Genetic Characterization of a Panel of Diverse HIV-1 Isolates at Seven International Sites.
HIV-1 subtypes and drug resistance are routinely tested by many international surveillance groups. However, results from different sites often vary. A systematic comparison of results from multiple sites is needed to determine whether a standardized protocol is required for consistent and accurate data analysis. A panel of well-characterized HIV-1 isolates (N = 50) from the External Quality Assurance Program Oversight Laboratory (EQAPOL) was assembled for evaluation at seven international sites. This virus panel included seven subtypes, six circulating recombinant forms (CRFs), nine unique recombinant forms (URFs) and three group O viruses. Seven viruses contained 10 major drug resistance mutations (DRMs). HIV-1 isolates were prepared at a concentration of 107 copies/ml and compiled into blinded panels. Subtypes and DRMs were determined with partial or full pol gene sequences by conventional Sanger sequencing and/or Next Generation Sequencing (NGS). Subtype and DRM results were reported and decoded for comparison with full-length genome sequences generated by EQAPOL. The partial pol gene was amplified by RT-PCR and sequenced for 89.4%-100% of group M viruses at six sites. Subtyping results of majority of the viruses (83%-97.9%) were correctly determined for the partial pol sequences. All 10 major DRMs in seven isolates were detected at these six sites. The complete pol gene sequence was also obtained by NGS at one site. However, this method missed six group M viruses and sequences contained host chromosome fragments. Three group O viruses were only characterized with additional group O-specific RT-PCR primers employed by one site. These results indicate that PCR protocols and subtyping tools should be standardized to efficiently amplify diverse viruses and more consistently assign virus genotypes, which is critical for accurate global subtype and drug resistance surveillance. Targeted NGS analysis of partial pol sequences can serve as an alternative approach, especially for detection of low-abundance DRMs.
Duke Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.
Bhavna
B
Hora
Blood Systems Research Institute, San Francisco, California, United States of America.|Department of Laboratory Medicine, University of California, San Francisco, California, United States of America.
Sheila M
SM
Keating
Duke Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.
Yue
Y
Chen
Duke Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.
Ana M
AM
Sanchez
Instituto de Medicina Tropical, Sao Paolo Brazil.
Ester
E
Sabino
National Institute of Communicable Diseases, Johannesburg, South Africa.
Gillian
G
Hunt
National Institute of Communicable Diseases, Johannesburg, South Africa.
Johanna
J
Ledwaba
Abbott Laboratories, Infectious Diseases Research, Abbott Park, Illinois, United States of America.
John
J
Hackett
Abbott Laboratories, Infectious Diseases Research, Abbott Park, Illinois, United States of America.
Priscilla
P
Swanson
Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Springs, Maryland, United States of America.
Indira
I
Hewlett
Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Springs, Maryland, United States of America.
Viswanath
V
Ragupathy
Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Springs, Maryland, United States of America.
Sai
S
Vikram Vemula
Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu, China.
Peibin
P
Zeng
Centre of Excellence for Research in AIDS, Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Kok-Keng
KK
Tee
Centre of Excellence for Research in AIDS, Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
Wei Zhen
WZ
Chow
National HIV & Retrovirology Laboratories at JC Wilt Infectious Diseases Research Center, Public Health Agency of Canada, Winnipeg, Canada.
Hezhao
H
Ji
National HIV & Retrovirology Laboratories at JC Wilt Infectious Diseases Research Center, Public Health Agency of Canada, Winnipeg, Canada.
Paul
P
Sandstrom
Duke Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.
Thomas N
TN
Denny
Blood Systems Research Institute, San Francisco, California, United States of America.|Department of Laboratory Medicine, University of California, San Francisco, California, United States of America.
Michael P
MP
Busch
Duke Human Vaccine Institute and Department of Medicine, Duke University Medical Center, Durham, North Carolina, United States of America.
Feng
F
Gao
D024882:Drug Resistance, Viral / Q000235:genetics; D015751:Genes, pol / Q000235:genetics*; D005838:Genotype; D015658:HIV Infections / Q000235:genetics* / Q000821:virology; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics* / Q000472:pathogenicity; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D011995:Recombination, Genetic