Development and Validation of Three Automated High-Throughput Molecular Tests to Detect Monkeypox Virus Infections.

BACKGROUND: The 2022 outbreak of the clade IIb monkeypox virus and subsequent global spread lead to an urgent need for the development of high-throughput, sensitive, and reproducible diagnostic tests. METHODS: We developed 3 assays to detect monkeypox virus, 2 (MPXV+ and MPXV) for m2000 RealTime and 1 (MPXV) for Alinity m platforms. Dual targets in E9L and B6R (MPXV+) and J2L and B7R (MPXV) increased mutation resistance. In silico prediction indicates MPXV+ cross-reactivity with orthopox viruses and specific monkeypox virus detection with MPXV. RESULTS: m2000 RealTime MPXV+ and MPXV assay sensitivity was determined to be 3.2 plaque-forming units/mL using a reference virus culture diluted into universal transport medium (UTM). Alinity m MPXV lower limit of detection was 200 copies/mL using monkeypox virus plasmids in pooled UTM matrix. m2000 RealTime MPXV+ and MPXV assays were validated with lesion swabs in UTM and 1:1 saliva to UTM mixtures. Commercially available and remnant clinical lesion specimens in UTM were tested with RealTime MPXV+, RealTime MPXV and Alinity m MPXV assays and demonstrated high agreement to known mpox (MPX)-positive specimens. CONCLUSIONS: RealTime MPXV+, RealTime MPXV, and Alinity MPXV are high throughput and sensitive assays used for the detection of monkeypox virus. These assays maybe useful during MPX outbreaks.

Purifying selection decreases the potential for Bangui orthobunyavirus outbreaks in humans.

Pathogens carried by insects, such as bunyaviruses, are frequently transmitted into human populations and cause diseases. Knowing which spillover events represent a public health threat remains a challenge. Metagenomic next-generation sequencing (mNGS) can support infectious disease diagnostics by enabling the detection of any pathogen from clinical specimens. mNGS was performed on blood samples to identify potential viral coinfections in human immunodeficiency virus (HIV)-positive individuals from Kinshasa, the Democratic Republic of the Congo (DRC), participating in an HIV diversity cohort study. Time-resolved phylogenetics and molecular assay development assisted in viral characterization. The nearly complete genome of a novel orthobunyavirus related to Nyangole virus, a virus previously identified in neighboring Uganda, was assembled from a hepatitis B virus-positive patient. A quantitative polymerase chain reaction assay was designed and used to screen >2,500 plasma samples from Cameroon, the DRC, and Uganda, failing to identify any additional cases. The recent sequencing of a US Center for Disease Control Arbovirus Reference Collection revealed that this same virus, now named Bangui virus, was first isolated in 1970 from an individual in the Central African Republic. Time-scaled phylogenetic analyses of Bangui with the related Anopheles and Tanga serogroup complexes indicate that this virus emerged nearly 10,000 years ago. Pervasive and episodic models further suggest that this virus is under purifying selection and that only distant common ancestors were subject to positive selection events. This study represents only the second identification of a Bangui virus infection in over 50 years. The presumed rarity of Bangui virus infections in humans can be explained by its constraint to an avian host and insect vector, precluding efficient transmission into the human population. Our results demonstrate that molecular phylogenetic analyses can provide insights into the threat posed by novel or re-emergent viruses identified by mNGS.

More DNA and RNA of HBV SP1 splice variants are detected in genotypes B and C at low viral replication.

HBV produces unspliced and spliced RNAs during replication. Encapsidated spliced RNA is converted into DNA generating defective virions that are detected in plasma and associated with HCC development. Herein we describe a quantitative real-time PCR detection of splice variant SP1 DNA/RNA in HBV plasma. Three PCR primers/probe sets were designed detecting the SP1 variants, unspliced core, or X gene. Plasmids carrying the three regions were constructed for the nine HBV genotypes to evaluate the three sets, which were also tested on DNA/RNA extracted from 193 HBV plasma with unknown HCC status. The assay had an LOD of 80 copies/ml and was equally efficient for detecting all nine genotypes and three targets. In testing 84 specimens for both SP1 DNA (77.4%) and RNA (82.1%), higher viral loads resulted in increased SP1 levels. Most samples yielded < 1% of SP1 DNA, while the average SP1 RNA was 3.29%. At viral load of ≤ 5 log copies/ml, the detectable SP1 DNA varied by genotype, with 70% for B, 33.3% for C, 10.5% for E, 4% for D and 0% for A, suggesting higher levels of splicing in B and C during low replication. At > 5 log, all samples regardless of genotype had detectable SP1 DNA.

Emergence of a Distinct Picobirnavirus Genotype Circulating in Patients Hospitalized with Acute Respiratory Illness.

Picobirnaviruses (PBV) are found in a wide range of hosts and typically associated with gastrointestinal infections in immunocompromised individuals. Here, a divergent PBV genome was assembled from a patient hospitalized for acute respiratory illness (ARI) in Colombia. The RdRp protein branched with sequences previously reported in patients with ARI from Cambodia and China. Sputa from hospitalized individuals (n = 130) were screened by RT-qPCR which enabled detection and subsequent metagenomic characterization of 25 additional PBV infections circulating in Colombia and the US. Phylogenetic analysis of RdRp highlighted the emergence of two dominant lineages linked to the index case and Asian strains, which together clustered as a distinct genotype. Bayesian inference further established capsid and RdRp sequences as both significantly associated with ARI. Various respiratory-tropic pathogens were detected in PBV+ patients, yet no specific bacteria was common among them and four individuals lacked co-infections, suggesting PBV may not be a prokaryotic virus nor exclusively opportunistic, respectively. Competing models for the origin and transmission of this PBV genotype are presented that attempt to reconcile vectoring by a bacterial host with human pathogenicity. A high prevalence in patients with ARI, an ability to reassort, and demonstrated global spread indicate PBV warrant greater public health concern.

Circulating Pregenomic Hepatitis B Virus RNA Is Primarily Full-length in Chronic Hepatitis B Patients Undergoing Nucleos(t)ide Analogue Therapy.

Hepatitis B virus RNA is detectable in the serum of infected patients; however, the RNA species has been questioned. We tested 1827 specimens using a quantitative dual-target quantitative polymerase chain reaction assay and determined that full-length pregenomic RNA is the primary source. These results clarify the major identity of circulating HBV RNA species.

Author Correction: Development and performance of prototype serologic and molecular tests for hepatitis delta infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of a putative novel genotype 3/rabbit hepatitis E virus (HEV) recombinant.

Hepatitis E virus (HEV) is a viral pathogen transmitted by the fecal-oral route and is a major cause of waterborne acute hepatitis in many developing countries. In addition to infecting humans, HEV has been identified in swine, wild boars, rabbits and other mammals; with swine and wild boars being main reservoirs for zoonotic transmission of HEV. There are four major HEV genotypes known to infect humans; genotypes 1 (HEV-1) and 2 (HEV-2) are restricted to humans, and genotypes 3 (HEV-3) and 4 (HEV-4) are zoonotic. Herein, three human HEV strains originating in France were sequenced and near full-length genomes were characterized. Phylogenetic analysis showed that two strains were genotype 3 and closely grouped (a 100% bootstrap value) with subtype 3i reference strains. In percent nucleotide identities, these two strains were 94% identical to each other, 90-93% identical to subtype 3i strains, 82-86% identical to other HEV-3, and 77-79% identical to rabbit HEV strains excluding the two divergent strains KJ013414 and KJ013415 (74%); these two strains were less than 77% identical to strains of HEV genotypes 1, 2 and 4. The third strain was found distinct from any known HEV strains in the database, and located between the clusters of HEV-3 and rabbit HEV strains. This unique strain was 74-75% identical to HEV-1, 73% to HEV-2, 81-82% to HEV-3, 77-79% to rabbit HEV again excluding the two divergent strains KJ013414 and KJ013415 (74%), and 74-75% to HEV-4, suggesting a novel unclassified strain associated with HEV-3 and rabbit HEV. SimPlot and BootScan analyses revealed a putative recombination of HEV-3 and rabbit HEV sequences at four breakpoints. Phylogenetic trees of the five fragments of the genome confirmed the presence of two HEV-3 derived and three unclassified sequences. Analyses of the amino acid sequences of the three open reading frames (ORF1-3) encoded proteins of these three novel strains showed that some amino acid residues specific to rabbit HEV strains were found solely in this unclassified strain but not in the two newly identified genotype 3i strains. The results obtained by SimPlots, BootScans, phylogenetic analyses, and amino acid sequence comparisons in this study all together appear to suggest that this novel unclassified strain is likely carrying a mosaic genome derived from HEV-3 and rabbit HEV sequences, and is thus designated as a putative genotype 3/rabbit HEV recombinant.

Hepatitis B Virus Serum DNA andRNA Levels in Nucleos(t)ide Analog-Treated or Untreated Patients During Chronic and Acute Infection.

Treatment of chronic hepatitis B (CHB) patients with nucleos(t)ide analogs (NAs) suppresses hepatitis B virus (HBV) DNA synthesis but does not affect synthesis of HBV pregenomic RNA (pgRNA). Hepatitis B virus pgRNA is detectable in the serum during NA treatment and has been proposed as a marker of HBV covalently closed circular DNA activity within the infected hepatocyte. We developed an automated assay for the quantification of serum HBV pgRNA using a dual-target real-time quantitative PCR approach on the Abbott m2000sp/rt system. We demonstrate accurate detection and quantification of serum HBV RNA. Hepatitis B virus DNA was quantified using the Abbott RealTime HBV viral load assay. We further compared serum nucleic acid levels and kinetics in HBV-positive populations. Samples included on-therapy CHB samples (n = 16), samples (n = 89) from 10 treatment naïve CHB subjects receiving 12 weeks of NA treatment with 8-week follow-up, hepatitis B surface antigen-positive blood donor samples (n = 102), and three seroconversion series from plasmapheresis donors (n = 79 samples). Conclusion: During NA treatment of CHB subjects, we observed low correlation of HBV DNA to pgRNA levels; pgRNA concentration was generally higher than HBV DNA concentrations. In contrast, when NA treatment was absent we observed serum pgRNA at concentrations that correlated to HBV DNA and were approximately 2 log lower than HBV DNA. Importantly, we observe this trend in untreated subject samples from both chronic infections and throughout seroconversion during acute infection. Results demonstrate that the presence of pgRNA in serum is part of the HBV lifecycle; constant relative detection of pgRNA and HBV DNA in the serum is suggestive of a linked mechanism for egress for HBV DNA or pgRNA containing virions.

Development and performance of prototype serologic and molecular tests for hepatitis delta infection.

Worldwide, an estimated 5% of hepatitis B virus (HBV) infected people are coinfected with hepatitis delta virus (HDV). HDV infection leads to increased mortality over HBV mono-infection, yet HDV diagnostics are not widely available. Prototype molecular (RNA) and serologic (IgG) assays were developed for high-throughput testing on the Abbott m2000 and ARCHITECT systems, respectively. RNA detection was achieved through amplification of a ribozyme region target, with a limit of detection of 5 IU/ml. The prototype serology assay (IgG) was developed using peptides derived from HDV large antigen (HDAg), and linear epitopes were further identified by peptide scan. Specificity of an HBV negative population was 100% for both assays. A panel of 145 HBsAg positive samples from Cameroon with unknown HDV status was tested using both assays: 16 (11.0%) had detectable HDV RNA, and 23 (15.7%) were sero-positive including the 16 HDV RNA positive samples. Additionally, an archival serial bleed panel from an HDV superinfected chimpanzee was tested with both prototypes; data was consistent with historic testing data using a commercial total anti-Delta test. Overall, the two prototype assays provide sensitive and specific methods for HDV detection using high throughput automated platforms, allowing opportunity for improved diagnosis of HDV infected patients.

Utility of Metagenomic Next-Generation Sequencing for Characterization of HIV and Human Pegivirus Diversity.

Given the dynamic changes in HIV-1 complexity and diversity, next-generation sequencing (NGS) has the potential to revolutionize strategies for effective HIV global surveillance. In this study, we explore the utility of metagenomic NGS to characterize divergent strains of HIV-1 and to simultaneously screen for other co-infecting viruses. Thirty-five HIV-1-infected Cameroonian blood donor specimens with viral loads of >4.4 log10 copies/ml were selected to include a diverse representation of group M strains. Random-primed NGS libraries, prepared from plasma specimens, resulted in greater than 90% genome coverage for 88% of specimens. Correct subtype designations based on NGS were concordant with sub-region PCR data in 31 of 35 (89%) cases. Complete genomes were assembled for 25 strains, including circulating recombinant forms with relatively limited data available (7 CRF11_cpx, 2 CRF13_cpx, 1 CRF18_cpx, and 1 CRF37_cpx), as well as 9 unique recombinant forms. HPgV (formerly designated GBV-C) co-infection was detected in 9 of 35 (25%) specimens, of which eight specimens yielded complete genomes. The recovered HPgV genomes formed a diverse cluster with genotype 1 sequences previously reported from Ghana, Uganda, and Japan. The extensive genome coverage obtained by NGS improved accuracy and confidence in phylogenetic classification of the HIV-1 strains present in the study population relative to conventional sub-region PCR. In addition, these data demonstrate the potential for metagenomic analysis to be used for routine characterization of HIV-1 and identification of other viral co-infections.

A cloud-compatible bioinformatics pipeline for ultrarapid pathogen identification from next-generation sequencing of clinical samples.

Unbiased next-generation sequencing (NGS) approaches enable comprehensive pathogen detection in the clinical microbiology laboratory and have numerous applications for public health surveillance, outbreak investigation, and the diagnosis of infectious diseases. However, practical deployment of the technology is hindered by the bioinformatics challenge of analyzing results accurately and in a clinically relevant timeframe. Here we describe SURPI ("sequence-based ultrarapid pathogen identification"), a computational pipeline for pathogen identification from complex metagenomic NGS data generated from clinical samples, and demonstrate use of the pipeline in the analysis of 237 clinical samples comprising more than 1.1 billion sequences. Deployable on both cloud-based and standalone servers, SURPI leverages two state-of-the-art aligners for accelerated analyses, SNAP and RAPSearch, which are as accurate as existing bioinformatics tools but orders of magnitude faster in performance. In fast mode, SURPI detects viruses and bacteria by scanning data sets of 7-500 million reads in 11 min to 5 h, while in comprehensive mode, all known microorganisms are identified, followed by de novo assembly and protein homology searches for divergent viruses in 50 min to 16 h. SURPI has also directly contributed to real-time microbial diagnosis in acutely ill patients, underscoring its potential key role in the development of unbiased NGS-based clinical assays in infectious diseases that demand rapid turnaround times.

In-depth investigation of archival and prospectively collected samples reveals no evidence for XMRV infection in prostate cancer.

XMRV, or xenotropic murine leukemia virus (MLV)-related virus, is a novel gammaretrovirus originally identified in studies that analyzed tissue from prostate cancer patients in 2006 and blood from patients with chronic fatigue syndrome (CFS) in 2009. However, a large number of subsequent studies failed to confirm a link between XMRV infection and CFS or prostate cancer. On the contrary, recent evidence indicates that XMRV is a contaminant originating from the recombination of two mouse endogenous retroviruses during passaging of a prostate tumor xenograft (CWR22) in mice, generating laboratory-derived cell lines that are XMRV-infected. To confirm or refute an association between XMRV and prostate cancer, we analyzed prostate cancer tissues and plasma from a prospectively collected cohort of 39 patients as well as archival RNA and prostate tissue from the original 2006 study. Despite comprehensive microarray, PCR, FISH, and serological testing, XMRV was not detected in any of the newly collected samples or in archival tissue, although archival RNA remained XMRV-positive. Notably, archival VP62 prostate tissue, from which the prototype XMRV strain was derived, tested negative for XMRV on re-analysis. Analysis of viral genomic and human mitochondrial sequences revealed that all previously characterized XMRV strains are identical and that the archival RNA had been contaminated by an XMRV-infected laboratory cell line. These findings reveal no association between XMRV and prostate cancer, and underscore the conclusion that XMRV is not a naturally acquired human infection.

Absence of XMRV and closely related viruses in primary prostate cancer tissues used to derive the XMRV-infected cell line 22Rv1.

The 22Rv1 cell line is widely used for prostate cancer research and other studies throughout the world. These cells were established from a human prostate tumor, CWR22, that was serially passaged in nude mice and selected for androgen independence. The 22Rv1 cells are known to produce high titers of xenotropic murine leukemia virus-related virus (XMRV). Recent studies suggested that XMRV was inadvertently created in the 1990's when two murine leukemia virus (MLV) genomes (pre-XMRV1 and pre-XMRV-2) recombined during passaging of the CWR22 tumor in mice. The conclusion that XMRV originated from mice and not the patient was based partly on the failure to detect XMRV in early CWR22 xenografts. While that deduction is certainly justified, we examined the possibility that a closely related virus could have been present in primary tumor tissue. Here we report that we have located the original prostate tumor tissue excised from patient CWR22 and have assayed the corresponding DNA by PCR and the tissue sections by fluorescence in situ hybridization for the presence of XMRV or a similar virus. The primary tumor tissues lacked mouse DNA as determined by PCR for intracisternal A type particle DNA, thus avoiding one of the limitations of studying xenografts. We show that neither XMRV nor a closely related virus was present in primary prostate tissue of patient CWR22. Our findings confirm and reinforce the conclusion that XMRV is a recombinant laboratory-generated mouse virus that is highly adapted for human prostate cancer cells.

No evidence of murine-like gammaretroviruses in CFS patients previously identified as XMRV-infected.

Members of the gammaretroviruses--such as murine leukemia viruses (MLVs), most notably XMRV [xenotropic murine leukemia virus (X-MLV)-related virus--have been reported to be present in the blood of patients with chronic fatigue syndrome (CFS). We evaluated blood samples from 61 patients with CFS from a single clinical practice, 43 of whom had previously been identified as XMRV-positive. Our analysis included polymerase chain reaction and reverse transcription polymerase chain reaction procedures for detection of viral nucleic acids and assays for detection of infectious virus and virus-specific antibodies. We found no evidence of XMRV or other MLVs in these blood samples. In addition, we found that these gammaretroviruses were strongly (X-MLV) or partially (XMRV) susceptible to inactivation by sera from CFS patients and healthy controls, which suggested that establishment of a successful MLV infection in humans would be unlikely. Consistent with previous reports, we detected MLV sequences in commercial laboratory reagents. Our results indicate that previous evidence linking XMRV and MLVs to CFS is likely attributable to laboratory contamination.

Characterization of antibodies elicited by XMRV infection and development of immunoassays useful for epidemiologic studies.

BACKGROUND: Xenotropic Murine Leukemia Virus-related Virus (XMRV) is a human gammaretrovirus recently identified in prostate cancer tissue and in lymphocytes of patients with chronic fatigue syndrome. To establish the etiologic role of XMRV infection in human disease requires large scale epidemiologic studies. Development of assays to detect XMRV-specific antibodies would greatly facilitate such studies. However, the nature and kinetics of the antibody response to XMRV infection have yet to be determined. RESULTS: Three rhesus macaques were infected with XMRV to determine the dynamics of the antibody responses elicited by infection with XMRV. All macaques developed antibodies to XMRV during the second week of infection, and the predominant responses were to the envelope protein gp70, transmembrane protein p15E, and capsid protein p30. In general, antibody responses to gp70 and p15E appeared early with higher titers than to p30, especially in the early period of seroconversion. Antibodies to gp70, p15E and p30 persisted to 158 days and were substantially boosted by re-infection, thus, were identified as useful serologic markers. Three high-throughput prototype assays were developed using recombinant proteins to detect antibodies to these viral proteins. Both gp70 and p15E prototype assays demonstrated 100% sensitivity by detecting all Western blot (WB) positive serial bleeds from the XMRV-infected macaques and good specificity (99.5-99.9%) with blood donors. Seroconversion sensitivity and specificity of the p30 prototype assay were 92% and 99.4% respectively. CONCLUSIONS: This study provides the first demonstration of seroconversion patterns elicited by XMRV infection. The nature and kinetics of antibody responses to XMRV in primates were fully characterized. Moreover, key serologic markers useful for detection of XMRV infection were identified. Three prototype immunoassays were developed to detect XMRV-specific antibodies. These assays demonstrated good sensitivity and specificity; thus, they will facilitate large scale epidemiologic studies of XMRV infection in humans.

Near full-length genome characterization of an HIV type 1 CRF25_cpx strain from Cameroon.

Recombinant forms of HIV-1 contribute significantly to the ongoing epidemic. In the present study, we characterized the near full-length genome of one candidate HIV-1 CRF25_cpx strain originating in Cameroon, 06CM-BA-040. Viral RNA was extracted from plasma, and the genome was obtained using RT-PCR amplification to generate 10 overlapping fragments. Bootscanning, recombination breakpoint analysis, and phylogenetic trees confirmed that 06CM-BA-040 had a genomic structure consistent with two available CRF25_cpx reference sequences. The CRF25_cpx mosaic composition consisted of nine segments derived from subtypes A and G as well as unclassified (U) regions. Subtype G and CRF25_cpx clusters diverged from each other with long branch lengths but were distinct from other known subtypes with high bootstrap support (94%). The epidemiological significance of CRF25_cpx strains is unknown; however, the availability of additional genomic sequences will improve our understanding of the overall genetic diversity within this recombinant form of HIV-1.

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.

Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR.

Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5'-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3'-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.

Partially double-stranded linear DNA probes: novel design for sensitive detection of genetically polymorphic targets.

Genetically polymorphic targets present a significant challenge to the reliability of detection and quantification by nucleic acid-based assays. A probe system with enhanced mismatch tolerance would be advantageous for such applications. The present study introduces a novel class of DNA probes, designated as partially double-stranded linear probes, composed of a long target-specific strand 5' labeled with a fluorophore and a markedly shorter quencher strand, complementary to the 5' end of the target-specific strand, that is 3' end-labeled with a quencher moiety. The utility of this probe system for sensitive detection of amplification products was demonstrated in a real-time PCR format. Comparison of multiple partially double-stranded linear probe combinations revealed that increased asymmetry in strand length was associated with improved mismatch tolerance. Notably, for a 45-mer/11-mer combination, the difference in threshold cycle values obtained for a perfectly matched target and one containing six mismatches was <1.5 cycles. The capacity for superior mismatch tolerance, ease of design, simplicity and flexibility of application are characteristics that make this new class of probes a desirable alternative for homogeneous detection of targets with a high level of genetic heterogeneity.

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.