Moloney murine leukemia virus

Vero cells were seeded in 6-well plates 1 day prior to the assay. The cells were inoculated with equal amount of HP strain or CCA strain, followed by incubation at 4°C for 1 hour. After synchronized viral adsorption, the cells were washed twice with PBS to remove the unbound virions. Then the cells were incubated at 37°C in the medium of DMEM. The virus infected cells were collected by Trizol reagent at indicated time points post infection. For detection of replication of negative strand genomic RNA, the total RNAs of the infected cell samples were extracted and subjected to RT-PCR quantification as described previously. In brief, cDNA was synthesized with reverse transcriptase of Moloney murine leukemia virus (Takara) using the primer 1F (5’-ttaaaacagcctgtgggttg-3’) specific to the negative strand genomic RNA. Then the cDNA of negative strand genomic RNA was quantified by real time PCR (SsoFast EvaGreen Supermix, Takara) using the primers EV71-RT-F (5’-gcagcccaaaagaacttcac-3’) and EV71-RT-R (5’-atttcagcagcttggagtgc-3’) that are located in the capsid protein VP1 gene. The gene of GAPDH was used as the internal control to normalize the total RNA of the sample. The negative strand RNA amount were expressed as fold increase relative to that of sample collected at 0.5 hour post infection.

RT-qPCR was performed using the same batch of specimens. The RNAs were extracted using TRIzol reagent, reverse transcribed using Moloney murine leukemia virus (Takara Biotechnology Co., Ltd., Shiga, Japan), and separately subjected to PCR amplification. The primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.) using GAPDH as the reference gene. The primer sequences, amplification length and annealing temperature are reported previously [10 (link)]. The reaction procedure was as follows: Initial denaturation at 94°C for 5 min; followed by 35 cycles for denaturation at 94°C for 30 sec, annealing for 30 sec and elongation at 72°C for 30 sec; each sample was assayed in triplicate. A temperature range of 65–95°C was selected for drawing melting curves.