Emilio E. Espínola

Sequence and phylogenetic analysis of the VP4 gene of human rotaviruses isolated in Paraguay

Nucleotide and amino acid analyzes of the VP4 gene of human rotaviruses isolated both in Paraguay and worldwide were carried out in order to increase our knowledge about the complex pattern of evolution of this virus in nature. Paraguayan strains bearing the P[8] genotype were grouped in the lineages P[8]-1, P[8]-2, and P[8]-3. Regardless of the year of detection, all of the G4 and G9 strains were related to lineage P[8]-3, whereas the G1 strains were related to the three lineages detected in Paraguay; this fact reinforces the notion of the existence of constraints within specific populations of rotavirus strains except for the G1 strains. In addition, we propose a phylogenetic classification for the P[4] strains in five different lineages (i.e. P[4]-1 to P[4]-5). The findings presented in this paper reinforce the importance of a continuous surveillance of rotavirus strains in order to predict the possible variants that will circulate in a country, and ultimately improve current vaccination programs.

Viral load of human bocavirus-1 in stools from children with viral diarrhoea in Paraguay

Since their discovery, four species of human bocavirus (HBoV) have been described in patients with respiratory and gastrointestinal diseases. However, a clear causal association between HBoV-1 and gastroenteritis has not been demonstrated. In this study, we describe the detection and quantification of HBoV-1 in stools from children with acute non-bacterial gastroenteritis using quantitative polymerase chain reaction. HBoV-1 genome was detected in 10.6% of stools with frequent association with rotavirus and norovirus. The median of HBoV-1 viral load was 1.88 × 104 genome/ml, lower than previously shown in secretions of patients with respiratory infections, without any obvious association between high viral load and presence of HBoV as single agent. Thus, although HBoV-1 was frequently detected in these patients, there is no clear causal association of this agent with diarrhoea. Indeed, HBoV-1 DNA in stools of patients with gastroenteritis without respiratory symptoms may be a remnant of previous infections or associated with prolonged shedding of virus in the respiratory or digestive tracts.

Microorganisms Associated With Pneumonia in Children <5 Years of Age in Developing and Emerging Countries: The GABRIEL Pneumonia Multicenter, Prospective, Case-Control Study

Summary In a multicenter, prospective case-control study involving 1758 children aged <5 years in developing and emerging countries, the main microorganisms associated with pneumonia were Streptococcus pneumoniae, human metapneumovirus, rhinovirus, and respiratory syncytial virus.

Viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances IP-10: a translational study

Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.

Genome Stability of Pandemic Influenza A (H1N1) 2009 Based on Analysis of Hemagglutinin and Neuraminidase Genes

Influenza A virus (H1N1), which arose in 2009, constituted the fourth pandemic after the cases of 1918, 1957, and 1968. This new variant was formed by a triple reassortment, with genomic segments from swine, avian, and human influenza origins. The objective of this study was to analyze sequences of hemagglutinin (n=2038) and neuraminidase (n=1273) genes, in order to assess the extent of diversity among circulating 2009-2010 strains, estimate if these genes evolved through positive, negative, or neutral selection models of evolution during the pandemic phase, and analyze the worldwide percentage of detection of important amino acid mutations that could enhance the viral performance, such as transmissibility or resistance to drugs. A continuous surveillance by public health authorities will be critical to monitor the appearance of new influenza variants, especially in animal reservoirs such as swine and birds, in order to prevent the potential animal-human transmission of viruses with pandemic potential.

Phylogeny-based classification of human rhinoviruses detected in hospitalized children with acute lower respiratory infection in Paraguay, 2010–2011

Human rhinovirus (HRV), a single‐stranded, positive‐sense RNA virus, is associated with mild upper respiratory tract infections in children. The aim of this study was to carry out a molecular characterization and phylogeny‐based classification of the circulating genotypes of HRV in hospitalized children with clinical manifestations of acute lower respiratory infection in Paraguay. Nasopharyngeal aspirates were collected from 101 children under 5 years of age, hospitalized with symptoms of acute lower respiratory infection, between May 2010 and December 2011, at the largest public pediatric hospital in the Central Department of Paraguay. Detection was performed by a real‐time polymerase chain reaction, followed by conventional amplification of the VP4/VP2 genomic region, sequencing, and phylogenetic analysis. Rhinovirus was detected in 33.7% of the samples. Amplification of 18 samples showed the presence of all three species (HRV‐A, ‐B, and ‐C). Different genotypes were found for each species: 11 for HRV‐A (‐9, ‐12, ‐22, ‐30, ‐36, ‐43, ‐59, ‐61, ‐68, ‐88, and ‐89), one for HRV‐B (‐4), and four for HRV‐C (‐C2, ‐C3, ‐C6, and ‐C9). In South America, information about HRV diversity is scarce. This is the first report on HRV genotype diversity in South America. J. Med. Virol. 85:1645–1651, 2013.

Computational analysis of a species D human adenovirus provides evidence of a novel virus

A human adenovirus (HAdV) species D, was isolated from a hospitalised child with severe lower respiratory infection. It was initially detected in the nasopharyngeal aspirate of the child followed by conventional PCR amplification of the hexon, penton base, and fibre genes. Sanger DNA sequencing and phylogenetic analyses showed characteristics of a recombinant genome not described before. Next Generation Sequencing analysis was performed to reconstruct its complete DNA genome after viral isolation in adenocarcinoma human cell line (A549). A complete genomic sequence of 35.2 kb in length, with a G+C content of 57 % was obtained, related to HAdV-D29 (96 % identity). Imputed serology analysis demonstrated its novel type with a nucleotide sequence identity of 95.3 % (hexon loop 1) and 96 % (hexon loop 2) to HAdV-D9. The penton base gene showed a novel sequence, distantly related to HAdV-D44. The E3 and E4 regions evolved significantly from their ancestors. The fibre gene was almost identical to the knob region of HAdV-D15 but showed an unrelated shaft sequence. In conclusion the genomics of this novel HAdV, designated the HAdV-D83 [P83H9F15] prototype and bearing a new penton base gene, supports the importance of viral evolution to understand modified tissue tropism, enhanced transmission, or altered virulence.

Influenza A H1N1pdm 2009 Virus in Paraguay: Nucleotide Point Mutations in Hemagglutinin and Neuraminidase Genes are not Associated with Drug Resistance.

Influenza virus is associated with upper respiratory tract infections. The fourth influenza pandemic was declared in 2009. The aim of this study was to determine the genetic variability of the 2009 H1N1 pandemic virus circulating in Paraguay. Nasal swabs were collected from 181 patients with flu symptoms managed at the Hospital of the Medical School in Asunción, Paraguay, between August and October 2009. Virus detection was carried out by real-time reverse transcription-polymerase chain reaction, followed by sequencing of the hemagglutinin and neuraminidase genes, and phylogenetic analysis. H1N1pdm09 was detected in 14.9% (27/181) of the suspected cases. Analysis of 13 samples showed that these viruses the clustered in a single genetic group. Neither the mutation related to exacerbation of disease (D239G in hemagglutinin) nor that related to antiviral resistance (H275Y in neuraminidase), both detected in neighboring countries, were found. This genetic analysis of H1N1pdm09 will help to understand the spread of the disease.

Presence of ON1 and BA HRSV-genotypes in Paraguay, 2010-2013.

Human respiratory syncytial virus (HRSV), a member of the aramyxoviridae family, is a nonsegmented, single-stranded RNA irus [1]. HRSV is the main viral cause of acute and severe lower resiratory tract infections in children worldwide. It has a single type, ith two subgroups (A and B) that are further divided into several enotypes based on the variability of the G-protein gene. For HRSV , 11 genotypes have been described, which are GA1–GA7, NA1, A2, SAA1, and ON1 [2]. HRSV B has 17 genotypes, designated as B1–GB4, SAB1–SAB3, and BA1–BA10 [3]. In Paraguay, we have previously found that children with severe cute respiratory infection required longer time of hospitalization 5–15 days) when HRSV was present alone or in co-infection with denovirus, rhinovirus, or coronavirus [4]. The lack of genetic inforation about these Paraguayan HRSV isolates led us to carry out molecular characterization of the circulating genotypes in the entral Department. A total of 36HRSV were detected out of 280 nasopharyngeal spirates, collected from children under 5 years of age with sympoms of severe acute respiratory infection, between May 2010 nd December 2013, admitted to the Hospital General Pediátrico iños de Acosta Ñu. HRSV detection was performed by real-time CR (Fast-Track Respiratory Pathogens Plus Kit, FTD, Luxembourg). enetic amplification of the attachment G-protein gene was caried out by Polymerase Chain Reaction. Phylogenetic relationships ere reconstructed by the neighbor-joining method with Kimura’s wo-parameters as the model of nucleotide substitution, as incororated in MEGA v5 [5]. Partial nucleotide sequences obtained in his study were deposited in the GenBank database (accession numers: KM508816–KM508827). Sequencing/phylogenetic analyses of 12 samples showed the resence of HRSV subgroups A (11 samples) and B (1 sample) Figs. 1 and 2). Concerning HRSV subgroup A, genotype NA1 cirulated between 2010 and 2012 (three seasons). In 2012 both enotypes NA1 and GA5 co-circulated. In 2013 the novel genotype N1 circulated (Fig. 1); this genotype has a 72-nt duplication of