Comparative genomic analysis of Pseudomonas aeruginosa strains susceptible and resistant to carbapenems and aztreonam isolated from patients with healthcare-associated infections in a Mexican hospital.

Pseudomonas aeruginosa (PA) is an important opportunistic pathogen that causes different infections on immunocompromised patients. Within PA accessory genome, differences in virulence, antibiotic resistance and biofilm formation have been described between strains, leading to the emergence of multidrug-resistant strains. The genome sequences of 17 strains isolated from patients with healthcare-associated infections in a Mexican hospital were genomically and phylogenetically analyzed and antibiotic resistance genes, virulence genes, and biofilm formation genes were detected. Fifteen of the 17 strains were resistant to at least two of the carbapenems meropenem, imipenem, and the monobactam aztreonam. The antibiotic resistance (mexA, mexB, and oprM) and the biofilm formation (pslA and pslD) genes were detected in all strains. Differences were found between strains in accessory genome size. The strains had different sequence types, and seven strains had sequence types associated with global high risk epidemic PA clones. All strains were represented in two groups among PA global strains. In the 17 strains, horizontally acquired resistance genes to aminoglycosides and beta-lactams were found, mainly, and between 230 and 240 genes that encode virulence factors. The strains under study were variable in terms of their accessory genome, antibiotic resistance, and virulence genes. With these characteristics, we provide information about the genomic diversity of clinically relevant PA strains.

Case Report of a Young Adult with Fatal COVID-19 and Abundant SARS-CoV-2 Nucleocapsid Protein and Lipofuscin Accumulation in Tissues.

This is a case report of a young adult who died of COVID-19 twelve days after admission, with coronavirus nucleocapsid protein and lipofuscin found in the heart and kidney tissues, providing further evidence of the role of SARS-CoV-2 in cellular senescence.

Current knowledge in the use of bacteriophages to combat infections caused by Pseudomonas aeruginosa in cystic fibrosis.

Pseudomonas aeruginosa (PA) is considered the first causal agent of morbidity and mortality in people with cystic fibrosis (CF) disease. Multi-resistant strains have emerged due to prolonged treatment with specific antibiotics, so new alternatives have been sought for their control. In this context, there is a renewed interest in therapies based on bacteriophages (phages) supported by several studies suggesting that therapy based on lytic phages and biofilm degraders may be promising for the treatment of lung infections in CF patients. However, there is little clinical data about phage studies in CF and the effectiveness and safety in patients with this disease has not been clear. Therefore, studies regarding on phage characterization, selection, and evaluation in vitro and in vivo models will provide reliable information for designing effective cocktails, either using mixed phages or in combination with antibiotics, making a great progress in clinical research. Hence, this review focuses on the most relevant and recent findings on the activity of lytic phages against PA strains isolated from CF patients and hospital environments, and discusses perspectives on the use of phage therapy on the treatment of PA in CF patients.

Correction to: A systematic review on the role of MSC­derived exosomal miRNAs in the treatment of heart failure.

BACKGROUND: Heart failure (HF) is a deeply serious clinical problem that remains unsolved. Many reports highlight the cardioprotective properties of mesenchymal stem cells (MSCs), the factors contained in their secretome being particularly important for this function; these include the exosomal miRNAs (exo-miRNAs) secreted with or without stimulus which can modulate various biological processes. In search of new paradigms in heart failure, we wondered whether MSC-derived exosomal microRNAs could play a role in the management of clinical patients. METHODS: To analyze whether there is sufficient evidence to support this hypothesis we designed a systematic review of studies on exo-miRNAs in heart diseases causing HF during the last decade. RESULTS: The cardioprotective functions of twenty-four exo-miRNAs derived from bone marrow MSCs (BM-MSCs) are known and the functions of exo-miRNAs from other MSC sources such as adipose tissue, trophoblasts, amniotic fluid, and endometrium were also determined. Inhibition of apoptosis is the most reported biological function and the targets for thirty exo-miRNAs are known. CONCLUSIONS: The results from this systematic review support the initial hypothesis and encourage us to test it in future experimental research works but more importantly, we seek to encourage other researchers in the field to propose other hypotheses aimed at the possible use of exo-miRNAs in HF secondary to cardiac disease.

A systematic review on the role of MSC-derived exosomal miRNAs in the treatment of heart failure.

BACKGROUND: Heart failure (HF) is a deeply serious clinical problem that remains unsolved. Many reports highlight the cardioprotective properties of mesenchymal stem cells (MSCs), the factors contained in their secretome being particularly important for this function; these include the exosomal miRNAs (exo-miRNAs) secreted with or without stimulus which can modulate various biological processes. In search of new paradigms in heart failure, we wondered whether MSC-derived exosomal microRNAs could play a role in the management of clinical patients. METHODS: To analyze whether there is sufficient evidence to support this hypothesis we designed a systematic review of studies on exo-miRNAs in heart diseases causing HF during the last decade. RESULTS: The cardioprotective functions of twenty-four exo-miRNAs derived from bone marrow MSCs (BM-MSCs) are known and the functions of exo-miRNAs from other MSC sources such as adipose tissue, trophoblasts, amniotic fluid, and endometrium were also determined. Inhibition of apoptosis is the most reported biological function and the targets for thirty exo-miRNAs are known. CONCLUSIONS: The results from this systematic review support the initial hypothesis and encourage us to test it in future experimental research works but more importantly, we seek to encourage other researchers in the field to propose other hypotheses aimed at the possible use of exo-miRNAs in HF secondary to cardiac disease.

Fetal and placental infection with SARS-CoV-2 in early pregnancy.

To date, mother-to-fetus transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the coronavirus disease 2019 (COVID-19) pandemic, remains controversial. Although placental COVID-19 infection has been documented in some cases during the second- and third-trimesters, no reports are available for the first trimester of pregnancy, and no SARS-CoV-2 protein has been found in fetal tissues. We studied the placenta and fetal organs from an early pregnancy miscarriage in a COVID-19 maternal infection by immunohistochemical, reverse transcription quantitative real-time polymerase chain reaction, immunofluorescence, and electron microscopy methods. SARS-CoV-2 nucleocapsid protein, viral RNA, and particles consistent with coronavirus were found in the placenta and fetal tissues, accompanied by RNA replication revealed by double-stranded RNA (dsRNA) positive immunostain. Prominent damage of the placenta and fetal organs were associated with a hyperinflammatory process identified by histological examination and immunohistochemistry. The findings provided in this study document that congenital SARS-CoV-2 infection is possible during the first trimester of pregnancy and that fetal organs, such as lung and kidney, are targets for coronavirus. The infection and multi-organic fetal inflammation produced by SARS-CoV-2 during early pregnancy should alert clinicians in the assessment and management of pregnant women for possible fetal consequences and adverse perinatal outcomes.

miR-927 has pro-viral effects during acute and persistent infection with dengue virus type 2 in C6/36 mosquito cells.

Dengue virus (DENV) is an important flavivirus that is transmitted to humans by Aedes mosquitoes, where it can establish a persistent infection underlying vertical and horizontal transmission. However, the exact mechanism of persistent DENV infection is not well understood. Recently miR-927 was found to be upregulated in C6/36-HT cells at 57 weeks of persistent infection (C6-L57), suggesting its participation during this type of infection. The aim of this study was to determine the role of miR-927 during infection with DENV type 2. The results indicate an overexpression of miR-927 in C6-L57 cells and acutely infected cells according to the time of infection and the m.o.i. used. The downregulation of miR-927 in C6-L57 cells results in a reduction of both viral titre and viral genome copy number. The overexpression of miR-927 in C6-L40 and C6/36 cells infected at an m.o.i. of 0.1 causes an increase in both viral titre and viral genome copy number, suggesting a pro-viral activity of miR-927. In silico prediction analysis reveals target mRNAs for miR-927 are implicated in post-translational modifications (SUMO), translation factors (eIF-2B), the innate immune system (NKIRAS), exocytosis (EXOC-2), endocytosis (APM1) and the cytoskeleton (FLN). The expression levels of FLN were the most affected by both miR-927 overexpression and inhibition, and FLN was determined to be a direct target of miR-927 by a dual-luciferase gene reporter assay. FLN has been associated with the regulation of the Toll pathway and either overexpression or downregulation of miR-927 resulted in expression changes of antimicrobial peptides (Cecropins A and G, and Defensin D) involved in the Toll pathway response.

Natural Vertical Transmission of Zika Virus in Larval Aedes aegypti Populations, Morelos, Mexico.

We characterized natural vertical transmission of Zika virus in pools of Aedes aegypti larvae hatched from eggs collected in Jojutla, Morelos, Mexico. Of the 151 pools analyzed, 17 tested positive for Zika virus RNA; infectious Zika virus was successfully isolated from 1 of the larvae pools (31N) in C6/36 cells. Real-time quantitative PCR and indirect immunofluorescence assays confirmed the identity of the isolate, named Zika virus isolate 31N; plaque assays in Vero cells demonstrated the isolate's infectivity in a mammalian cell line. We obtained the complete genome of Zika virus isolate 31N by next-generation sequencing and identified 3 single-nucleotide variants specific to Zika virus isolate 31N using the meta-CATS tool. These results demonstrate the occurrence of natural vertical transmission of Zika virus in wild Ae. aegypti mosquitoes and suggest that this transmission mode could aid in the spread and maintenance of Zika virus in nature.

Complete genome of DENV2 isolated from mosquitoes in Mexico.

Dengue virus is the most prevalent arbovirus in Mexico, and although the diversity of this virus has been studied, the vast majority of sequences have been derived from viruses isolated from the human host. In this work, we aimed to sequence and to analyze DENVs derived from wild mosquitoes captured in Acapulco Guerrero, Mexico. We succeeded in determining three full genome sequences of such viruses and were able to compare them with other reported sequences from human and mosquito-derived DENVs. We found 15 nonsynonymous and 88 synonymous substitutions that were present more frequently in mosquito viruses than what would be expected by chance, although the limited number of genomes reported so far puts a constraint on the conclusions that can be derived from these analyses. Also, given the high depth of coverage attained in one of the genomes a variant analysis was carried out, finding 68 polymorphic sites in this genome. Interestingly, six of them corresponded to SNV that were detected as potentially differential between mosquitoes and humans, indicating that a that at least some positions may be maintained as polymorphic, which may facilitate host transmission.

Congenital Zika Syndrome and Extra-Central Nervous System Detection of Zika Virus in a Pre-term Newborn in Mexico.

BACKGROUND: During pregnancy, the Zika virus (ZIKV) replicates in the placenta and central nervous system (CNS) of infected fetuses; nevertheless, the ability of ZIKV to replicate in other fetal tissues has not been extensively characterized. METHODS: We researched whether dissemination of congenitally-acquired ZIKV outside the CNS exists by searching for the accumulation of the viral envelope protein, ZIKV ribonucleic acid (RNA), and infectious viral particles in different organs of a deceased newborn with Congenital Zika Syndrome. A real-time qualitative polymerase chain reaction (qPCR) was used to detect ZIKV RNA in the brain, thymus, lungs, kidneys, adrenal glands, spleen, liver, and small intestine. The same tissues were analyzed by indirect immunofluorescence and immunoperoxidase assays using the monoclonal antibody 4G2 to detect ZIKV envelope antigens. Isolation of infectious ZIKV in a cell culture was carried out using brain and kidney samples. RESULTS: A postmortem, virological analysis of multiple organs, such as the kidneys (epithelial cells in the renal tubules), lungs (bronchial epithelia), thymus (epithelial cells inside the Hassall's corpuscles), and brain (neurons, ependymal cells, and macrophages) revealed the presence of ZIKV RNA and envelope antigens. Other tissues of the deceased newborn tested positive by qPCR for Epstein-Barr virus and human herpesvirus 6, including the brain cortex (Epstein-Barr) and the thymus, kidneys, and adrenal glands (human herpesvirus 6). The kidneys were identified as a significant niche for viral replication, given that infectious particles were successfully isolated from renal tissues. CONCLUSIONS: Our findings demonstrate the ability of congenitally-acquired ZIKV to produce disseminated infections and the viral tropism towards epithelial cells.

Dengue virus induced changes in Ca2+ homeostasis in human hepatic cells that favor the viral replicative cycle.

The role of Ca2+ during dengue virus (DENV) replication is unknown; thus, changes in Ca2+ homeostasis in DENV infected human hepatic HepG2 and Huh-7 cells were analyzed. Infected HepG2 cells, but not Huh-7 cells, showed a significant increase in plasma membrane permeability to Ca2+, while both cell lines showed marked reduced levels of Ca2+ stored in the endoplasmic reticulum. While the expression levels of STIM1 and ORAI1 showed no changes, STIM1 and ORAI1 were shown to co-localized in infected cells, indicating activation of the store-operated Ca2+ entry (SOCE) pathway. Finally, manipulation in the infected cells of the intra and extracellular Ca2+ levels by chelators (BAPTA-AM and EGTA), SOC inhibitor (SKF96365), IP3 Receptor antagonist (2APB) or increase of extracellular [Ca2+], significantly reduced DENV yield, but not vesicular stomatitis virus yield, used as a control. These results show that DENV infection alters cell Ca2+ homeostasis and that such changes favor viral replication.

Analysis of the miRNA profile in C6/36 cells persistently infected with dengue virus type 2.

Dengue virus (DENV) is the most important arbovirus in the world; DENV is transmitted by the Aedes genus of mosquitoes and can establish a life-long persistent infection in mosquitoes. However, the exact mechanism by which persistent infection is established remains unknown. In this study the differential expression of miRNAs was analysed by deep sequencing and RT-qPCR using a previously established C6/36-HT cell line persistently infected with DENV 2 (C6-L) as a model. miR-927, miR-87, miR-210, miR-2a-3p, miR-190 and miR-970 were up-regulated, whereas miR-252, miR-263a-3p, miR-92b, miR-10-5p miR-9a-5p, miR-9a-1, miR-124, miR-286a and miR-286b were down-regulated in C6-L cells compared with C6/36 cells acutely infected with the same virus or mock-infected cells. Deep sequencing results were validated by RT-qPCR for the highly differentially expressed miR-927 and miR-9a-5p, which were up- and down-regulated, respectively, compared with both acutely and mock-infected C6/36 cells. The putative targets of these miRNAs include components of the ubiquitin conjugation pathway, vesicle-mediated transport, autophagy, and the JAK-STAT cascade as well as proteins with endopeptidase activity. Other putative targets include members of the Toll signalling pathway and proteins with kinase, ATPase, protease, scavenger receptor or Lectin C-type activity or that participate in fatty acid biosynthesis or oxidative stress. Our results suggest that several specific miRNAs help regulate the cellular functions that maintain equilibrium between viral replication and the antiviral response during persistent infection of mosquito cells. This study is the first report of a global miRNA profile in a mosquito cell line persistently infected with DENV.

Complete Genome Sequences, before and after Mammalian Cell Culture, of Zika Virus Isolated from the Serum of a Symptomatic Male Patient from Oaxaca, Mexico.

Zika virus (ZIKV) is an emerging arthropod-borne flavivirus associated with severe congenital malformations and neurological complications. Although the ZIKV genome is well characterized, there is limited information regarding changes after cell isolation and culture adaptation. We isolated, and passaged in Vero cells, ZIKV from the serum of a symptomatic male patient and compared the viral genomes before and after culture. Single nucleotide polymorphisms were characteristic among serum-circulating genomes, while such diversity decreased after cell culture.

Human Virome.

The human virome is composed by the set of all viruses, eukaryotic and prokaryotic, present in the human body; as each body compartment constitutes a different microenvironment, the virome varies with the body part. Additionally, other factors influence the virome composition, such as age, diet, and the presence of other components of the microbiome. The study of the virome takes advantage of the development of next generation sequencing, and has allowed the discovery of novel viruses, and the characterization of the virome in healthy and diseased individuals, allowing the association of viruses with specific diseases. Perhaps the most interesting development of the study of the virome is the interplay that viruses can have with other components of the microbiome, specifically bacteria, that can either up- or down-regulate the antiviral immune response and can therefore modulate viral infectivity. This relationship is reciprocal since viruses can in turn modulate bacterial infections. The complex interactions of the virome with other members of the microbiome in the context of host genetics, and their influence in the health status of the patient have just begun to be investigated and are not completely understood, but the findings so far indicate that the regulation of the immune response by viruses and other members of the microbiome can affect the outcome of infections.

Low Proteolytic Clipping of Histone H3 in Cervical Cancer.

Chromatin in cervical cancer (CC) undergoes chemical and structural changes that alter the expression pattern of genes. Recently, a potential mechanism, which regulates gene expression at transcriptional levels is the proteolytic clipping of histone H3. However, until now this process in CC has not been reported. Using HeLa cells as a model of CC and human samples from patients with CC, we identify that the H3 cleavage was lower in CC compared with control tissue. Additionally, the histone H3 clipping was performed by serine and aspartyl proteases in HeLa cells. These results suggest that histone H3 clipping operates as part of post-translational modification system in CC.

Negative effect of heat shock on feline calicivirus release from infected cells is associated with the control of apoptosis.

FCV infection causes rapid cytopathic effects, and its replication results in the induction of apoptosis changes in cultured cells. It is well established that the survival of apoptotic cells can be enhanced by the expression of heat-shock proteins (Hsp) to prevent damage or facilitate recovery. Hsps can act as molecular chaperones, but they can also have anti-apoptotic roles by binding to apoptotic proteins and inhibiting the activation of caspases, the primary mediators of apoptosis. Because apoptosis occurs during FCV infection and heat shock (HS) treatment has a cytoprotective role due to the expression of Hsps, we studied the effect of the HS response to hyperthermia during FCV infection in cultured cells. We found that FCV infection does not inhibit the expression of Hsp70 induced by HS and that non-structural and structural protein synthesis was not modified during HS treatment. However, HS caused a delay in the appearance of a cytopathic effect in infected cells, as well as a reduction in the extracellular but not in the cell-associated viral yield. This antiviral effect of HS correlates with the inhibition of caspase-3 activation. Thus, the HS-induced reduction in virus production appeared to be associated with the control of apoptosis, supporting previous data that indicate that apoptosis is necessary for FCV release.

Let-7c overexpression inhibits dengue virus replication in human hepatoma Huh-7 cells.

MicroRNAs (miRNAs) constitute an important class of non-coding RNA implicated in gene expression regulation. More than 1900 miRNA molecules have been identified in humans and their modulation during viral infection and it is recognized to play a role in latency regulation or in establishing an antiviral state. The liver cells are targets during DENV infection, and alteration of liver functions contributes to severe disease. In this work the miRNAs expression profile of the human hepatoma cell line, Huh-7, infected with DENV-2 was determined using microarray and real-time PCR. Let-7c is one of the miRNAs up-regulated during DENV infection in the hepatic Huh-7 as well as in the macrophage-monocytic cell line U937-DC-SIGN. Let-7c overexpression down-regulates both DENV-2 and DENV-4 infection. Additionally, we found that the transcription factor BACH1, a let-7c target, is also down-regulated during DENV infection. In accordance with this finding, HO-1, the main responsive factor of BACH1 was found up-regulated. The up-regulation of HO-1 may contribute to the stress oxidative response in infected cells.

Nucleolin interacts with the feline calicivirus 3' untranslated region and the protease-polymerase NS6 and NS7 proteins, playing a role in virus replication.

Cellular proteins play many important roles during the life cycle of all viruses. Specifically, host cell nucleic acid-binding proteins interact with viral components of positive-stranded RNA viruses and regulate viral translation, as well as RNA replication. Here, we report that nucleolin, a ubiquitous multifunctional nucleolar shuttling phosphoprotein, interacts with the Norwalk virus and feline calicivirus (FCV) genomic 3' untranslated regions (UTRs). Nucleolin can also form a complex in vitro with recombinant Norwalk virus NS6 and -7 (NS6/7) and can be copurified with the analogous protein from feline calicivirus (p76 or NS6/7) from infected feline kidney cells. Nucleolin RNA levels or protein were not modified during FCV infection; however, as a consequence of the infection, nucleolin was seen to relocalize from the nucleoli to the nucleoplasm, as well as to the perinuclear area where it colocalizes with the feline calicivirus NS6/7 protein. In addition, antibodies to nucleolin were able to precipitate viral RNA from feline calicivirus-infected cells, indicating a direct or indirect association of nucleolin with the viral RNA during virus replication. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a reduction of the cytopathic effect and virus yield in CrFK cells. Taken together, these results demonstrate that nucleolin is a nucleolar component that interacts with viral RNA and NS6/7 and is required for feline calicivirus replication.

Mosquito La protein binds to the 3' untranslated region of the positive and negative polarity dengue virus RNAs and relocates to the cytoplasm of infected cells.

The untranslated regions (UTRs) of the positive and negative strand RNAs of several viruses are major binding sites for cellular and viral proteins. Human La autoantigen is one of the cellular proteins that interacts with various positive strand RNA viral genomes including that of dengue virus (DEN) within the 5'- and 3'-UTRs of positive (+) and the 3'-UTR of negative strand (-) RNA, and with the nonstructural proteins NS3 and NS5, that form DEN replicase complex. Since DEN replicates in human and mosquito cells, some functional interactions have to be conserved in both hosts. In the present report, we demonstrate that mosquito La protein interacts with the 3'-UTRs of (+) and (-) polarity viral RNAs. The localization of La protein, examined by confocal microscopy, indicates that La protein is redistributed in DEN-infected cells. Furthermore, the presence of La protein in an in vitro replication system inhibited RNA synthesis in a dose-dependent manner, suggesting that La protein plays an important role in dengue virus replicative cycle.