Generation of HIV-1 primary isolates representative of plasma variants using the U87.CD4 cell line.
In order to obtain HIV-1 primary isolates in settings with limited access to donor PBMCs, a culture method was developed where patient PBMCs infected with HIV-1 were cultured together with U87.CD4 cells. Using this non-laborious method, it is possible to harvest virus solely on the basis of syncytia formation and circumventing monitoring of viral replication by CA-p24 ELISA. Primary isolates from 23 out of 33 patients (70%) were isolated successfully. From PCR amplification and sequencing of the V1V5 region of the viral gp120 envelope gene, primary isolates were compared with variants obtained from plasma and PBMCs of 13 patients. The primary isolates of seven patients (54%) resembled closely the plasma viral quasispecies, whereas different variants were isolated from the other patients (46%). Three patients harboured a dual infection, while this remained unnoticed from sequencing the plasma or PBMC compartment. The primary isolates were highly infectious for TZM-bl cells and could infect CD4-enriched lymphocytes. This study demonstrates that it is possible to grow viral isolates using a non-laborious and simple method. These isolates may be used in the field for studies on antiretroviral therapy or for vaccine trials.
Heeregrave
Edwin J
EJ
Laboratory of Experimental Virology, Department of Medical Microbiology, Center of Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, The Netherlands.
Ampofo
William K
WK
Tetteh
John K A
JK
Ofori
Michael
M
Ofori
Sampson B
SB
Shah
Akram S
AS
Pollakis
Georgios
G
Paxton
William A
WA
D044822:Biodiversity; D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D016000:Cluster Analysis; D015699:HIV Envelope Protein gp120; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000145:classification* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D010949:Plasma / Q000821:virology*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA; D014773:Virology / Q000379:methods*; D014776:Virus Cultivation / Q000379:methods
D015699:HIV Envelope Protein gp120; C514793:gp120 protein, Human immunodeficiency virus 1