César González-Bonilla

Infection and death from influenza A H1N1 virus in Mexico: a retrospective analysis

BACKGROUND In April, 2009, the first cases of influenza A H1N1 were registered in Mexico and associated with an unexpected number of deaths. We report the timing and spread of H1N1 in cases, and explore protective and risk factors for infection, severe disease, and death. METHODS We analysed information gathered by the influenza surveillance system from April 28 to July 31, 2009, for patients with influenza-like illness who attended clinics that were part of the Mexican Institute for Social Security network. We calculated odds ratios (ORs) to compare risks of testing positive for H1N1 in those with influenza-like illness at clinic visits, the risk of admission for laboratory-confirmed cases of H1N1, and of death for inpatients according to demographic characteristics, clinical symptoms, seasonal influenza vaccine status, and elapsed time from symptom onset to admission. FINDINGS By July 31, 63 479 cases of influenza-like illness were reported; 6945 (11%) cases of H1N1 were confirmed, 6407 (92%) were outpatients, 475 (7%) were admitted and survived, and 63 (<1%) died. Those aged 10-39 years were most affected (3922 [56%]). Mortality rates showed a J-shaped curve, with greatest risk in those aged 70 years and older (10.3%). Risk of infection was lowered in those who had been vaccinated for seasonal influenza (OR 0.65 [95% CI 0.55-0.77]). Delayed admission (1.19 [1.11-1.28] per day) and presence of chronic diseases (6.1 [2.37-15.99]) were associated with increased risk of dying. INTERPRETATION Risk communication and hospital preparedness are key factors to reduce mortality from H1N1 infection. Protective effects of seasonal influenza vaccination for the virus need to be investigated. FUNDING None.

Establishment and cryptic transmission of Zika virus in Brazil and the Americas

Transmission of Zika virus (ZIKV) in the Americas was first confirmed in May 2015 in northeast Brazil. Brazil has had the highest number of reported ZIKV cases worldwide (more than 200,000 by 24 December 2016) and the most cases associated with microcephaly and other birth defects (2,366 confirmed by 31 December 2016). Since the initial detection of ZIKV in Brazil, more than 45 countries in the Americas have reported local ZIKV transmission, with 24 of these reporting severe ZIKV-associated disease. However, the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of this information for interpreting observed trends in reported microcephaly. Here we address this issue by generating 54 complete or partial ZIKV genomes, mostly from Brazil, and reporting data generated by a mobile genomics laboratory that travelled across northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Analyses of viral genomes with ecological and epidemiological data yield an estimate that ZIKV was present in northeast Brazil by February 2014 and is likely to have disseminated from there, nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates for the international spread of ZIKV from Brazil indicate the duration of pre-detection cryptic transmission in recipient regions. The role of northeast Brazil in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the basic reproduction number of the virus.

Expression of the Plasmodium falciparum Immunodominant Epitope (NANP) 4 on the Surface of Salmonella enterica Using the Autotransporter MisL

ABSTRACT Gram-negative bacterial proteins which are exported from the cytosol to the external environment by the type V secretion system are also known as autotransporters. Once translocated to the periplasmic compartment by the sec-dependent general secretory pathway, their C-terminal domain forms a pore through which the N-terminal domain travels to the outer membrane without the need of other accessory proteins. MisL (protein of membrane insertion and secretion) is a protein of unknown function located in the pathogenicity island SPI-3 of Salmonella enterica and classified as an autotransporter due to its high homology to Escherichia coli AIDA-I. In the present work, the MisL C-terminal translocator domain was used to display the immunodominant B-cell epitope of the circumsporozoite protein (CSP) from Plasmodium falciparum on the surface of Salmonella enterica serovar Typhimurium (serovar Typhimurium SL3261) and serovar Typhi (serovar Typhi CVD 908). The MisL β domain was predicted by alignment with AIDA-I, amplified from serovar Typhimurium SL3261, cloned in a plasmid fused to four repeats of the tetrapeptide NANP behind the Escherichia coli heat-labile enterotoxin B subunit signal peptide to ensure periplasmic traffic, and expressed under the control of the anaerobically inducible nirB promoter. The fusion protein was translocated to the outer membrane of both bacterial strains, although the foreign epitope was displayed more efficiently in serovar Typhimurium SL3261, which elicited a better specific antibody response in BALB/c mice. More importantly, antibodies were able to recognize the native CSP in P. falciparum sporozoites. These results confirm that MisL is indeed an autotransporter and that it can be used to express foreign immunogenic epitopes on the surface of gram-negative bacteria.

Cross-talk between Rac1 and Cdc42 GTPases regulates formation of filopodia required for dengue virus type-2 entry into HMEC-1 cells.

Infection with dengue virus type-2 (DENV-2) begins with virus adherence to cell surface receptors. In endothelial cells (HMEC-1), a cell model for DENV-2 infection, alpha 5 beta 3 integrin has been identified as a putative receptor for the virus. Previous work had suggested that the actin cytoskeleton of HMEC-1 cells plays an important role in virus entry and infection. In the present work, fixed and living HMEC-1 cells expressing enhanced green fluorescent protein-actin were monitored for actin reorganization after virus inoculation, utilizing fluorescence and time lapse microscopy. Cell infection and production of infective viruses were quantified using an anti-E protein antibody and by measuring the p.f.u. ml(-1). Specific drugs that antagonize actin organization and regulate actin-signalling pathways were tested in viral adhesion and infection assays, as were the expression of dominant-negative Rac1 and Cdc42 proteins. Disorganization of actin precluded infection, while microtubule depolymerization had no effect. Activation of Rac1 and Cdc42 signalling, which occurs upon virus binding, induced reorganization of actin to form filopodia in the cellular periphery. Formation of filopodia was a requirement for virus entry and further cell infection. Expression of the dominant-negative proteins Rac1 and Cdc42 confirmed the role of these GTPases in the actin reorganization that is required to form filopodia. In addition, inhibition of the ATPase activity of myosin II greatly decreased infection, suggesting its participation in filopodial stability. We show here, for the first time, that internalization of DENV-2 into endothelial cells requires viral induction of dynamic filopodia regulated by Rac1 and Cdc42 cross-talk and myosin II motor activities.

In vitro and in vivo sensitization of SW620 metastatic colon cancer cells to CDDP-induced apoptosis by the nitric oxide donor DETANONOate: Involvement of AIF

Tumor cells develop mechanisms that dysregulate apoptotic pathways resulting in resistance to cytotoxic stimuli. Primary SW480 and metastatic SW620 colon cancer cells are resistant to CDDP-induced apoptosis. Apoptosis-inducing factor (AIF) was significantly downregulated in SW620 compared to SW480 cells; while apoptotic mediators such as Bax, Bcl-2, and Bcl(XL) were not altered in these cell lines. Examination of tumor tissues from patients with colon cancer demonstrated a significant downregulation of AIF in patients with advanced disease. The role of AIF expression in resistance was examined. Several lines of evidence suggest the involvement of AIF expression level in the sensitivity of SW620 to CDDP-induced apoptosis: (1) sensitization of SW620 by the NO donor DETANONOate to CDDP-induced apoptosis correlated with the induction of AIF as assessed by RT-PCR and Western blot analysis, (2) treatment of SW620 cells with siRNA AIF, but not with control siRNAs, inhibited DETANONOate-induced sensitization to CDDP apoptosis, (3) sensitization by DETANONOate observed in vitro was corroborated in vivo in nude mice bearing SW620 tumor xenografts and treated with the combination of DETANONOate and CDDP, and (4) tumor tissues derived from the SW620 xenografts revealed significant upregulation of AIF and increased apoptosis by DETANONOate and CDDP combination treatment. Altogether, these findings underscore the potential therapeutic application of NO donors and subtoxic chemotherapeutic drugs in the treatment of advanced colon cancer resistant to conventional chemotherapeutic agents.

Display and release of the Plasmodium falciparum circumsporozoite protein using the autotransporter MisL of Salmonella enterica

The Salmonella enterica MisL (protein of membrane insertion and secretion) is an autotransporter with high homology to AIDA-I (adhesin involved in diffuse adherence) of enteropathogenic Escherichia coli. Considering that it has been reported that the MisL beta translocator domain is able to display heterologous passenger peptides to the bacterial surface, we developed a system to display proteins and release them to the external environment by means of proteolytic cleavage. Plasmids were constructed encoding 8 or 53 repeats of the NANP (Asp-Ala-Asp-Pro) tetrapeptide, which is the main B cell epitope of the Plasmodium falciparum circumsporozoitic protein (CSP), fused to the the MisL beta-domain and including the recognition cleavage sequence from the E. coli OmpT surface protease. E. coli XL-10Gold and BL21(DE3) (OmpT positive and negative, respectively) and Salmonella enterica serovar Typhimurium SL3261 (Aro A(-)) were transformed with the plasmids and, both expression and localization of the fusion proteins were assessed by Western blot, indirect immunofluorescence, and flow cytometry, using a monoclonal antibody against (NANP)(3). Higher expression of the (NANP)(8) and (NANP)(53) fusion proteins was demonstrated on the bacterial surface of the OmpT negative E. coli strains and the (NANP)(53) in the culture supernatant of E. coli XL-10Gold indicating a protease mediated cleavage. The flow cytometry analysis suggested 71 and 98% cleavage efficiency for the (NANP)(8) and (NANP)(53), respectively, in E. coli XL-10Gold. Similar results were obtained in S. enterica serovar Typhimurium SL3261, suggesting the involvement of other proteases related to OmpT. These results demonstrate that MisL may be used for the autodisplay and release of passenger proteins in attenuated Salmonella or E. coli strains, which may have several applications in vaccine design.

2-Methoxyestradiol (2-ME) reduces the airway inflammation and remodeling in an experimental mouse model.

Patients with asthma experience airway structural changes, termed airway remodeling, in response to persistent inflammation. 2-Methoxyestradiol (2-ME) is an anti-angiogenic agent and downregulates hypoxia-inducible factor 1 (HIF-1) and inhibits HIF-1alpha-induced transcriptional activation of vascular endothelial growth factor (VEGF) expression. We hypothesized that 2-ME may interfere with the development of the clinical manifestations of asthma. We used a chronic murine model of allergic airway inflammation with subepithelial fibrosis in BALB/c mice. Mice were sensitized with ovalbumin (OVA) that was administered intraperitoneally at days 0-5 and challenged intratracheally (IT) with OVA on days 12-22. The mice received 2-ME IT at days 24, 26 and 28 and sacrificed at day 32. The sensitized/challenged mice developed an extensive cell inflammatory response of the airways. 2-ME administration significantly reduced the cellular infiltrate in the perivascular and peribronchial lung tissues, reduced goblet mucous production, reduced airway fibrosis and thickness of smooth muscle and blood vessels, and reduced eosinophil infiltration. Mice treated with 2-ME had a significant decrease of HIF-1 and VEGF expression in the perivascular, peribronchial, and interstitium of lung tissues. Collagen IV expression was also significantly reduced in 2-ME treated mice compared to untreated mice. The 2-ME treatment was associated with a significant decrease of OVA-specific IgE antibodies. These findings provide the first indication that IT administration of 2-ME is effective in preventing and reversing antigen-induced airway remodeling in the OVA allergen inflammatory murine model. The potential role of 2-ME in patients is discussed.

Epidemiological Characterization of a Fourth Wave of Pandemic A/H1N1 Influenza in Mexico, Winter 2011–2012: Age Shift and Severity

BACKGROUND AND AIMS A substantial recrudescent wave of pandemic influenza A/H1N1 affected the Mexican population from December 1, 2011-March 20, 2012 following a 2-year period of sporadic transmission. METHODS We analyzed demographic and geographic data on all hospitalizations with severe acute respiratory infection (SARI) and laboratory-confirmed A/H1N1 influenza, and inpatient deaths, from a large prospective surveillance system maintained by a Mexican social security medical system during April 1, 2009-March 20, 2012. We also estimated the reproduction number (R) based on the growth rate of the daily case incidence by date of symptoms onset. RESULTS A total of 7569 SARI hospitalizations and 443 in-patient deaths (5.9%) were reported between December 1, 2011, and March 20, 2012 (1115 A/H1N1-positive inpatients and 154 A/H1N1-positive deaths). The proportion of laboratory-confirmed A/H1N1 hospitalizations and deaths was higher among subjects ≥60 years of age (χ(2) test, p <0.0001) and lower among younger age groups (χ(2) test, p <0.04) for the 2011-2012 pandemic wave compared to the earlier waves in 2009. The reproduction number of the winter 2011-2012 wave in central Mexico was estimated at 1.2-1.3, similar to that reported for the fall 2009 wave, but lower than that of spring 2009. CONCLUSIONS We documented a substantial increase in the number of SARI hospitalizations during the period December 2011-March 2012 and an older age distribution of laboratory-confirmed A/H1N1 influenza hospitalizations and deaths relative to 2009 A/H1N1 pandemic patterns. The gradual change in the age distribution of A/H1N1 infections in the post-pandemic period is consistent with a build-up of immunity among younger populations.

New approaches to improve a peptide vaccine against porcine Taenia solium cysticercosis.

Cysticercosis caused by Taenia solium frequently affects human health and rustic porciculture. Cysticerci may localize in the central nervous system of humans causing neurocysticercosis, a major health problem in undeveloped countries. Prevalence and intensity of this disease in pigs and humans are related to social factors (poor personal hygiene, low sanitary conditions, rustic rearing of pigs, open fecalism) and possibly to biological factors such as immunity, genetic background, and gender. The indispensable role of pigs as an obligatory intermediate host in the life cycle offers the possibility of interfering with transmission through vaccination of pigs. An effective vaccine based on three synthetic peptides against pig cysticercosis has been successfully developed and proved effective in experimental and field conditions. The well-defined peptides that constitute the cysticercosis vaccine offer the possibility to explore alternative forms of antigen production and delivery systems that may improve the cost/benefit of this and other vaccines. Encouraging results were obtained in attempts to produce large amounts of these peptides and increased its immunogenicity by expression in recombinant filamentous phage (M13), in transgenic plants (carrots and papaya), and associated to bacterial immunogenic carrier proteins.

Epidemiological characteristics and underlying risk factors for mortality during the autumn 2009 pandemic wave in Mexico.

Background Elucidating the role of the underlying risk factors for severe outcomes of the 2009 A/H1N1 influenza pandemic could be crucial to define priority risk groups in resource-limited settings in future pandemics. Methods We use individual-level clinical data on a large series of ARI (acute respiratory infection) hospitalizations from a prospective surveillance system of the Mexican Social Security medical system to analyze clinical features at presentation, admission delays, selected comorbidities and receipt of seasonal vaccine on the risk of A/H1N1-related death. We considered ARI hospitalizations and inpatient-deaths, and recorded demographic, geographic, and medical information on individual patients during August-December, 2009. Results Seasonal influenza vaccination was associated with a reduced risk of death among A/H1N1 inpatients (OR = 0.43 (95% CI: 0.25, 0.74)) after adjustment for age, gender, geography, antiviral treatment, admission delays, comorbidities and medical conditions. However, this result should be interpreted with caution as it could have been affected by factors not directly measured in our study. Moreover, the effect of antiviral treatment against A/H1N1 inpatient death did not reach statistical significance (OR = 0.56 (95% CI: 0.29, 1.10)) probably because only 8.9% of A/H1N1 inpatients received antiviral treatment. Moreover, diabetes (OR = 1.6) and immune suppression (OR = 2.3) were statistically significant risk factors for death whereas asthmatic persons (OR = 0.3) or pregnant women (OR = 0.4) experienced a reduced fatality rate among A/H1N1 inpatients. We also observed an increased risk of death among A/H1N1 inpatients with admission delays >2 days after symptom onset (OR = 2.7). Similar associations were also observed for A/H1N1-negative inpatients. Conclusions Geographical variation in identified medical risk factors including prevalence of diabetes and immune suppression may in part explain between-country differences in pandemic mortality burden. Furthermore, access to care including hospitalization without delay and antiviral treatment and are also important factors, as well as vaccination coverage with the 2008–09 trivalent inactivated influenza vaccine.