Phenotypic and Genotypic Characteristics of Novel Mouse Cell Line (NIH/3T3)-Adapted Human Enterovirus 71 Strains (EV71:TLLm and EV71:TLLmv)

Abstract

Since its identification in 1969, Enterovirus 71 (EV71) has been causing periodic outbreaks of infection in children worldwide and most prominently in the Asia-Pacific Region. Understanding the pathogenesis of Enterovirus 71 (EV71) is hampered by the virus’s inability to infect small animals and replicate in their derived in vitro cultured cells. This manuscript describes the phenotypic and genotypic characteristics of two selected EV71 strains (EV71:TLLm and EV71:TLLmv), which have been adapted to replicate in mouse-derived NIH/3T3 cells, in contrast to the original parental virus which is only able to replicate in primate cell lines. The EV71:TLLm strain exhibited productive infection in all primate and rodent cell lines tested, while EV71:TLLmv exhibited greater preference for mouse cell lines. EV71:TLLmv displayed higher degree of adaptation and temperature adaptability in NIH/3T3 cells than in Vero cells, suggesting much higher fitness in NIH/3T3 cells. In comparison with the parental EV71:BS strain, the adapted strains accumulated multiple adaptive mutations in the genome resulting in amino acid substitutions, most notably in the capsid-encoding region (P1) and viral RNA-dependent RNA polymerase (3D). Two mutations, E167D and L169F, were mapped to the VP1 canyon that binds the SCARB2 receptor on host cells. Another two mutations, S135T and K140I, were located in the VP2 neutralization epitope spanning amino acids 136–150. This is the first report of human EV71 with the ability to productively infect rodent cell lines in vitro.

Enterovirus 71 infection of motor neuron-like NSC-34 cells undergoes a non-lytic exit pathway

“…To determine whether mSCARB2 mediates EV71 entry into NSC-34 cells, an in vitro competition assay was performed using a commercially available anti-mouse SCARB-2 (mSCARB-2) antibody. The mouse-adapted EV71:TLLm strain which was previously shown to enter mouse cells via mSCARB-2 receptor36, was used as a positive control. Expectedly, incubation of NSC-34 cells with mSCARB-2 antibody prior to infection with EV71:TLLm strain led to significant reduction of virus titers in the culture supernatant (Fig.…”

A clinically authentic mouse model of enterovirus 71 (EV-A71)-induced neurogenic pulmonary oedema

“…Mice were also provided with food pellets and distilled water ad libitum , and were allowed to mate to obtain newborn mice. EV-A71 strains used for inoculation (EV71:BS, EV71:TLLm, and EV71:TLLmv) had been described and characterized previously 38 . Virus stocks of known titer and matching lot were stored at −80 °C and thawed at 4 °C before use.…”

“…Our group generated two novel EV-A71 strains (EV71:TLLm and EV71:TLLmv; GenBank accession numbers KF514879 and KF514880, respectively) adapted through serial passage of a clinical isolate (EV71:BS) in a mouse embryonic fibroblast (NIH/3T3, ATCC CRL-1658) cell line 38 . Unlike EV-A71 clinical isolates, these adapted strains productively infected and replicated to high titres in both rodent and primate cell lines.…”

“…Unlike EV-A71 clinical isolates, these adapted strains productively infected and replicated to high titres in both rodent and primate cell lines. Their genomes also accumulated multiple non-synonymous mutations, most notably in the capsid (P1) and RNA-dependent RNA polymerase (3D) genes 38 . Of these, three amino acid substitutions in VP1 protein—K98E, E145A, and L169F—that were common to both strains were demonstrated to facilitate viral infection of murine cells by enabling the virus to utilize the form of SCARB2 protein expressed on rodent cells (Victorio et al ., 2016.…”

Cooperative effect of the VP1 amino acids 98E, 145A and 169F in the productive infection of mouse cell lines by enterovirus 71 (BS strain)

“…Through serial passage of a clinical isolate (EV71:BS) in this murine cell line, we generated 2 strains—EV71:TLLm (60 infection cycles) and EV71:TLLmv (100 infection cycles)—that productively infected a range of rodent cell lines. 40 Previous viral RNA transfection studies have demonstrated that EV71:BS infection of murine cells is blocked at an early stage during cellular entry, and the adapted strains (EV71:TLLm and EV71:TLLmv) overcame this, presumably by adaptive mutations. Genome sequence comparison of the three strains revealed that EV71:TLLm and EV71:TLLmv accumulated multiple non-synonymous mutations in the capsid-encoding (P1) region, suggesting a possible correlation between the capsid mutations and the novel ability to infect rodent cell lines.…”