Many viruses evolve rapidly. This is due, in part, to their high mutation rates. Mutation rate estimates for over 25 viruses are currently available. Here, we review the population genetics of virus mutation rates. We specifically cover the topics of mutation rate estimation, the forces that drive the evolution of mutation rates, and how the optimal mutation rate can be context-dependent.

Human dipeptidyl peptidase 4 (hDPP4) was recently identified as the receptor for Middle East respiratory syndrome coronavirus (MERS-CoV) infection, suggesting that other mammalian DPP4 orthologs may also support infection. We demonstrate that mouse DPP4 cannot support MERS-CoV infection. However, employing mouse DPP4 as a scaffold, we identified two critical amino acids (A288L and T330R) that regulate species specificity in the mouse. This knowledge can support the rational design of a mouse-adapted MERS-CoV for rapid assessment of therapeutics. Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently identified betacoronavirus that can infect the lower respiratory airway of humans, leading to acute respiratory distress syndrome (ARDS) with ϳ43% mortality in hospitalized individuals (1). Disease symptoms associated with MERS-CoV are similar to those of severe acute respiratory syndrome coronavirus (SARS-CoV); however, MERS-CoV is phylogenetically more closely related to the bat coronaviruses HKU4 and HKU5 (2, 3). MERS-CoV also differs from SARS-CoV in terms of receptor usage, where MERS-CoV utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor (4). Given the importance of MERS-CoV as an emerging pathogen, there is a clear need for the development of new therapeutics, which requires the appropriate animal models. However, to date, nonhuman primates are the only reported animal model for MERS-CoV replication, while traditional small animal models, such as ferrets (5), hamsters (6), and mice (7), are nonpermissive. Given that species-specific differences in DPP4 may confound animal model development, it is important to identify determinants in DPP4 that govern MERS-CoV host range. Knowledge of DPP4 determinants may provide novel insights into interactions between DPP4 and MERS-CoV spike receptor binding domain (RBD), as well as support development of new small animal models.To test whether mouse DPP4 (mDPP4) is capable of acting as an entry receptor for MERS-CoV, we compared mDPP4 with human DPP4 (hDPP4). An ectopic expression system was utilized to constitutively express mDPP4 and hDPP4 in human embryonic kidney 293T (HEK 293T) cells, which lack detectable expression of endogenous hDPP4 (data not shown). Human DPP4 and mouse DPP4 were expressed either as full-length proteins or as fusions to the Venus protein at the carboxy terminus. HEK 293T cells were transfected with 3 g of the indicated DPP4 expression plasmid, and at ϳ20 h posttransfection, cells were infected at a multiplicity of infection (MOI) of 5 with a recombinant MERSCoV strain designed to express tomato red fluorescent protein (rMERS-CoV-red) (Fig. 1). The rMERS-CoV-red virus is derived from the EMC2012 substrain and was previously shown to infect and replicate in a manner similar to wild-type MERS virus (8). Transfection of the DPP4-Venus fusion constructs resulted in high transfection efficiency (nearly 100%) (Fig. 1A and B). Control HEK 293T cells were poorly permissive for MERS-CoV, while cells overexpressing hDPP4 were readily ...

Zika virus (ZIKV), a mosquito-borne flavivirus discovered in 1947, has only recently caused large outbreaks and emerged as a significant human pathogen. In 2015, ZIKV was detected in Brazil, and the resulting epidemic has spread throughout the Western Hemisphere. Severe complications from ZIKV infection include neurological disorders such as Guillain-Barré syndrome in adults and a variety of fetal abnormalities, including microcephaly, blindness, placental insufficiency, and fetal demise. There is an urgent need for tools and reagents to study the pathogenesis of epidemic ZIKV and for testing vaccines and antivirals. Using a reverse genetics platform, we generated six ZIKV infectious clones and derivative viruses representing diverse temporal and geographic origins. These include three versions of MR766, the prototype 1947 strain (with and without a glycosylation site in the envelope protein), and H/PF/2013, a 2013 human isolate from French Polynesia representative of the virus introduced to Brazil. In the course of synthesizing a clone of a circulating Brazilian strain, phylogenetic studies identified two distinct ZIKV clades in Brazil. We reconstructed viable clones of strains SPH2015 and BeH819015, representing ancestral members of each clade. We assessed recombinant virus replication, binding to monoclonal antibodies, and virulence in mice. This panel of molecular clones and recombinant virus isolates will enable targeted studies of viral determinants of pathogenesis, adaptation, and evolution, as well as the rational attenuation of contemporary outbreak strains to facilitate the design of vaccines and therapeutics.

Coronaviruses have frequently expanded their host range in recent history, with two events resulting in severe disease outbreaks in human populations. Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2003 in Southeast Asia and rapidly spread around the world before it was controlled by public health intervention strategies. The 2012 Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak represents another prime example of virus emergence from a zoonotic reservoir. Here, we review the current knowledge of coronavirus cross-species transmission, with particular focus on MERS-CoV. MERS-CoV is still circulating in the human population, and the mechanisms governing its cross-species transmission have been only partially elucidated, highlighting a need for further investigation. We discuss biochemical determinants mediating MERS-CoV host cell permissivity, including virus spike interactions with the MERS-CoV cell surface receptor dipeptidyl peptidase 4 (DPP4), and evolutionary mechanisms that may facilitate host range expansion, including recombination, mutator alleles, and mutational robustness. Understanding these mechanisms can help us better recognize the threat of emergence for currently circulating zoonotic strains.

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor. Mouse DPP4 (mDPP4) does not support MERS-CoV entry; however, changes at positions 288 and 330 can confer permissivity. Position 330 changes the charge and glycosylation state of mDPP4. We show that glycosylation is a major factor impacting DPP4 receptor function. These results provide insight into DPP4 species-specific differences impacting MERS-CoV host range and may inform MERS-CoV mouse model development. Coronaviruses are a diverse family of single-stranded, positivesense RNA viruses that have frequently undergone host range expansion events. While coronaviruses have expanded their host range into humans multiple times over the course of their evolutionary history, two recent events have resulted in the emergence of highly pathogenic epidemic strains. First, severe acute respiratory syndrome coronavirus (SARS-CoV) emerged into the human population in 2003 and infected over 8,000 people before finally being contained by aggressive public health intervention strategies. More recently in 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged from its zoonotic host species into humans, resulting in severe disease and a 38% mortality rate. MERS-CoV likely originated from a bat reservoir species, as evidenced by the identification of closely related MERS-CoV-like viruses in bats (1, 2), although current hypotheses suggest that a camel intermediate host also played an important role in the host range expansion event.The functional receptor for MERS-CoV was recently identified as dipeptidyl peptidase 4 (DPP4) (3). Interestingly, while MERSCoV can utilize human, bat, and camel DPP4 (14, 15), traditional small animal models are nonpermissive, including mice (4, 5), ferrets (6), and hamsters (7,14). The relevance of MERS-CoV as an emerging pathogen and the importance of small animal models for studying pathogenesis and for developing vaccines and therapeutics led us to identify the determinants of interactions between the MERS-CoV receptor binding domain (RBD) and mouse DPP4 (mDPP4). Interactions between DPP4 and the MERS-CoV RBD are primarily restricted to blades IV and V of the DPP4 Nterminal ␤-propeller domain (8, 9). Recently, we found that two key residues in mDPP4 (A288L and T330R) could permit infection by MERS-CoV when mutated to the human DPP4 (hDPP4) amino acids (4). These residues lie within blades IV and V of the ␤-propeller domain (8, 9). The importance of A288L can be understood by recognizing that there is a strong hydrophobic region in the MERS-CoV RBD that engages the equivalent hDPP4 residue (L294) (9). In fact, all permissive DPP4 orthologs have a leucine residue at this site (i.e., bat, camel, human, and marmoset). This interaction, however, is altered in mDPP4, potentially making this hydrophobic region less amenable to interacting with the MERS-CoV RBD.On blade IV, the T330R substitution in mDPP4 regulates two potentially critical virus-host cell receptor interaction ...