Marina Siljic

mTOR-independent autophagy counteracts apoptosis in herpes simplex virus type 1-infected U251 glioma cells.

We investigated the role of autophagy, a stress-inducible lysosomal self-digestion of cellular components, in modulation of herpes simplex virus type 1 (HSV-1)-triggered death of U251 human glioma cells. HSV-1 caused apoptotic death in U251 cells, characterized by phosphatidylserine externalization, caspase activation and DNA fragmentation. HSV-1-induced apoptosis was associated with the induction of autophagic response, as confirmed by the conversion of cytosolic LC3-I to autophagosome-associated LC3-II, increase in intracellular acidification, presence of autophagic vesicles, and increase in proteolysis of the selective autophagic target p62. HSV-1-triggered autophagy was not associated with the significant increase in the expression of proautophagic protein beclin-1 or downregulation of the major autophagy suppressor mammalian target of rapamycin (mTOR). Moreover, the phosphorylation of mTOR and its direct substrate p70 S6 kinase was augmented by HSV-1 infection, while the mTOR stimulator Akt and inhibitor AMPK-activated protein kinase (AMPK) were accordingly activated and suppressed, respectively. An shRNA-mediated knockdown of the autophagy-essential LC3β, as well as pharmacological inhibition of autophagy with bafilomycin A1 or 3-methyladenine, markedly accelerated apoptotic changes and ensuing cell death in HSV-1-infected glioma cells. These data indicate that AMPK/Akt/mTOR-independent autophagy could prolong survival of HSV-1-infected U251 glioma cells by counteracting the coinciding apoptotic response.

Evidence of recombination in Tula virus strains from Serbia.

Tula hantavirus (TULV) belongs to Bunyaviridae family, with negative sense RNA genome. Segmented nature of hantaviral genome allows for genetic reassortment, but the evidence of homologous recombination also exists. In this study we analyzed TULV sequences isolated in Serbia on different occasions and from different rodent hosts: 1987 strain from Microtus subterraneus and 2007 strain from Microtus arvalis. Phylogenetic analysis of both L and S segment sequences is suggestive of geographically related clustering, as previously shown for majority of hantaviruses. Reconstruction of phylogenetic tree for TULV S segment showed that both sequences from Serbia clustered together with sequences from East Slovakia, which had previously been shown to be recombinants (Kosice strain). Exploratory recombination analysis, supported by phylogenetic and amino acid pattern analysis, revealed the presence of recombination in the S segment sequences from Serbia, resulting in mosaic-like structure of TULV S segment similar to the one of Kosice strain. Although recombination is considered a rare event in molecular evolution of negative strand RNA viruses, obtained molecular data in this study support evidence of recombination in TULV, in geographically distant regions of Europe.

Molecular typing of the local HIV-1 epidemic in Serbia.

Worldwide HIV-1 pandemic is becoming increasingly complex, with growing heterogeneity of subtypes and recombinant viruses. Previous studies have documented HIV-1 subtype B as the predominant one in Serbia, with limited presence and genetic diversity of non B subtypes. In recent years, MSM transmission has become the most frequently reported risk for HIV infection among newly diagnosed patients in Serbia, but very little is known of the network structure and dynamics of viral transmission in this and other risk groups. To gain insight about the HIV-1 subtypes distribution pattern as well as characteristics of HIV-1 transmission clusters in Serbia, we analyzed the genetic diversity of the pol gene segment in 221 HIV-1-infected patients sampled during 2002-2011. Subtype B was found to still be the most prevalent one in Serbia, accounting for over 90% of samples, while greater diversity of other subtypes was found than previously reported, including subtypes G, C, A, F, CRF01 and CRF02. In total, 41.3% of analyzed subtype B sequences were found associated in transmission clusters/network, that are highly related with MSM transmission route.

Forensic application of phylogenetic analyses – Exploration of suspected HIV-1 transmission case

Transmission of human immunodeficiency virus (HIV) between individuals may have important legal implications and therefore may come to require forensic investigation based upon phylogenetic analysis. In criminal trials results of phylogenetic analyses have been used as evidence of responsibility for HIV transmission. In Serbia, as in many countries worldwide, exposure and deliberate transmission of HIV are criminalized. We present the results of applying state of the art phylogenetic analyses, based on pol and env genetic sequences, in exploration of suspected HIV transmission among three subjects: a man and two women, with presumed assumption of transmission direction from one woman to a man. Phylogenetic methods included relevant neighbor-joining (NJ), maximum likelihood (ML) and Bayesian methods of phylogenetic trees reconstruction and hypothesis testing, that has been shown to be the most sensitive for the reconstruction of epidemiological links mostly from sexually infected individuals. End-point limiting-dilution PCR (EPLD-PCR) assay, generating the minimum of 10 sequences per genetic region per subject, was performed to assess HIV quasispecies distribution and to explore the direction of HIV transmission between three subjects. Phylogenetic analysis revealed that the viral sequences from the three subjects were more genetically related to each other than to other strains circulating in the same area with the similar epidemiological profile, forming strongly supported transmission chain, which could be in favour of a priori hypothesis of one of the women infecting the man. However, in the EPLD based phylogenetic trees for both pol and env genetic region, viral sequences of one subject (man) were paraphyletic to those of two other subjects (women), implying the direction of transmission opposite to the a priori assumption. The dated tree in our analysis confirmed the clustering pattern of query sequences. Still, in the context of unsampled sequences and inherent limitations of the applied methods, we cannot unambiguously prove that HIV-1 transmission occurred directly between two individuals. Further exploration of the known and suspected transmission cases is needed in order to define methodologies and establish their reliability.

Substitution rate and natural selection in parvovirus B19

The aim of this study was to estimate substitution rate and imprints of natural selection on parvovirus B19 genotype 1. Studied datasets included 137 near complete coding B19 genomes (positions 665 to 4851) for phylogenetic and substitution rate analysis and 146 and 214 partial genomes for selection analyses in open reading frames ORF1 and ORF2, respectively, collected 1973–2012 and including 9 newly sequenced isolates from Serbia. Phylogenetic clustering assigned majority of studied isolates to G1A. Nucleotide substitution rate for total coding DNA was 1.03 (0.6–1.27) x 10−4 substitutions/site/year, with higher values for analyzed genome partitions. In spite of the highest evolutionary rate, VP2 codons were found to be under purifying selection with rare episodic positive selection, whereas codons under diversifying selection were found in the unique part of VP1, known to contain B19 immune epitopes important in persistent infection. Analyses of overlapping gene regions identified nucleotide positions under opposite selective pressure in different ORFs, suggesting complex evolutionary mechanisms of nucleotide changes in B19 viral genomes.

A13 Social affects phylogeny: Exploration of ongoing and active transmission chains

from children to mothers through breast-feeding. Since then the infection was transferred both vertically and horizontally. The source of the outbreak was the husband of one of the infected mothers who was previously infected in Congo. Before the end of 1990s, all samples of HIV subtype G in Russia were considered to originate from that outbreak. Here, we study the phylogenetics of HIV subtype G in Russia. The dataset consisted of 1,169 HIV-1 subtype G sequences of protease and reverse transcriptase, including 12 Russian sequences of unknown origin and three Russian sequences from the Elista outbreak sequenced in the Central Research Institute of Epidemiology (CRIE), and 122 sequences of Russian origin from the Los Alamos National Laboratory (LANL) HIV database (lanl.hiv.gov). Sequences were aligned with Clustal Omega, and the alignment was then manually edited in AliView. A maximum likelihood tree was reconstructed using RAxML. In the resulting phylogeny, the 120 Russian LANL sequences and the three CRIE sequences from the Elista outbreak fall into a clade nested within the sequences from Democratic Republic of Congo as expected for the 1988 Elista outbreak. By contrast, the remaining thirteen other Russian sequences, including twelve CRIE sequences and one LANL sequence, formed a strongly supported clade nested within sequences from Portugal, next to 2003–2004 sequences from Denmark. This phylogenetic evidence suggests a mixed origin of the subtype G, with a previously unreported influx of subtype G in Russia in mid-2000s from Western Europe. About eight out of twelve subtype G sequences of presumably Western European origin were later fully sequenced. Recombination analysis performed with jpHMM has shown that all of them share the same recombination with subtype B (positions 790– 1,160 based on HXB2 numbering). In contrast, analysis of fullgenome subtype G sequences from the Elista outbreak has not shown any recombination events. These results also support the hypothesis of mixed origin of subtype G in Russia.

High Frequency of Human Leukocyte Antigen-B*57:01 Allele Carriers among HIV-Infected Patients in Serbia

Abacavir is an effective antiretroviral drug and one of the most commonly used nucleoside reverse transcriptase inhibitors in Serbia. А percentage of the treated patients experience a potentially life-threatening hypersensitivity reaction, which was shown to be associated with the presence of the class I MHC allele, HLA-B*57:01; hence genotyping for HLA-B*57:01 prior to starting abacavir is nowadays recommended in international HIV treatment guidelines. In Serbia, this testing became available in 2013. This study was designed to estimate the prevalence of the HLA-B*57:01 allele in Serbian HIV-1-infected patients. The presence of the HLA-B*57:01 allele was analyzed in 273 HIV-1-infected patients aged 18 years or more, who were abacavir naïve. Buccal swab samples were obtained from all participants and assayed for the presence of HLA-B*57:01 using a commercially available HLA-B*57:01 real-time PCR kit. The presence of the HLA-B*57:01 allele was found in 22 of 273 tested individuals (8%; 95% CI 5.4-11.9%). This is the first study that estimated the HLA-B*57:01 prevalence among HIV-infected patients in Serbia. The very high prevalence of HLA-B*57:01 found in our study strongly supports HLA-B*57:01 genotyping, which should be implemented prior to the initiation of an abacavir-containing therapy to reduce the risk of potentially life-threatening hypersensitivity reactions.

Locally advanced rectal cancers with simultaneous occurrence of KRAS mutation and high VEGF expression show invasive characteristics

In this study, we investigated the mutation status of KRAS gene in pretherapeutic and preoperative biopsies in 63 specimens of locally advanced rectal cancers in order to evaluate its potential predictive and/or prognostic role. Regions of interest of KRAS exon 2 were amplified and visualized on 2% agarose gel. Obtained PCR products were subjected to direct sequencing. KRAS mutations were detected in 35% of patients, 91% of which were located in codon 12 and 9% in codon 13. In general, KRAS mutation status did not affect the response to neoadjuvant chemoradiotherapy (CRT). However, patients harboring mutated KRAS gene, simultaneously with high vascular endothelial growth factor (VEGF) expression, exhibited a worse response to CRT (p=0.030), a more frequent appearance of local recurrences and distant metastasis (p=0.003), and shorter overall survival (p=0.001) compared to all others. On the contrary, patients with GGT>GCT KRAS mutation exhibited a significantly better response to CRT than those with any other type of KRAS mutation (p=0.017). Moreover, the presence of GGT>GCT mutation was associated with low VEGF and Ki67 expression (p=0.012 in both cases), parameters related to less aggressiveness of the disease. Our results suggest that KRAS mutation status could have some predictive and prognostic importance in rectal cancer when analyzed together with other parameters, such as VEGF and Ki67 expression. In addition, it seems that not only the presence but the type of KRAS mutation is important for examining its impact on CRT response.

Forensic application of phylogenetic analysis – exploration of suspected epidemiological linkage

Phylogenetic analysis may serve as a valuable tool in assessing the epidemiological relation between viral DNA sequences. In order to increase the likelihood of observing phylogenetic separation of sequences as well as to give strong forensic evidence regarding transmission, phylogenetic analysis needs to be performed on appropriate local control sequences and by sequencing of at least two genetic regions of reasonable length, depending on the gene under investigation. The aim of this study was to explore the suspected epidemiological linkage between DNA sequences isolated from three HIV-1 infected patients by means of phylogenetic analyses. Phylogenetic analysis was performed on investigated sequences together with a number of local controls, that are viral sequences from infected individuals in the same location and diagnosed in a similar time period. Two genetic regions, of 1,6 kb for pol and 800 bp for env, were amplified and sequenced from viral RNA extracted from plasma. Transmission clusters were assigned as those phylogenetic clades consisting of three or more sequences, fulfilling the conditions of genetic distance of 1.5% or less with posterior probability higher than 0.9 in the Bayesian analyses, for both genetic region. Maximum likelihood (ML) phylogenetic analysis was performed as implemented in Phylogenetic Analysis Using Parsimony (PAUP), using the evolutionary model selected by jModeltest software. Phylogenetic analysis revealed the presence of a single transmission cluster that accomplished all predefined sets of criteria. This cluster was composed of three viral sequences isolated from patients whose epidemiological linkage was under investigation. The mean pairwise nucleotide divergence among all observed pol sequences was 7.1% (range 0.2-12.8%) while among sequences under investigation it was 0.8% (0.3-1.1%). Regarding env sequences, nucleotide divergence among all was 16.1% (range 0.3-26.3%), while among sequences under investigation it was 1.3% (1.1-1.5%). Our results strongly support the epidemiological linkage between sequences under investigation. Viral phylogenies can help to evaluate proposed epidemiological linkage and to infer the ancestral relationships of infections. However phylogenetic analysis cannot unambiguously prove that HIV-1 transmission occurred directly between two individuals, since any transmission chain may contain additional sequences not included in the analysis.

The prevalence of RT 245 codon polymorphisms and its association with duration of infection among HIV-1 patients in Serbia

Amino acid (aa) substitutions at position 245 of HIV-1 reverse transcriptase (RT) have been described to be associated to the presence of human leukocyte antigen (HLA)–B*5701 allele in the host, in particular in subtype B infection. Preliminary data show HLA–B*5701 prevalence of around 6.5% in Serbian population. In this study, we investigated the prevalence of RT codon 245 substitutions among HIV infected patients in Serbia. Furthermore, we analyzed the association between RT 245 aa substitutions with duration of infection, estimated by proportion of ambiguous basecalls per sequence. The study included 184 consenting, subtype B HIV infected patients aged 18 or more. The majority of patients were newly diagnosed, 150/184, while 34/184 patients were on treatment. Pol region sequences, covering protease and minimally 250 RT codons, were obtained within routine drug resistance testing. The fraction of ambiguous nucleotides in each sequence was calculated for samples drawn from naive patients. We used ambiguity percentage of 0.47% as a cut-off value delimiting recent (less than 1 year) vs. chronic infection (longer than 1 year). In total, predominant aa at RT codon 245 was the wild type valin (V) found in 118/184 (64.1%), hence 35.9% (66/184) contained mutation at this position (among naive patients this percentage was 38% (57/150). The most common substitution at RT codon 245 was methionine (M) 29/184 (15.7%), followed by glutamic acid (E) 20/184 (10.9%) and others. Based on the percentage of ambiguous basecalls, a total of 55.3% of naive samples (83/150) were classified as recent infection, while among these, 57.8% (48/83) had V at position 245. A total of 45.3% (68/150) were classified as chronic infection, with the presence of V at RT codon 245 found in 69.1% (47/68). We did not find statistically significant association between polymorphisms at codon 245 and duration of infection (p=0.2080). The frequency of RT 245 substitution found in our study exceeds the estimated prevalence HLA–B*5701 in Serbian population. This may be related to the presence of other, similar HLA alleles, limiting specificity of the correlation between HLA-B*5701 and RT codon 245 variation. Furthermore, no statistically significant difference was found in the prevalence of RT 245 substitutions between recent and chronic infection. This may suggest early fixation of an HLA induced selective imprint, during viral evolution in an infected patient.