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

Chua KB (2014) Phenotypic and Genotypic Characteristics of Novel Mouse Cell Line (NIH/3T3)-Adapted Human Enterovirus 71 Strains (EV71:TLLm and EV71:TLLmv). PLoS ONE 9(3): e92719. doi:10.1371/journal.pone.0092719 Phenotypic and Genotypic Characteristics of Novel Mouse Cell Line (NIH/3T3)-Adapted Human Enterovirus 71 Strains (EV71:TLLm and EV71:TLLmv) Carla Bianca Luena Victorio 0 Yishi Xu 0 Qimei Ng 0 Vincent T. K. Chow 0 Kaw Bing Chua 0 Herman Tse, The University of Hong Kong, Hong Kong 0 1 Temasek Lifesciences Laboratory , 1 Research Link , National University of Singapore, Singapore, 2 Host and Pathogen Interactivity Laboratory, Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore , Singapore 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. - Funding: The research was fully funded by Temasek LifeSciences Laboratory (TLL). The funder had no role in study design, data collection and analysis, or preparation of the manuscript. The authors have obtained permission from the institute to publish these results. Competing Interests: The authors have declared that no competing interests exist. Enterovirus 71 (EV71) is a small non-enveloped virus approximately 30 nm in diameter. The viral capsid exhibits icosahedral symmetry and is comprised of 60 identical units (protomers), with each consisting of four viral structural proteins VP1VP4. The capsid surrounds a core of a single-stranded positive-sense RNA genome of 7,450 nucleotides (nt) long. The genome contains a single open reading frame which encodes a polyprotein of 2193 amino acids (aa) and is flanked by a long 59 untranslated region (UTR) of 745 nt and a shorter 39 UTR of 85 nt with a poly-A tract of variable length at its 39 terminus. The polyprotein is divided into three regions, i.e. P1, P2 and P3. P1 encodes four viral structural proteins 1A-1D (VP4, VP2, VP3 and VP1); P2 and P3 encode seven non-structural proteins 2A-2C and 3A-3D [13]. EV71 causes an array of clinical diseases including hand, foot and mouth disease (HFMD), aseptic meningitis, encephalitis and poliomyelitis-like paralysis mainly in infants and young children [4,5]. The virus was first isolated from a child with acute encephalitis in California, USA in 1969, and subsequently characterized as a new serotype of the genus Enterovirus in 1974 [6]. Outbreaks of HFMD with or without neurologic complications and deaths were reported in various parts of the world [7 20]. Since 1997, EV71 infections have been a major public health burden and of constant epidemiologic concern in the Asia-Pacific Region. An HFMD outbreak due to highly neurovirulent EV71 emerged in Malaysia resulting in 48 deaths in 1997 [21,22], followed by a larger outbreak that occurred in Taiwan in 1998 with more than 129,000 cases of HFMD, 405 severe infections and 78 deaths due to acute brainstem encephalomyelitis with neurogenic cardiac failure and pulmonary edema [2326]. In Peoples Republic of China, 488,955 HFMD cases with 126 deaths were recorded in 2008 [27] and increased to 1,155,525 cases with 353 fatalities in 2009 [28]. In 2010, China experienced the largest ever HFMD outbreak with more than 1.7 million cases, 27,000 patients with severe neurologic complications, and 905 deaths [29]. Similar to other human enteroviruses, EV71 is unable to infect animals other than humans, although rhesus and cynomolgous monkeys can be experimentally infected [3032]. Understanding its pathogenesis and development of specific therapeutics against the virus are hampered by the lack of suitable small animal models, because EV71 is unable to naturally infect small rodents. Attempts to establish mouse models of EV71 infection and disease have been made, mostly through virus adaptation by serial passages in young suckling mice [3339]. Although some models were able to recapitulate symptoms of clinical illness, none has been reported to cause disease in immune-competent mice aged 2 weeks old or older. Moreover, clinical features of disease and pathology of EV71 infections in humans and experimental monkeys could not be replicated in mice, with the exception of the immunocompromised interferon receptor-deficient AG129 mice [35]. RNA viruses, by virtue of their error-prone replication and high mutation rates [4042], replicate as a swarm of related variant sequences known as quasispecies [43,44]. It is comprised of a master species exhibiting the highest fitness in a certain environment, and of a mutant spectrum composed of a collection of closely related mutant sequences with a certain probability distribution [44,45]. These endow RNA viruses with genome plasticity, which is reflected in their ability to quickly adapt to changing environments. Despite these endowments, there has been no report of human EV71 that possesses the ability to productively infect rodent-derived cell lines, until now. This manuscript details the phenotypic and genotypic characteristics of two selected mouse cell line (NIH/3T3)-adapted EV71 strains (EV71:TLLm and EV71:TLLmv), which have gained the ability to cause productive infection in cultured rodent cells due to adaptive mutations in the viral genomes. This is the first report of human-derived EV71 that have been successfully adapted to productively infect cell lines of mouse, hamster, and rat origin. EV71:TLLm and EV71:TLLmv represent novel EV71 strains with unique genomic sequences, which can be further applie (...truncated)