Small hydrophobic (SH) gene is one of the mostly diverse genomic regions of human respiratory syncytial virus (HRSV). Its coding region constitutes less than 50% of the complete gene length, enabling ...SH gene to be highly variable and the SH protein highly conserved. In standard HRSV molecular epidemiology studies, solely sequences of the second hypervariable region of the glycoprotein gene (HVR2) are analyzed. To what extent do the strains identical in HVR2 differ elsewhere in genomes is rarely investigated. Our goal was to investigate whether diversity and inter-genotype differences observed for HVR2 are also present in the SH gene.
We sequenced 198 clinical samples collected within a limited area and time frame. In this HRSV collection, rapid and significant changes in HVR2 occurred.
Over 20% of strains from this pool (containing HRSV genotypes NA1, ON1, GA5, BA9 and BA10) would be incorrectly assumed to be identical to another strain if only the HVR2 region was analysed. The majority of differences found in SH gene were located in the 5' untranslated region (UTR). Seven indels were detected, one was genotype GA5 specific. An in-frame deletion of 9 nucleotides (coding for amino acids 49-51) was observed in one of group A strains. Fifteen different SH protein sequences were detected; 68% of strains possessed the consensus sequence and most of others differed from the consensus in only one amino acid (only 4 strains differed in 2 amino acids). The majority of differing amino acids in group A viruses had the same identity as the corresponding amino acids in group B strains. When analysis was restricted to strains with identical HVR2 nucleotide sequences and differing SH protein sequences, 75% of differences observed in the SH ectodomain were located within region coding for amino acids 49-51.
Basing HRSV molecular epidemiology studies solely on HVR2 largely underestimates the complexity of circulating virus populations. In strain identification, broadening of the genomic target sequence to SH gene would provide a more comprehensive insight into viral pool versatility and its evolutionary processes.
Human metapneumovirus (HMPV) is recognized as a global and frequent cause of acute respiratory tract infections among people of all ages. The objectives of this study were molecular epidemiology and ...evolutionary analysis of HMPV strains which produced moderate and severe acute respiratory tract infections in children in Croatia during four consecutive seasons (2011‐2014). A total of 117 HMPV‐positive samples collected from hospitalized pediatric patients presenting with acute respiratory tract infections and tested by direct immunofluorescence assay were first analyzed by amplifying a part of the F gene. Sixteen samples were further analyzed based on complete F, G, and SH gene sequences. HMPV genome was identified in 92 of 117 samples (78%) and the circulation of multiple lineages of HMPV was confirmed. In 2011, 2012, and 2014, subgroups A2 and B2 co‐circulated, while B1 gained prevalence in 2013 and 2014. The study established the presence of a novel subcluster A2c in Croatia. This subcluster has only recently been detected in East and Southeast Asia. This study provides new insights into epidemiology and genetic diversity of HMPV in this part of Europe.
This study investigated the HPIV3 circulating strains in Croatia and whether the other parts of HPIV3 genome (F gene and HN 582 nucleotides fragment) could be equally suitable for genetic and ...phylogenetic analysis.
Clinical materials were collected in period 2011-2015 from children suffering from respiratory illnesses. In positive HPIV3 samples viral genome was partially amplified and sequenced for HN and F genes. Obtained sequences were analysed by phylogenetic analysis and genetic characterization was performed.
All samples from this study belonged to subcluster C and over a short period of time, genetic lineage C3a gained prevalence over the other C genetic lineages, from 39 % in 2011 to more than 90 % in 2013 and 2014. Phylogenetic classifications of HPIV3 based on the entire HN gene, HN 582 nt fragment and entire fusion (F) gene showed identical classification results for Croatian strains and the reference strains. Molecular analysis of the F and HN glycoproteins, showed their similar nucleotide diversity (Fcds P=0.0244 and HNcds P=0.0231) and similar Ka/Ks ratios (F Ka/Ks=0.0553 and HN Ka/Ks=0.0428). Potential N-glycosylation sites, cysteine residues and antigenic sites are generally strongly conserved in HPIV3 glycoproteins from both our and the reference samples.
The HPIV3 subclaster C3 (genetic lineage C3a) became the most detected circulating HPIV3 strain in Croatia. The results indicated that the HN 582 nt and the entire F gene sequences were as good for phylogenetic analysis as the entire HN gene sequence.
Human respiratory syncytial virus (HRSV) causes common respiratory tract infections in infants, young children and the elderly. The diversity of HRSV strains circulating in Croatia was investigated ...throughout a period of four consecutive years from March 2011–March 2014. The analysis was based on sequences from the second hypervariable region of the G gene. A predominance of HRSV group A was observed in the first three years of the study, while group B became slightly predominant during the first few months of 2014. Overall, 76% of viruses belonged to group A including the genotypes NA1, ON1 and GA5. NA1 was by far the most common genotype within group A in 2011–2013; however, only ON1 and a few GA5 viruses were detected in the first three months of 2014. The majority of group B strains were of genotype BA9 (97%), and a few BA10 genotypes were detected. BA9 had the highest substitution rate of all the detected genotypes, followed by ON1.
Multiple analyses showed that HRSV group A strains were more diverse than group B strains. Gly at residue 232 (previously described to be specific for ON1) was also detected in three NA1 strains, which were phylogenetically placed on separate branches within the NA1 genotype. For all genotypes, the diversity was higher at the amino acid level than at the nucleotide level, although positive selection of mutations was shown for only a few sites using four different methods of codon-based analysis of selective pressure. More codons were predicted to be negatively selected.
The complexity of the HRSV pools present during each epidemic peak was determined and compared to previous epidemiological data. In addition to presenting genetic versatility of HRSV in this geographic region, the collected sequences provide data for further geographical and temporal comparative analyses of HRSV and its evolutionary pathways.
•Diversity of HRSV in Croatia, March 2011–March 2014, is described.•Samples were obtained from infants hospitalized due to acute respiratory infections.•Co-circulation of NA1, ON1, GA5, BA9 and BA10 genotypes was detected.•Different bioinformatics analytical tools were used for genetic characterisation.•HRSV A strains (NA1 in particular) were found to be more diverse than HRSV B.
Molecular epidemiology of human parainfluenza viruses type 1 (HPIV1) was investigated. Samples were collected from patients hospitalized in Croatia during the three consecutive epidemic seasons ...(2011-2014). Results indicated co-circulation of two major genetic clusters of HPIV1. Samples from the current study refer to clades II and III in a phylogenetic tree of haemagglutinin-neuraminidase (HN) gene. Additional phylogenetic trees of fusion (F) and phosphoprotein (P) genes confirmed the topology. Analysis of nucleotide diversity of entire P, F and HN genes demonstrated similar values: 0.0255, 0.0236 and 0.0237, respectively. However, amino acid diversity showed F protein to be the most conserved, while P protein was the most tolerant to mutations. Potential N- and O-glycosylation sites suggested that HPIV1 HN protein is abundantly glycosylated, and a specific N-glycosylation pattern could distinguish between clades II and III. Analysis of potential O-glycosylation sites in F protein indicated that samples from this study have two potential O-glycosylation sites, while publicly available sequences have five potential sites. This study provides data on the molecular characterization and epidemic pattern of HPIV1 in Croatia.
Comparative genomics of human rubulavirus 2 Šantak, Maja; Mlinarić-Galinović, Gordana; Vilibić-Čavlek, Tatjana ...
Archives of virology,
11/2018, Volume:
163, Issue:
11
Journal Article
Peer reviewed
Although human rubulavirus 2 (HPIV2) is an important respiratory pathogen, little is known about its molecular epidemiology. We performed a comparative analysis of the full-length genomes of fourteen ...HPIV2 isolates belonging to different genotypes. Additionally, evolutionary analyses (phylogenetic reconstruction, sequence identity, detection of recombination and adaptive evolution) were conducted. Our study presents a systematic comparative genetic analysis that complements prior analyses and utilizes full-length HPIV2 genomes to provide a basis for future work on the clinical significance, molecular variation and conservation, and evolution of HPIV2.
There are >50 adenovirus (ADV) serotypes that are divided into 7 species (A-G). The aim of this study was to characterize ADV serotypes and species in hospitalized infants and children in the City of ...Zagreb and Zagreb County and to describe clinical features and laboratory findings of ADV infections according to the causative ADV serotype.
During the 3-year period from January 2006 to November 2008, 135 children (<10 years of age) with ADV respiratory infection, based on virus isolation, were treated at 2 hospitals in Zagreb. Demographics, clinical presentations and laboratory findings were evaluated.
Of the 135 ADV isolates, 77 (57.0%) were type 2, followed by 26 (19.3%) of type 1, 15 (11.1%) isolates of type 3, 2 (1.5%) of type 6 and only 1 (0.7%) was type 7. Male-to-female ratio was 3.2:1 (103 boys and 32 girls). The mean age was 22.9 months. The most common symptoms were fever (98%), rhinorrhea (89%) and cough (71%). The mean peak body temperature was 39.8°C. Tonsillitis was present in 79 (59%) and acute otitis media in 37 (28%) patients. Leukocytosis (>15.0×10⁹/L) was noted in 103 (77%) patients. Serum C-reactive protein was >40 mg/L in 74 patients (56%). The erythrocyte sedimentation rate was ≥30 mm/h in 91 (71%) of the 127 patients tested.
In this study, the most common isolated serotype was ADV type 2. Most affected children were younger than 3 years. ADV infections in young children can present with prolonged fever, leukocytosis and significantly elevated C-reactive protein and erythrocyte sedimentation rate, mimicking bacterial infections.
The main objective of this study is to determine the prevalence of trichomoniasis in men with and without symptoms of urethritis, with concomitant analysis of sociodemographic and behavioural ...specificities of both groups. Also, the objective is to evaluate laboratory methods used in the diagnostics of this parasitic disease.
A total of 500 men with and 200 without urethritis symptoms were included in the study. Every respondent filled out a questionnaire asking for some general data, specific information about habits, sexual behaviour and symptoms. Sediment of first void urine was analysed by wet mount microscopy, cultivation in Diamond's medium and real-time PCR.
In the symptomatic group, Trichomonas vaginalis infection was documented in 2.4% of respondents by wet mount microscopy, in 4.8% by cultivation and in 8.2% by real-time PCR. In the asymptomatic group, infection was proven using the same methods in 1.0%, 1.5% and 2.0% of the respondents, respectively. Trichomoniasis prevalence was statistically significantly higher in the respondents manifesting urethritis symptoms when cultivation (χ2=4.20, p=0.041) and real-time PCR (χ2=9.20, p=0.002) were used. Several epidemiological risk factors were identified, and greater sensitivity of real-time PCR was found in comparison with microscopy and culture.
Trichomonas infection was statistically more frequent in men with urethritis syndrome. Assuming that the samples found positive by any laboratory technique are truly positive, it can be concluded that the real-time PCR showed the greatest sensitivity of all the methods used in this study.
During July 2009 an outbreak in neonates represented with gastrointestinal and respiratory symptoms was observed at the Neonatal Postintensive Care Unit, Clinical Hospital Center, Zagreb. Human ...parechovirus type 1 (HPeV1) was isolated from seven patients, one of whom was asymptomatic. All but one were premature neonates with serious underlying conditions, and all recovered fully after several days. In order to characterize the HPeV1s, sequencing of the VP1/2A region was conducted on six isolates from the outbreak and four isolates detected in Croatia in 2008 and 2007. The analysis of sequence similarity showed that the nucleotide identity between the prototype strain (Harris) and HPeV1 isolated in Croatia was 76.5-77.5%. Croatian strains from 2007 and 2009 clustered together with strains from the Netherlands and Germany detected in 2003 and 2006, respectively, while strains from 2008 clustered with the strain from Finland detected in 2000. Change of the dominant strains each year may suggest antigenic variation as a result of viral response to specific immunity of the target population. J. Med. Virol. 83:137-141, 2011.
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