Alicia S. Mistchenko

Natural History of Human Respiratory Syncytial Virus Inferred from Phylogenetic Analysis of the Attachment (G) Glycoprotein with a 60-Nucleotide Duplication

ABSTRACT A total of 47 clinical samples were identified during an active surveillance program of respiratory infections in Buenos Aires (BA) (1999 to 2004) that contained sequences of human respiratory syncytial virus (HRSV) with a 60-nucleotide duplication in the attachment (G) protein gene. This duplication was analogous to that previously described for other three viruses also isolated in Buenos Aires in 1999 (A. Trento et al., J. Gen. Virol. 84:3115-3120, 2003). Phylogenetic analysis indicated that BA sequences with that duplication shared a common ancestor (dated about 1998) with other HRSV G sequences reported worldwide after 1999. The duplicated nucleotide sequence was an exact copy of the preceding 60 nucleotides in early viruses, but both copies of the duplicated segment accumulated nucleotide substitutions in more recent viruses at a rate apparently higher than in other regions of the G protein gene. The evolution of the viruses with the duplicated G segment apparently followed the overall evolutionary pattern previously described for HRSV, and this genotype has replaced other prevailing antigenic group B genotypes in Buenos Aires and other places. Thus, the duplicated segment represents a natural tag that can be used to track the dissemination and evolution of HRSV in an unprecedented setting. We have taken advantage of this situation to reexamine the molecular epidemiology of HRSV and to explore the natural history of this important human pathogen.

Molecular epidemiology of adenovirus acute lower respiratory infections of children in the south cone of south America (1991-1994)

A collection of 165 adenovirus strains isolated from nasopharyngeal aspirates of children hospitalized for acute lower respiratory infection in Argentina, Chile, and Uruguay between 1991 and 1994 was studied by restriction enzyme analysis (work performed in the Department of Virology, University of Umeå). Of the isolates, 71% (n = 117) were identified as members of subgenus B. Of these, 101 (61.2%) corresponded to genome type 7h, four (2.4%) to genome type 3p2, four (2.4%) to genome type 11a, one (0.6%) to genome type 7b, and one (0.6%) to genome type 7c. Two isolates that were neutralized as serotype 3 and four isolates that were neutralized as serotype 7 exhibited novel BamHI cleavage profiles corresponding to three new genome types denominated 3x, 7i, and 7j.

Respiratory syncytial virus: Changes in prevalence of subgroups A and B among Argentinian children, 1990–1996

The frequency of respiratory syncytial virus (RSV) and the distribution of subgroups A and B strains during 7 consecutive years (1990–1996) were examined in two cities of Argentina. Nasopharyngeal aspirates from 1,304 children less than 2 years of age hospitalized with acute lower respiratory infection were studied by indirect immunofluorescence. RSV was detected in 352 cases (26.9%), and the peak activity was observed in midwinter. Subgroup characterization was performed with two monoclonal antibodies against the F protein on nasopharyngeal aspirate smears. Of 195 samples, 174 (89.2%) were identified as subgroup A strains and 21 (10.8%) as subgroup B. Both strains cocirculated during 5 of 7 years studied with subgroup A predominating. Subgroup A occurred at least 8 times as often in all years except for 1994–1995. Children infected by subgroup A were younger than those infected by subgroup B (P < 0.05). The association of subgroup A infection with bronchiolitis and subgroup B with pneumonia was statistically significant (P < 0.03). J. Med. Virol. 61:275–279, 2000.

Fatal adenovirus infection associated with new genome type.

The first fatal case caused by the new genome type 7i is described in an 8‐month‐old boy requiring long‐term respiratory support who developed Reyes syndrome, acute respiratory distress, and bronchiolitis obliterans with fatal evolution. Adenovirus was detected in nasopharyngeal secretions and was persistently positive during hospitalization. IgM and IgG adenovirus antibody titers measured in serum by enzyme‐linked immunoassay (EIA) were 1:32 and 1:800, respectively. Serum interleukins (IL) and interferons (IFN) measured by EIA were as follows: IL‐2, 110 pg/ml; IL‐6, 300 pg/ml; IL‐8, 7,000 pg/ml; TNF‐α, 35 pg/ml, IL‐1 and IL‐4 undetectable, IFN‐α 2,200 pg/ml, and IFN‐γ 700 pg/ml. Virologic studies showed that adenovirus isolated belonged to subgenus B, and digestion of viral DNA with Bam HI, Sma I, Bgl II, and Hind III identified the isolate as belonging to genome type 7i. Autopsy showed bronchiolitis obliterans with diffuse alveolar damage and perivenular fatty degeneration with polymorphonuclear infiltrates in the periportal spaces. The difficulty in obtaining adequate oxygenation with minimization of iatrogenic oxygen injury is discussed.J. Med. Virol. 54:233–236, 1998.

Measles resurgence in Argentina: 1997-8 outbreak.

Epidemiological and clinical findings from 1162 serologically confirmed measles cases occurring in Buenos Aires, Argentina in 1997 and 1998 were retrospectively reviewed. From 90 hospitalized children, measles virus was detected by direct RT-PCR from nasopharyngeal secretions. Patients were grouped as follows: (i) not vaccinated: infants < 12 months; (ii) regularly vaccinated: children 1-4 years not covered by the last catch-up; (iii) catch-up vaccinated: patients 5-19 years immunized during the 1993 campaign. Most cases were recorded in non-vaccinated infants (54%), and the lowest in catch-up vaccinated children (16%). Mean age of the 90 hospitalized children was 11.3 months. Pneumonia was the major hospitalization cause followed by pneumonitis. Two children required intensive care and one died. The 1993 catch-up campaign seemed to reduce the number of cases in the 5- to 19-year-old group. Lack of timely follow-up probably led to the accumulation of susceptible individuals allowing measles re-emergence. Direct viral detection by RT-PCR proved to be a sensitive tool for molecular epidemiology surveillance.

Sequence analysis of measles virus hemagglutinin isolated in Argentina during the 1997-1998 outbreak.

Sequence analysis was performed on 50 measles viruses (MV) isolated in Argentina. Forty‐six were obtained during the current outbreak (1997–1998), three from the previous outbreak (1991) and one sporadic case (1994). A 377‐bp fragment of the hemagglutinin (H) gene was directly amplified by RT‐PCR from nasopharyngeal secretions. Nucleotides 8152 to 8417 were sequenced and subjected to phylogenetic analysis. Multiple silent changes and point mutations were found in all MVs. In 1991, substitutions affected the third base in codons resulting in silent changes. In 1994 an A→C substitution at position 8321 changed amino acids 351 (Leu→Ile). In 1997–1998, an A→G substitution at position 8339 changed amino acids 357 (Val→Ile). In 3/46 viruses, guanine deletion at position 8205 changed the reading frame and insertion of an extra cytosine at nucleotide 8235 shifted it back to the original frame. Phylogenetic analysis revealed that viruses leading to the last two major outbreaks are clustered into two separate branches. MVs that prevailed until 1994 were related to genotype C1 and MVs of the current outbreak to D6. Random drift mutations rendered a 0.5 ratio of nonsilent over silent mutations in most of the MVs analyzed. However, in those showing a reading frame shift, the ratio was greater than 1, suggesting that it was driven by immune selection. J. Med. Virol. 60:91–96, 2000.

Acute and Chronic Human Adenovirus Pneumonia: Cellular and Extracellular Matrix Components

We present a comparative histopathological study of both acute and chronic human adenovirus pneumonia, with reference to the cellular and extracellular matrix components. Seventeen lungs from autopsied patients whose ages ranged from 2 to 60 months were studied. Adenovirus types 1, 2, 3, 5, and 7 were isolated from 15 patients with acute lung disease, and types 2 and 7 were isolated from the other two patients with chronic pulmonary illness. The results indicated the occurrence of two basic patterns of adenovirus interstitial pneumonia (1) classic pattern (acute), characterized by necrosis and degeneration and many type II pneumocytes with intranuclear inclusion bodies, which were positive for adenovirus DNA by in situ hybridization, and (2) proliferative or proliferative-productive pattern (chronic), which presented with diffuse pulmonary fibrosis and the interstitial proliferation of fibroblast-like cells, compatible with myofibroblasts (positive for vimentin and alpha smooth muscle actin), and increase in collagen types I and III, elastic fibers, and proteoglycans. Alveolar collapse appears to be an important pathogenetic mechanism in the development of this pattern.

Wild-type Measles Virus in Brain Tissue of Children with Subacute Sclerosing Panencephalitis, Argentina

We studied eight children who had measles at 6 to 10 months of age during the 1998 Argentine measles outbreak and in whom subacute sclerosing panencephalitis developed 4 years later. We report the genetic characterization of brain tissue–associated measles virus samples from three patients. Phylogenetic relationships clustered these viruses with the wild-type D6 genotype isolated during the 1998 outbreak. The children received measles vaccine; however, vaccinal strains were not found.

Sixteen years of evolution of human respiratory syncytial virus subgroup A in Buenos Aires, Argentina: GA2 the prevalent genotype through the years

Human respiratory syncytial virus (HRSV) is the main viral cause of acute lower respiratory tract infections (LRTI) in children worldwide. In recent years, several preclinical trials with vaccine candidates have been reported. It is in this sense that molecular epidemiological studies become important. Understanding viral dispersion patterns before and after the implementation of a vaccine can provide insight into the effectiveness of the control strategies. In this work we analyzed the molecular epidemiology of HRSV-A over a period of sixteen years (1999-2014) in Buenos Aires. By bioinformatic tools we analyzed 169 sequences of the G glycoprotein gene from hospitalized pediatric patients with LRTI. We found that GA2 was the most prevalent genotype (73.35%). GA5 genotype co-circulated in our region until 2009 when it was no longer detected, except in 2011. The recently globally emerging ON1 lineage with a 72-nt duplication increased its frequency to become the only lineage detected in Buenos Aires in 2014. By discrete phylogeographic analysis of global ON1 strains we could determine that Panama could be the location of the MRCA dated June 20, 2010; and this lineage could be introduced in Argentina from Spain in April 2011. This analysis also showed temporary and geographical clustering of ON1 strains observed as phylogenetic clades with strains exclusively associated from a single country, nevertheless among our 44 ON1 strains from three outbreaks (2012-2014) we could also detect posterior reintroductions and circulation from United States, Cuba, South Korea, and Spain. The continuous phylogeographic analysis of one sublineage of Argentine ON1 strains allowed us to establish that there could be a local clustering of some strains even in neighborhoods. This work shows the potential of this type of bioinformatic tools in the context of a future vaccine surveillance network to trace the spread of new genetic lineages in human populations.

Phylogenetic and molecular analyses of human parainfluenza type 3 virus in Buenos Aires, Argentina, between 2009 and 2013: The emergence of new genetic lineages

Despite that human parainfluenza type 3 viruses (HPIV3) are one of the leading causes of acute lower respiratory tract infections in children under five, there is no licensed vaccine and there is limited current information on the molecular characteristics of regional and global circulating strains. The aim of this study was to describe the molecular characterization of HPIV3 circulating in Buenos Aires. We performed a genetic and phylogenetic analysis of the HN glycoprotein gene. Between 2009 and 2013, 124 HPIV3-positive samples taken from hospitalized pediatric patients were analyzed. Four new genetic lineages were described. Among them, C1c and C3d lineages showed local circulation patterns, whereas C3e and C3f comprised sequences from very distant countries. Despite the diversity of the described genotypes, C3a and C3d predominated over the others, the latter was present during the first years of the study and it was progressively replaced by C3a. Molecular analyses showed 28 non-synonymous substitutions; of these, 13 were located in potentially predicted B-cell epitopes. Taken together, the emergence of genetic lineages and the information of the molecular characteristics of HN protein may contribute to the general knowledge of HPIV3 molecular epidemiology for future vaccine development and antiviral therapies.