Identification of the Near Full-Length Genome of a Novel HIV-1 CRF01_AE/CRF07_BC Recombinant in Hebei Province, China.

To analyze the genetic structure and recombination characteristics of a newly discovered HIV-1 unique recombinant form (URF) isolated in Hebei Province, China, viral RNA was extracted from the plasma sample of the infected individual and reverse transcribed to cDNA. Two overlapping segments of the HIV-1 genome were amplified using a near-endpoint dilution method. Recombinant breakpoints were determined using RIP, jpHMM, and SimPlot 3.5.1 software. MEGA 6.0 software was used to construct a neighbor-joining phylogenetic tree. The near full-length genome sequence (8,862 bp) of a recombinant of CRF01_AE/CRF07_BC was obtained. The genome comprised at least seven overlapping segments, including four CRF01_AE and three CRF07_BC segments, with CRF01_AE as the backbone. A URF virus between CRF01_AE and CRF07_BC was amplified and characterized in this study. Parental viruses were homologous with HIV-1 strains prevalent among men who have sex with men in northern China and may originate from sexual transmission of local HIV-1 strains in Hebei Province.

Genetic and pathogenic characterizations of a naturally occurring reassortant and homologous recombinant strain of the classical infectious bursal disease virus re-emerging in chickens in southern China.

Infectious bursal disease (IBD) classical virus strain (cIBDV) can cause morbidity and mortality in young chickens with severe long-term immunosuppression. However, since the emergence and widespread prevalence of very virulent strain (vvIBDV) in China from 1991, reports of cIBDV have become rare. A novel reassortant and recombinant strain GXYL211225 (genotype A1aB1a) with segment A originating from the classical strain (A1a) and segment B from the attenuated vaccine strain (B1a) was characterized in the study. Notably, segment A resulted from recombination between the cIBDV strains 150127-0.2 and Faragher52-70, expressing as a backbone from 150127-0.2, where a fragment located at the position of nucleotide (nt) 519-1 410 was replaced by the corresponding region of Faragher52-70. The infection of GXYL211225 caused mortality in SPF chicken embryos, despite lacking the critical amino acid (aa) residues 253H, 279 N and 284A associated with the cellular tropism, and induced significant cytopathic effect (CPE) on a wide range of cells, confirming its natural cell-adapted character. Furthermore, the challenge experiment of GXYL211225 was performed on the commercial Three-yellow chickens of 4-week-old, and with the vvIBDV HLJ-0504-like strain NN1172 and the novel variant (nv) IBDV strain QZ191002 as the comparison. All the challenged birds experienced reduced body-weight gain. QZ191002 infected birds showed no obvious clinical symptoms or mortality, while those of NN1172 and GXYL211225 showed typical IBD symptoms and resulted in 20% (2/10) and 10% (1/10) of mortality rates, respectively. At 7 days post-challenge (dpc), the damages of bursal of Fabricius (BF) varied among groups, with NN1172 causing the most severe lesions, followed by GXYL211225, and then QZ191002. It was also found that the pathogenicity was correlated positively with the viral load, aligning with the histopathological severity in BF. The study confirms the rapid and diverse evolution of the re-emerged classical strains in the field and emphasizes the need to monitor the changes of IBDV on both the genetic and pathogenic aspects for the effective control of the disease.

Three Novel CRF01_AE/CRF07_BC Recombinant Forms of HIV-1 Identified in Shijiazhuang City, China.

BACKGROUND: Hebei, a province with a low Human Immunodeficiency Virus (HIV) prevalence, is also a region with the most abundant HIV-1 genetic diversity. HIV-1 recombinant forms have been the key factor influencing the effectiveness of HIV-1 control and therapy. OBJECTIVES: We aimed to study inter-subtype recombinant structures of new HIV-1-second generation recombinant forms. METHODS: Monitoring the HIV-1 subtype by phylogenetic and recombinant breakpoint analyses are the two most frequent methods among men who have sex with men (MSM). Here, three near full-length genomes (NFLGs) were obtained from HIV-1 seropositive MSM in Shijiazhuang City, China, who have never received antiretroviral therapy in 2021. RESULTS: Phylogenetic analysis indicated that three NFLGs were novel inter-subtype recombinant forms between CRF07_BC and CRF01_AE. For the NFLG 21S009, four CRF07_BC gene fragments were inserted into the pol, vif-vpr, vpu-env, and nef-3` LTR gene regions within a CRF01_ AE backbone, respectively. For the NFLG 21S095, four breakpoints were identified in HIV-1 pol and vpu regions. The NFLG 21S370 contained four gene recombinant breakpoints within HIV-1 pol and vpu-env gene regions. Of these three NFLGs, the NFLG 21S009 contained the most breakpoints, distributed in the pol, vif, vpr, vpu, env, and nef regions, respectively. In the gag-pol regions, three NFLGs had only one CRF07_BC gene fragment inserted into gene points between 4250 and 4792. CONCLUSION: Our findings provide strong evidence that the surveillance of novel recombinant forms is necessary for the increase in better control of HIV.

Severe pneumonia and pathogenic damage in human airway epithelium caused by Coxsackievirus B4.

Coxsackievirus B4 (CVB4) has one of the highest proportions of fatal outcomes of other enterovirus serotypes. However, the pathogenesis of severe respiratory disease caused by CVB4 infection remains unclear. In this study, 3 of 42 (7.2%, GZ-R6, GZ-R7 and GZ-R8) patients with severe pneumonia tested positive for CVB4 infection in southern China. Three full-length genomes of pneumonia-derived CVB4 were sequenced and annotated for the first time, showing their high nucleotide similarity and clustering within genotype V. To analyze the pathogenic damage caused by CVB4 in the lungs, a well-differentiated human airway epithelium (HAE) was established and infected with the pneumonia-derived CVB4 isolate GZ-R6. The outcome was compared with that of a severe hand-foot-mouth disease (HFMD)-derived CVB4 strain GZ-HFM01. Compared with HFMD-derived CVB4, pneumonia-derived CVB4 caused more intense and rapid disruption of HAE polarity, leading to tight-junction barrier disruption, loss of cilia, and airway epithelial cell hypertrophy. More pneumonia-derived CVB4 were released from the basolateral side of the HAE than HFMD-derived CVB4. Of the 18 cytokines tested, only IL-6 and IL-1b secretion significantly increased on bilateral sides of HAE during the early stage of pneumonia-derived CVB4 infection, while multiple cytokine secretions significantly increased in HFMD-derived CVB4-infected HAE. HFMD-derived CVB4 exhibited stronger neurovirulence in the human neuroblastoma cells SH-SY5Y than pneumonia-derived CVB4, which is consistent with the clinical manifestations of patients infected with these two viruses. This study has increased the depth of our knowledge of severe pneumonia infection caused by CVB4 and will benefit its prevention and treatment.

Identification of the Near Full-length Genome of a Novel HIV-1 CRF01_AE/CRF07_BC Recombinant with a Complex Genomic Structure Isolated in Hebei Province, China.

BACKGROUND: During HIV genotypic drug resistance testing of patient samples in Baoding, Hebei Province, China, in 2022, a recombinant fragment was detected in the pol region of an HIV-1 strain. OBJECTIVE: The objective of the study was to analyze the near full-length genome of a novel HIV-1 CRF01_AE/CRF07_BC recombinant with a complex genomic structure. METHODS: Viral RNA was extracted from the blood of the infected individual and reverse transcribed to cDNA. Two overlapping segments of the HIV-1 genome were amplified using a nearendpoint dilution method and sequenced. Recombinant breakpoints were determined using RIP, jpHMM, and SimPlot 3.5.1 software. MEGA 6.0 software was used to construct a neighbor-joining phylogenetic tree. RESULTS: We obtained the near full-length genome sequence (8680 bp) of a novel HIV-1 CRF01_AE/CRF07_BC recombinant. Recombination analysis showed that the genome comprised at least 12 overlapping segments, including six CRF07_BC and six CRF01_AE segments, with CRF07_BC as the backbone. The emergence of CRF01_AE/CRF07_BC recombinant strains indicated that HIV-1 co-infection is common. However, the increasing genetic complexity of the HIV-1 epidemic in China warrants continued investigation. CONCLUSION: The increase in CRF01_AE/CRF07_BC recombinant viruses suggests that HIV-1 has a high genetic mutation rate in Hebei, China. This highlights the need for close monitoring of HIV-1 molecular epidemiologic changes to provide accurate, up-to-date information for effective disease control.

Phylogenetic and antigenic analysis of infectious bronchitis virus isolated from commercial and backyard chickens in Pakistan, 2015-2018.

Infectious bronchitis virus (IBV) is a rapidly evolving virus affecting both vaccinated and unvaccinated poultry flocks and is responsible for significant economic losses globally; hence, it is imperative to obtain a deeper understanding of this pathogen. In this study, seven IBV strains were isolated from commercial and backyard poultry flocks during 2015-2018. We obtained full-length IBV genomes of two viruses using the Illumina sequencing method, while five additional viruses were genetically characterized through full-length spike (S1) gene sequencing. Phylogenetic and distance analysis based on complete S1 gene and full-length genome sequences revealed that one IBV isolate belonged to genotype GI-1 and six viruses were clustered within genotype GI-13. Deduced amino acid sequences of GI-13 strains exhibited 31.8-37.2 % divergence with the commonly used classic vaccine strains (M41) and 2.7-12.6 % with variant vaccine strains (4/91) in Pakistan. High evolutionary distances suggest that the IBV viruses circulating in Pakistan are under continuous evolutionary pressure. Moreover, ch/IBV/Pak/AW-2/2017 was found to have originated from an intra-genotypic recombination event between the variant group (GI-23 lineage as a major parent) and variant vaccine strain (4/91-like as a minor parent) and is the first example of recombination within genotype GI-13 in Pakistan. Together, these findings provide genetic and evolutionary insights into the currently circulating IBV genotypes in Pakistan, which could help to better understand the origin, spread and evolution of IBVs, and to ascertain the importance of disease monitoring as well as re-evaluation forof currently used vaccines and vaccination programmes.

Genomic Characterization of a Novel HIV-1 Second-Generation Recombinant (CRF01_AE/CRF07_BC) Identified from a Men Who Have Sex with Men (MSM) Patient in Hebei, China.

Along with the co-circulation of dominant HIV-1 strains (CRF01_AE and CRF07_BC) in China, an increasing number of second-generation recombinants are being detected, especially among men who have sex with men (MSM). In this study, we identified a unique CRF01_AE/CRF07_BC recombinant from a HIV-1-positive man (BDD015A) infected through homosexual transmission in Baoding city, Hebei Province. Analysis of the near full-length genome sequence of the recombinant revealed five segments divided by four breakpoints, with two regions of CRF07_BC inserted into the pol and env regions of the CRF01_AE backbone. Three CRF01_AE segments (I, III, and V) clustered within the cluster 4 lineage, which mainly circulated among MSM in China. This recombinant differed from previously reported CRF01_ AE and CRF07_BC recombinant forms. The continuous emergence of novel recombinants increases the genetic complexity of HIV-1 in Hebei. Further measures are needed to monitor the molecular epidemiological characteristics of HIV-1 to help control the spread of infections.

Characterization of two novel HIV-1 second-generation recombinants (CRF01_AE/CRF07_BC) identified in Hebei Province, China.

Introduction: The unique recombinant forms (URFs) of HIV-1 consist of a mixture of subtypes, and each URF has a unique breakpoint. In this study, we identified the near fulllength genome (NFLG) sequences of two novel HIV-1 URFs (Sample ID: BDD034A and BDL060) isolated during HIV-1 molecular surveillance in 2022 in Baoding city, Hebei Province, China. Methods: The two sequences were aligned with subtype reference sequences and CRFs from China using MAFFT v7.0, and the alignments were adjusted manually using BioEdit (v7.2.5.0). Phylogenetic and subregion trees were constructed using MEGA11 with the neighbor-joining (N-J) method. Recombination breakpoints were identified by SimPlot (v3.5.1) based on Bootscan analyses. Results: Recombinant breakpoint analysis revealed that the NFLGs of BDD034A and BDL060 were composed of CRF01_AE and CRF07_BC, containing seven segments, respectively. For BDD034A, three CRF01_AE fragments were inserted into the CRF07_BC main framework, whereas for BDL060, three CRF07_BC fragments were inserted into the CRF01_AE main framework. Discussion: The emergence of the CRF01_AE/CRF07_BC recombinant strains indicates that HIV-1 co-infection is common. The increasing genetic complexity of the HIV-1 epidemic in China warrants continued investigation.

Genetic characteristics of a novel HIV-1 recombinant lineage (CRF103_01B) and its prevalence in northern China.

During the routine surveillance of HIV-1 pretreatment drug resistance in Beijing, five men who have sex with men (MSM) and a woman were observed to get infected by newly identified CRF103_01B strain. To elucidate the genetic characteristics, the near full-length genome (NFLG) was obtained. Phylogenetic inference indicated that CRF103_01B NFLG was composed of six mosaic segments. Segments IV and V of CRF103_01B were located among the clusters subtype B and CRF01_AE (group 5), respectively. The CRF103_01B strain was deduced to originate from Beijing MSM population around 2002.3-2006.4 and continued to spread among MSM population at a low level, then to the general population via heterosexual contact in northern China. Molecular epidemiology surveillance of CRF103_01B should be reinforced.

Identification of two near-identical novel HIV-1 unique recombinant forms (CRF01_AE/B) among men who have sex with men in baoding, hebei, China.

Men who have sex with men (MSM) are the most frequent infection route of the human immunodeficiency virus (HIV) in Baoding, China, creating chances for the occurrence of unique recombinant forms (URFs) of the virus, i.e., recombination of different subtypes caused by co-circulation of multiple subtypes. In this report, two near-identical URFs (BDD002A and BDD069A) isolated from MSM in Baoding were identified. Phylogenetic tree analysis based on nearly full-length genomes (NFLGs) revealed that the two URFs formed a distinct monophyletic cluster with a bootstrap value of 100%. Recombinant breakpoints analysis identified that the NFLGs of BDD002A and BDD069A were both composed of CRF01_AE and subtype B, with six subtype B mosaic segments inserted into the CRF01_AE backbone. The CRF01_AE segments of the URFs clustered closely with the CRF01_AE reference sequences, and the B subregions clustered with the B reference sequences. The recombinant breakpoints of the two URFs were almost identical. These results suggest that effective interventions are urgently needed to prevent the formation of complex HIV-1 recombinant forms in Baoding, China.

Genetic Characteristics of Two Unique Recombinant Forms of HIV-1 CRF01_AE/CRF07_BC by Near Full-Length Genome from Men Who Have Sex with Men in Beijing, China.

Two HIV-1 infections with unassigned genotypes were identified during HIV-1 pretreatment drug resistance surveillance. The near full-length genome sequences of BL5040-00 and BL5085-00 were obtained and were classified as unique recombinant forms (URFs) between CRF01_AE and CRF07_BC. Simplot (version 3.5) analyses showed that the two URFs shared similar recombinant forms, and in the backbone belonging to CRF01_AE, the gag-pol, vpu, env, and nef gene fragments were genetically substituted by CRF07_BC. BL5040-00, with 10 breakpoints, had 6 CRF07_BC fragments and 5 CRF01_AE fragments, whereas BL5085-00, with 6 breakpoints, had 4 CRF07_BC fragments and 3 CRF01_AE fragments. BL5040-00 strain had two additional recombination breakpoints in pol-vif gene. The presence of URFs suggests that the men who have sex with men population in Beijing has an active HIV epidemic and the genetic diversity of HIV-1 is complex, emphasizing molecular epidemiology and disease progression monitoring should be strengthened.

Identification and genomic characterization of a novel HIV-1 unique recombinant form (CRF01_AE/CRF07_BC) in Zhejiang Province, China.

Mutation and recombination are important mechanisms leading to the frequent evolution and genetic diversity of viruses as HIV-1. In this study, we identified the near full-length genomic characterization of a novel HIV-1 unique recombinant form (URF) strain (Sample ID: ZJ20202195/ZJ/CHN/2020, hereafter referred to as ZJ20202195) isolated during the HIV-1 molecular surveillance in 2020 in Zhejiang Province, China, through different recombination analysis tools and phylogenetic analysis. Our results amply proved that the near full-length genome (NFLG) sequence of ZJ20202195 was a novel HIV-1 unique recombinant form (URF) consisting of CRF01_AE and CRF07_BC subtype, and delimited three recombinant segments, of which the Segment I (HXB2:776-5559 nucleotide (nt)) and Segment III (HXB2:6224-9412 nt) were mainly originated from CRF01_AE cluster g4a strains prevalent in China and Segment II (HXB2:5560-6223 nt) was from CRF07_BC subtype. Overall, our findings provide insight and a scientific basis in the genetic diversity and accurate determination of HIV-1 recombinant strains in China.

Tracing the origin of an imported HIV-1 sub-subtype A6 strain first identified in Zhejiang Province, China.

Human migration and mobility have been identified as key drivers of HIV dissemination among nations, which increases the problem of genetic diversity. Here, we report the near full-length genome of HIV-1 A6 identified in a female patient in the remote mountain area of Lishui, Zhejiang Province, which is the first time A6 has been reported in China. The near full-length genome was amplified with two large amplicons of 5.5 kb and 3.7 kb, and then target PCR products were sequenced by Sanger sequencing. The A6 strain was confirmed by the Basic Local Alignment Search Tool (BLAST) and a maximum-likelihood (ML) phylogenetic tree. The time to the most recent common ancestor (tMRCA) was inferred to be 2004 (95% HPD interval: 2003-2006). The sequence harbored the L74I mutation, which is a key characteristic genetic marker of A6. Combining the above evidence with epidemiological investigations, this A6 strain was determined to be from Ukraine, which was supported by phylogenetic analysis. This study identified a foreign imported strain, indicating a trend of increasing complication in the HIV-1 epidemic in Zhejiang, China.

Identification of a New HIV-1 Circulating Recombinant Form CRF112_01B Strain in Baoding City, Hebei Province, China.

BACKGROUND: A large number of HIV-1 recombinants that originated from CRF01_AE and B strains are constantly emerging in men who have sex with men populations in China and deserve more attention and further monitoring. OBJECTIVE: To analyze the near-full-length genome structure and recombination characteristics of a new HIV-1 strain (BD226AJ) detected in Baoding City and determine its subtype. CASE REPRESENTATION: Viral RNA was extracted from a blood sample collected from an infected individual and reverse transcribed to cDNA. Two overlapping segments of the HIV-1 genome were amplified using a near-endpoint dilution method and sequenced. Recombinant breakpoints were determined using RIP, jpHMM, and SimPlot 3.5.1 software. MEGA v6.0 was used to construct a neighbor-joining phylogenetic tree to determine the homology relationships of this strain. RESULTS AND DISCUSSION: We obtained 8830 nucleotides (nt) of the HIV-1 genome sequence by amplification and sequencing, and four recombinant fragments were identified by recombination analysis, namely CRF01_AE (HXB2, 823-4224 nt), subtype B (HXB2, 4225-5991 nt), CRF01_AE (HXB2, 5992-9295 nt), and subtype B (HXB2, 9296-9406 nt). The BLAST results showed that 96% of the sequence was similar to CRF112_01B. The jpHMM results confirmed that BD226AJ was the CRF112_01B strain. CONCLUSION: Our results confirm the first epidemic of CRF112_01B in Hebei Province. This finding suggests that HIV-1 CRF112_01B may have been introduced into Hebei by men who have sex with men and indicates that the epidemic trend of this strain should be closely monitored.

Identification and genetic characterization of a minor norovirus genotype, GIX.1[GII.P15], from China.

BACKGROUND: Human noroviruses, single-stranded RNA viruses in the family Caliciviridae, are a leading cause of nonbacterial acute gastroenteritis in people of all ages worldwide. Despite three decades of genomic sequencing and epidemiological norovirus studies, full-length genome analyses of the non-epidemic or minor norovirus genotypes are rare and genomic regions other than ORF2 and 3'-end of ORF1 have been largely understudied, which hampers a better understanding of the evolutionary mechanisms of emergence of new strains. In this study, we detected a rare norovirus genotype, GIX.1[GII.P15], in a vomit sample of a 60 year old woman with acute gastroenteritis using Raji cells and sequenced the complete genome. RESULTS: Using electron microscopy, a morphology of spherical and lace-like appearance of norovirus virus particles with a diameter of approximately 30 nm were observed. Phylogenetic analysis of VP1 and the RdRp region indicated that the KMN1 strain could be genotyped as GIX.1[GII.P15]. In addition, the VP1 region of KMN1 strain had 94.15% ± 3.54% percent nucleotide identity (PNI) compared to 26 genomic sequences available in GenBank, indicating a higher degree similarity between KMN1 and other GIX.1[GII.P15] strains. Further analysis of the full genome sequence of KMN1 strain showed that a total of 96 nucleotide substitutions (63 in ORF1, 25 in ORF2, and 8 in ORF3) were found across the genome compared with the consensus sequence of GIX.1[GII.P15] genome, and 6 substitutions caused amino acid changes (4 in ORF1, 1 in ORF2, and 1 in ORF3). However, only one nucleotide substitution results in the amino acid change (P302S) in the VP1 protein and the site was located near one of the predicted conformational B epitopes on the dimer structure. CONCLUSIONS: The genomic information of the new GIX.1[GII.P15] strain KMN1, which was identified in Kunming, China could provide helpful insights for the study of the genetic evolution of the virus.

Genomic Characterization of Chicken Anemia Virus in Broilers in Shandong Province, China, 2020-2021.

Chicken infectious anemia (CIA), caused by chicken anemia virus (CAV), is an immunosuppressive disease characterized by growth retardation, aplastic anemia, lymphoid depletion, and immunodepression in young chickens. In this study, 33 CAV strains were isolated from broilers in Shandong Province during 2020-2021. Phylogenetic analysis of full-length genome sequences showed that most CAV strains isolated in this study were scattered across different branches, but mainly clustered in two genotypes, indicating a certain regional characteristic. Analysis of VP1 protein identified several amino acid substitutions which were relevant with the virulence and virus spread efficiency. Interestingly, four putative DNA recombination events were detected in the genomes of novel isolated CAV strains. In summary, this study demonstrated a genomic diversity of CAV in broilers isolated in Shandong Province during 2020-2021, and provided information for the further study of CAV molecular epidemiology and viral evolution.

Complete genome and pathogenesis of a novel recombinant Senecavirus A isolate in P.R. China.

Senecavirus A (SVA), formerly called Seneca Valley virus (SVV) was first isolated from the USA in 2002. This study isolated an SVA strain from a pig herd in Shandong Province, PR China and designated it SVA-CH-SDGT-2017. The full-length genome, excluding the poly(A) tails of the SVA isolates, was 7280 nucleotides long, with the genomic organization resembling and sharing high nucleotide identities of 90.7-96.9 % with other previously reported SVA isolates. To investigate the pathogenicity of the SVA isolates, experimental infections of pigs were performed. The SVA strains successfully infected the pigs, as evidenced by the presence of virus shedding and robust serum neutralizing antibody responses. In addition, the contact-exposed experiment showed that the virus shedding of the contact-exposed pigs was approximately a 100-fold reduced compared to that of the inoculated group, indicating that the virus is capable of transmission to pigs. Our findings provide useful data for studying the pathogenesis and transmission of SVA in pigs.

New Approaches to Multi-Parametric HIV-1 Genetics Using Multiple Displacement Amplification: Determining the What, How, and Where of the HIV-1 Reservoir.

Development of potential HIV-1 curative interventions requires accurate characterization of the proviral reservoir, defined as host-integrated viral DNA genomes that drive rebound of viremia upon halting ART (antiretroviral therapy). Evaluation of such interventions necessitates methods capable of pinpointing the rare, genetically intact, replication-competent proviruses within a background of defective proviruses. This evaluation can be achieved by identifying the distinct integration sites of intact proviruses within host genomes and monitoring the dynamics of these proviruses and host cell lineages over longitudinal sampling. Until recently, molecular genetic approaches at the single proviral level have been generally limited to one of a few metrics, such as proviral genome sequence/intactness, host-proviral integration site, or replication competency. New approaches, taking advantage of MDA (multiple displacement amplification) for WGA (whole genome amplification), have enabled multiparametric proviral characterization at the single-genome level, including proviral genome sequence, host-proviral integration site, and phenotypic characterization of the host cell lineage, such as CD4 memory subset and antigen specificity. In this review, we will examine the workflow of MDA-augmented molecular genetic approaches to study the HIV-1 reservoir, highlighting technical advantages and flexibility. We focus on a collection of recent studies in which investigators have used these approaches to comprehensively characterize intact and defective proviruses from donors on ART, investigate mechanisms of elite control, and define cell lineage identity and antigen specificity of infected CD4+ T cell clones. The highlighted studies exemplify how these approaches and their future iterations will be key in defining the targets and evaluating the impacts of HIV curative interventions.

Increased Frequency of Inter-Subtype HIV-1 Recombinants Identified by Near Full-Length Virus Sequencing in Rwandan Acute Transmission Cohorts.

Most studies of HIV-1 transmission have focused on subtypes B and C. In this study, we determined the genomic sequences of the transmitted founder (TF) viruses from acutely infected individuals enrolled between 2005 and 2011 into IAVI protocol C in Rwanda and have compared these isolates to viruses from more recent (2016-2019) acute/early infections in three at risk populations - MSM, high risk women (HRW), and discordant couples (DC). For the Protocol C samples, we utilized near full-length single genome (NFLG) amplification to generate 288 HIV-1 amplicons from 26 acutely infected seroconverters (SC), while for the 21 recent seroconverter samples (13 from HRW, two from DC, and six from MSM), we PCR amplified overlapping half-genomes. Using PacBio SMRT technology combined with the MDPseq workflow, we performed multiplex sequencing to obtain high accuracy sequences for each amplicon. Phylogenetic analyses indicated that the majority of recent transmitted viruses from DC and HRW clustered within those of the earlier Protocol C cohort. However, five of six sequences from the MSM cohort branched together and were greater than 97% identical. Recombination analyses revealed a high frequency (6/26; 23%) of unique inter-subtype recombination in Protocol C with 19% AC and 4% CD recombinant viruses, which contrasted with only 6.5% of recombinants defined by sequencing of the pol gene previously. The frequency of recombinants was significantly higher (12/21; 57%) in the more recent isolates, although, the five related viruses from the MSM cohort had identical recombination break points. While major drug resistance mutations were absent from Protocol C viruses, 4/21 of recent isolates exhibited transmitted nevirapine resistance. These results demonstrate the ongoing evolution and increased prevalence of recombinant and drug resistant transmitted viruses in Rwanda and highlight the importance of defining NFLG sequences to fully understand the nature of TF viruses and in particular the prevalence of unique recombinant forms (URFs) in transmission cohorts.

HIV-1 proviral landscape characterization varies by pipeline analysis.

INTRODUCTION: HIV rebounds after cessation of antiretroviral therapy, representing a barrier to cure. To better understand the virus reservoir, analysis pipelines have been developed that categorize proviral sequences as intact or defective, and further determine the precise nature of the sequence defects that may be present. We investigated the effects that different analysis pipelines had on the characterization of HIV-1 proviral sequences. METHODS: We used single genome amplification to generate near full-length (NFL) HIV-1 proviral DNA sequences, defined as amplicons greater than 8000 base pairs in length, isolated from peripheral blood mononuclear cells (PBMC) of treated suppressed participants with HIV-1. Amplicons underwent direct next-generation single genome sequencing and were analysed using four HIV-1 proviral characterization pipelines. Sequences were characterized as intact or defective; defective sequences were assessed for the number and types of defects present. To confirm and extend our findings, 691 proviruses from the Proviral Sequence Database (PSD) were analysed and the ProSeq-IT tool of the PSD was used to characterize both the participant and PSD proviruses. RESULTS AND DISCUSSION: Virus sequences derived from thirteen ART-treated virologically suppressed participants with HIV were studied. A total of 693 HIV-1 proviral sequences were generated, 282 of which were NFL. An average of 53 sequences per participant was analysed. We found that proviruses often harbour multiple sequence defect types (mean 2.7, 95% confidence interval [CI] 2.5, 3.0); the elimination order used by each pipeline affected the percentage of proviruses allotted into each defect category. These differences varied between participants, depending on the number of defect categories present in a given provirus sequence. Pipeline-specific differences in characterizing the HIV-1 5' untranslated region (5' UTR) led to an overestimation of the number of intact NFL proviral sequences, a finding corroborated in the independent PSD analysis. A comparison of the four published pipelines to ProSeq-IT found that ProSeq IT was more likely to characterize proviruses as intact. CONCLUSIONS: The choice of pipeline used for HIV-1 provirus landscape analysis may bias the classification of defective sequences. To improve the comparison of provirus characterizations across research groups, the development of a consensus elimination pipeline should be prioritized.