Evaluation of a recombination-resistant coronavirus as a broadly applicable, rapidly implementable vaccine platform

ABSTRACT Emerging and re-emerging zoonotic viral diseases are major threats to global health, economic stability, and national security. Vaccines are key for reducing coronaviral disease

burden; however, the utility of live-attenuated vaccines is limited by risks of reversion or repair. Because of their history of emergence events due to their prevalence in zoonotic pools,

designing live-attenuated coronavirus vaccines that can be rapidly and broadly implemented is essential for outbreak preparedness. Here, we show that coronaviruses with completely rewired

transcription regulatory networks (TRNs) are effective vaccines against SARS-CoV. The TRN-rewired viruses are attenuated and protect against lethal SARS-CoV challenge. While a 3-nt rewired

TRN reverts via second-site mutation upon serial passage, a 7-nt rewired TRN is more stable, suggesting that a more extensively rewired TRN might be essential for avoiding growth selection.

In summary, rewiring the TRN is a feasible strategy for limiting reversion in an effective live-attenuated coronavirus vaccine candidate that is potentially portable across the Nidovirales

order. SIMILAR CONTENT BEING VIEWED BY OTHERS RAPID DEVELOPMENT OF ATTENUATED IBV VACCINE CANDIDATES THROUGH A VERSATILE BACKBONE APPLICABLE TO VARIANTS Article Open access 28 March 2025

ENGINEERING SARS-COV-2 USING A REVERSE GENETIC SYSTEM Article 29 January 2021 ABROGATION OF MAREK’S DISEASE VIRUS REPLICATION USING CRISPR/CAS9 Article Open access 02 July 2020 INTRODUCTION

Emerging and re-emerging zoonotic viral diseases are major threats to global human health, economic stability, and national security1,2,3,4,5,6. The incidence of human zoonotic disease is

estimated to surpass 1 billion cases per year, with novel emerging infectious diseases accruing hundreds of billions of dollars in economic losses7,8, losses that are greatly magnified when

new emerging viruses such as coronaviruses (CoVs) devastate economically critical livestock populations across the globe. With the continued encroachment of human populations into animal

habitats and our close contact with domesticated animals, zoonoses will continue to increase as the human and livestock population numbers and density expand over the next century. In fact,

a recent study recognized that the majority of emerging infectious disease events have origins in wildlife3,8, underscoring the importance of developing broadly applicable strategies for

vaccine design for virus families that are harbored within extensive zoonotic pools. Vaccines are well established in their capacity to reduce viral disease burden. Live-attenuated vaccines,

because they can elicit balanced innate and adaptive—and often lifelong—protective immune responses, including lactogenic immunity, are ideal candidates for vaccine development in humans

and animals1. However, their utility as broadly applicable vaccine platforms has long been limited by risks of reversion of attenuated vaccine strains to virulence, largely because the

stability of the attenuation cannot be clearly evaluated or assured. The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle-East Respiratory Syndrome

Coronavirus (MERS-CoV) in the 21st century emphasizes the threat of pandemic viral infections originating from cross-species transmission events9,10,11. These highly pathogenic variants are

prime models for the development of broad-based strategies for evaluating live virus vaccines for the Nidovirales order. Coronaviruses (CoVs) all reproduce with conserved replication

strategies, emphasizing the strength and rapidly adaptable potential of a vaccine design platform that takes advantage of this biology. CoVs replicate and transcribe subgenomic RNAs (sgRNAs)

via a discontinuous transcription mechanism mediated by transcription regulatory sequences (TRSs), a series of conserved nucleotide sequences positioned near the 5′-end of the genome and at

several locations immediately 5′ of each downstream open reading frame (ORF) (Fig. 1). These TRSs regulate a transcription attenuation program via base-pairing interactions between the

leader TRS and body TRSs that results in the production of sgRNAs, from which downstream ORFs are translated. Within the TRS, a 6- to 8-nt core sequence (ACGAAC for SARS-CoV) guides

base-pairing and duplex formation between nascent RNA and the leader TRS12,13,14. While this collective TRS arrangement, the transcription regulatory network (TRN), is conserved, our

laboratory has shown that it is possible to rewire the guide sequence of the SARS-CoV TRN and produce infectious virus13. We also showed that recombination between this rewired TRN virus and

wild-type (WT) SARS-CoV was not viable, indicating that recombination-mediated reversion of a CoV vaccine platform featuring a rewired TRN is highly unlikely. Based on conservation of the

TRN biology across CoVs, this report further explores the feasibility of the development of a stably attenuated vaccine platform featuring a completely rewired TRN as a candidate strategy

for a broadly applicable, rapidly implementable CoV vaccine platform that is highly resistant to recombination repair and stably attenuated in both young and highly vulnerable mouse models

of human disease. RESULTS THE 3-NT TRN MUTANT IS ATTENUATED FOR VIRULENCE In a previous study in our laboratory, we demonstrated that the SARS-CoV TRN could be reprogrammed, provided the

individual TRSs were replaced with matching sequences13. The rewired TRN replaced the conserved 6-nt TRS with a 6-nt cassette that is not used in any other characterized CoVs, encoding a net

change of 3 nts (ACGAAC to CCGGAU). Our previous work showed that this rewired TRN was refractory to recombination with WT genomes13. Therefore, we tested its replication and pathogenesis

in young and aged BALB/c mice. Consistent with earlier reports, WT SARS-CoV only caused weight loss in aged animals. In contrast, CRG3 replicated but caused no weight loss in young

(10-week-old) mice (Fig. 2a) and minimal weight loss in aged (12-month-old) BALB/c mice (Fig. 2b) (young mice: _P_ _=_ 0.25 for titer, Wilcoxon test, _P_ _=_ 0.37 for weight loss,

Mann-Whitney test; old mice: _P_ _=_ 0.25 for titer, _P_ _<_ 0.001 for weight loss). Both WT and recombinant viruses replicated to high titers that were detectable on days 2 and 4

post-infection (p.i.) in both young and aged animals, with titers beginning to clear by day 7 p.i. (Fig. 2c, d), as is usually observed in mice infected with SARS-CoV1. VACCINATION WITH CRG3

PROTECTS MICE AGAINST CHALLENGE The high replication titer and low or absent virulence in young and aged mouse models paired with the inherently recombination-refractory genome suggested

that the CRG3 virus would be an ideal vaccine candidate. To test CRG3’s efficacy in protecting against homologous and heterologous challenge, the virus was administered to mice in a

single-dose vaccination, alongside viral replicon particles (VRPs) expressing the viral Spike attachment protein (VRP-S) as a control15. On day 22 post-vaccination, mice were then challenged

with either mouse-adapted SARS-CoV (MA15—homologous challenge) or SARS-CoV expressing the Spike gene from the Himalayan palm civet (_Paguma larvata_) strain HCSZ6103 (heterologous

challenge). Mice were then observed for morbidity and mortality, and surviving animals were euthanized on day 4 p.i. Upon homologous challenge, CRG vaccination was protective against weight

loss and mortality in young and aged mice, with no detectable viral titer in the lungs at 4 days p.i., in contrast to PBS and VRP-S subcutaneous vaccination (Fig. 3a–d, Supplementary Fig. 

1A-B). Importantly, against heterologous challenge, while both CRG3 and VRP-S vaccines were protective in young mice, only CRG3 was protective in aged mice against replication and mortality,

with no detectable titer in the lungs at 4 days p.i. (Fig. 3e–h, Supplementary Fig. 1C–D). CRG vaccination induced SARS-CoV neutralizing antibody titers approaching 4 log10 in both young

and aged mice, nearly 2-fold higher than the titers induced by VRP-S vaccination (_P_ < 0.0001, two-way ANOVA) (Supplementary Fig. 1E). Moreover, CRG vaccination largely protected aged

mice from the extensive inflammatory cell infiltration, tissue damage, perivascular cuffing, edema, and septal thickening observed in PBS- and VRP-S-vaccinated mice (Supplementary Fig. 2).

CRG3 REVERTS WITH PANDEMIC-ASSOCIATED MUTATIONS A live-attenuated vaccine candidate should demonstrate phenotypic stability in infected host populations. Therefore, to test its resistance to

reversion to virulence, CRG3 was subjected to five independent serial passages in parallel with WT SARS-CoV in aged (14-month-old) BALB/c mice. Over the course of six 4-day passages, all

CRG3 passaged viruses acquired a virulent phenotype, with weight losses and percent survival curves mirroring the kinetic rates of the emergence of virulence seen with WT passaged viruses

(Supplementary Fig. 3). To attempt to identify the genotypic causes of this phenotypic reversion to virulence, the genomes of 5 independent plaque isolates of the passage 6 viruses (WT P6

and CRG3 P6) were submitted to Sanger sequencing. Surprisingly, and contrary to expectations, no mutations in either the Spike or the Membrane protein—both of which were targets for

mutational selection in 3 separate adaptations of SARS-CoV Urbani to mice (Fig. 4a)—were identified in WT or CRG3 revertants. Instead, 4 of 5 CRG3 P6 isolates showed evidence of often large

deletions in the accessory ORFs 7b, 8a, and 8b (Fig. 4a, b). These deletions, often in-frame, were reminiscent of several incidences of host range-associated deletions identified in human

isolates from the 2003 SARS-CoV epidemic, including a 29-nt deletion in ORF8 relative to the Himalayan palm civet strain16 and 82-nt17 and 386-nt18 deletions in this same region. In

contrast, deletions in accessory ORFs were rare in virulent WT revertants. Rather, scattered nonsynonymous mutations were identified in ORF1a, ORF3a, ORF8a, and ORF9. Notably, only 1 WT

revertant exhibited a small deletion in ORF7b. Collectively, these findings demonstrated that this region of the SARS-CoV genome is inherently unstable when subjected to replication

pressure, especially in aged animals, and indicated that a TRN-rewired virus would require additional stabilizing mutations to be feasible as a vaccine candidate. AN ATTENUATED SARS-COV

MUTANT WITH A 7-NT TRN REPLACEMENT The demonstration of second-site reversion to virulence upon passage of CRG3 suggested that a more ideal TRN-rewired vaccine candidate would be one that

would be less likely to phenotypically revert in vivo. CoV TRS networks are finely tuned and regulate the expression of both highly abundant and low-frequency mRNA transcripts; thus, small

changes in the TRS might subtly alter the regulation of subgenomic transcripts13. Moreover, the exact regulatory milieu around each TRS is uncertain beyond the recognition that both, up- and

downstream sequences can influence mRNA expression efficiency, perhaps in a TRN sequence-specific manner19,20. We hypothesized that more extensive remodeling of the TRS network may better

disrupt the finely tuned network of abundant and low-frequency transcripts, altering the natural regulation of global gene expression, and thereby leading to decreased virulence. In

addition, it was possible that the virulence observed in CRG3 P6 isolates was due, at least in part, to mouse adaptation during passage. Therefore, to evaluate the effects of more extensive

TRN rewiring in a lethal pathogenic model and to further stabilize the TRN against recombination repair and mutation selection, a TRN mutant was constructed that featured the following: (1)

the set of 6 mouse-adapted mutations present in the SARS-MA15 virus (see Fig. 4a); and (2) a newly designed TRN consisting of a 7-nt complete replacement of the 9 SARS-CoV TRS loci (ACGAAC

to UGGUCGC – CRG7-MA – Fig. 1). These replacements yielded infectious CRG7-MA virus that was capable of replicating to titers equivalent to those of the mouse-adapted WT background virus,

SARS-MA15, though with a pinpoint plaque phenotype that remained stable after tissue culture passage. CRG3-MA (9 CRG3 replacements in the virulent SARS-MA15 backbone) was also generated as a

control TRN-rewired virus. northern blot analysis of CRG7-MA versus CRG3 and SARS-MA15 revealed that the virus produced the expected bands at similar proportions to WT virus, although some

additional low-abundance transcripts were also noted in CRG7-MA (Supplementary Fig. 4). These viruses were then evaluated for replication and virulence in young (10-week-old) and aged

(12-month-old) BALB/c mice in comparison with SARS-MA15. In young mice, CRG7-MA was attenuated compared with CRG3-MA and SARS-MA15, causing a weight loss of ~5% (_P_ _<_ 0.0001, _t_ test,

on days 2–5 p.i. for CRG7-MA vs. CRG3-MA), though replicating to high titers (approximately 7 log10 PFU for CRG7-MA vs. 8 log10 PFU for CRG3-MA and SARS-MA15) at 2 days p.i. Further,

CRG7-MA showed evidence of more rapid clearance kinetics, with a nearly 3-log10 difference in titer compared with the CRG3-MA and SARS-MA15 controls at 4 days p.i. (Fig. 5a–c) (_P_ < 

0.001, _t_ test, for both 2 and 4 days p.i. for CRG3-MA vs CRG7-MA). While morbidity and mortality were more pronounced in old mice, CRG7-MA was still attenuated compared with CRG3-MA and

SARS-MA15 in terms of weight loss (less weight loss on days 2–4 p.i., _P_ < 0.05 on days 2 and 4 p.i., _t_ test, CRG3-MA vs. CRG7-mA) and lung titer (8 log10 PFU for CRG7-MA vs. >9

log10 PFU for CRG3-MA and SARS-MA15 on day 2 p.i. (_P_ < 0.0001, _t_ test, CRG3-MA vs. CRG7-MA); ~2.5-log10 PFU difference in titer on day 4 p.i. (_P_ < 0.05, _t_ test, CRG3-MA vs.

CRG7-MA) (Fig. 5d–f). Infection of aged BALB/c mice with 2 log10, 3 log10, or 4 log10 PFU of CRG7-MA or SARS-MA15 emphasized the attenuated virulence phenotype, with the 2-log10 PFU

infection of CRG7-MA causing almost no weight loss over the course of the infection, despite replicating to the same titers as the 3 log10 and 4 log10 PFU infections (Fig. 5g–h). Under

identical conditions, SARS-MA15 mice lost more body weight (>20%) and experienced 60% mortality rates. Survival was markedly different between CRG7-MA and SARS-MA15 infections, with 100%

of mice infected with 2 log10 or 3 log10 PFU and 60% of mice infected with 4 log10 PFU surviving the course of CRG7-MA infection. In contrast, only 40% of mice infected with 2 log10 PFU of

SARS-MA15 survived infection; mice infected with 3 log10 or 4 log10 PFU survived only until days 6 or 3 p.i. maximum, respectively (_P_ < 0.0001, log-rank test) (Fig. 5i). When CRG7-MA

was serially passaged in young (10-week-old) BALB/c mice, in contrast to the rapid phenotypic reversion observed with CRG3, CRG7-MA remained phenotypically stable with passage, with mice

losing a minimal (~5%) amount of their starting weight over the 3-day infections throughout all 4 passages (Supplementary Fig. 5A). Viral titer also remained stable over passage, with >5

log10 PFU detectable at 3 days p.i. in each passage (Supplementary Fig. 5B). Furthermore, when CRG7-MA was serially passaged independently three times for six passages in aged mice,

replicating the passage conditions in which CRG3 reverted to virulence, CRG7-MA did not exhibit an increase in virulence, causing <20% weight loss in infected animals and ~60% mortality

upon infection of 12-month-old BALB/c mice with 105 PFU of CRG7-MA post-passage, essentially replicating the mortality shown in aged mice infected with non-passaged CRG7-MA virus and

distinct from the 100% mortality caused by wild-type SARS-MA15 infection of aged mice (see Fig. 5i, Supplementary Fig. 5C-D, and Supplementary Table 1). Finally, CRG7-MA was evaluated for

its capacity to protect against lethal SARS-MA15 challenge in aged (12-month-old) mice. Mice were vaccinated with 2.5 log10 PFU of either CRG7-MA or ExoN-MA, which we previously demonstrated

to be an effective vaccine in the SARS-MA15 backbone in the aged BALB/c mouse model of SARS-CoV pathogenesis1. On day 22 post-vaccination, mice were then challenged with a lethal dose (5

log10 PFU) of SARS-MA15 and evaluated for weight loss. Mice lost only minimal amounts (~5%) of their starting weights with both vaccines (_P_ = 0.125 for both viruses versus PBS vaccination,

Wilcoxon test), indicating that CRG7-MA and ExoN-MA were equally protective against lethal SARS-MA15 challenge (Fig. 6). DISCUSSION Live-attenuated vaccines remain key players in reducing

the global disease burden associated with viral infections in humans, critically important livestock, and companion animals. Historically and contemporarily, live-attenuated vaccines have

been used with success to help control measles, mumps, rubella, polio, yellow fever, and chickenpox infections and outbreaks1,21,22. However, live-attenuated vaccines are also associated

with the risk of reversion by either mutation- or recombination-driven processes, which can cause dangerous outbreaks in unvaccinated populations, including animals22. For example, highly

pathogenic porcine epidemic diarrhea virus (PEDV) strains emerged in China in 2012, circumventing existing vaccines, and RNA recombination events between wild-type and live-attenuated PEDV

and between avian infectious bronchitis virus (IBV) strains have seeded new outbreaks23,24,25,26. Therefore, measures are needed to stabilize live-attenuated vaccines against reversion under

selective pressure, particularly for viruses like CoVs, which employ recombination as a standard feature of their replication cycle, as incidental recombination events in the context of a

co-infection could unintentionally introduce alleles with enhanced virulence into an attenuated vaccine genome, with consequences that could be difficult to predict13,27,28. Several groups,

including our own, have developed novel strategies in fidelity regulation and control that attenuate RNA virus pathogenesis, and in the case of CoVs, the development of mutants that prevent

reversion repair to virulence1,29. Recombination repair is a well-characterized process essential for genome evolution in many biological systems and plays critical roles in spread,

virulence, and pathogenesis30. In general, strategies to engineer recombination-resistant RNA viruses have been limited to CoVs and, to a lesser extent, enteroviruses13,31. In the context of

a CoV infection, recombination occurs when the viral RNA-dependent RNA polymerase (RdRp) switches templates during nascent RNA synthesis, using the nascent RNA itself as a primer once the

RdRp reassociates with the template genome. The likelihood of a recombination event occurring is regulated by several viral factors, including replication rates, RNA secondary structure,

genome size, and the nature of the replicase/transcriptase protein complex; however, the heritability of a recombination, once it has occurred, is mediated by the replication fitness of the

resulting progeny genome13,32,33,34,35. Several reports have described the emergence of CoVs with enhanced virulence that, upon genome analysis, clearly originated from recombination events

of related viruses in avian and mammalian hosts, including SARS-CoV27,28,36,37,38. Furthermore, reservoir species, such as bats, from which the prevailing evidence suggests that both

potentially lethal human CoVs, SARS-CoV and MERS-CoV, emerged, have been shown to harbor multiple CoV species, and recombinant RNA genomes have been frequently identified within colonies and

among individual infected animals, increasing the likelihood of recombination-driven alterations in species specificity and virulence16,27,39,40. Thus, engineering elements to render a

genome recombination-refractory is an essential step towards ensuring that a live-attenuated vaccine candidate cannot regain virulence during an incidental co-infection with another

TRN-compatible CoV genome. SARS-CoV is a highly pathogenic pneumo-enteric pathogen that captures many disease features seen among other CoVs. The TRN conserved sequence (CS) motifs utilized

in the CRG3 and CRG7 backgrounds, CCGGAU and UGGUCGC, respectively, are both unique sequence motifs when compared with all known CoV genome TRN sequences, greatly reducing and likely

eliminating the possibility of recombination with unmodified genomes at the canonical TRS loci. Our previous work demonstrated that introducing mismatched TRSs was lethal for RNA recombinant

virus replication13. Furthermore, here, we showed that CRG3 TRN replacement did not revert at the primary sites of mutation, indicating that the rewired TRN is stable and not under

sufficient selective pressure to revert, even in vivo. As coordinated interactions are required for TRN function, TRN reversion is unlikely, given the requirement for nearly simultaneous

reversions at multiple sites across the genome. Larger-sized TRS CSs (>7 nts) within the TRN may not prove effective, as evolution appears to have selected for a CoV RNA polymerase that

is heavily focused on recognizing a 5- to 7-nt TRS CS within the TRN to regulate subgenomic transcription. The attenuation resulting from TRN rewiring is most likely attributed to

alterations in the viral transcription profile, low-abundance transcripts that either encode or reduce the expression of previously unidentified out-of-frame ORFs, or viral or host factor

interactions with the viral genome. The rewired TRN produces obvious novel viral RNA species (Supplementary Fig. 4). These novel RNA species may serve as functional mRNAs, producing

noncanonical viral protein products that attenuate viral replication or pathogenesis. Alternatively, these novel RNA species may compete with canonical viral RNA species for replication and

transcription, attenuating viral replication and/or pathogenesis due to the altered availability of transcripts encoding bona fide viral virulence factors. Furthermore, the rewired TRN

itself may serve as an attenuating factor, as the virus’ discontinuous transcription program alters programmed RNA-protein interactions (involving both viral and host proteins) either

directly (i.e., by altering the bases required for RNA-protein interactions) or indirectly (i.e., through changes in the RNA’s secondary structure that affect the steric availability of

RNA-protein interaction sites). Moreover, Di et al., in an arterivirus model, used next-generation sequencing analysis to show that noncanonical transcripts are produced in the course of

wild-type infection, indicating that the coding capacity of nidoviruses is actually much larger than what has been characterized using Sanger sequencing and biochemical detection methods.

Their findings suggest that attenuation via the TRN may be able to target noncanonical RNA species, which might be able to impact pathogenesis with fewer effects on replication and

structural protein production41. These fascinating possibilities will be the focus of future studies on the mechanism of TRN-related attenuation. The hypothesis that alterations in the

expression profiles of canonical viral RNA species and protein products attenuate pathogenesis is further strengthened when paired with observations of the types of mutations that were

selected upon passage of CRG3 in aged mice: mutation profiles were different if selection occurred in young versus aged populations. Frieman et al. previously demonstrated that mutations in

Spike and nonstructural protein 9 (nsp9) were repetitively selected in several independent passages and that these mutations conferred virulence in young mice, suggesting that Spike–viral

receptor interactions and nsp9–replicase protein interactions were most important for virulence42. In severely ill SARS-CoV-infected humans (predominantly over 50 years of age), deletions in

and around ORF8 were identified, as discussed earlier16,17,18,39. Deletions of different accessory ORFs were the most frequently observed changes after selection in aged animals.

Furthermore, in aged mice, nearly any combination of 2 SARS-MA15-identified alleles (shown in Fig. 4a) also conferred increased virulence, most likely reflecting the increased susceptibility

of aged mice to lethal outcomes. These data suggest that CoV adaptation to virulence is different in young and aged animals, especially when coupled with variations in virus pathogenic

determinants. While immune senescence can enhance virus virulence43, our data support the novel hypothesis that virulent zoonotic coronaviruses may emerge more quickly after in vivo passage,

especially in the aged, where multiple evolutionary pathways exist that can program virus virulence. The disproportionate identification of mutations in the accessory ORFs following passage

of the CRG3 TRN virus in aged animals may reflect the roles these accessory ORFs are hypothesized to play in the modulation of host immunity44. Aged animals’ immune systems respond

differently, and usually more severely, to microbial challenge, with the immune response skewed towards severe innate immune effects and defects in adaptive immunity45. However, in the

context of CoV infection, this augmented immune response, when presented in the context of accessory ORF deletion, may serve as a virulence fulcrum, with the balance poised between essential

immune recognition and severe disease, possibly due to defects in spread and persistence. These potential effects on host immunity argue that a stable vaccine candidate should consider the

potential for changes in accessory ORFs. A well-designed vaccine candidate should include genetic traps that are either independently attenuating or triggered by recombination events. We

have previously demonstrated both stable attenuation and protection against lethal challenge with inactivations of the nsp14 exonuclease1 and the nsp16 2′-O-methyltransferase46 activities.

In addition, Züst et al. demonstrated that a partial deletion of the murine hepatitis virus (MHV) nsp1 replicase protein could protect against homologous and heterologous challenge47.

Combining alleles that render the virus recombination-refractory, alter RNA replication fidelity, and that result in an altered host immune response could produce stable, reversion-proof,

live-attenuated viruses that induce robust neutralizing immunity. Attenuating alleles, coupled with a rewired TRN, are anticipated to increase the stability of the attenuation and to

minimize the chances for RNA recombination repair. The strategies reported herein, coupled with the availability of new molecular clones for CoVs that cause severe disease in livestock

populations, provide a vehicle for improved live virus vaccine design48,49,50,51,52,53. It may also be possible to “tune” the CoV TRN, with attenuating changes concentrated on virulence

alleles, such as accessory genes known to impact pathogenesis. Such alterations have the potential to greater enhance the stability of a vaccine candidate, particularly if considering TRS

context outside of the 5- to 7-nt core sequence. Such studies would benefit the most if the RNA structure is also considered, as TRS accessibility is most likely modulated by structural

elements in the CoV RNA genome. Such studies will be the focus of future work. With the continuing identification of zoonotic pools of CoVs that genetically resemble lethal human and animal

CoVs, often with only a few percentage points of difference between the zoonotic and lethal human sequences10,54,55,56,57, the necessity for a rapidly implementable, universal attenuation

platform for CoV live-attenuated vaccine design is underscored. In this report, we described the design and implementation of a CoV attenuation strategy that can be easily and rapidly

adapted to any CoV genome. The presence of 8–9 characterized TRSs within any CoV genome, the CSs of which are 6–7 nts each, offers too large and complex a target for primary site reversion

to be a likely event. As most single recombination events would decouple TRN expression networks, these recombinants would be lethal. Therefore, this attenuation strategy, when paired with

alleles that can resist selection events that lead to second-site reversion, could bring live-attenuated CoV vaccines within the reach of realization in the face of the ever-growing threat

of new human and animal CoV-based epidemics. METHODS VIRUSES AND CELLS All virus stocks were propagated in Vero-E6 cells as described in58. All virus work was performed in a biological

safety cabinet in a biosafety level 3 laboratory. CONSTRUCTION OF SARS PLASMIDS AND VIRUSES TRN3-based plasmids were constructed in13. TRN7 mutations were introduced into SARS plasmids A and

F using cassettes generated by BioBasic. To generate SARS-F plasmids containing both TRN mutations and the mouse-adapted mutation at nt 2663, the mouse-adapted mutation was cloned into the

F TRN plasmids via a PCR and restriction digestion strategy. To generate SARS-F plasmids containing both TRN mutations and the mouse-adapted mutation at nt 2663, the mouse-adapted mutation

was cloned into the F TRN plasmids via a PCR and restriction digestion strategy. The plasmids F-BstZ (5′-GGAGGCGCAATTTTTGTACCTCTATGCCTTG-3′), R-MAmut

(5′-AGCTATCGTCTCCGCTTCTCAACGGTAATAGTACCGTTGTCTG-3′), F-MAmut (5′-AGCTATCGTCTCCAAGCTTAAACAACTCCTGGAACAATGGAAC-3′), and R-Msc (5′-GTGGCTTAGCTACTTCGTTGCTTCCTTCAGGC-3′) were used to generate 2

amplicons. The resulting amplicons were restriction-digested with _Bsm_B I, ligated, and purified, after which the ligated amplicons and the parent vectors were restriction-digested with

_BstZ17_ I and _Msc_ I and ligated. Ligated vectors were transformed into TopTen _E_. _coli_ cells, and the resulting colonies were screened and sequence-verified. Viruses were then

constructed as described in ref. 58. NORTHERN BLOT ANALYSIS Intracellular RNA was isolated using RiboPure reagents (Ambion, Austin, TX) 12 h post-infection13. The mRNA was then isolated

using a Qiagen Oligotex mRNA isolation kit, treated with glyoxal, and separated an agarose gel using NorthernMax-Gly (Ambion). The RNA was then transferred to BrightStar-Plus membrane

(Ambion) for 5 h, cross-linked using UV light, prehybridized, and probed with an N gene-specific oligonucleotide probe (5′-CTTGACTGCCGCCTCTGCTbTbCCCTbCTbGCb-3′; biotinylated nucleotides are

denoted with a superscripted “b”). The blot was hybridized overnight and washed with low- and high-stringency buffers and was then incubated with phosphatase-conjugated streptavidin. The

blot was then incubated with CDP-STAR, overlaid with film, and developed. MOUSE INFECTIONS WITH SARS-COV AND MUTANTS All experimental protocols involving mice were reviewed and approved by

the institutional animal care and use committee at the University of North Carolina, Chapel Hill, NC, USA. The following mice were used: 10-week-old female BALB/c (Charles River

Laboratories, Wilmington, MA, USA) and 14-month-old female BALB/c (Harlan Laboratories, Indianapolis, IN, USA). Mice were lightly anesthetized and infected intranasally with varying doses

(102–106 PFU, depending on the experiment) of SARS-CoV, MA15, or TRN mutants. Mice were weighed daily, and on certain days specified in each experiment, mouse lungs were harvested for virus

titer and/or RNA. Serial passages were inoculated as above for passage 1; subsequent passages were inoculated with 50 μL of clarified lung homogenate (lungs were homogenized in 1 mL of PBS)

from the previous passage. All experiments used a minimum of _n_ = 5 mice per virus per dosage/condition (if applicable) per timepoint. For infection–challenge studies, mice were infected

with 102–103 PFU of the indicated vaccine virus, weighed for the 7 days following initial infection, and then challenged with a lethal dose (106 PFU) of MA15 for the challenge infection.

DETERMINATION OF VIRUS TITER IN INFECTED MOUSE LUNGS Lungs harvested for virus titer were weighed and homogenized in 1.0 mL of PBS at 6000 rpm for 60 s in a MagnaLyser (Roche, Basel,

Switzerland). Virus titers were determined by plaque assay on Vero cells. DETERMINATION OF VIRAL NEUTRALIZATION ANTIBODY TITERS Mouse sera were heat-inactivated for 30 min at 55 °C and were

then serially diluted to 1:100, 1:200, 1:400, 1:800, and 1:1600 in PBS to a volume of 125 μL. Next, 125 μL of PBS containing low-concentration SARS-CoV MA15 (40 PFU) or high-concentration

SARS-CoV MA15 (240 PFU) was added to each serum dilution. The virus-serum mixtures were incubated at 37 °C for 30 min. Following incubation, virus titers of the mixtures were determined by

plaque assay. Finally, we calculated the 50% plaque reduction neutralization titer (PRNT50) values, the serum dilutions at which plaque formation was reduced by 50% relative to that of virus

stock not treated with serum. VIRAL GENOME SEQUENCING To determine the sequences of viral genomes present in mouse lungs after passage, plaques were isolated from lung samples as described

above. Briefly, individual viral plaques were harvested by collecting the agarose plugs above them using a 200-μL pipette tip. Each agarose plug was dropped in 0.5 mL PBS, allowed to diffuse

for 24 h at 4 °C, and then applied to ~70% confluent monolayers of Vero-E6 cells in T25 flasks and incubated for 48 h at 37 °C. Infected cell monolayers were then harvested in 1 mL of

TRIzol. First-strand cDNA was generated as described in ref. 59. Amplicons of the viral genomes were generated as described in ref. 1. Sequence results were analyzed using Geneious R11

(Biomatters, Auckland, New Zealand) and Serial Cloner 2.6.1 (SerialBasics, http://serialbasics.free.fr/Home/Home.html). STATISTICAL ANALYSES Statistical analyses were performed using

GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA). The tests run depended on the experimental design and are specified in the text. Significance was set at _P_ < 0.05. DATA

AVAILABILITY The viral genome sequence datasets are available via the NCBI GenBank with accession identifiers MK062179 through MK062184. All other data generated during and/or analyzed

during the current study are available from the corresponding author on reasonable request. REFERENCES * Graham, R. L. et al. A live, impaired-fidelity coronavirus vaccine protects in an

aged, immunocompromised mouse model of lethal disease. _Nat. Med_ 18, 1820–1826 (2012). Article  CAS  Google Scholar  * Graham, R. L., Donaldson, E. F. & Baric, R. S. A decade after

SARS: strategies for controlling emerging coronaviruses. _Nat. Rev. Microbiol._ 11, 836–848 (2013). Article  CAS  Google Scholar  * Jones, K. E. et al. Global trends in emerging infectious

diseases. _Nature_ 451, 990–993 (2008). Article  CAS  Google Scholar  * Li, Y. et al. On the origin of smallpox: correlating variola phylogenics with historical smallpox records. _Proc. Natl

Acad. Sci. USA_ 104, 15787–15792 (2007). Article  CAS  Google Scholar  * Morens, D. M. & Fauci, A. S. The 1918 influenza pandemic: insights for the 21st century. _J. Infect. Dis._ 195,

1018–1028 (2007). Article  Google Scholar  * Sessa, R., Palagiano, C., Scifoni, M. G., di Pietro, M. & Del Piano, M. The major epidemic infections: a gift from the Old World to the New?

_Panminerva Med._ 41, 78–84 (1999). CAS  PubMed  Google Scholar  * Karesh, W. B. et al. Ecology of zoonoses: natural and unnatural histories. _Lancet_ 380, 1936–1945 (2012). Article  Google

Scholar  * Kreuder Johnson, C. et al. Spillover and pandemic properties of zoonotic viruses with high host plasticity. _Sci. Rep._ 5, 14830 (2015). Article  CAS  Google Scholar  * Al-Tawfiq,

J. A. et al. Surveillance for emerging respiratory viruses. _Lancet Infect. Dis._ 14, 992–1000 (2014). Article  Google Scholar  * Menachery, V. D. et al. SARS-like WIV1-CoV poised for human

emergence. _Proc. Natl Acad. Sci. USA_ 113, 3048–3053 (2016). Article  CAS  Google Scholar  * Peiris, J. S., Guan, Y. & Yuen, K. Y. Severe acute respiratory syndrome. _Nat. Med._ 10,

S88–S97 (2004). Article  CAS  Google Scholar  * Sola, I., Moreno, J. L., Zuniga, S., Alonso, S. & Enjuanes, L. Role of nucleotides immediately flanking the transcription-regulating

sequence core in coronavirus subgenomic mRNA synthesis. _J. Virol._ 79, 2506–2516 (2005). Article  CAS  Google Scholar  * Yount, B., Roberts, R. S., Lindesmith, L. & Baric, R. S.

Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: engineering a recombination-resistant genome. _Proc. Natl Acad. Sci. USA_ 103, 12546–12551

(2006). Article  CAS  Google Scholar  * Zuniga, S., Sola, I., Alonso, S. & Enjuanes, L. Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis.

_J. Virol._ 78, 980–994 (2004). Article  CAS  Google Scholar  * Sheahan, T. et al. Successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and

heterologous severe acute respiratory syndrome coronavirus. _J. Virol._ 85, 217–230 (2011). Article  CAS  Google Scholar  * Graham, R. L. & Baric, R. S. Recombination, reservoirs, and

the modular spike: mechanisms of coronavirus cross-species transmission. _J. Virol._ 84, 3134–3146 (2010). Article  CAS  Google Scholar  * Consortium, C. S. M. E. Molecular evolution of the

SARS coronavirus during the course of the SARS epidemic in China. _Sci. (New Y., N. Y.)_ 303, 1666–1669 (2004). Article  Google Scholar  * Tang, J. W. et al. The large 386-nt deletion in

SARS-associated coronavirus: evidence for quasispecies? _J. Infect. Dis._ 194, 808–813 (2006). Article  Google Scholar  * Mateos-Gomez, P. A., Zuniga, S., Palacio, L., Enjuanes, L. &

Sola, I. Gene N proximal and distal RNA motifs regulate coronavirus nucleocapsid mRNA transcription. _J. Virol._ 85, 8968–8980 (2011). Article  CAS  Google Scholar  * Mateos-Gomez, P. A.,

Morales, L., Zuniga, S., Enjuanes, L. & Sola, I. Long-distance RNA-RNA interactions in the coronavirus genome form high-order structures promoting discontinuous RNA synthesis during

transcription. _J. Virol._ 87, 177–186 (2013). Article  CAS  Google Scholar  * Lauring, A. S., Jones, J. O. & Andino, R. Rationalizing the development of live attenuated virus vaccines.

_Nat. Biotechnol._ 28, 573–579 (2010). Article  CAS  Google Scholar  * Vignuzzi, M., Wendt, E. & Andino, R. Engineering attenuated virus vaccines by controlling replication fidelity.

_Nat. Med_ 14, 154–161 (2008). Article  CAS  Google Scholar  * Feng, K. et al. Epidemiology and characterization of avian infectious bronchitis virus strains circulating in southern China

during the period from 2013-2015. _Sci. Rep._ 7, 6576 (2017). Article  Google Scholar  * Quinteros, J. A. et al. Full genome analysis of Australian infectious bronchitis viruses suggests

frequent recombination events between vaccine strains and multiple phylogenetically distant avian coronaviruses of unknown origin. _Vet. Microbiol._ 197, 27–38 (2016). Article  CAS  Google

Scholar  * Sun, R. Q. et al. Outbreak of porcine epidemic diarrhea in suckling piglets, China. _Emerg. Infect. Dis._ 18, 161–163 (2012). Article  Google Scholar  * Zhang, Y. et al. Complete

genome sequence and recombination analysis of infectious bronchitis virus attenuated vaccine strain H120. _Virus Genes_ 41, 377–388 (2010). Article  CAS  Google Scholar  * Wang, L. et al.

Discovery and genetic analysis of novel coronaviruses in least horseshoe bats in southwestern China. _Emerg. Microbes Infect._ 6, e14 (2017). Article  Google Scholar  * Wang, L., Junker, D.

& Collisson, E. W. Evidence of natural recombination within the S1 gene of infectious bronchitis virus. _Virology_ 192, 710–716 (1993). Article  CAS  Google Scholar  * Menachery, V. D.,

et al. Combination attenuation offers strategy for live-attenuated coronavirus vaccines. _J. Virol._ 92, e00710–18 (2018). * Xiao, Y. et al. RNA recombination enhances adaptability and is

required for virus spread and virulence. _Cell Host Microbe_ 19, 493–503 (2016). Article  CAS  Google Scholar  * Runckel, C., Westesson, O., Andino, R. & DeRisi, J. L. Identification and

manipulation of the molecular determinants influencing poliovirus recombination. _PLoS Pathog._ 9, e1003164 (2013). Article  CAS  Google Scholar  * Cheng, C. P., Serviene, E. & Nagy, P.

D. Suppression of viral RNA recombination by a host exoribonuclease. _J. Virol._ 80, 2631–2640 (2006). Article  CAS  Google Scholar  * Figlerowicz, M., Nagy, P. D. & Bujarski, J. J. A

mutation in the putative RNA polymerase gene inhibits nonhomologous, but not homologous, genetic recombination in an RNA virus. _Proc. Natl Acad. Sci. USA_ 94, 2073–2078 (1997). Article  CAS

  Google Scholar  * Nagy, P. D., Pogany, J. & Simon, A. E. RNA elements required for RNA recombination function as replication enhancers in vitro and in vivo in a plus-strand RNA virus.

_EMBO J._ 18, 5653–5665 (1999). Article  CAS  Google Scholar  * Nagy, P. D. & Simon, A. E. New insights into the mechanisms of RNA recombination. _Virology_ 235, 1–9 (1997). Article  CAS

  Google Scholar  * Terada, Y. et al. Emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses. _PloS ONE_ 9, e106534 (2014). Article

  Google Scholar  * Truyen, U., Parrish, C. R., Harder, T. C. & Kaaden, O. R. There is nothing permanent except change. _Émerg. New Virus Dis. Vet. Microbiol._ 43, 103–122 (1995).

Article  CAS  Google Scholar  * Hu, B. et al. Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus. _PLoS Pathog._ 13,

e1006698 (2017). Article  Google Scholar  * Tang, X. C. et al. Prevalence and genetic diversity of coronaviruses in bats from China. _J. Virol._ 80, 7481–7490 (2006). Article  CAS  Google

Scholar  * Tao, Y., et al. Surveillance of Bat coronaviruses in kenya identifies relatives of human coronaviruses NL63 and 229E and their recombination history. _J. Virol._ 91, e01953-16

(2017). * Di, H. et al. Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus. _Proc. Natl Acad. Sci. USA_ 114, E8895–E8904 (2017). Article  CAS  Google Scholar  *

Frieman, M. et al. Molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease. _J. Virol._ 86, 884–897

(2012). Article  CAS  Google Scholar  * Goldstein, D. R. Aging, imbalanced inflammation and viral infection. _Virulence_ 1, 295–298 (2010). Article  Google Scholar  * Totura, A. L. &

Baric, R. S. SARS coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon. _Curr. Opin. Virol._ 2, 264–275 (2012). Article  CAS  Google Scholar  * Rockx, B.

et al. Early upregulation of acute respiratory distress syndrome-associated cytokines promotes lethal disease in an aged-mouse model of severe acute respiratory syndrome coronavirus

infection. _J. Virol._ 83, 7062–7074 (2009). Article  CAS  Google Scholar  * Menachery, V. D. et al. Attenuation and restoration of severe acute respiratory syndrome coronavirus mutant

lacking 2′-o-methyltransferase activity. _J. Virol._ 88, 4251–4264 (2014). Article  Google Scholar  * Zust, R. et al. Coronavirus non-structural protein 1 is a major pathogenicity factor:

implications for the rational design of coronavirus vaccines. _PLoS Pathog._ 3, e109 (2007). Article  Google Scholar  * Beall, A. et al. Characterization of a pathogenic full-length cDNA

clone and transmission model for porcine epidemic diarrhea virus strain PC22A. _MBio_ 7, e01451 (2016). 01415. Article  CAS  Google Scholar  * Gonzalez, J. M., Penzes, Z., Almazan, F.,

Calvo, E. & Enjuanes, L. Stabilization of a full-length infectious cDNA clone of transmissible gastroenteritis coronavirus by insertion of an intron. _J. Virol._ 76, 4655–4661 (2002).

Article  CAS  Google Scholar  * Meulenberg, J. J., Bos-de Ruijter, J. N., van de Graaf, R., Wensvoort, G. & Moormann, R. J. Infectious transcripts from cloned genome-length cDNA of

porcine reproductive and respiratory syndrome virus. _J. Virol._ 72, 380–387 (1998). CAS  PubMed  PubMed Central  Google Scholar  * Youn, S., Leibowitz, J. L. & Collisson, E. W. In vitro

assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication. _Virology_ 332, 206–215 (2005). Article  CAS  Google

Scholar  * Zhou, H. et al. Identification of a novel recombinant virulent avian infectious bronchitis virus. _Vet. Microbiol._ 199, 120–127 (2017). Article  CAS  Google Scholar  * Zhou, Y.

S. et al. Establishment of reverse genetics system for infectious bronchitis virus attenuated vaccine strain H120. _Vet. Microbiol._ 162, 53–61 (2013). Article  CAS  Google Scholar  * Ge, X.

Y. et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. _Nature_ 503, 535–538 (2013). Article  CAS  Google Scholar  * Ge, X. Y. et al.

Coexistence of multiple coronaviruses in several bat colonies in an abandoned mineshaft. _Virol. Sin._ 31, 31–40 (2016). Article  CAS  Google Scholar  * Yang, X. L. et al. Isolation and

characterization of a novel Bat coronavirus closely related to the direct progenitor of severe acute respiratory syndrome coronavirus. _J. Virol._ 90, 3253–3256 (2015). Article  Google

Scholar  * Menachery, V. D. et al. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. _Nat. Med_ 21, 1508–1513 (2015). Article  CAS  Google Scholar  *

Yount, B. et al. Reverse genetics with a full length infectious cDNA of the severe acute respiratory syndrome coronavirus. _Proc. Natl Acad. Sci. USA_ 100, 12995–13000 (2003). Article  CAS 

Google Scholar  * Becker, M. M. et al. Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice. _Proc. Natl Acad. Sci. USA_ 105, 19944–19949 (2008).

Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This research was supported by NIH NIAID grants U19-AI107810, R01-AI108197 and U54-AI057157 to R.S.B. and 5F32AI080148 to

R.L.G. AUTHOR INFORMATION Author notes * Damon J. Deming Present address: Food and Drug Administration, 10933 New Hampshire Avenue, Bldg 22, Rm 6170, Silver Spring, MD, 20993, USA * Meagan

E. Deming Present address: University of Maryland Medical Center, Department of Medicine, Division of Infectious Disease, Institute of Human Virology, 725 West Lombard Street, Room 211A,

Baltimore, MD, 21201, USA AUTHORS AND AFFILIATIONS * Department of Epidemiology, The University of North Carolina at Chapel Hill, 2107 McGavran-Greenberg, CB 7435, Chapel Hill, NC, 27599,

USA Rachel L. Graham, Damon J. Deming, Boyd L. Yount & Ralph S. Baric * Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC,

27599, USA Damon J. Deming, Meagan E. Deming & Ralph S. Baric Authors * Rachel L. Graham View author publications You can also search for this author inPubMed Google Scholar * Damon J.

Deming View author publications You can also search for this author inPubMed Google Scholar * Meagan E. Deming View author publications You can also search for this author inPubMed Google

Scholar * Boyd L. Yount View author publications You can also search for this author inPubMed Google Scholar * Ralph S. Baric View author publications You can also search for this author

inPubMed Google Scholar CONTRIBUTIONS R.L.G. designed and performed experiments, analyzed data and wrote and edited the paper. D.J.D., M.E.D. and B.L.Y. designed and performed experiments

and analyzed data. R.S.B. analyzed data and wrote and edited the paper. CORRESPONDING AUTHOR Correspondence to Ralph S. Baric. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

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and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Graham, R.L., Deming, D.J., Deming, M.E. _et al._ Evaluation of a recombination-resistant coronavirus as a broadly applicable, rapidly

implementable vaccine platform. _Commun Biol_ 1, 179 (2018). https://doi.org/10.1038/s42003-018-0175-7 Download citation * Received: 31 January 2018 * Accepted: 19 September 2018 *

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