Full-genome evolutionary analysis of the novel corona virus (2019-nCoV) rejects the hypothesis of emergence as a result of a recent recombination event.

BACKGROUND: A novel coronavirus (2019-nCoV) associated with human to human transmission and severe human infection has been recently reported from the city of Wuhan in China. Our objectives were to characterize the genetic relationships of the 2019-nCoV and to search for putative recombination within the subgenus of sarbecovirus. METHODS: Putative recombination was investigated by RDP4 and Simplot v3.5.1 and discordant phylogenetic clustering in individual genomic fragments was confirmed by phylogenetic analysis using maximum likelihood and Bayesian methods. RESULTS: Our analysis suggests that the 2019-nCoV although closely related to BatCoV RaTG13 sequence throughout the genome (sequence similarity 96.3%), shows discordant clustering with the Bat_SARS-like coronavirus sequences. Specifically, in the 5'-part spanning the first 11,498 nucleotides and the last 3'-part spanning 24,341-30,696 positions, 2019-nCoV and RaTG13 formed a single cluster with Bat_SARS-like coronavirus sequences, whereas in the middle region spanning the 3'-end of ORF1a, the ORF1b and almost half of the spike regions, 2019-nCoV and RaTG13 grouped in a separate distant lineage within the sarbecovirus branch. CONCLUSIONS: The levels of genetic similarity between the 2019-nCoV and RaTG13 suggest that the latter does not provide the exact variant that caused the outbreak in humans, but the hypothesis that 2019-nCoV has originated from bats is very likely. We show evidence that the novel coronavirus (2019-nCov) is not-mosaic consisting in almost half of its genome of a distinct lineage within the betacoronavirus. These genomic features and their potential association with virus characteristics and virulence in humans need further attention.

Prevalence of drug resistance among HIV-1 treatment-naive patients in Greece during 2003-2015: Transmitted drug resistance is due to onward transmissions.

BACKGROUND: The prevalence of HIV-1 drug resistance among treatment-naïve patients ranges between 8.3% and 15% in Europe and North America. Previous studies showed that subtypes A and B were the most prevalent in the Greek HIV-1 epidemic. Our aim was to estimate the prevalence of resistance among drug naïve patients in Greece and to investigate the levels of transmission networking among those carrying resistant strains. METHODS: HIV-1 sequences were determined from 3428 drug naïve HIV-1 patients, in Greece sampled during 01/01/2003-30/6/2015. Transmission clusters were estimated by means of phylogenetic analysis including as references sequences from patients failing antiretroviral treatment in Greece and sequences sampled globally. RESULTS: The proportion of sequences with SDRMs was 5.98% (n=205). The most prevalent SDRMs were found for NNRTIs (3.76%), followed by N(t)RTIs (2.28%) and PIs (1.02%). The resistance prevalence was 22.2% based on all mutations associated with resistance estimated using the HIVdb resistance interpretation algorithm. Resistance to NNRTIs was the most common (16.9%) followed by PIs (4.9%) and N(t)RTIs (2.8%). The most frequently observed NNRTI resistant mutations were E138A (7.7%), E138Q (4.0%), K103N (2.3%) and V179D (1.3%). The majority of subtype A sequences (89.7%; 245 out of 273) with the dominant NNRTI resistance mutations (E138A, K103N, E138Q, V179D) were found to belong to monophyletic clusters suggesting regional dispersal. For subtype B, 68.1% (139 out of 204) of resistant strains (E138A, K103N, E138Q V179D) belonged to clusters. For N(t)RTI-resistance, evidence for regional dispersal was found for 27.3% and 21.6% of subtype A and B sequences, respectively. CONCLUSIONS: The TDR rate based on the prevalence of SDRM is lower than the average rate in Europe. However, the prevalence of NNRTI resistance estimated using the HIVdb approach, is high in Greece and it is mostly due to onward transmissions among drug-naïve patients.

The application of HIV molecular epidemiology to public health.

HIV is responsible for one of the largest viral pandemics in human history. Despite a concerted global response for prevention and treatment, the virus persists. Thus, urgent public health action, utilizing novel interventions, is needed to prevent future transmission events, critical to eliminating HIV. For public health planning to prove effective and successful, we need to understand the dynamics of regional epidemics and to intervene appropriately. HIV molecular epidemiology tools as implemented in phylogenetic, phylodynamic and phylogeographic analyses have proven to be powerful tools in public health planning across many studies. Numerous applications with HIV suggest that molecular methods alone or in combination with mathematical modelling can provide inferences about the transmission dynamics, critical epidemiological parameters (prevalence, incidence, effective number of infections, Re, generation times, time between infection and diagnosis), or the spatiotemporal characteristics of epidemics. Molecular tools have been used to assess the impact of an intervention and outbreak investigation which are of great public health relevance. In some settings, molecular sequence data may be more readily available than HIV surveillance data, and can therefore allow for molecular analyses to be conducted more easily. Nonetheless, classic methods have an integral role in monitoring and evaluation of public health programmes, and should supplement emerging techniques from the field of molecular epidemiology. Importantly, molecular epidemiology remains a promising approach in responding to viral diseases.

Phylogenetic clustering of hepatitis C virus among people who inject drugs in Vancouver, Canada.

UNLABELLED: Little is known about factors associated with hepatitis C virus (HCV) transmission among people who inject drugs (PWID). Phylogenetic clustering and associated factors were evaluated among PWID in Vancouver, Canada. Data were derived from the Vancouver Injection Drug Users Study. Participants who were HCV antibody-positive at enrolment and those with HCV antibody seroconversion during follow-up (1996 to 2012) were tested for HCV RNA and sequenced (Core-E2 region). Phylogenetic trees were inferred using maximum likelihood analysis and clusters were identified using ClusterPicker (90% bootstrap threshold, 0.05 genetic distance threshold). Factors associated with clustering were assessed using logistic regression. Among 655 eligible participants, HCV genotype prevalence was: G1a: 48% (n=313), G1b: 6% (n=41), G2a: 3% (n=20), G2b: 7% (n=46), G3a: 33% (n=213), G4a: <1% (n=4), G6a: 1% (n=8), G6e: <1% (n=1), and unclassifiable: 1% (n=9). The mean age was 36 years, 162 (25%) were female, and 164 (25%) were HIV+. Among 501 participants with HCV G1a and G3a, 31% (n=156) were in a pair/cluster. Factors independently associated with phylogenetic clustering included: age <40 (versus age≥40, adjusted odds ratio [AOR]=1.64; 95% confidence interval [CI] 1.03, 2.63), human immunodeficiency virus (HIV) infection (AOR=1.82; 95% CI 1.18, 2.81), HCV seroconversion (AOR=3.05; 95% CI 1.40, 6.66), and recent syringe borrowing (AOR 1.59; 95% CI 1.07, 2.36). CONCLUSION: In this sample of PWID, one-third demonstrated phylogenetic clustering. Factors independently associated with phylogenetic clustering included younger age, recent HCV seroconversion, prevalent HIV infection, and recent syringe borrowing. Strategies to enhance the delivery of prevention and/or treatment strategies to those with HIV and recent HCV seroconversion should be explored, given an increased likelihood of HCV transmission in these subpopulations.

Prevalence and clinical manifestations of ankylosing spondylitis in young Greek males.

Increased awareness and sensitivity of general physicians have increased the early diagnoses of seronegative arthritis in young patients, while new agents such as anti-TNF blockers have significantly changed the treatment of the disease. To investigate the prevalence, the clinical manifestations, and the ability for military service of young Greek males (18-30 years old) with ankylosing spondylitis (AS) in the pre-anti-TNF era. We retrospectively studied the AS cases recorded from 1989 to 1995 of the rheumatology department of the largest General Military Hospital in Greece; the diagnosis was based on the modified New York criteria for AS. A total of 285 AS cases were diagnosed among 357,184 young men. The overall prevalence of AS on December 1995 was estimated at 8.2 cases per 10,000 young men (95 % C.I. 7.2-9.2). All the patients had chronic back pain. Two hundred forty (84 %, 95 % C.I. 79-88 %) patients presented sacroiliitis of whom 163 (68 %, 95 % C.I. 62-73 %) were bilateral. Two hundred five patients (72 %, 95 % C.I. 66-77 %) had peripheral joint involvement. Thirty-one patients presented with anterior uveitis (11 %, 95 % C.I. 8-15 %). One patient had IgA nephropathy. None had gut involvement. HLA-B27 antigen was found in 257 patients (90 %, 95 % C.I. 86-93 %). Ninety-one patients (32 %, 95 % C.I. 27-38 %) had permanent discharge from the military service, while 128 (45 %, 95 % C.I. 39-51 %) were able for auxiliaries attendances. The prevalence of AS for the age group 18-30 years old in this young Greek men cohort was significantly lower than in other Caucasian European populations, and the clinical manifestations were considered as mild.

Mediterranean diet intervention for patients with hyperuricemia: a pilot study.

Dietary interventions have been suggested to be a safe cost-efficient way to control hyperuricemia. The aim of the study is to assess the potential of mediterranean diet as intervention to control the level of urate in patients with hyperuricemia in a small sample of patients. Patients with asymptomatic hyperuricemia were recruited from outpatient clinics and were enrolled into personal Mediterranean diet-based programs. Body mass index (BMI), serum urate, lipid profile and indirect calorimetry were measured at the beginning and then monthly for the first 3 months and then at the sixth month. At the same time, patients' compliance with the Mediterranean diet was assessed by a formal interview and standard questionnaire. Only six out of twelve patients managed to complete the diet (dropout rate 50 %). Their BMI remained constant during the trial period in the level of 1st degree obesity (BMI = 31.46). The mean value of serum urate at the beginning of the study was 9.12 mg/dl. After the first month, there was a reduction in urate by 20 % with mean urate at 6.92 mg/dl. The second, third and sixth month mean urate levels were 6.32, 6.1 and 6.4 mg/dl, respectively. The effect of the mediterranean diet was rapid at the first month and remained constant throughout the dietary intervention, suggesting that it might have a clinically significant effect on urate level thus providing a cost-efficient and safe alternative to pharmaceutical intervention as first-line treatment of hyperuricemia.

Quantitative detection of the M204V hepatitis B virus minor variants by amplification refractory mutation system real-time PCR combined with molecular beacon technology.

Mutations in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif are frequently associated with resistance to antivirals and represent a major concern in the treatment of hepatitis B virus (HBV) infection. Conventional methods fail to detect minority populations of drug-resistant viral quasispecies if they represent less than 25% of the total sample virus population. The amplification refractory mutation system real-time PCR (ARMS RT-PCR) was combined with molecular beacon technology using the LightCycler system. The samples from HBV patients selected for assay evaluation included (i) 57 samples from treatment-naïve patients for biological discriminatory ability (cutoff) estimation, (ii) 12 samples from patients with treatment failure that were M204V positive by sequencing, and (iii) 13 samples from patients with treatment failure that were negative for mutation at codon 204 by sequencing. The discriminatory ability of the assay was 0.25% when tested with laboratory-synthesized DNA target sequences. The median mutant-to-wild-type ratio for samples from naive patients tested positive for the wild type and for mutant variants was 0.01% (5th and 95th percentiles = 0.0001 and 0.04%, respectively). A value of 0.04% was selected as the biological cutoff of the assay of clinical samples. In all samples M204V positive by sequencing (12/12), the mutant variant was detected as the predominant population (range, 82.76 to 99.43%). Interestingly, in 5 (38%) of 13 samples negative by sequencing, the M204V variant was detected at a ratio above the biological cutoff (0.05 to 28%). The assay represents an efficient technique for the early detection and quantification of M204V variants before mutant strains emerge to dominate the population.

The phylogenetic information profile of HIV-1 and the degradation effect of recombination.

HIV-1, while known to recombine frequently and evolve rapidly, is one of the most sequenced organisms. The availability of many and long sequences (almost full-length) renders HIV-1 as a good model for studying theoretical predictions linked to evolution and phylogenetic inferences. Here we study the effects of rapid and through-recombination evolution on phylogenetic information in order to confirm theoretical predictions of the characteristics of phylogenetic information on a real dataset. Firstly we study the fluctuation of the phylogenetic information along the HIV-1 genome showing that genomic regions such as the first part and the last part of the pol gene contain less phylogenetic information, while the vpr, vpu and the first exon of the tat gene contain more phylogenetic information compared to the rest of the genome. Moreover, we provide evidence that phylogenetic information is correlated to the sequence similarity of the dataset used and is degraded by the effect of recombination.

Analysing the evolutionary history of HCV: puzzle of ancient phylogenetic discordance.

Though recombination is an important evolutionary strategy in RNA viruses, only two cases of HCV recombinant strains have been reported. Our objective was to analyze the evolutionary history of the HCV genotypes aiming to obtain evidence of significant phylogenetic discordance due to either recombination or selective forces leading to convergent/divergent evolution. The data support an evolutionary preservation of the interferon-resistance related genomic region (ISDR) for the genotypes 1 and 4. On the other hand, there was no evidence that recombination has occurred in the past with the possible exception of genotype 4. Moreover, it is evidenced that genotypes 3 and 10 split more recently than genotypes 6-9 and 11. This analysis reverberates a commonly found pattern in rapidly evolving viruses, that is the strongly disturbed evolutionary history which deforms the uniform distribution of the phylogenetic relationships across the genome, and introduces a conservative inference framework for approaching this kind of data.

Molecular epidemiology of hepatitis C virus (HCV) in Greece: temporal trends in HCV genotype-specific incidence and molecular characterization of genotype 4 isolates.

This study aimed to estimate the overall HCV genotype distribution and to reconstruct the HCV genotype-specific incidence in Greece during the recent decades. It also focused at the identification of genotype 4 subtype variability in Greek isolates. A total of 1686 chronically infected HCV patients with detectable serum HCV RNA by RT-PCR, belonging to different risk groups were studied. Amplified products from the 5'-noncoding region were typed using a commercially available assay based on the reverse hybridization principle. The HCV genotype-specific incidence was estimated using a previously described back calculation method. HCV genotype 1 was the most prevalent (46.9%) followed by genotype 3 (28.1%), 4 (13.2%), 2 (6.9%) and 5 (0.4%). A high prevalence of genotype 1 (66.3%) in haemophilia patients was recorded whereas HCV genotype 3 was found mainly among patients infected by I.V. drug use (58.2%). Data on the temporal patterns of HCV genotype-specific incidence in Greece revealed a moderate increase (1.3-1.6 times) for genotypes 1 and 4, and a decrease (1.5 times) for genotype 2 from 1970 to 1990, whereas there was a sharp (13-fold) increase for genotype 3. The molecular characterization of 41 genotype 4 HCV isolates belonging to various risk groups revealed that, subtype 4a was the most frequently detected (78%). Phylogenetic comparison of the Greek 4a isolates with all HCV-4a isolates reported worldwide so far revealed a topology which does not discriminate Greek isolates from the others. HCV-4 does not represent a recent introduction in Greece.

Re-analysis of 34 full-length HIV-1 intersubtype recombinant sequences.

One of the main characteristics of the HIV-1 is its extensive genetic heterogeneity. Intersubtype recombination was first described in 1995 and since then a significant proportion of the HIV-1 isolates was found to comprise mosaic sequences. Re-analysis of 34 full-length HIV-1 intersubtype recombinants, including all "pure" HIV-1 subtypes revealed that 19 of the 34 analyzed mosaics consist of a more complex mosaic pattern than initially described. These findings indicate that the complexity of the HIV-1 recombinants is much greater than previously estimated.

SlidingBayes: exploring recombination using a sliding window approach based on Bayesian phylogenetic inference.

We developed a software tool (SlidingBayes) for recombination analysis based on Bayesian phylogenetic inference. Sliding-Bayes provides a powerful approach for detecting potential recombination, especially between highly divergent sequences and complex HIV-1 recombinants for which simpler methods like neighbor joining (NJ) may be less powerful. SlidingBayes guides Markov Chain Monte Carlo (MCMC) sampling performed by MrBayes in a sliding window across the alignment (Bayesian scanning). The tool can be used for nucleotide and amino acid sequences and combines all the modeling possibilities of MrBayes with the ability to plot the posterior probability support for clustering of various combinations of taxa.

Phylogenetic analysis of the full-length SARS-CoV sequences: evidence for phylogenetic discordance in three genomic regions.

The origin of the severe acute respiratory syndrome-coronavirus (SARS-CoV) remains unclear. Evidence based on Bayesian scanning plots and phylogenetic analysis using maximum likelihood (ML) and Bayesian methods indicates that SARS-CoV, for the largest part of the genome ( approximately 80%), is more closely related to Group II coronaviruses sequences, whereas in three regions in the ORF1ab gene it shows no apparent similarity to any of the previously characterized groups of coronaviruses. There is discordant phylogenetic clustering of SARS-CoV and coronaviruses sequences, throughout the genome, compatible with either ancient recombination events or altered evolutionary rates in different lineages, or a combination of both.

Mutations associated with genotypic resistance to antiretroviral therapy in treatment naïve HIV-1 infected patients in Greece.

The widespread use of antiviral drugs against HIV has increased the prevalence of HIV-1 resistant strains among naïve individuals due to transmission of resistant strains. The purpose of this study was to investigate the presence of HIV-1 strains harboring resistance mutations in naïve patients in Greece. Blood samples were collected from 25 individuals. The DNA sequence of protease and partial reverse transcriptase regions (codons 41-223) were obtained by direct sequencing. Our results showed the absence of any primary resistance mutations in the study population. However, we were able to identify high prevalence of sequence polymorphisms at positions in reverse transcriptase region associated mainly with resistance to NNRTIs. Moreover, in protease region several secondary mutations were detected, suggesting the higher genetic variability of this region. The clinical significance of the polymorphisms associated with reduced susceptibility to NNRTIs remains to be clarified.

Molecular characterization of a complex, recombinant human immunodeficiency virus type 1 (HIV-1) isolate (A/G/J/K/?): evidence to support the existence of a novel HIV-1 subtype.

Recombination is one of several factors that contribute to the great genetic diversity of human immunodeficiency virus type 1 (HIV-1). In the current study, analysis of the full-length genome of a novel complex mosaic HIV-1 isolate (99GR303) from a Greek sailor who was possibly infected in Sierra Leone, Africa is presented. The 99GR303 isolate was found to comprise genomic regions belonging to subtypes A, G, J and K as well as of regions of a subtype that remains unclassified. For a partial region of env as well as vpr, no apparent similarity to the known HIV-1 subtypes or to any of the circulating recombinant forms was found. In fact, in the partial env gene, including the C2-V3 region, the 99GR303 isolate formed a new clade, suggesting the existence of an additional HIV-1 subtype. Thus, novel recombinants embody partial genomic regions which may have originated either from subtypes that existed in the past and became extinct or from contemporary subtypes that are extremely rare.