Oropouche virus as an emerging cause of acute febrile illness in Colombia.

Arbovirus infections are frequent causes of acute febrile illness (AFI) in tropical countries. We conducted health facility-based AFI surveillance at four sites in Colombia (Cucuta, Cali, Villavicencio, Leticia) during 2019-2022. Demographic, clinical and risk factor data were collected from persons with AFI that consented to participate in the study (n = 2,967). Serologic specimens were obtained and tested for multiple pathogens by RT-PCR and rapid test (Antigen/IgM), with 20.7% identified as dengue positive from combined testing. Oropouche virus (OROV) was initially detected in serum by metagenomic next-generation sequencing (mNGS) and virus target capture in a patient from Cúcuta. Three additional infections from Leticia were confirmed by conventional PCR, sequenced, and isolated in tissue culture. Phylogenetic analysis determined there have been at least two independent OROV introductions into Colombia. To assess OROV spread, a RT-qPCR dual-target assay was developed which identified 87/791 (10.9%) viremic cases in AFI specimens from Cali (3/53), Cucuta (3/19), Villavicencio (38/566), and Leticia (43/153). In parallel, an automated anti-nucleocapsid antibody assay detected IgM in 27/503 (5.4%) and IgG in 92/568 (16.2%) patients screened, for which 24/68 (35.3%) of PCR positives had antibodies. Dengue was found primarily in people aged <18 years and linked to several clinical manifestations (weakness, skin rash and petechiae), whereas Oropouche cases were associated with the location, climate phase, and odynophagia symptom. Our results confirm OROV as an emerging pathogen and recommend increased surveillance to determine its burden as a cause of AFI in Colombia.

A high prevalence of potential HIV elite controllers identified over 30 years in Democratic Republic of Congo.

BACKGROUND: In-depth analysis of the HIV pandemic at its epicenter in the Congo basin has been hampered by 40 years of political unrest and lack of functional public health infrastructure. In recent surveillance studies (2017-18), we found that the prevalence of HIV in Kinshasa, Democratic Republic of Congo (11%) far exceeded previous estimates. METHODS: 10,457 participants were screened in Kinshasa with rapid tests from 2017-2019. Individuals confirmed as reactive by the Abbott ARCHITECT HIV Ag/Ab Combo assay (n=1968) were measured by the Abbott RealTime HIV-1 viral load assay. Follow up characterization of samples was performed with alternate manufacturer viral load assays, qPCR for additional blood borne viruses, unbiased next generation sequencing, and HIV Western blotting. FINDINGS: Our data suggested the existence of a significant cohort (n=429) of HIV antibody positive/viral load negative individuals. We systematically eliminated collection site bias, sample integrity, and viral genetic diversity as alternative explanations for undetectable viral loads. Mass spectroscopy unexpectedly detected the presence of 3TC antiviral medication in approximately 60% of those tested (209/354), and negative Western blot results indicated false positive serology in 12% (49/404). From the remaining Western blot positives (n=53) and indeterminates (n=31) with reactive Combo and rapid test results, we estimate 2.7-4.3% of infections in DRC to be potential elite controllers. We also analyzed samples from the DRC collected in 1987 and 2001-03, when antiretroviral drugs were not available, and found similarly elevated trends. INTERPRETATION: Viral suppression to undetectable viral loads without therapy occurs infrequently in HIV-1 infected patients around the world. Mining of global data suggests a unique ability to control HIV infection arose early in central Africa and occurs in <1% of founder populations. Identification of this group of elite controllers presents a unique opportunity to study potentially novel genetic mechanisms of viral suppression. FUNDING: Abbott Laboratories funded surveillance in DRC and subsequent research efforts. Additional funding was received from a MIZZOU Award from the University of Missouri. Research was supported in part by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH.

Advanced molecular surveillance approaches for characterization of blood borne hepatitis viruses.

Defining genetic diversity of viral infections directly from patient specimens is the ultimate goal of surveillance. Simple tools that can provide full-length sequence information on blood borne viral hepatitis viruses: hepatitis C, hepatitis B and hepatitis D viruses (HCV, HBV and HDV) remain elusive. Here, an unbiased metagenomic next generation sequencing approach (mNGS) was used for molecular characterization of HCV infections (n = 99) from Israel which yielded full-length HCV sequences in 89% of samples, with 7 partial sequences sufficient for classification. HCV genotypes were primarily 1b (68%) and 1a (19%), with minor representation of genotypes 2c (1%) and 3a (8%). HBV/HDV coinfections were characterized by suppressed HBV viral loads, resulting in sparse mNGS coverage. A probe-based enrichment approach (xGen) aiming to increase HBV and HDV coverage was validated on a panel of diverse genotypes, geography and titers. The method extended HBV genome coverage a median 61% (range 8-84%) and provided orders of magnitude boosts in reads and sequence depth for both viruses. When HBV-xGen was applied to Israeli samples, coverage was improved by 28-73% in 4 samples and identified HBV genotype A1, A2, D1 specimens and a dual B/D infection. Abundant HDV reads in mNGS libraries yielded 18/26 (69%) full genomes and 8 partial sequences, with HDV-xGen only providing minimal extension (3-11%) of what were all genotype 1 genomes. Advanced molecular approaches coupled to virus-specific capture probes promise to enhance surveillance of viral infections and aid in monitoring the spread of local subtypes.

Brief Report: Complete Genome Sequence of CG-0018a-01 Establishes HIV-1 Subtype L.

BACKGROUND: The full spectrum of HIV-1 diversity can be found in Central Africa, including 2 divergent HIV-1 strains collected in 1983 and 1990 in Democratic Republic of Congo (DRC) that were preliminarily classified as group M subtype L. However, a third epidemiologically distinct subtype L genome must be identified to designate L as a true subtype. METHODS: Specimen CG-0018a-01 was collected in 2001 in DRC as part of an HIV diversity study. Previous subgenomic HIV-1 sequences from this specimen branched closely with proposed subtype L references. Metagenomic next-generation sequencing (mNGS) and HIV-specific target-enriched (HIV-xGen) libraries were combined for NGS to extend genome coverage. mNGS reads were analyzed for the presence of other coinfections with the sequence-based ultrarapid pathogen identification bioinformatics pipeline. RESULTS: A complete HIV-1 genome was generated with an average coverage depth of 47,783×. After bioinformatic analysis also identified hepatitis B virus reads, a complete hepatitis B virus genotype A genome was assembled with an average coverage depth of 73,830×. The CG-0018a-01 HIV-1 genome branched basal to the 2 previous putative subtype L strains with strong bootstrap support of 100. With no evidence of recombination present, the strain was classified as subtype L. CONCLUSIONS: The CG-0018a-01 HIV-1 genome establishes subtype L and confirms ongoing transmission in DRC as recently as 2001. Since CG-0018a-01 is more closely related to an ancestral strain than to isolates from 1983 to 1990, additional strains are likely circulating in DRC and possibly elsewhere.

Universal Target Capture of HIV Sequences From NGS Libraries.

Background: Global surveillance of viral sequence diversity is needed to keep pace with the constant evolution of HIV. Recent next generation sequencing (NGS) methods have realized the goal of sequencing circulating virus directly from patient specimens. Yet, a simple, universal approach that maximizes sensitivity and sequencing capacity remains elusive. Here we present a novel HIV enrichment strategy to yield near complete genomes from low viral load specimens. Methodology: A non-redundant biotin-labeled probe set (HIV-xGen; n = 652) was synthesized to tile all HIV-1 (groups M, N, O, and P) and HIV-2 (A and B) strains. Illumina Nextera barcoded libraries of either gene-specific or randomly primed cDNA derived from infected plasma were hybridized to probes in a single pool and unbound sequences were washed away. Captured viral cDNA was amplified by Illumina adaptor primers, sequenced on a MiSeq, and NGS reads were demultiplexed for alignment with CLC Bio software. Results: HIV-xGen probes selectively captured and amplified reads spanning the entirety of the HIV phylogenetic tree. HIV sequences clearly present in unenriched libraries of specimens but previously not observed due to high host background levels, insufficient sequencing depth or the extent of multiplexing, were now enriched by >1,000-fold. Thus, xGen selection not only substantially increased the depth of existing sequence, but also extended overall genome coverage by an average of 40%. We characterized 50 new, diverse HIV strains from clinical specimens and demonstrated a viral load cutoff of approximately log 3.5 copies/ml for full length coverage. Genome coverage was <20% for 5/10 samples with viral loads 90% for 35/40 samples with higher viral loads. Conclusions: Characterization of >20 complete genomes at a time is now possible from a single probe hybridization and MiSeq run. With the versatility to capture all HIV strains and the sensitivity to detect low titer specimens, HIV-xGen will serve as an important tool for monitoring HIV sequence diversity.

ARCHITECT HIV Combo Ag/Ab and RealTime HIV-1 Assays Detect Diverse HIV Strains in Clinical Specimens.

Periodic evaluation of the impact of viral diversity on diagnostic tests is critical to ensure current technologies are keeping pace with viral evolution. To determine whether HIV diversity impacts the ARCHITECT HIV Combo Ag/Ab (HIV Combo) or RealTime HIV-1 (RT) assays, a set of N = 199 HIV clinical specimens from Cameroon, Senegal, Saudi Arabia, and Thailand were sequenced and tested in both assays. The panel included historical groups N and P specimens and a newly identified group N specimen. These and specimens classified as H, U (unclassified)/URF (unique recombinant form), CRF (circulating recombinant form) 01, 02, 06, 09, 11, 13, 18, 22, 37, and 43 were detected by both the RT assay (1.75-6.84 log copies/ml) and the HIV Combo assay (3.26-1121.96 sample to cutoff ratios). Sequence alignment identified 3 or fewer mismatches to the RT assay oligos in 82.4% of samples. Altogether, these data demonstrate the HIV Combo and RT assays detect diverse strains of HIV in clinical specimens.

HIV-2 Surveillance with Next-Generation Sequencing Reveals Mutations in a Cytotoxic Lymphocyte-Restricted Epitope Involved in Long-Term Nonprogression.

HIV-2 exhibits a natural history of infection distinct from HIV-1. Primarily found in West Africa and in only 10%-20% of HIV infections in this region, patients with HIV-2 typically exhibit a slower progression to AIDS, lower viral loads, and decreased rates of transmission. Here, we used next-generation sequencing to determine the sequence and phylogenetic classification of nine HIV-2 genomes. We identified a patient with a series of mutations in an invariant cytotoxic lymphocyte (CTL)-restricted gag epitope required for retroviral structure and replication and implicated in long-term nonprogression to AIDS. The presence of wild-type sequence argues these mutations are involved in immune escape, whereas its reversion to a sequence seen only in the sooty mangabey reservoir suggests an alternate means of controlling infection. Surveillance and molecular characterization of circulating strains are essential for continued development of monitoring tools and may provide greater insight into the reduced pathogenicity of HIV-2.

A Pan-HIV Strategy for Complete Genome Sequencing.

Molecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e.,switchingmechanismat 5' end ofRNAtranscript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ∼2,000× depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of ≤4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance.

ARCHITECT® HIV Ag/Ab Combo assay: correlation of HIV-1 p24 antigen sensitivity and RNA viral load using genetically diverse virus isolates.

BACKGROUND: HIV antigen/antibody (Ag/Ab) combination assays represent a significant advancement in assays used for diagnosing HIV infection based on their ability to detect acute and chronic infections. During acute HIV infection (AHI), detection depends on assay sensitivity for p24 Ag. OBJECTIVE: To directly compare the Ag sensitivity of the ARCHITECT(®) HIV Ag/Ab Combo assay to RNA viral load using cell culture supernatants of virus isolates. HIV-1 isolates allow correlation in the total absence of an antibody response to infection and across genetically diverse HIV-1 group M strains. METHODS: Thirty-five HIV-1 isolates comprising subtypes A-D, F and G, CRF01_AE, CRF02_AG, and unique recombinant forms were evaluated. Cell-free culture supernatant for each isolate was diluted to four levels and tested in the HIV Combo assay to determine a signal to cutoff ratio and the RealTime(®) HIV-1 assay to quantify RNA. The RNA copies/mL at the HIV Combo assay cutoff was determined. RESULTS: The median RNA copies/mL at the HIV Combo assay cutoff was 57,900 for individual virus isolates (range 26,440-102,400). A single plot of all the data gave a value of 58,500RNA copies/mL. An analysis of data published for acute HIV infection in human subjects gave a similar result; HIV Combo detected 97% of AHIs with RNA copies/mL > 30,700. CONCLUSIONS: Based on analysis of virus isolates, the ARCHITECT HIV Combo assay can detect p24 Ag when RNA is above approximately 58,000copies/mL. The correlation of viral load and Ag sensitivity was consistent across genetically diverse HIV-1 group M strains.

Confirmation of putative HIV-1 group P in Cameroon.

We report the second human immunodeficiency virus (HIV) belonging to the new HIV type 1 (HIV-1) group P lineage that is closely related to the simian immunodeficiency virus found in gorillas. This virus was identified in an HIV-seropositive male hospital patient in Cameroon, confirming that the group P virus is circulating in humans. Results from screening 1,736 HIV-seropositive specimens collected in Cameroon indicate that HIV-1 group P infections are rare, accounting for only 0.06% of HIV infections. Despite its rarity, group P shows evidence of adaptation to humans.

Near full-length sequence of HIV type 1 subtype J strain 04CMU11421 from Cameroon.

Although Cameroon, in west central Africa, has a relatively low HIV prevalence of 5-6%, all HIV-1 groups (M, N, O, and P), nearly all HIV-1 group M subtypes, and numerous intersubtype recombinant forms have been identified in Cameroon. In this report, we describe the near full-length sequence of 04CMU11421, an HIV-1 group M subtype J strain collected in Cameroon in 2004. Phylogenetic analysis of the genome sequence shows high bootstrap support with three subtype J reference sequences in the HIV Sequence database. Therefore, 04CMU11421 represents a fourth pure subtype J isolate and the first reported in Cameroon.

Expanded evaluation of blood donors in the United States for human immunodeficiency virus type 1 non-B subtypes and antiretroviral drug-resistant strains: 2005 through 2007.

BACKGROUND: In a previous study of 66 human immunodeficiency virus (HIV)-infected US blood donors from 1999 to 2005, HIV-1 non-B and antiretroviral drug-resistant strains accounted for 4.7 and 6.5% of HIV infections, respectively. This study was expanded to include an additional 11 recently acquired infections and 197 established infections collected from January 2005 through December 2007. STUDY DESIGN AND METHODS: HIV-infected donors were detected using FDA-licensed assays. Drug resistance profiles for protease and reverse transcriptase (RT) genes were determined using a genotyping system (ViroSeq, Celera Diagnostics); genetic subtype was determined by phylogenetic analysis of these sequences. RESULTS: Drug resistance profiles were obtained for 203 of 208 specimens; 9.9% had mutations that confer drug resistance. Ten showed resistance to a single drug class: nine to nonnucleoside RT inhibitors (NNRTIs) and one to nucleoside RT inhibitors (NRTIs). Eight showed two drug class resistance: five NRTI plus NNRTI, two NRTI plus protease inhibitor (PI), and one NNRTI plus PI. Two showed three drug class resistance. Non-B strains were identified in 2.5% of donors and consisted of subtypes A1 and D, CRF02_AG, CRF43-02G, and URF_BF. CONCLUSIONS: Data from this and the previous study show that antiretroviral drug-resistant HIV-1 is present in 9.1% of HIV-infected donors from 1999 through 2007; 9.3% of established infections and 6.9% of recent infections. Diverse HIV-1 non-B strains presently account for 3.0% of HIV infections in US donors.

HIV type 1 group M subtype G in Cameroon: five genome sequences.

Near full-length viral genome sequences were obtained for five putative subtype G candidates identified in HIV-infected Cameroonian blood donors, based on partial genome sequences for the gag, pol, and env regions. Phylogenetic analysis of the genome sequences shows that all five strains are pure subtype G with no indication of intersubtype recombination. The Cameroon subtype G sequences did not form a geographically based subcluster and were intermixed within the subtype G branch with isolates from several different countries. HIV-1 group M subtype G accounts for only 4.5% of HIV infections in Cameroon. However, genome segments of subtype G are present in 67% of all infections and 80% of infections due to intersubtype recombinant strains in Cameroon. The addition of five subtype G genome sequences to the HIV database may contribute to a better understanding of the origins and classification of HIV-1 subtypes and CRFs.

Identification of human immunodeficiency virus type 1 non-B subtypes and antiretroviral drug-resistant strains in United States blood donors.

BACKGROUND: In this study, human immunodeficiency virus type 1 (HIV-1)-infected blood donors were evaluated for genetic subtype and drug resistance to determine the prevalence of divergent HIV strains in the US donor population. STUDY DESIGN AND METHODS: Subtype was determined by phylogenetic analysis of viral sequences amplified by reverse transcription-polymerase chain reaction. The drug resistance profile of the protease and reverse transcriptase (RT) genes was determined using an HIV-1 genotyping system (ViroSeq). RESULTS: From 1999 through 2005, 26 recently infected donors, defined as HIV-1 RNA-positive, antibody-negative (RNA+/Ab-), were identified (yield, 1:1.61 million). Over the same period, the frequency of anti-HIV-positive donors was 1:34,700. Twenty RNA+/Ab- specimens were evaluated; all were infected with HIV-1 subtype B. Drug resistance profiles obtained for 18 donors identified one strain with protease mutation L90M that confers resistance to nelfinavir and one with RT mutation Y188H that confers resistance to nevirapine. Genetic subtype was determined for 44 of 46 HIV antibody-reactive and confirmed-positive (Ab+) specimens. Three infections (6.8%) were due to circulating recombinant forms: 2 CRF01_AE and 1 CRF02_AG. In the Ab+ group, one strain was resistant to all nucleoside RT inhibitors and one had mutations that confer resistance to protease inhibitors. CONCLUSION: The data show that antiretroviral drug-resistant HIV strains are being transmitted in the United States. Overall 6.5 percent (4 of 62) of HIV-1-infected donors harbored drug-resistant strains. HIV-1 non-B strains accounted for 4.7 percent (3 of 64) of the infections in donors. HIV-1 subtype B is still the predominant strain in the United States; however, non-B strains are increasing.

Identification of new CRF43_02G and CRF25_cpx in Saudi Arabia based on full genome sequence analysis of six HIV type 1 isolates.

Recently, we reported a high level of HIV-1 strain diversity in patients at the King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia. Based on phylogenetic analysis of gag p24, pol integrase, and env gp41 sequences, subtypes A, B, C, D, and G, and CRF02_AG, as well as unique recombinant forms were identified. Subtype G accounted for 25% of the infections in the Saudi population and this high prevalence was unexpected. Although subtype G is found in west central Africa, pure subtype G strains are uncommon. To further characterize the subtype G infections in Saudi Arabia, six strains that appeared to be pure subtype G were selected for full genome sequencing. Near full-length genomes were obtained using RT-PCR amplification to generate overlapping fragments from viral RNA extracted from plasma. The six strains are not subtype G throughout their entire genome. Four isolates have a recombinant structure composed of CRF02_AG and subtype G and share three identical breakpoints. This recombinant form defines a new CRF designated CRF43_02G. The remaining two isolates are CRF25_cpx, a circulating recombinant form identified in Cameroon composed of subtypes A and G and unclassified segments. Reanalysis of the previously reported Saudi HIV-1 partial genome sequences revealed additional isolates classified as CRF43_02G and CRF25_cpx and one isolate was reclassified to CRF22_01A. Identification of CRF43_02G in Saudi Arabia could indicate a transmission network within the country. Alternatively, the new CRF could have been introduced from an external source where this CRF is not yet recognized.

The prevalence of diverse HIV-1 strains was stable in Cameroonian blood donors from 1996 to 2004.

OBJECTIVE: The HIV epidemic in Cameroon is characterized by a high level of strain diversity despite a relatively low prevalence of infection. In this study, HIV strains infecting blood donors in Cameroon were characterized to determine the prevalence of subtypes and intersubtype recombinants and if strain prevalence was changing over time. METHODS: From 1996 through 2004, 676 HIV-infected blood donations were collected at blood banks in Douala and Yaoundé, Cameroon. A subset of the HIV-1 group M strains (n = 574) were classified based on phylogenetic analysis of viral sequences from the gag p24, pol integrase, and env gp41 regions. RESULTS: HIV-1 group M accounted for 97.3% (n = 658) of infections, whereas group O was present in 2.2% (n = 15) and HIV-2 in 0.4% (n = 3). Within the group M infections, 14 subtypes and circulating recombinant forms (CRFs) and unique recombinant forms (URFs) were identified. Overall, CRFO2_AG accounted for 58.2% of infections, URFs 14.8%, and levels of subtypes, A, B, C, D, F2, and G, and CRFs, 01, 06, 09, 11, 13, 22, and 37, varied from 0.2% to 6.1%. Evaluation of HIV strains present in the donor population over this 9-year period showed no substantial changes in the proportion of infections caused by each subtype and CRF, the percentage of intersubtype recombinants, or the strain composition of the URFs. CONCLUSIONS: HIV-1 strain diversity in Cameroon did not significantly change, suggesting a mature and relatively stable epidemic.

HIV type 2 intergroup recombinant identified in Cameroon.

A unique HIV-2 intergroup recombinant strain was identified in Cameroon. The virus, CM-03-510-03, was amplified from blood collected from a 47-year-old female patient in Douala, Cameroon in 2003 who was seroreactive for HIV-2. A near full-length genome 9089 nucleotides in length was amplified from proviral DNA. The genome for CM-03-510-03 is composed of segments of HIV-2 groups A and B with four recombination break-points and has open reading frames for all the structural and regulatory genes. A comparison of CM-03-510-03 to the only previously reported HIV-2 intergroup recombinant shows that the two strains share one recombination breakpoint but are otherwise distinct from each other. Similar to HIV-1, HIV-2 intergroup recombination is an indication that coinfection with more than one strain has occurred in individuals and is a mechanism that increases strain genetic diversity.

Evaluation of a new, fully automated immunoassay for detection of HTLV-I and HTLV-II antibodies.

Screening blood donations for human T-lymphotropic virus types I and II (HTLV-I/II) continues to be important in protecting the safety of blood products and controlling the global spread of these retroviruses. We have developed a fully automated, third generation chemiluminescent immunoassay, ARCHITECT rHTLV-I/II, for detection of antibodies to HTLV-I/II. The assay utilizes recombinant proteins and synthetic peptides and is configured in a double antigen sandwich assay format. Specificity of the assay was 99.98% (9,254/9,256, 95% CI = 99.92-100%) with the negative specimens from the general population including blood donors, hospital patients and pregnant women from the US, Japan and Nicaragua. The assay demonstrated 100% sensitivity by detecting 498 specimens from individuals infected with HTLV-I (n = 385) and HTLV-II (n = 113). ARCHITECT rHTLV-I/II results were in complete agreement with the Murex HTLV-I/II reference assay and 99.7% agreement with the Genelabs HTLV Blot 2.4 confirmatory assay. Analytical sensitivity of the assay was equivalent to Murex HTLV-I/II assay based on end point dilutions. Furthermore, using a panel of 397 specimens from Japan, the ARCHITECT rHTLV-I/II assay exhibited distinct discrimination between the antibody negative (Delta Value = -7.6) and positive (Delta Value = 7.6) populations. Based on the excellent specificity and sensitivity, the new ARCHITECT rHTLV-I/II assay should be an effective test for the diagnosis of HTLV-I/II infection and also for blood donor screening.

Identification and characterization of HIV type 1 subtypes present in the Kingdom of Saudi Arabia: high level of genetic diversity found.

Saudi Arabia has a very low prevalence of HIV infections and nothing is known about HIV strains present in the population. Here specimens were collected from 62 HIV-1-infected patients at the King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia. Viral sequences were PCR amplified using primers for HIV-1 group M in gag p24, pol integrase, and env gp41 and genetic subtype was determined by phylogenetic analysis. HIV-1 viral sequences were amplified from 56 of the 62 specimens. Based on phylogenetic analysis of viral sequences, subtype C was the most common subtype present and accounted for 39.3% of the infections followed by subtype G (25%), subtype B (17.9%), subtype D (3.6%), and subtypes A and CRF02_AG (1.8% each). In addition, for six specimens subtype classifications were discordant between gag, pol, and/or env; these intersubtype recombinant viruses account for 10.7% of the infections and consisted of recombinants of subtypes A/CRF01, A/CRF02, A/G, B/G, and D/CRF02. The high HIV-1 strain diversity suggests that there have been multiple introductions of HIV-1 into Saudi Arabia from several sources. Within the study population, there were five husband/wife pairs. For each pair, the viral sequences obtained were closely related to each other showing that heterosexual transmission occurred.

Near full-length genome characterization of three additional HIV type 1 CRF13_cpx strains from Cameroon.

Recombinant forms of HIV-1 contribute significantly to the ongoing epidemic. In the present study, we characterized the near full-length genomes of three candidate HIV-1 CRF13_cpx strains originating in Cameroon, 04CM-173-9, 04CM-632-28, and 02CM-A1394. Bootscanning, recombination breakpoint analysis, and phylogenetic trees confirmed similar genomic structures with identical breakpoint positions compared to the three available CRF13_cpx sequences. The candidate and reference sequences formed a distinct cluster well separated from other group M subtypes and had a mosaic structure derived from subtypes A1, G, J, and CRF01_AE. The similarity in genomic composition and position of recombination breakpoints suggest that these isolates share a common ancestor. The epidemiological significance of CRF13_cpx strains in Cameroon is unknown; however, the availability of three additional genomic sequences will improve our understanding of the overall genetic diversity within this recombinant form of HIV-1.