Marko Kolenc

Zika Virus Associated with Microcephaly

A widespread epidemic of Zika virus (ZIKV) infection was reported in 2015 in South and Central America and the Caribbean. A major concern associated with this infection is the apparent increased incidence of microcephaly in fetuses born to mothers infected with ZIKV. In this report, we describe the case of an expectant mother who had a febrile illness with rash at the end of the first trimester of pregnancy while she was living in Brazil. Ultrasonography performed at 29 weeks of gestation revealed microcephaly with calcifications in the fetal brain and placenta. After the mother requested termination of the pregnancy, a fetal autopsy was performed. Micrencephaly (an abnormally small brain) was observed, with almost complete agyria, hydrocephalus, and multifocal dystrophic calcifications in the cortex and subcortical white matter, with associated cortical displacement and mild focal inflammation. ZIKV was found in the fetal brain tissue on reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay, with consistent findings on electron microscopy. The complete genome of ZIKV was recovered from the fetal brain.

High Similarity of Novel Orthoreovirus Detected in a Child Hospitalized with Acute Gastroenteritis to Mammalian Orthoreoviruses Found in Bats in Europe

ABSTRACT Mammalian orthoreoviruses (MRVs) are known to cause mild enteric and respiratory infections in humans. They are widespread and infect a broad spectrum of mammals. We report here the first case of an MRV detected in a child with acute gastroenteritis, which showed the highest similarity to an MRV reported recently in European bats. An examination of a stool sample from the child was negative for most common viral and bacterial pathogens. Reovirus particles were identified by electron microscopic examination of both the stool suspension and cell culture supernatant. The whole-genome sequence was obtained with the Ion Torrent next-generation sequencing platform. Prior to sequencing, the stool sample suspension and cell culture supernatant were pretreated with nucleases and/or the convective interaction medium (CIM) monolithic chromatographic method to purify and concentrate the target viral nucleic acid. Whole-genome sequence analysis revealed that the Slovenian SI-MRV01 isolate was most similar to an MRV found in a bat in Germany. High similarity was shared in all genome segments, with nucleotide and amino acid identities between 93.8 to 99.0% and 98.4 to 99.7%, respectively. It was shown that CIM monolithic chromatography alone is an efficient method for enriching the sample in viral particles before nucleic acid isolation and next-generation sequencing application.

Whole genome sequence analysis of bovine G6P[11] rotavirus strain found in a child with gastroenteritis

During the rotavirus strain surveillance in Slovenia, G6P[11] bovine rotavirus strain was detected in a 5 months old boy with gastroenteritis. The strain was enrolled in a whole genome sequence analysis to determine its genome segment composition and genetic characteristics. Genotype composition for the whole genome was G6-P[11]-I2-R2-C2-M2-A13-N2-T6-E2-H3, reflecting similarities with bovine rotavirus strains. The bovine origin of the strain was confirmed in all genome segments, showing the highest nucleotide identity with bovine rotavirus strains and clustering of the RVA/Human-wt/SVN/SI-R56/07/2007/G6P[11] together with bovine rotavirus strains in phylogenetic analysis. This is the first bovine G6P[11] rotavirus strain with the whole genome analysis and the first report on rotavirus G6P[11] genotype detected in humans.

Molecular characterization of rotavirus strains from pre- and post-vaccination periods in a country with low vaccination coverage: the case of Slovenia.

Rotavirus vaccination started in Slovenia in 2007 on a voluntarily basis. The vaccination rate is relatively low (up to 27%) and no increasing trend is observed. We present rotavirus genotype distribution among children hospitalized for rotavirus gastroenteritis in Slovenia. Eight consecutive rotavirus seasons were followed, from 2005/06 to 2012/13, and 113 strains of the most common rotavirus genotypes were randomly selected for molecular characterization of rotavirus VP7 and VP4 (VP8(∗)) genome segments. During the vaccine introduction period, from 2007 to 2013, rotavirus genotype prevalences changed, with G1P[8] decreasing from 74.1% to 8.7% between 2007/08 and 2010/11 seasons, replaced by G4P[8] and G2P[4], with up to 52.0% prevalence. Comparable analysis of VP7 and VP8(∗) genome fragments within G1P[8] genotype lineages revealed considerable differences for rotavirus strains circulating before and during the vaccination period. The G1P[8] rotavirus strains from the pre-vaccination period clustered in a phylogenetic tree within Rotarix®-like VP7 and VP8(∗) lineages. However, since 2007, the majority of G1P[8] strains have shifted to distant genetic lineages with lower nucleotide (88.1-94.0% for VP7 and 86.6-91.1% for VP8(∗)) and amino acid (93.8-95.2% for VP7 and 85.3-94.6% for VP8(∗)) identities to the vaccine Rotarix® strain. This change also resulted in a different deduced amino acid profile at the major VP7 and VP8(∗) antigenic epitopes.

Narrowing of the Diagnostic Gap of Acute Gastroenteritis in Children 0-6 Years of Age Using a Combination of Classical and Molecular Techniques, Delivers Challenges in Syndromic Approach Diagnostics.

Background: Twenty-five percent to 50% of acute gastroenteritis (AGE) cases remain etiologically undiagnosed. Our main aim was to determine the most appropriate list of enteric pathogens to be included in the daily diagnostics scheme of AGE, ensuring the lowest possible diagnostic gap. Methods: Two hundred ninety seven children ⩽6 years of age, admitted to hospital in Slovenia, October 2011 to October 2012, with AGE, and 88 ⩽6 years old healthy children were included in the study. A broad spectrum of enteric pathogens was targeted with molecular methods, including 8 viruses, 6 bacteria and 2 parasites. Results: At least one enteric pathogen was detected in 91.2% of cases with AGE and 27.3% of controls. Viruses were the most prevalent (82.5% and 15.9%), followed by bacteria (27.3% and 10.2%) and parasites (3.0% and 1.1%) in cases and controls, respectively. A high proportion (41.8%) of mixed infections was observed in the cases. For cases with undetermined etiology (8.8%), stool samples were analyzed with next generation sequencing, and a potential viral pathogen was detected in 17 additional samples (5.8%). Conclusions: Our study suggests that tests for rotaviruses, noroviruses genogroup II, adenoviruses 40/41, astroviruses, Campylobacter spp. and Salmonella sp. should be included in the initial diagnostic algorithm, which revealed the etiology in 83.5% of children tested. The use of molecular methods in diagnostics of gastroenteritis is preferable because of their high sensitivity, specificity, fast performance and the possibility of establishing the concentration of the target. The latter may be valuable for assessing the clinical significance of the detected enteric, particularly viral pathogens.

Distribution network development based on stochastic modelling approach

The paper analyses the influence of distributed generation (DG) on network planning methods in low voltage (LV) networks. Due to environmental considerations, legal constraints, price, and introduction of new elements into the network, classical network planning is not any more sufficient. To form the basis of planning decisions approach which provides information on the probabilities of attributes such as voltage limits or power losses, should be provided. The paper presents statistical Monte Carlo approach which can be used in LV distribution network planning. The method enables evaluation of the future voltage conditions and therefore the comparison of different possible network development strategies, taking into account the stochastic natures of future DG location and loads consumption. The results are demonstrated on an actual LV Slovenian distribution network model.

A Novel Strain of Porcine Adenovirus Detected in Urinary Bladder Urothelial Cell Culture

Contamination of cell cultures is the most common problem encountered in cell culture laboratories. Besides the secondary cell contaminations often occurring in the cell laboratories, the contaminations originating from donor animal or human tissue are equally as common, but usually harder to recognize and as such require special attention. The present study describes the detection of porcine adenovirus (PAdV), strain PAdV-SVN1 in cultures of normal porcine urothelial (NPU) cells isolated from urinary bladders of domestic pigs. NPU cell cultures were evaluated by light microscopy (LM), polymerase chain reaction (PCR), and additionally assessed by transmission electron microscopy (TEM). Characteristic ultrastructure of virions revealed the infection with adenovirus. The adenoviral contamination was further identified by the sequence analysis, which showed the highest similarity to recently described PAdV strain PAdV-WI. Additionally, the cell ultrastructural analysis confirmed the life-cycle characteristic for adenoviruses. To closely mimic the in vivo situation, the majority of research on in vitro models uses cell cultures isolated from human or animal tissue and their subsequent passages. Since the donor tissue could be a potential source of contamination, the microbiological screening of the excised tissue and harvested cell cultures is highly recommended.

Intrahost Norovirus Evolution in Chronic Infection Over 5 Years of Shedding in a Kidney Transplant Recipient

Noroviruses are the leading cause of acute gastroenteritis, and they can affect humans of all age groups. In immunocompromised patients, norovirus infections can develop into chronic diarrhea or show prolonged asymptomatic virus shedding. Chronic norovirus infections are frequently reported for solid organ transplant recipients, with rapid intrahost norovirus evolution seen. In this report, we describe a case of chronic norovirus infection in an immunocompromised patient who was followed up for over 5 years. The purpose of the study was to specify the norovirus evolution in a chronically infected immunocompromised host and identify possible selection sites in norovirus capsid protein. During the follow-up period, 25 sequential stool samples were collected and nine of them were selected to generate amplicons covering viral RNA-dependent RNA polymerase (RdRp) and viral capsid protein (VP1) genes. Amplicons were sequenced using next-generation sequencing. Single nucleotide polymorphisms were defined, which demonstrated a nearly 3-fold greater mutation rate in the VP1 genome region compared to the RdRp genome region (7.9 vs. 2.8 variable sites/100 nucleotides, respectively). This indicates that mutations in the virus genome were not accumulated randomly, but are rather the result of mutant selection during the infection cycle. Using ShoRAH software we were able to reconstruct haplotypes occurring in each of the nine selected samples. The deduced amino-acid haplotype sequences were aligned and the positions were analyzed for selective pressure using the Datamonkey program. Only 12 out of 25 positive selection sites were within the commonly described epitopes A, B, C, and D of the VP1 protein. New positive selection sites were determined that have not been described before and might reflect adaptation of the norovirus toward optimal histo-blood-group antigen binding, or modification of the norovirus antigenic properties. These data provide new insights into norovirus evolutionary dynamics and indicate new putative epitope “hot-spots” of modified and optimized norovirus–host interactions.