Proficiency testing for the detection of Middle East respiratory syndrome coronavirus demonstrates global capacity to detect Middle East respiratory syndrome coronavirus.

The first reported case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection was identified in Saudi Arabia in September 2012, since which time there have been over 2000 laboratory-confirmed cases, including 750 deaths in 27 countries. Nucleic acid testing (NAT) is the preferred method for the detection of MERS-CoV. A single round of a Proficiency Testing Program (PTP) was used to assess the capability of laboratories globally to accurately detect the presence of MERS-CoV using NAT. A panel of eleven lyophilized specimens containing different viral loads of MERS-CoV, common coronaviruses, and in vitro RNA transcripts was distributed to laboratories in all six World Health Organization regions. A total of 96 laboratories from 79 countries participating in the PTP, with 76 of 96 (79.2%) reporting correct MERS-CoV results for all nine scored specimens. A further 10 laboratories (10.4%) scored correctly in eight of nine specimens of the PTP. The majority of laboratories demonstrated satisfactory performance in detecting the presence of MERS-CoV using NAT. However, some laboratories require improved assay sensitivity, reduced cross contamination of samples, and improved speciation of coronavirus subtypes for potentially complex clinical specimens. Further PTP and enhanced links with expert laboratories globally may improve the laboratory performance.

RBM3 regulates temperature sensitive miR-142-5p and miR-143 (thermomiRs), which target immune genes and control fever.

Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40 °C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142-5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40 °C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142-5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142-5p and miR-143.

Ebola Preparedness: Diagnosis Improvement Using Rapid Approaches for Proficiency Testing.

The unprecedented 2015 Ebolavirus (EBOV) outbreak in West Africa was declared a public health emergency, making diagnosis and quality of testing a global issue. The accuracy of laboratory diagnostic capacity for EBOV was assessed in 2014 to 2016 using a proficiency testing (PT) strategy developed by the Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) in Biosecurity. Following a literature search, EBOV-specific gene targets were ranked according to the frequency of their use in published methods. The most commonly used gene regions (nucleoprotein [NP], glycoprotein [GP], and RNA-dependent RNA polymerase [L]) were selected for the design of in vitro RNA transcripts to be included in the simulated EBOV specimens used for EBOV detection with PCR-based assays. Specimens were tested for stability and found to be stable on long-term storage (1 year) at -80°C and on shorter-term storage in lyophilized form (1 week at ambient temperature and a subsequent week at -80°C). These specimens were used in three EBOV PTs offered from April 2014 to March 2016. In the first and third PTs, all laboratories (3/3 and 9/9, respectively) correctly identified specimens containing EBOV RNA transcripts, while in the second PT, all but one laboratory (5/6) correctly confirmed the presence of EBOV. The EBOV PT panel was useful for ensuring the competency of laboratories in detecting EBOV in the absence of readily available clinical samples. The simulated EBOV specimen was safe, stable, and reliable and can be used in lyophilized form for future EBOV PT programs, allowing simplicity of transport.

Epigenetic modifications of splicing factor genes in myelodysplastic syndromes and acute myeloid leukemia.

Somatic mutations in splicing factor genes have frequently been reported in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Although aberrant epigenetic changes are frequently implicated in blood cancers, their direct role in suppressing one or both alleles of critical splicing factors has not been previously examined. Here, we examined promoter DNA hypermethylation of nine splicing factors, SF3B1, SRSF2, U2AF1, ZRSR2, SF3A1, HNRNPR, MATR3, ZFR, and YBX3 in 10 leukemic cell lines and 94 MDS or AML patient samples from the Australasian Leukemia and Lymphoma Group Tissue Bank. The only evidence of epigenetic effects was hypermethylation of the YBX3 promoter in U937 cells in conjunction with an enrichment of histone marks associated with gene silencing. In silico analysis of DNA methylation data for 173 AML samples generated by the Cancer Genome Atlas Research Network revealed promoter hypermethylation of the gene encoding Y box binding protein 3, YBX3, in 11/173 (6.4%) AML cases, which was significantly associated with reduced mRNA expression (P < 0.0001). Hypermethylation of the ZRSR2 promoter was also detected in 7/173 (4%) cases but was not associated with decreased mRNA expression (P = 0.1204). Hypermethylation was absent at the promoter of seven other splicing factor genes in all cell lines and patient samples examined. We conclude that DNA hypermethylation does not frequently silence splicing factors in MDS and AML. However, in the case of YBX3, promoter hypermethylation-induced downregulation may contribute to the pathogenesis or maintenance of AML.

Polymorphism in endothelial connexin40 enhances sensitivity to intraluminal pressure and increases arterial stiffness.

OBJECTIVE: To determine whether impairment of endothelial connexin40 (Cx40), an effect that can occur in hypertension and aging, contributes to the arterial dysfunction and stiffening in these conditions. APPROACH AND RESULTS: A new transgenic mouse strain, expressing a mutant Cx40, (Cx40T202S), specifically in the vascular endothelium, has been developed and characterized. This mutation produces nonfunctional hemichannels, whereas gap junctions containing the mutant are electrically, but not chemically, patent. Mesenteric resistance arteries from Cx40T202S mice showed increased sensitivity of the myogenic response to intraluminal pressure in vitro, compared with wild-type mice, whereas transgenic mice overexpressing native Cx40 (Cx40Tg) showed reduced sensitivity. In control and Cx40Tg mice, the sensitivity to pressure of myogenic constriction was modulated by both NO and endothelium-derived hyperpolarization; however, the endothelium-derived hyperpolarization component was absent in Cx40T202S arteries. Analysis of passive mechanical properties revealed that arterial stiffness was enhanced in vessels from Cx40T202S mice, but not in wild-type or Cx40Tg mice. CONCLUSIONS: Introduction of a mutant form of Cx40 in the endogenous endothelial Cx40 population prevents endothelium-derived hyperpolarization activation during myogenic constriction, enhancing sensitivity to intraluminal pressure and increasing arterial stiffness. We conclude that genetic polymorphisms in endothelial Cx40 can contribute to the pathogenesis of arterial disease.

Continued improvement in the development of the SARS-CoV-2 whole genome sequencing proficiency testing program.

Application of whole genome sequencing (WGS) has allowed monitoring of the emergence of variants of concern (VOC) of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) globally. Genomic investigation of emerging variants and surveillance of clinical progress has reduced the public health impact of infection during the COVID-19 pandemic. These steps required developing and implementing a proficiency testing program (PTP), as WGS has been incorporated into routine reference laboratory practice. In this study, we describe how the PTP evaluated the capacity and capability of one New Zealand and 14 Australian public health laboratories to perform WGS of SARS-CoV-2 in 2022. The participants' performances in characterising a specimen panel of known SARS-CoV-2 isolates in the PTP were assessed based on: (1) genome coverage, (2) Pango lineage, and (3) sequence quality, with the choice of assessment metrics refined based on a previously reported assessment conducted in 2021. The participants' performances in 2021 and 2022 were also compared after reassessing the 2021 results using the more stringent metrics adopted in 2022. We found that more participants would have failed the 2021 assessment for all survey samples and a significantly higher fail rate per sample in 2021 compared to 2022. This study highlights the importance of choosing appropriate performance metrics to reflect better the laboratories' capacity to perform SARS-CoV-2 WGS, as was done in the 2022 PTP. It also displays the need for a PTP for WGS of SARS-CoV-2 to be available to public health laboratories ongoing, with continuous refinements in the design and provision of the PTP to account for the dynamic nature of the COVID-19 pandemic as SARS-CoV-2 continues to evolve.

Bioinformatic investigation of discordant sequence data for SARS-CoV-2: insights for robust genomic analysis during pandemic surveillance.

The COVID-19 pandemic has necessitated the rapid development and implementation of whole-genome sequencing (WGS) and bioinformatic methods for managing the pandemic. However, variability in methods and capabilities between laboratories has posed challenges in ensuring data accuracy. A national working group comprising 18 laboratory scientists and bioinformaticians from Australia and New Zealand was formed to improve data concordance across public health laboratories (PHLs). One effort, presented in this study, sought to understand the impact of the methodology on consensus genome concordance and interpretation. SARS-CoV-2 WGS proficiency testing programme (PTP) data were retrospectively obtained from the 2021 Royal College of Pathologists of Australasia Quality Assurance Programmes (RCPAQAP), which included 11 participating Australian laboratories. The submitted consensus genomes and reads from eight contrived specimens were investigated, focusing on discordant sequence data and findings were presented to the working group to inform best practices. Despite using a variety of laboratory and bioinformatic methods for SARS-CoV-2 WGS, participants largely produced concordant genomes. Two participants returned five discordant sites in a high-Cτ replicate, which could be resolved with reasonable bioinformatic quality thresholds. We noted ten discrepancies in genome assessment that arose from nucleotide heterogeneity at three different sites in three cell-culture-derived control specimens. While these sites were ultimately accurate after considering the participants' bioinformatic parameters, it presented an interesting challenge for developing standards to account for intrahost single nucleotide variation (iSNV). Observed differences had little to no impact on key surveillance metrics, lineage assignment and phylogenetic clustering, while genome coverage <90 % affected both. We recommend PHLs bioinformatically generate two consensus genomes with and without ambiguity thresholds for quality control and downstream analysis, respectively, and adhere to a minimum 90 % genome coverage threshold for inclusion in surveillance interpretations. We also suggest additional PTP assessment criteria, including primer efficiency, detection of iSNVs and minimum genome coverage of 90 %. This study underscores the importance of multidisciplinary national working groups in informing guidelines in real time for bioinformatic quality acceptance criteria. It demonstrates the potential for enhancing public health responses through improved data concordance and quality control in SARS-CoV-2 genomic analysis during pandemic surveillance.

Proficiency testing for SARS-CoV-2 in assuring the quality and overall performance in viral RNA detection in clinical and public health laboratories.

Diagnostic testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has undergone significant changes over the duration of the pandemic. In early 2020, SARS-CoV-2 specific nucleic acid testing (NAT) protocols were predominantly in-house assays developed based on protocols published in peer reviewed journals. As the pandemic has progressed, there has been an increase in the choice of testing platforms. A proficiency testing program for the detection of SARS-CoV-2 by NAT was provided to assist laboratories in assessing and improving test capabilities in the early stages of the pandemic. This was vital in quality assuring initial in-house assays, later commercially produced assays, and informing the public health response. The Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) offered three rounds of proficiency testing for SARS-CoV-2 to Australian and New Zealand public and private laboratories in March, May, and November 2020. Each round included a panel of five specimens, consisting of positive (low, medium or high viral loads), inconclusive (technical specimen of selected SARS-CoV-2 specific genes) and negative specimens. Results were received for round 1 from 16, round 2 from 97 and round 3 from 101 participating laboratories. Improvement in the accuracy over time was shown, with the concordance of results in round 1 being 75.0%, in round 2 above 95.0% for all samples except one, and for round 3 above 95.0%. Overall, participants demonstrated high capabilities in detecting SARS-CoV-2, even in samples of low viral load, indicating excellent testing accuracy and therefore providing confidence in Australian and New Zealand public and private laboratories test results.

Proficiency testing for SARS-CoV-2 whole genome sequencing.

Extensive studies and analyses into the molecular features of severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2) have enhanced the surveillance and investigation of its clusters and transmission worldwide. The whole genome sequencing (WGS) approach is crucial in identifying the source of infection and transmission routes by monitoring the emergence of variants over time and through communities. Varying SARS-CoV-2 genomics capacity and capability levels have been established in public health laboratories across different Australian states and territories. Therefore, laboratories performing SARS-CoV-2 WGS for public health purposes are recommended to participate in an external proficiency testing program (PTP). This study describes the development of a SARS-CoV-2 WGS PTP. The PTP assessed the performance of laboratories while providing valuable insight into the current state of SARS-CoV-2 genomics in public health across Australia. Part 1 of the PTP contained eight simulated SARS-CoV-2 positive and negative specimens to assess laboratories' wet and dry laboratory capacity. Part 2 involved the analysis of a genomic dataset that consisted of a multi-FASTA file of 70 consensus genomes of SARS-CoV-2. Participating laboratories were required to (1) submit raw data for independent bioinformatics analysis, (2) analyse the data with their processes, and (3) answer relevant questions about the data. The performance of the laboratories was commendable, despite some variation in the reported results due to the different sequencing and bioinformatics approaches used by laboratories. The overall outcome is positive and demonstrates the critical role of the PTP in supporting the implementation and validation of SARS-CoV-2 WGS processes. The data derived from this PTP will contribute to the development of SARS-CoV-2 bioinformatic quality control (QC) and performance benchmarking for accreditation.

Proficiency testing for bacterial whole genome sequencing in assuring the quality of microbiology diagnostics in clinical and public health laboratories.

The adoption of whole genome sequencing (WGS) data over the past decade for pathogen surveillance, and decision-making for infectious diseases has rapidly transformed the landscape of clinical microbiology and public health. However, for successful transition to routine use of these techniques, it is crucial to ensure the WGS data generated meet defined quality standards for pathogen identification, typing, antimicrobial resistance detection and surveillance. Further, the ongoing development of these standards will ensure that the bioinformatic processes are capable of accurately identifying and characterising organisms of interest, and thereby facilitate the integration of WGS into routine clinical and public health laboratory setting. A pilot proficiency testing (PT) program for WGS of infectious agents was developed to facilitate widely applicable standardisation and benchmarking standards for WGS across a range of laboratories. The PT participating laboratories were required to generate WGS data from two bacterial isolates, and submit the raw data for independent bioinformatics analysis, as well as analyse the data with their own processes and answer relevant questions about the data. Overall, laboratories used a diverse range of bioinformatics tools and could generate and analyse high-quality data, either meeting or exceeding the minimum requirements. This pilot has provided valuable insight into the current state of genomics in clinical microbiology and public health laboratories across Australia. It will provide a baseline guide for the standardisation of WGS and enable the development of a PT program that allows an ongoing performance benchmark for accreditation of WGS-based test processes.

Laboratory biosafety measures involving SARS-CoV-2 and the classification as a Risk Group 3 biological agent.

The current public health emergency surrounding the COVID-19 pandemic, that is the illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in thousands of cases in Australia since 25 January 2020 when the first case was diagnosed. This emerging virus presents particular hazards to researchers and laboratory staff in a clinical setting, highlighted by rapid and widespread global transmission. Based on the epidemiological and clinical data that have become available in mid-2020, we propose the interim classification of SARS-CoV-2 as a Risk Group 3 organism is reasonable, and discuss establishing Biosafety Level 3 (BSL-3) regulations accordingly. Despite its global spread, the reported mortality rate of SARS-CoV-2 ranging from 0.13% to 6.22% is considerably less than that of other Risk Group 4 agents including Ebola and Marburg viruses with fatality rates as high as 90%. In addition, studies have demonstrated that approximately 86% of patients presenting with severe courses of the disease are aged 70 years or above, with the presence of comorbid conditions such as cardiovascular and respiratory system diseases in the majority of all fatal cases. In contrary to recent discussions surrounding the protective and administrative measures needed in a laboratory, the emerging evidence surrounding mortality rate, distinct demographics of severe infections, and the presence of underlying diseases does not justify the categorisation of SARS-CoV-2 as a Risk Group 4 organism. This article summarises biosafety precautions, control measures and appropriate physical containment facilities required to minimise the risk of laboratory-acquired infections with SARS-CoV-2.

Emerging trends of HIV epidemiology in Asia.

The main molecular trait of HIV-1 is the inherent capacity to vary, recombine, and diversify, which gives it a clear edge to evade the human immune system and survive through the generation of complex molecular forms, termed recombinants. In a setting of coinfection, molecular and biological interactions between diverse HIV-1 subtypes may promote the emergence of circulating recombinant forms through the shuffling of viral genomes, which results in increased intra- and inter- host viral variation and altered biological properties. The focus of this review is on Asia, which has the highest proportion of HIV-1 recombinants circulating worldwide, with the top in South and Southeast Asia, amounting to 89% of its total HIV-1 infection. The HIV-1 strains which are spreading in this geographic area are CRF01_AE, subtypes B and C. Given the rapid spread and active establishment of some of the recombinant forms in Asia, it is essential to understand how they differ from their parental strains, the acquisition of certain molecular traits, and their biological attributes upon recombination, which give these strains an epidemiologic edge. The current epidemic provides strong evidence that the parental subtypes are being replaced via competition with possibly more versatile HIV-1 recombinant forms. This appears to be an ongoing phenomenon and has resulted in an HIV-1 epidemic shift, with the expansion and dissemination of a wide variety of HIV-1 forms within this geographic region.

Near full-length sequence analysis of a Unique CRF01_AE/B recombinant from Kuala Lumpur, Malaysia.

A new HIV-1 circulating recombinant form (CRF), CRF33_01B, has been identified in Malaysia. Concurrently we found a unique recombinant form (URF), that is, the HIV-1 isolate 06MYKLD46, in Kuala Lumpur, Malaysia. It is composed of B or a Thai variant of the B subtype (B') and CRF01_AE. Here, we determined the near full-length genome of the isolate 06MYKLD46 and performed detailed phylogenetic and bootscanning analyses to characterize its mosaic composition and to further confirm the subtype assignments. Although the majority of the 06MYKLD46 genome is CRF01_AE, we found three short fragments of B or B' subtype inserted along the genome. These B or B' subtype regions were 716 and 335 bp, respectively, in the protease-reverse transcriptase (PR-RT) region, similar to those found in CRF33_01B, as well as an extra 590 bp in the env gene region. Thus we suggest that 06MYKLD46 is a possible second-generation HIV-1 recombinant derived from CRF33_01B.

Identification of nuclear-enriched miRNAs during mouse granulopoiesis.

BACKGROUND: MicroRNAs (miRNAs) are coordinators of cellular differentiation, including granulopoiesis. Although differential expression of many miRNAs is associated with the maturation of granulocytes, analysis of differentially expressed miRNAs and their cellular localization across all stages of granulopoiesis, starting from hemopoietic stems cells, is not well characterized. METHODS: We analyzed whole cell miRNA and mRNA expression during granulopoiesis using Taqman low-density and Affymetrix arrays respectively. We also performed nuclear and cytoplasmic fractionation followed by Taqman low-density array and/or quantitative PCR to identify nuclear-enriched miRNAs in hemopoietic stem/progenitor cells, promyelocytes, myelocytes, granulocytes and several hemopoietic cell lines. Anti-correlation between the expression of miRNA and target pairs was used to determine putative miRNA targets. RESULTS: Analyses of our array data revealed distinct clusters of differentially expressed miRNAs that are specific to promyelocytes and granulocytes. While the roles of many of these miRNAs in granulopoiesis are not currently known, anti-correlation of the expression of miRNA/mRNA target pairs identified a suite of novel target genes. Clusters of miRNAs (including members of the let-7 and miR-17-92 families) are downregulated in hemopoietic stem/progenitor cells, potentially allowing the expression of target genes known to facilitate stem cell proliferation and homeostasis. Additionally, four miRNAs (miR-709, miR-706, miR-690 and miR-467a*) were found to be enriched in the nucleus of myeloid cells and multiple hemopoietic cell lines compared to other miRNAs, which are predominantly cytoplasmic-enriched. Both miR-709 and miR-706 are nuclear-enriched throughout granulopoiesis and have putative binding sites of extensive complementarity downstream of pri-miRNAs. Nuclear enrichment of miR-467a* is specific to hemopoietic stem/progenitors and promyelocytes. These miRNAs are also nuclear-enriched in other hemopoietic cell lines, where nuclear sequestering may fine-tune the expression of cytoplasmic mRNA targets. CONCLUSIONS: Overall, we have demonstrated differentially expressed miRNAs that have not previously been associated with hemopoietic differentiation and provided further evidence of regulated nuclear-enrichment of miRNAs. Further studies into miRNA function in granulocyte development may shed light on fundamental aspects of regulatory RNA biology and the role of nuclear miRNAs.

Current Trends of HIV Recombination Worldwide.

One of the major characteristics of HIV-1 is its high genetic variability and extensive heterogeneity. This characteristic is due to its molecular traits, which in turn allows it to vary, recombine, and diversify at a high frequency. As such, it generates complex molecular forms, termed recombinants, which evade the human immune system and so survive. There is no sequence constraint to the recombination pattern as it appears to occur at inter-group (between groups M and O), as well as interand intra-subtype within group M. Rapid emergence and active global transmission of HIV-1 recombinants, known as circulating recombinant forms (CRFs) and unique recombinant forms (URFs), requires urgent attention. To date, 55 CRFs have been reported around the world. The first CRF01_AE originated from Central Africa but spread widely in Asia. The most recent CRF; CRF55_01B is a recombinant form of CRF01_AE and subtype B, although its origin is yet to be publicly disclosed. HIV-1 recombination is an ongoing event and plays an indispensable role in HIV epidemics in different regions. Africa, Asia and South America are identified as recombination hot-spots. They are affected by continual emergence and cocirculation of newly emerging CRFs and URFs, which are now responsible for almost 20% of HIV-1 infections worldwide. Better understanding of recombinants is necessary to determine their biological and molecular attributes.

Evidence for possible biological advantages of the newly emerging HIV-1 circulating recombinant form from Malaysia - CRF33_01B in comparison to its progenitors - CRF01_AE and subtype B.

In Malaysia, co-circulation of CRF01_AE and subtype B has resulted in the emergence of the second generation derivative; CRF33_01B in approximately 20% of its HIV-1 infected individuals. Our objective was to identify possible biological advantages that CRF33_01B possesses over its progenitors. Biological and molecular comparisons of CRF33_01B against its parental subtypes clearly show that CRF33_01B replicated better in activated whole peripheral blood mononuclear cells (PBMCs) and CD4+ T-lymphocytes, but not monocyte-derived macrophages (MDMs). Also, its acquired fitness was greater than CRF01_AE but not subtype B. Moreover, CRF33_01B has higher rate of apoptotic cell death and syncytia induction compared to subtype B. These adaptive and survival abilities could have been acquired by CRF33_01B due to the incorporation of subtype B fragments into the gag-RT region of its full-length genome. Our studies confirm the previously held belief that HIV-1 strains may harbor enhanced biological fitness upon recombination. We therefore estimate a possible gradual replacement of the current predominance of CRF01_AE, as well as wider dissemination of CRF33_01B, together with the identification of other new CRF01_AE/B inter-subtype recombinants in Malaysia.

Continuous crossover(s) events of HIV-1 CRF01_AE and B subtype strains in Malaysia: evidence of rapid and extensive HIV-1 evolution in the region.

The Asian HIV epidemic appears to be complex, characterized by the prevalence of multiple subtypes and circulating recombinant forms with gradual replacement of pure HIV-1 subtypes in several geographical regions. The main objectives of the present study are to identify and analyse the full-length viral genomes of three unique recombinant forms (URFs); the HIV-1 isolates 07MYKLD47, 07MYKLD48 and 07MYKLD49 from Malaysia. Long-range polymerase chain reaction (PCR) amplification of seven overlapping reading frames was used to derive near full-length HIV-1 genomes. Detailed phylogenetic and bootscanning analyses were performed to determine phylogenetic associations and subtypic assignments. We further confirmed the mosaic composition of these CRF01_AE/B inter-subtype recombinant forms, which are composed of B-subtype fragment(s) in the backbone of CRF01_AE. Both 07MYKLD47 and 07MYKLD48 have an insertion of B subtype (880 bp and 532 bp) in the gag-pol and gp41-env gene regions, respectively. Whereas the isolate 07MYKLD49 has three B-subtype fragments inserted in different gene region along the genome; one each in the gag-pol (1862 bp) and pol-vif (1935 bp) regions, and a short B-subtype insertion (541 bp) in the 5' LTR-gag region. This highlights the public health relevance of newly emerging second generation HIV-1 recombinant forms and their dispersal, along with their rapid and continuous evolution in the region.

Complex patterns of the HIV-1 epidemic in Kuala Lumpur, Malaysia: evidence for expansion of circulating recombinant form CRF33_01B and detection of multiple other recombinants.

The HIV protease-reverse transcriptase (PR-RT) (1047 bp), gp120-env (891 bp) and gp41-env (547 bp) regions from the plasma of 115 HIV-1-infected patients in Kuala Lumpur (KL), Malaysia were sequenced. Detailed phylogenetic and bootscanning analyses were performed to determine the mosaic structure of the HIV-1 strains and their recombination breakpoint(s). Among the 50 patient samples in which all three regions could be amplified, the HIV-1 CRF01_AE subtype (46%) was predominant followed by subtypes B (10%) and B' (6%). A total of 9/50 (18%) patients were infected with a CRF01_AE/B inter-subtype recombinant, displaying a recombinant form (RF)(PR-RT), CRF01_AE(gp120-env) and CRF01_AE(gp41-env). This RF was derived from the Thai variants of CRF01_AE and B' subtype, with two distinct B' subtype segments in the backbone of CRF01_AE, similar to the newly identified CRF33_01B. In addition, one sample demonstrated a close structural relationship with the new CRF33_01B in the PR-RT region but displayed B' segment in part of the env region (RF(PR-RT), CRF01_AE/B'(gp120-env) and B'(gp41-env)) indicating continuing evolution of CRF33_01B. The remaining 18% of samples were identified as unique recombinant forms (URFs).