Kirsi Liitsola

HIV-1 genetic subtype A/B recombinant strain causing an explosive epidemic in injecting drug users in Kaliningrad

Objectives:To investigate the molecular epidemiology and genetic structure of the virus strain(s) causing an outbreak of HIV-1 infection in the Kaliningrad province of the Russian Federation and to investigate the relationship of this outbreak to some other emerging HIV-1 epidemics in the countries of the former Soviet Union. Design:A molecular epidemiological investigation was conducted in the city of Kaliningrad amongst individuals recently diagnosed as HIV-1-positive. Samples were also collected from neighbouring Lithuania and from the Ukraine. Methods:Incident and population data was collected from official health statistics in Kaliningrad. A standardized questionnaire was administered to newly diagnosed individuals to assess risk factors for HIV-1 infection. For genotyping, two regions of the virus (env C2-V3 and gag NCp7) were directly sequenced. Results:The number of newly diagnosed individuals testing seropositive for HIV-1 infection in Kaliningrad rose from less than one per month to more than 100 per month during the period of July-October 1996. A total of 1335 new infections were identified between 1 July 1996 and 30 June 1997. The main reported risk factor for HIV-1 infection (80%) was injecting drug use, in particular with a locally produced opiate. Sequence analysis of patient viruses in Kaliningrad (n = 50) showed that the epidemic was caused by a highly homogenous HIV-1 strain, recombinant between the genetic subtypes A and B. Comparison with subtype A strains prevalent amongst injecting drug users (IDU) in the Ukraine showed that one of these strains was the direct subtype A parent of the epidemic A/B recombinant strain in Kaliningrad. Conclusions:The HIV-1 epidemic in Kaliningrad probably started from a single source, with rapid spread of the virus through the IDU population. The origin of the epidemic strain is a recombination event occurring between the subtype A strain virus prevalent among IDU in some southern CIS countries, and a subtype B strain of unknown origin.

HIV-1 subtype distribution and its demographic determinants in newly diagnosed patients in Europe suggest highly compartmentalized epidemics

BackgroundUnderstanding HIV-1 subtype distribution and epidemiology can assist preventive measures and clinical decisions. Sequence variation may affect antiviral drug resistance development, disease progression, evolutionary rates and transmission routes.ResultsWe investigated the subtype distribution of HIV-1 in Europe and Israel in a representative sample of patients diagnosed between 2002 and 2005 and related it to the demographic data available. 2793 PRO-RT sequences were subtyped either with the REGA Subtyping tool or by a manual procedure that included phylogenetic tree and recombination analysis. The most prevalent subtypes/CRFs in our dataset were subtype B (66.1%), followed by sub-subtype A1 (6.9%), subtype C (6.8%) and CRF02_AG (4.7%). Substantial differences in the proportion of new diagnoses with distinct subtypes were found between European countries: the lowest proportion of subtype B was found in Israel (27.9%) and Portugal (39.2%), while the highest was observed in Poland (96.2%) and Slovenia (93.6%). Other subtypes were significantly more diagnosed in immigrant populations. Subtype B was significantly more diagnosed in men than in women and in MSM > IDUs > heterosexuals. Furthermore, the subtype distribution according to continent of origin of the patients suggests they acquired their infection there or in Europe from compatriots.ConclusionsThe association of subtype with demographic parameters suggests highly compartmentalized epidemics, determined by social and behavioural characteristics of the patients.

An AB recombinant and its parental HIV type 1 strains in the area of the former Soviet Union : Low requirements for sequence identity in recombination

In the former Soviet Union (SU) increasing numbers of HIV-1 infections among injecting drug users (IDU) have been reported, especially in the Ukraine. The main subtype transmitted among the IDUs seems to be subtype A, but limited numbers of subtype B cases have also been reported. In Kaliningrad, Russia, an AB recombinant strain was earlier shown to be responsible for the local outbreak. Here we describe the genetic relationship of HIV-1 strains circulating among IDUs in the former SU. For subtype A and the AB recombinant strains nearly full-length genomes were sequenced, and for one subtype B strain the entire envelope gene was cloned. The relationship between the AB recombinant strain and the subtype A and subtype B strains and the mosaic structure of the recombinant was studied by phylogenetic analysis. Ukrainian A and B strains were shown to be the probable parental viruses of the Kaliningrad AB recombinant strain. In the envelope gene the recombination breakpoint could also be precisely mapped to a region of similarity of only 14 base pairs. This suggests that only short stretches of absolute sequence identity may be needed for efficient RNA recombination between HIV-1 subtypes.

Transmission of HIV Drug Resistance and the Predicted Effect on Current First-line Regimens in Europe

Transmitted human immunodeficiency virus drug resistance in Europe is stable at around 8%. The impact of baseline mutation patterns on susceptibility to antiretroviral drugs should be addressed using clinical guidelines. The impact on baseline susceptibility is largest for nonnucleoside reverse transcriptase inhibitors.

Patterns of transmitted HIV drug resistance in Europe vary by risk group

Background In Europe, a continuous programme (SPREAD) has been in place for ten years to study transmission of drug resistant HIV. We analysed time trends of transmitted drug resistance mutations (TDRM) in relation to the risk behaviour reported. Methods HIV-1 patients newly diagnosed in 27 countries from 2002 through 2007 were included. Inclusion was representative for risk group and geographical distribution in the participating countries in Europe. Trends over time were calculated by logistic regression. Results From the 4317 patients included, the majority was men-having-sex-with-men -MSM (2084, 48%), followed by heterosexuals (1501, 35%) and injection drug users (IDU) (355, 8%). MSM were more often from Western Europe origin, infected with subtype B virus, and recently infected (<1 year) (p<0.001). The prevalence of TDRM was highest in MSM (prevalence of 11.1%), followed by heterosexuals (6.6%) and IDU (5.1%, p<0.001). TDRM was predominantly ascribed to nucleoside reverse transcriptase inhibitors (NRTI) with a prevalence of 6.6% in MSM, 3.3% in heterosexuals and 2.0% in IDU (p = 0.001). A significant increase in resistance to non- nucleoside reverse transcriptase inhibitors (NNRTIs) and a decrease in resistance to protease inhibitors was observed in MSM (p = 0.008 and p = 0.006, respectively), but not in heterosexual patients (p = 0.68 and p = 0.14, respectively). Conclusions MSM showed to have significantly higher TDRM prevalence compared to heterosexuals and IDU. The increasing NNRTI resistance in MSM is likely to negatively influence the therapy response of first-line therapy, as most include NNRTI drugs.

Serodiagnosis of Primary Infections with Human Parvovirus 4, Finland

To determine the prevalence of parvovirus 4 infection and its clinical and sociodemographic correlations in Finland, we used virus-like particle–based serodiagnostic procedures (immunoglobulin [Ig] G, IgM, and IgG avidity) and PCR. We found 2 persons with parvovirus 4 primary infection who had mild or asymptomatic clinical features among hepatitis C virus–infected injection drug users.

Virtually Full-Length Sequences of HIV Type 1 Subtype J Reference Strains

293 THE RAPID VIRAL TURNOVER and high mutation rate of HIV1 result in remarkable genetic variation among strains obtained from different parts of the world. This has led to the development of evolutionary lineages (genetic subtypes) that can be identified by phylogenetic analysis of gene sequences.2,3 Eight genetic subtypes, named A through H in group M (major) and a group of other strains, group O (outlier), have previously been determined by phylogenetic analysis, mainly of gag and env genes. The genetic subtype classification is based on consistent clustering of the sequences in different parts of the genome, and approximate equidistance to strains belonging to other subtypes. Two additional subtypes, I and J, have been proposed, on the basis of analysis of gene subfragments. Sequences from these strains were shown to group as independent, monophyletic lineages in phylogenetic comparisons to representative reference strains of previously described subtypes A through H.2,3 However, the definition of a genetic subtype is dependent on consistent clustering of sequences independent of the genome fragment analyzed (provided that enough informative variation is present in the region). This means that the classification results must be similar regardless of the genome region examined. Since HIV-1 strains of different subtypes have been shown to recombine, a classification based on a single subgenomic fragment does not necessarily indicate the genetic subtype of the complete genome. When a putative new genetic subtype is found the complete genome should be analyzed to verify that all regions of the virus cluster independently. In this study we have cloned and sequenced the first virtually complete proviruses of two independently obtained, epidemiologically unlinked primary isolates of HIV-1 subtype J. They form an independent cluster, approximately equidistant to the other subtypes over all of the genome, verifying their classification as a new subtype. Blood samples SE9173 and SE9280 for virus isolation were collected in Sweden from two previously identified HIV-1-infected individuals carrying the putative new subtype J viruses.8 Both individuals were immigrants from the Democratic Republic of Congo (formerly Zaire). A thorough epidemiological investigation, including contact tracing, did not reveal any direct or indirect contact between the two individuals. The initial identification of subtype J was based on direct sequencing and phylogenetic analysis of the gag p17 and env V3 regions amplified from uncultured patient lymphocytes. Samples SE9173 and SE9280 were obtained from the same individuals as the previously described samples SE7022 and SE7887, respectively. A seminested extended polymerase chain reaction (PCR) was used to amplify the virtually complete provirus from peripheral blood mononuclear cell (PBMC) cocultures essentially as has been previously described. Briefly, primers MSF12, binding to the tRNA-binding site located in the U5-gag leader junction of the 59 long terminal repeat (LTR), and MSR5, binding to the mRNA-polyadenylation signal site located in the R-U5 junction of the 3 9 LTR10 (Fig. 1), were used for first-round amplification. For second-round amplification one-tenth of the reaction was reamplified using primers MSF14 and MSR5. Second-round amplicons were cloned with a TA-cloning kit (pCR2.1 cloning kit; Invitrogen, San Diego, CA). Clones were sequenced on both strands by primer walking, using an ABI 377 automated sequencer and dye-terminator chemistry (Perkin-Elmer Applied Biosystems Division, Foster City, CA). The sequences have been submitted to GenBank with accession numbers AF082394 and AF082395. The lengths of the clones from strains SE9173 and SE9280 were 8953 and 8943 bp, respectively. The genome structure was similar to all other subtypes of HIV-1, with the gag, pol, and env structural genes, and reading frames for vif, vpr, vpu, and nef single exon regulatory/accessory genes (Table 1). The tat and rev exons 1 and 2 were also discernible at their usual positions. All reading frames appeared open and of complete length. The newly obtained SE9173 and SE9280 sequences were 94.2 and 97.6% similar to the SE7022 and SE7887 sequences in the V3 region, respectively.

Global Dispersal Pattern of HIV Type 1 Subtype CRF01_AE: A Genetic Trace of Human Mobility Related to Heterosexual Sexual Activities Centralized in Southeast Asia

BACKGROUND Human immunodeficiency virus type 1 (HIV-1) subtype CRF01_AE originated in Africa and then passed to Thailand, where it established a major epidemic. Despite the global presence of CRF01_AE, little is known about its subsequent dispersal pattern. METHODS We assembled a global data set of 2736 CRF01_AE sequences by pooling sequences from public databases and patient-cohort studies. We estimated viral dispersal patterns, using statistical phylogeographic analysis run over bootstrap trees estimated by the maximum likelihood method. RESULTS We show that Thailand has been the source of viral dispersal to most areas worldwide, including 17 of 20 sampled countries in Europe. Japan, Singapore, Vietnam, and other Asian countries have played a secondary role in the viral dissemination. In contrast, China and Taiwan have mainly imported strains from neighboring Asian countries, North America, and Africa without any significant viral exportation. DISCUSSION The central role of Thailand in the global spread of CRF01_AE can be probably explained by the popularity of Thailand as a vacation destination characterized by sex tourism and by Thai emigration to the Western world. Our study highlights the unique case of CRF01_AE, the only globally distributed non-B clade whose global dispersal did not originate in Africa.

Limited cross-border infections in patients newly diagnosed with HIV in Europe

BackgroundInternational travel plays a role in the spread of HIV-1 across Europe. It is, however, not known whether international travel is more important for spread of the epidemic as compared to endogenous infections within single countries. In this study, phylogenetic associations among HIV of newly diagnosed patients were determined across Europe.ResultsData came from the SPREAD programme which collects samples of newly diagnosed patients that are representative for national HIV epidemics. 4260 pol sequences from 25 European countries and Israel collected in 2002–2007 were included.We identified 457 clusters including 1330 persons (31.2% of all patients). The cluster size ranged between 2 and 28. A number of 987 patients (74.2%) were part of a cluster that consisted only of patients originating from the same country. In addition, 135 patients (10.2%) were in a cluster including only individuals from neighboring countries. Finally, 208 patients (15.6%) clustered with individuals from countries without a common border. Clustering with patients from the same country was less prevalent in patients being infected with B subtype (P-value <0.0001), in men who have sex with men (P-value <0.0001), and in recently infected patients (P-value =0.045).ConclusionsOur findings indicate that the transmission of HIV-1 in Europe is predominantly occurring between patients from the same country. This could have implications for HIV-1 transmission prevention programmes. Because infections through travelling between countries is not frequently observed it is important to have good surveillance of the national HIV-1 epidemics.

Analysis of HIV-1 Genetic Subtypes in Finland Reveals Good Correlation Between Molecular and Epidemiological Data

The molecular epidemiology of HIV-1 genetic subtypes was studied in a cross-sectional sample collected from HIV-infected individuals living in Finland between 1988 and 1994 and compared with independently collected epidemiological data. Subtypes were determined by sequencing and phylogenetic analysis of the gag NCp7 and the env coding regions of PBMC provirus. Finnish viruses belonging to 7 subtypes were found. Two thirds (n=70) of the sequences could be classified as subtype B, while others belonged to subtypes A, C, D, F and G and the circulating recombinant form AECM240 (n=25). There were significant differences in gender distribution and mode-of-transmission between B-type infections and infections with the other subtypes. Most subtype B strains in Finland were associated with homosexual transmission and about half of these were acquired in Finland, while most individuals harbouring non-B infections indicated heterosexual transmission and direct or indirect contact with Africa or Southeast Asia. The heterogeneity of genetic subtypes in the country was in good agreement with the epidemiological data suggesting that a significant proportion of infections were imported. HIV-1 subtype determination may prove to be a valuable tool for providing objective epidemiological data.The molecular epidemiology of HIV-1 genetic subtypes was studied in a cross-sectional sample collected from HIV-infected individuals living in Finland between 1988 and 1994 and compared with independently collected epidemiological data. Subtypes were determined by sequencing and phylogenetic analysis of the gag NCp7 and the env coding regions of PBMC provirus. Finnish viruses belonging to 7 subtypes were found. Two thirds (n = 70) of the sequences could be classified as subtype B, while others belonged to subtypes A, C, D, F and G and the circulating recombinant form AE(CM240) (n = 25). There were significant differences in gender distribution and mode-of-transmission between B-type infections and infections with the other subtypes. Most subtype B strains in Finland were associated with homosexual transmission and about half of these were acquired in Finland, while most individuals harbouring non-B infections indicated heterosexual transmission and direct or indirect contact with Africa or Southeast Asia. The heterogeneity of genetic subtypes in the country was in good agreement with the epidemiological data suggesting that a significant proportion of infections were imported. HIV-1 subtype determination may prove to be a valuable tool for providing objective epidemiological data.