Molecular characterization and epidemic history of hepatitis C virus using core sequences of isolates from Central Province, Saudi Arabia.

The source of HCV transmission in Saudi Arabia is unknown. This study aimed to determine HCV genotypes in a representative sample of chronically infected patients in Saudi Arabia. All HCV isolates were genotyped and subtyped by sequencing of the HCV core region and 54 new HCV isolates were identified. Three sets of primers targeting the core region were used for both amplification and sequencing of all isolates resulting in a 326 bp fragment. Most HCV isolates were genotype 4 (85%), whereas only a few isolates were recognized as genotype 1 (15%). With the assistance of Genbank database and BLAST, subtyping results showed that most of genotype 4 isolates were 4d whereas most of genotype 1 isolates were 1b. Nucleotide conservation and variation rates of HCV core sequences showed that 4a and 1b have the highest levels of variation. Phylogenetic analysis of sequences by Maximum Likelihood and Bayesian Coalescent methods was used to explore the source of HCV transmission by investigating the relationship between Saudi Arabia and other countries in the Middle East and Africa. Coalescent analysis showed that transmissions of HCV from Egypt to Saudi Arabia are estimated to have occurred in three major clusters: 4d was introduced into the country before 1900, the major 4a clade's MRCA was introduced between 1900 and 1920, and the remaining lineages were introduced between 1940 and 1960 from Egypt and Middle Africa. Results showed that no lineages seem to have crossed from Egypt to Saudi Arabia in the last 15 years. Finally, sequencing and characterization of new HCV isolates from Saudi Arabia will enrich the HCV database and help further studies related to treatment and management of the virus.

Evaluation of Viremia Frequencies of a Novel Human Pegivirus by Using Bioinformatic Screening and PCR.

Next-generation sequencing has critical applications in virus discovery, diagnostics, and environmental surveillance. We used metagenomic sequence libraries for retrospective screening of plasma samples for the recently discovered human hepegivirus 1 (HHpgV-1). From a cohort of 150 hepatitis C virus (HCV)-positive case-patients, we identified 2 persons with HHpgV-1 viremia and a high frequency of human pegivirus (HPgV) viremia (14%). Detection of HHpgV-1 and HPgV was concordant with parallel PCR-based screening using conserved primers matching groups 1 (HPgV) and 2 (HHPgV-1) nonstructural 3 region sequences. PCR identified 1 HHPgV-1-positive person with viremia from a group of 195 persons with hemophilia who had been exposed to nonvirally inactivated factor VII/IX; 18 (9%) were HPgV-positive. Relative to HCV and HPgV, active infections with HHpgV-1 were infrequently detected in blood, even in groups that had substantial parenteral exposure. Our findings are consistent with lower transmissibility or higher rates of virus clearance for HHpgV-1 than for other bloodborne human flaviviruses.

Epidemic History and Iatrogenic Transmission of Blood-borne Viruses in Mid-20th Century Kinshasa.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) pandemic was ignited in Léopoldville (now known as Kinshasa), in the former Belgian Congo. Factors that jump-started its early expansion remain unclear. Nonlethal hepatitis C virus (HCV) and human T-cell lymphotropic virus (HTLV-1) can be used to investigate past iatrogenic transmission. METHODS: We undertook a cross-sectional study of elderly inhabitants of Kinshasa, with serological assays, amplification, and sequencing. Risk factors were assessed through logistic regression. Phylogenetic methods reconstructed the genetic history of HCV. RESULTS: A total of 217 of 839 participants (25.9%) were HCV seropositive; 26 (3.1%) were HTLV-1-seropositive. Amplification products were obtained from 118 HCV-seropositive participants; subtypes 4k (in 47 participants) and 4r (in 38) were most common. Independent risk factors for HCV subtype 4r seropositivity were intramuscular tuberculosis therapy, intravenous injections at hospital A, intravenous injections before 1960, and injections at a colonial-era venereology clinic. Intravenous injections at hospital B and antimalarials were associated with HCV subtype 4k seropositivity. Risk factors for HTLV-1 seropositivity included intravenous injections at hospitals C or D and transfusions. Evolutionary analysis of viral sequences revealed independent exponential amplification of HCV subtypes 4r and 4k from the 1950s onward. CONCLUSIONS: Iatrogenic transmission of HCV and HTLV-1 occurred in mid-20th century Kinshasa, at the same time and place HIV-1 emerged. Iatrogenic routes may have contributed to the early establishment of the pandemic.

Characterization of Hepatitis C Virus Recombination in Cameroon by Use of Nonspecific Next-Generation Sequencing.

The importance of recombination in the evolution and genetic diversity of the hepatitis C virus (HCV) is currently uncertain. Only a small number of intergenotypic recombinants have been identified so far, and each has core and envelope genes classified as belonging to genotype 2. Here, we investigated two putative genotype 4/1 recombinants from southern Cameroon using a number of approaches, including standard Sanger sequencing, genotype-specific PCR amplification, and non-HCV-specific Illumina RNA sequencing (RNA-seq). Recombination between genotypes 1 and 4 was confirmed in both samples, and the parental lineages of each recombinant belong to HCV subtypes that are cocirculating at a high prevalence in Cameroon. Using the RNA-seq approach, we obtained a complete genome for one sample, which contained a recombination breakpoint at the E2/P7 gene junction. We developed and applied a new method, called Deep SimPlot, which can be used to visualize and identify viral recombination directly from the short sequence reads created by next-generation sequencing in conjunction with a consensus sequence.

Phylogeography and epidemic history of hepatitis C virus genotype 4 in Africa.

HCV genotype 4 is prevalent in many African countries, yet little is known about the genotype׳s epidemic history on the continent. We present a comprehensive study of the molecular epidemiology of genotype 4. To address the deficit of data from the Democratic Republic of the Congo (DRC) we PCR amplified 60 new HCV isolates from the DRC, resulting in 33 core- and 48 NS5B-region sequences. Our data, together with genotype 4 database sequences, were analysed using Bayesian phylogenetic approaches. We find three well-supported intra-genotypic lineages and estimate that the genotype 4 common ancestor existed around 1733 (1650-1805). We show that genotype 4 originated in central Africa and that multiple lineages have been exported to north Africa since ~1850, including subtype 4a which dominates the epidemic in Egypt. We speculate on the causes of the historical intra-continental spread of genotype 4, including population movements during World War 2.

Hepatitis C virus infections in the Democratic Republic of Congo exhibit a cohort effect.

The prevalence and genetic diversity of hepatitis C virus (HCV) and human pegivirus (HPgV) in many regions of sub-Saharan Africa is poorly characterized, including in the Democratic Republic of Congo--the largest country in the region and one of the most populous. To address this situation we conducted a molecular epidemiological survey of HCV and HPgV (previously named GB Virus C or hepatitis G virus) in samples collected in 2007 from 299 males from the DRC, whose ages ranged from 21 to 71 years old. Samples were tested for the presence of HCV antibodies by ELISA and reactive samples were subsequently tested for HCV RNA using RT-PCR in which both the HCV Core and NS5B genome regions were amplified. Remaining samples were tested for HPgV RNA and the HPgV NS3 genome region of positive samples was amplified. For HCV, 13.7% of the samples were seropositive (41/299) but only 3.7% were viremic (11/299). HPgV RNA was found in 12.7% (33/259) of samples. HCV viremia was strongly associated with age; the percentage of samples that contained detectable HCV RNA was ~0.5% in those younger than 50 and 13% in those older than 50. Our study represents the first systematic survey of HCV genetic diversity in the DRC. HCV sequences obtained belonged to diverse lineages of genotype 4, including subtypes 4c, 4 k, 4 l and 4r, plus one unclassified lineage that may constitute a new subtype. These data suggest that HCV in the DRC exhibits an age 'cohort effect', as has been recently reported in neighbouring countries, and are consistent with the hypothesis that HCV transmission rates were higher in the mid-twentieth century, possibly as a result of parenteral, iatrogenic, or other unidentified factors. Different HCV subtypes were associated with individuals of different ages, implying that HCV infection in the DRC may have arisen through multiple separate HCV epidemics with different causes.

The fitness cost of rifampicin resistance in Pseudomonas aeruginosa depends on demand for RNA polymerase.

Bacterial resistance to antibiotics usually incurs a fitness cost in the absence of selecting drugs, and this cost of resistance plays a key role in the spread of antibiotic resistance in pathogen populations. Costs of resistance have been shown to vary with environmental conditions, but the causes of this variability remain obscure. In this article, we show that the average cost of rifampicin resistance in the pathogenic bacterium Pseudomonas aeruginosa is reduced by the addition of ribosome inhibitors (chloramphenicol or streptomycin) that indirectly constrain transcription rate and therefore reduce demand for RNA polymerase activity. This effect is consistent with predictions from metabolic control theory. We also tested the alternative hypothesis that the observed trend was due to a general effect of environmental quality on the cost of resistance. To do this we measured the fitness of resistant mutants in the presence of other antibiotics (ciprofloxacin and carbenicillin) that have similar effects on bacterial growth rate but bind to different target enzymes (DNA gyrase and penicillin-binding proteins, respectively) and in 41 single-carbon source environments of varying quality. We find no consistent effect of environmental quality on the average cost of resistance in these treatments. These results show that the cost of rifampicin resistance varies with demand for the mutated target enzyme, rather than as a simple function of bacterial growth rate or stress.