesive and cytoskeletal proteins, in which the mechanical sensor vinculin is highly enriched. The vinculin-enriched structure senses mechanical force imposed on cells, as indicated by fluorescence resonance energy transfer (FRET) analysis, and is thus termed the mechanical ring (MR). The MR actively interacts with CA and the AJ to help establish and maintain polarized actomyosin that drives cell internalization. Vinculin depletion leads to compromised MR formation, CA depolarization, and subsequent CIC failure. In summary, we suggest that the vinculin-enriched MR, in addition to CA and AJ, is another core element essential for entosis.

Cholesterol inhibits entotic cell-in-cell formation and actomyosin contraction.

Cell-in-cell structure is prevalent in human cancer, and associated with several specific pathophysiological phenomena. Although cell membrane adhesion molecules were found critical for cell-in-cell formation, the roles of other membrane components, such as lipids, remain to be explored. In this study, we attempted to investigate the effects of cholesterol and phospholipids on the formation of cell-in-cell structures by utilizing liposome as a vector. We found that Lipofectamine-2000, the reagent commonly used for routine transfection, could significantly reduce entotic cell-in-cell formation in a cell-specific manner, which is correlated with suppressed actomyosin contraction as indicated by reduced ß-actin expression and myosin light chain phosphorylation. The influence on cell-in-cell formation was likely dictated by specific liposome components as some liposomes affected cell-in-cell formation while some others didn't. Screening on a limited number of lipids, the major components of liposome, identified phosphatidylethanolamine (PE), stearamide (SA), lysophosphatidic acid (LPA) and cholesterol (CHOL) as the inhibitors of cell-in-cell formation. Importantly, cholesterol treatment significantly inhibited myosin light chain phosphorylation, which resembles the effect of Lipofectamine-2000, suggesting cholesterol might be partially responsible for liposomes' effects on cell-in-cell formation. Together, our findings supporting a role of membrane lipids and cholesterol in cell-in-cell formation probably via regulating actomyosin contraction.

Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset.

Middle East respiratory syndrome coronavirus (MERS-CoV) infection in humans is highly lethal, with a fatality rate of 35%. New prophylactic and therapeutic strategies to combat human infections are urgently needed. We isolated a fully human neutralizing antibody, MCA1, from a human survivor. The antibody recognizes the receptor-binding domain of MERS-CoV S glycoprotein and interferes with the interaction between viral S and the human cellular receptor human dipeptidyl peptidase 4 (DPP4). To our knowledge, this study is the first to report a human neutralizing monoclonal antibody that completely inhibits MERS-CoV replication in common marmosets. Monotherapy with MCA1 represents a potential alternative treatment for human infections with MERS-CoV worthy of evaluation in clinical settings.

Hemagglutinin amino acids related to receptor specificity could affect the protection efficacy of H5N1 and H7N9 avian influenza virus vaccines in mice.

The continuous and sporadic human transmission of highly pathogenic avian H5N1 and H7N9 influenza viruses illustrates the urgent need for efficacious vaccines. However, all tested vaccines for the H5N1 and H7N9 viruses appear to be poorly immunogenic in mammals. In this study, a series of vaccines was produced using reverse genetic techniques that possess HA and NA genes from the H5N1 virus in the genetic background of the high-yield strain A/PR/8/34 (H1N1). Meanwhile, a group of H7N9 VLP vaccines that contain HA from H7N9 and NA and M1 from A/PR/8/34 (H1N1) was also produced. The HA amino acids of both the H5N1 and H7N9 vaccines differed at residues 226 and 228, both of which are critical for receptor specificity for an avian or mammalian host. Mice received two doses (3µg of HA each) of each vaccine and were challenged with lethal doses of wild type H5N1 or H7N9 viruses. The results showed that a recombinant H5N1 vaccine in which the HA amino acid G228 (avian specificity) was converted to S228 (mammalian specificity) resulted in higher HI titers, a lower viral titer in the lungs, and 100% protection in mice. However, a H7N9 VLP vaccine that contains L226 (mammalian specificity) and G228 (avian specificity) in HA showed better immunogenicity and protection efficacy in mice than VLP containing HA with either L226+S228 or Q226+S228. This observation indicated that specific HA residues could enhance a vaccine's protection efficacy and HA glycoproteins with both avian-type and human-type receptor specificities may produce better pandemic influenza vaccines for humans.

Isolation, Identification, and Sequencing of a Goose-Derived Newcastle Disease Virus and Determination of Its Pathogenicity.

In August 2010, geese in the Meihekou area of Jilin province in China were found to be infected by a pathogen that caused a disease similar to Newcastle disease. To determine the causative agent of the infections, a virus was isolated from liver tissues of infected geese, followed by a pathogenicity determination. The isolated virus was named NDV/White Goose/China/Jilin(Meihekou)/MHK-1/2010. Specific primers were designed to amplify the whole genome of the MHK-1 virus, followed by sequencing and splicing of the entire genome. Sequencing and phylogenetic analysis of MHK-1 showed that the isolate was a virulent strain of Newcastle disease virus. The MHK-1 genome is 15,192 nucleotides long, and it belongs to the class II branch of Newcastle disease viruses, as evidenced by the amino acid sequence (112R-R-Q-K-R-F117) of the F protein. The hemagglutinin titer was 1:128 to 1:512. The chicken embryo mean death time, the intracerebral pathogenicity index, and the median lethal dose of chicken embryos of MHK-1 were 43 hr, 1.63, and 10(9)/ml, respectively, which revealed that the newly isolated MHK-1 strain is strongly pathogenic to geese.

[Real-time PCR Detection Method for the Reston Subtype of the Ebola Virus].

We aimed to develop a real-time polymerase chain reaction (PCR) detection method for the Reston subtype of the Ebola virus. The NP gene of the Reston subtype of the Ebola virus was selected as the detection object. Sequences of different subtypes of Ebola viruses were aligned using Clustal W software. The most unique and conserved regions of the Reston subtype of the Ebola virus were recruited as candidate sequences for specific primers. Primer Express and Primer Premier 5. 0 software were used to filter the optimal pair of primers for detection. Real-time PCR was carried out using optimized parameters and positive DNA prepared by serial (tenfold) dilution of a recombinant plasmid and by plotting a standard curve. In addition, the reproducibility, accuracy, and specificity of the assay were tested. Results showed that the sensitivity of detection of the Reston subtype of the Ebola virus by real-time PCR could reached 10(2) copies/microL. The linear relationship (R2) reached 0.997, the slope of the standard curve was -0.3101, and amplification efficiency was 110.145%. A sharp and narrow melting peak appeared at 79.94 degrees C for all standards in different dilutions. In conclusion, a fast and sensitive real-time PCR detection system for the Reston subtype of the Ebola virus was developed. This system could be used as a supplementary diagnostic and monitoring approach for basic and clinical studies on the Reston subtype of the Ebola virus. The detection system does not require expensive technology or specialist operators.

Characterization of an H9N2 avian influenza virus from a Fringilla montifringilla brambling in northern China.

Avian H9N2 influenza viruses circulating in domestic poultry populations are occasionally transmitted to humans. We report the genomic characterization of an H9N2 avian influenza virus (A/Brambling/Beijing/16/2012) first isolated from a healthy Fringilla montifringilla brambling in northern China in 2012. Phylogenetic analyses revealed that this H9N2 virus belongs to the BJ/94-like sublineage. This virus had a low pathogenicity for chickens and was able to replicate at a low level in mouse lung tissue. Transmission studies in ferrets showed that this H9N2 strain shed high levels of the virus in nasal and throat swabs. In vitro receptor binding assays, the virus bound only to α-2,6 linkage receptors and not to the avian-type α-2,3 linkage receptors, suggesting that H9N2 influenza viruses present potential public health risks. Therefore, attention should be paid to H9N2 influenza viruses and the close surveillance of H9N2 viruses in poultry.

Combinations of oseltamivir and fibrates prolong the mean survival time of mice infected with the lethal H7N9 influenza virus.

The outbreak of human infections caused by the novel avian-origin H7N9 influenza viruses in China since March 2013 underscores the urgent need to find an effective treatment strategy against H7N9 infection in humans. In this study, we assessed the effectiveness of combinations of oseltamivir and two immunomodulators (simvastatin and fenofibrate) against H7N9 infection in a mouse model. Mice treated with oseltamivir plus fenofibrate exhibited the longest mean survival time, the largest reduction of viral titre in lung tissue, the highest levels of CD4(+) and CD8(+) T-lymphocytes, and the greatest decrease in pulmonary inflammation. Thus, the combination of oseltamivir plus fenofibrate improved the outcomes of mice infected with H7N9 virus by simultaneously reducing viral replication and normalizing the aberrant immune response. This drug combination should be considered in randomized controlled trials of treatments for H7N9 patients.

Rapid adaptation of avian H7N9 virus in pigs.

How the H7N9 avian influenza virus gained the distinct ability to infect humans is unclear. Pigs are an important host in influenza virus ecology because they are susceptible to infection with both avian and human influenza viruses and are often involved in interspecies transmission. Here, we passaged one avian isolate and one human isolate in pigs to examine the mammalian host adaptation of the H7N9 virus. The avian virus replicated to a high titer after one passage, whereas the human isolate replicated poorly after three passages in pig lungs. Sequence analysis found nine substitutions in the HA, NA, M and NS segments of the avian isolate, which enhanced the binding affinity for human-type receptors. These results indicate that avian H7N9 influenza viruses can be easily adapted to pigs and that pigs may act as an important intermediate host for the reassortment and transmission of such novel viruses.

An animal model of MERS produced by infection of rhesus macaques with MERS coronavirus.

In 2012, a novel coronavirus (CoV) associated with severe respiratory disease, Middle East respiratory syndrome (MERS-CoV; previously known as human coronavirus-Erasmus Medical Center or hCoV-EMC), emerged in the Arabian Peninsula. To date, 114 human cases of MERS-CoV have been reported, with 54 fatalities. Animal models for MERS-CoV infection of humans are needed to elucidate MERS pathogenesis and to develop vaccines and antivirals. In this study, we developed rhesus macaques as a model for MERS-CoV using intratracheal inoculation. The infected monkeys showed clinical signs of disease, virus replication, histological lesions, and neutralizing antibody production, indicating that this monkey model is suitable for studies of MERS-CoV infection.