Selective pressure acting on influenza virus neuraminidase protein and relation with development of resistance to antiviral drugs

Neuraminidase (NA) protein of influenza viruses has the particularity of being under antibody and antiviral drug selective pressure, as it is one of the main surface antigens and the target of neuraminidase inhibitors(NAIs). The aim of this study is to investigate the selective pressure(SP) acting on the NA of seasonal and pandemic influenza viruses. It comprises two objectives: (a)to evaluate the contribution of positive SP for the emergence of NAIs resistant viruses; and (b)to determine the impact of NAIs introduction into clinic and its wide use during pandemic on the SP acting on NA. For the 1st objective it will be analysed the SP acting on the sites associated with NAIs resistance or reduction in susceptibility. The 2nd objective implies a differential evolutionary pressure analysis according to time, with 3 sub-datasets of NA sequences being considered: (1)before worldwide introduction of NAIs into clinic(1999); (2)before wide use of oseltamivir during A(H1N1)2009 pandemic(2009); and (3)from 2009 to date. A large dataset of full-length NA coding sequences will be used for each (sub)type/variant, comprising sequences obtained at national level(since 2000/2001) and sequences available at GISAID and NCBI. A(H1N1)seasonal dataset was already created, including a total of 1523 sequences, from which 94 belong to 1st sub-dataset, 1094 to 2nd and 335 to 3rd. All SP analysis will be performed using the expertise acquired with this workshop. This study may contribute for understanding the role of antiviral drug selective pressure in NAIs resistance, patterns of emergency of resistant viruses and NA evoluti

Evaluation and Characterization of Influenza Antiviral Drug Resistance in Portugal: Major Results and Achievements of a 5-Year Study

In 2007 started to be carried out for the first time in Portugal a study focused on influenza antiviral drug resistance. Three main objectives were established:(1)to determine the antiviral profile of influenza viruses to oseltamivir, zanamivir and amantadine;(2)to determine and monitor the baseline level of susceptibility along winter seasons and for each influenza sub(type);(3)to analyse and characterize the whole genome of viruses that showed phenotypic levels of inhibition to neuraminidase inhibitors(NAIs). NAIs profile was determined phenotypically, using a fluorescence MUNUNA assay, and genotypically by NA and HA sequencing. A total of 340 seasonal viruses(117 A(H3N2),93 A(H1N1),130 B) were tested for oseltamivir and of 297(112 A(H3N2),68 A(H1N1),117 B) for zanamivir. Additionally, 142 A(H1N1)pdm09 viruses were evaluated for both NAIs. Whole genome sequencing was performed in 27 of the A(H1N1)pdm09 viruses. Amantadine profile was determined through M2 pyrosequencing or conventional sequencing in a total of 205 seasonal A viruses(138 A(H3N2),84 A(H1N1)) and of 117 A(H1N1)pdm09 viruses. Main results are: -Resistance to oseltamivir in 27 A(H1N1) seasonal viruses(29%,N=93) from 2007/2008 and 2008/2009 and in one A(H1N1)pdm09 virus(0.7%,N=142) from 2010/2011. These viruses exhibited a highly reduced level of inhibition to oseltamivir by phenotypic analysis (170-650 IC50 fold-change) and NA H275Y mutation; -One suspected case of clinical resistance to oseltamivir with a mixed population of H275Y viruses(73,8%H275,26.2%Y275); -No resistance to zanamivir; -Dual reduced susceptibility to oseltamivir and zanamivir in one B virus(0,85%,N=117) and in two A(H1N1)pdm09 viruses(1,41%,N=142). These viruses exhibited a 2-4 IC50 fold-change level of inhibition to both NAIs. A mixed population of D197N viruses was found in the B virus(56%D197,44%N197) and the two A(H1N1)pdm09 viruses shared NA I223V and PB2 V480I mutations; -Resistance to amantadine in 49 A(H3N2) viruses(35,5%,N=138) from 2005/2006 to 2008/2009(46 S31N,3 S31N+V27A), and in all A(H1N1)pdm09 viruses(S31N). This 5-year study allowed to establish a technical platform for influenza antiviral drug resistance evaluation, to timely detect the emergence of resistant viruses, to acquire know-how on the natural variation of virus susceptibility, and to contribute for the management of cases suspected of clinical resistance. Additionally, it allowed the gathering of a large amount of data that will be used in more advanced studies, focused on evolutionary analysis and on detailed characterization of specific mutations

Influenza A(H1N1)pdm09 Resistance and Cross-Decreased Susceptibility to Oseltamivir and Zanamivir Antiviral Drugs

Neuraminidase inhibitors (NAIs) oseltamivir and zanamivir are currently the only effective antiviral drugs available worldwide for the management of influenza. The potential development of resistance is continually threatening their use, rationalizing and highlighting the need for a close and sustained evaluation of virus susceptibility. This study aimed to analyze and characterize the phenotypic and genotypic NAIs susceptibility profiles of A(H1N1)pdm09 viruses circulating in Portugal from 2009 to 2010/2011. A total of 144 cases of A(H1N1)pdm09 virus infection from community and hospitalized patients were studied, including three suspected cases of clinical resistance to oseltamivir. Oseltamivir resistance was confirmed for two of the suspected cases. Neuraminidase (NA) H275Y resistant marker was found in viruses from both cases but for one it was only present in 26.2% of virus population, raising questions about the minimal percentage of resistant virus that should be considered relevant. Cross-decreased susceptibility to oseltamivir and zanamivir (2-4 IC50 fold-change) was detected on viruses from two potentially linked community patients from 2009. Both viruses harbored the NA I223V mutation. NA Y155H mutation was found in 18 statistical non-outlier viruses from 2009, having no impact on virus susceptibility. The mutations at NA N369K and V241I may have contributed to the significantly higher baseline IC50 value obtained to oseltamivir for 2010/2011 viruses, compared to viruses from the pandemic period. These results may contribute to a better understanding of the relationship between phenotype and genotype, which is currently challenging, and to the global assessment of A(H1N1)pdm09 virus susceptibility profile and baseline level to NAIs

Expansion of activated cxcr5+icos+ tfh cells and plasmablasts induced by seasonal influenza vaccine is impaired in anti-il-6r treated rheumatoid arthritis patients

Objectives: To investigate the importance of IL-6 for the in vivo differentiation of human Tfh cells, taking advantage of influenza vaccination in patients under anti-IL-6R therapy.info:eu-repo/semantics/publishedVersio

Global update on the susceptibility of humam influenza viruses to neuraminidase inhibitors 2012-2013

Emergence of influenza viruses with reduced susceptibility to neuraminidase inhibitors (NAIs) is sporadic, often follows exposure to NAIs, but occasionally occurs in the absence of NAI pressure. The emergence and global spread in 2007/2008 of A(H1N1) influenza viruses showing clinical resistance to oseltamivir due to neuraminidase (NA) H275Y substitution, in the absence of drug pressure, warrants continued vigilance and monitoring for similar viruses. Four World Health Organization (WHO) Collaborating Centres for Reference and Research on Influenza and one WHO Collaborating Centre for the Surveillance, Epidemiology and Control of Influenza (WHO CCs) tested 11,387 viruses collected by WHO-recognized National Influenza Centres (NIC) between May 2012 and May 2013 to determine 50% inhibitory concentration (IC50) data for oseltamivir, zanamivir, peramivir and laninamivir. The data were evaluated using normalized IC50 fold-changes rather than raw IC50 data. Nearly 90% of the 11,387 viruses were from three WHO regions: Western Pacific, the Americas and Europe. Only 0.2% (n=27) showed highly reduced inhibition (HRI) against at least one of the four NAIs, usually oseltamivir, while 0.3% (n=39) showed reduced inhibition (RI). NA sequence data, available from the WHO CCs and from sequence databases (n=3661), were screened for amino acid substitutions associated with reduced NAI susceptibility. Those showing HRI were A(H1N1)pdm09 with NA H275Y (n=18), A(H3N2) with NA E119V (n=3) or NA R292K (n=1) and B/Victoria-lineage with NA H273Y (n=2); amino acid position numbering is A subtype and B type specific. Overall, approximately 99% of circulating viruses tested during the 2012-2013 period were sensitive to all four NAIs. Consequently, these drugs remain an appropriate choice for the treatment and prophylaxis of influenza virus infections

Using Recombinant Proteins from Lutzomyia longipalpis Saliva to Estimate Human Vector Exposure in Visceral Leishmaniasis Endemic Areas

During the blood meal, female sand flies (insects that transmit the parasite Leishmania) inject saliva containing a large variety of molecules with different pharmacological activities that facilitate the acquisition of blood. These molecules can induce the production of anti-saliva antibodies, which can then be used as markers for insect (vector) biting or exposure. Epidemiological studies using sand fly salivary gland sonicate as antigens are hampered by the difficulty of obtaining large amounts of salivary glands. In the present study, we have investigated the use of two salivary recombinant proteins from the sand fly Lutzomyia longipalpis, considered the main vector of visceral leishmaniasis, as an alternative method for screening of exposure to the sand fly. We primarily tested the suitability of using the recombinant proteins to estimate positive anti-saliva ELISA test in small sets of serum samples. Further, we validated the assay in a large sample of 1,077 individuals from an epidemiological survey in a second area endemic for visceral leishmaniasis. Our findings indicate that these proteins represent a promising epidemiological tool that can aid in implementing control measures against leishmaniasis

Epidemiology of severe pediatric adenovirus lower respiratory tract infections in Manitoba, Canada, 1991-2005

<p>Abstract</p> <p>Background</p> <p>Most pediatric adenovirus respiratory infections are mild and indistinguishable from other viral causes. However, in a few children, the disease can be severe and result in substantial morbidity. We describe the epidemiologic, clinical, radiologic features and outcome of adenovirus lower respiratory tract infections (LRTI) in Aboriginal and Non-Aboriginal children in Manitoba, Canada during the years 1991 and 2005.</p> <p>Methods</p> <p>This was a retrospective study of 193 children who presented to the department of pediatrics at Winnipeg Children's Hospital, Manitoba, Canada with LRTI and had a positive respiratory culture for adenovirus. Patients' demographics, clinical and radiologic features and outcomes were collected. Adenovirus serotype distributions and temporal associations were described. Approximate incidence comparisons (detection rates) of adenovirus LRTI among Aboriginal and Non-Aboriginal children were estimated with 95% confidence intervals.</p> <p>Results</p> <p>Adenovirus infections occurred throughout the year with clusters in the fall and winter. Serotypes 1 to 3 were the predominant isolates (two thirds of the cases). The infection was more frequent among Canadian Aboriginals, as illustrated in 2004, where its incidence in children 0-4 years old was 5.6 fold higher in Aboriginals (13.51 vs. 2.39 per 10,000, <it>p </it>< 0.000). There were no significant differences in length of hospitalization and use of ventilator assistance between the two groups (<it>p </it>> 0.185 and <it>p </it>> 0.624, respectively) nor across serotypes (<it>p </it>> 0.10 and <it>p </it>> 0.05, respectively). The disease primarily affected infants (median age, 9.5 months). Most children presented with bronchiolitis or pneumonia, with multi-lobar consolidations on the chest x-ray. Chronic (residual) changes were documented in 16 patients, with eight patients showing bronchiectasis on the chest computerized tomography scan.</p> <p>Conclusions</p> <p>Adenovirus infection is associated with significant respiratory morbidities, especially in young infants. The infection appears to be more frequent in Aboriginal children. These results justify a careful follow-up for children with adenovirus LRTI.</p

Discovery of Markers of Exposure Specific to Bites of Lutzomyia longipalpis, the Vector of Leishmania infantum chagasi in Latin America

Leishmania parasites are transmitted by the bite of an infected vector sand fly that injects salivary molecules into the host skin during feeding. Certain salivary molecules can produce antibodies and can be used as an indicator of exposure to a vector sand fly and potentially the disease it transmits. Here we identified potential markers of specific exposure to the sand fly Lutzomyia longipalpis, the vector of visceral leishmaniasis in Latin America. Initially, we determined which of the salivary proteins produce antibodies in humans, dogs, and foxes from areas endemic for the disease. To identify potential specific markers of vector exposure, we produced nine different recombinant salivary proteins from Lu. longipalpis and tested for their recognition by individuals exposed to another human-biting sand fly, Lu. intermedia, that transmits cutaneous leishmaniasis and commonly occurs in the same endemic areas as Lu. longipalpis. Two of the nine salivary proteins were recognized only by humans exposed to Lu. longipalpis, suggesting they are immunogenic proteins and may be useful in epidemiological studies. The identification of specific salivary proteins as potential markers of exposure to vector sand flies will increase our understanding of vector–human interaction, bring new insights to vector control, and in some instances act as an indicator for risk of acquiring disease

Structure-Based High-Throughput Epitope Analysis of Hexon Proteins in B and C Species Human Adenoviruses (HAdVs)

Human adenoviruses (HAdVs) are the etiologic agent of many human infectious diseases. The existence of at least 54 different serotypes of HAdVs has resulted in difficulties in clinical diagnosis. Acute respiratory tract disease (ARD) caused by some serotypes from B and C species is particularly serious. Hexon, the main coat protein of HAdV, contains the major serotype-specific B cell epitopes; however, few studies have addressed epitope mapping in most HAdV serotypes. In this study, we utilized a novel and rapid method for the modeling of homologous proteins based on the phylogenetic tree of protein families and built three-dimensional (3D) models of hexon proteins in B and C species HAdVs. Based on refined hexon structures, we used reverse evolutionary trace (RET) bioinformatics analysis combined with a specially designed hexon epitope screening algorithm to achieve high-throughput epitope mapping of all 13 hexon proteins in B and C species HAdVs. This study has demonstrated that all of the epitopes from the 13 hexon proteins are located in the proteins' tower regions; however, the exact number, location, and size of the epitopes differ among the HAdV serotypes