The VLDLR entry receptor is required for the pathogenesis of multiple encephalitic alphaviruses.

The very-low-density lipoprotein receptor (VLDLR) has been reported as an entry receptor for Semliki Forest (SFV) and Eastern equine encephalitis (EEEV) alphaviruses in cell cultures. However, the role of VLDLR in alphavirus pathogenesis and the extent to which other alphaviruses can engage VLDLR remains unclear. Here, using a surface protein-targeted CRISPR-Cas9 screen, we identify VLDLR as a receptor for Western equine encephalitis virus (WEEV) and demonstrate that it promotes the infection of multiple viruses in the WEE antigenic complex. In vivo studies show that the pathogenicity of WEEV, EEEV, and SFV, but not the distantly related Venezuelan equine encephalitis virus, is markedly diminished in VLDLR-deficient mice and that mice treated with a soluble VLDLR-Fc decoy molecule are protected against disease. Overall, these results expand our understanding of the role of VLDLR in alphavirus pathogenesis and provide a potential path for developing countermeasures against alphaviruses from different antigenic complexes.

Nanopore sequencing provides snapshots of the genetic variation within salmonid alphavirus-3 (SAV3) during an ongoing infection in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta).

Frequent RNA virus mutations raise concerns about evolving virulent variants. The purpose of this study was to investigate genetic variation in salmonid alphavirus-3 (SAV3) over the course of an experimental infection in Atlantic salmon and brown trout. Atlantic salmon and brown trout parr were infected using a cohabitation challenge, and heart samples were collected for analysis of the SAV3 genome at 2-, 4- and 8-weeks post-challenge. PCR was used to amplify eight overlapping amplicons covering 98.8% of the SAV3 genome. The amplicons were subsequently sequenced using the Nanopore platform. Nanopore sequencing identified a multitude of single nucleotide variants (SNVs) and deletions. The variation was widespread across the SAV3 genome in samples from both species. Mostly, specific SNVs were observed in single fish at some sampling time points, but two relatively frequent (i.e., major) SNVs were observed in two out of four fish within the same experimental group. Two other, less frequent (i.e., minor) SNVs only showed an increase in frequency in brown trout. Nanopore reads were de novo clustered using a 99% sequence identity threshold. For each amplicon, a number of variant clusters were observed that were defined by relatively large deletions. Nonmetric multidimensional scaling analysis integrating the cluster data for eight amplicons indicated that late in infection, SAV3 genomes isolated from brown trout had greater variation than those from Atlantic salmon. The sequencing methods and bioinformatics pipeline presented in this study provide an approach to investigate the composition of genetic diversity during viral infections.

Alphavirus nsP3 organizes into tubular scaffolds essential for infection and the cytoplasmic granule architecture.

Alphaviruses, such as chikungunya virus (CHIKV), are mosquito-borne viruses that represent a significant threat to human health due to the current context of global warming. Efficient alphavirus infection relies on the activity of the non-structural protein 3 (nsP3), a puzzling multifunctional molecule whose role in infection remains largely unknown. NsP3 is a component of the plasma membrane-bound viral RNA replication complex (vRC) essential for RNA amplification and is also found in large cytoplasmic aggregates of unknown function. Here, we report the cryo-electron microscopy (cryo-EM) structure of the CHIKV nsP3 at 2.35 Å resolution. We show that nsP3 assembles into tubular structures made by a helical arrangement of its alphavirus unique domain (AUD). The nsP3 helical scaffolds are consistent with crown structures found on tomographic reconstructions of the mature viral RCs. In addition, nsP3 helices assemble into cytoplasmic granules organized in a network of tubular structures that contain viral genomic RNA and capsid as well as host factors required for productive infection. Structure-guided mutagenesis identified residues that prevent or disturb nsP3 assemblies, resulting in impaired viral replication or transcription. Altogether, our results reveal an unexpected nsP3-dependent molecular organization essential for different phases of alphavirus infection.

ß-enaminoester derivatives exhibit promising in vitro and in silico antiviral potential against Mayaro virus.

Mayaro virus (MAYV) is the causative agent of Mayaro fever, which is characterized mainly by acute fever and long-term severe arthralgia, common manifestations of other arbovirus infections, making the correct diagnosis a challenge. Besides, MAYV infections have been reported in South America, especially in Brazil. However, the lack of vaccines or specific antiviral drugs to control these infections makes the search for new antivirals an urgent need. Herein, we evaluated the antiviral potential of synthetic ß-enaminoesters derivatives against MAYV replication and their pharmacokinetic and toxicological (ADMET) properties using in vitro and in silico strategies. For this purpose, Vero cells were infected with MAYV at an MOI of 0.1, treated with compounds (50 µM) for 24 h, and virus titers were quantified by plaque reduction assays. Compounds 2b (83.33%) and 2d (77.53%) exhibited the highest activity with inhibition rates of 83.33% and 77.53%, respectively. The most active compounds 2b (EC50 = 18.92 µM; SI > 52.85), and 2d (EC50 = 14.52 µM; SI > 68.87) exhibited higher potency and selectivity than the control drug suramin (EC50 = 38.97 µM; SI > 25.66). Then, we investigated the mechanism of action of the most active compounds. None of the compounds showed virucidal activity, neither inhibited virus adsorption, but compound 2b inhibited virus entry (62.64%). Also, compounds 2b and 2d inhibited some processes involved with the release of new virus particles. Finally, in silico results indicated good ADMET parameters of the most active compounds and reinforced their promising profile as drug candidates against MAYV.

How structural biology has changed our understanding of icosahedral viruses.

Cryo-electron microscopy and tomography have allowed us to unveil the remarkable structure of icosahedral viruses. However, in the past few years, the idea that these viruses must have perfectly symmetric virions, but in some cases, it might not be true. This has opened the door to challenging paradigms in structural virology and raised new questions about the biological implications of "unusual" or "defective" symmetries and structures. Also, the continual improvement of these technologies, coupled with more rigorous sample purification protocols, improvements in data processing, and the use of artificial intelligence, has allowed solving the structure of sub-viral particles in highly heterogeneous samples and finding novel symmetries or structural defects. In this review, I initially analyzed the case of the symmetry and composition of hepatitis B virus-produced spherical sub-viral particles. Then, I focused on Alphaviruses as an example of "imperfect" icosahedrons and analyzed how structural biology has changed our understanding of the Alphavirus assembly and some biological implications arising from these discoveries.

The First Isolation of Insect-Specific Alphavirus (Agua Salud alphavirus) in Culex (Melanoconion) Mosquitoes in the Brazilian Amazon.

Advances in diagnostic techniques coupled with ongoing environmental changes have resulted in intensified surveillance and monitoring of arbovirus circulation in the Amazon. This increased effort has resulted in increased detection of insect-specific viruses among hematophagous arthropods collected in the field. This study aimed to document the first isolation of Agua Salud alphavirus in mosquitoes collected within the Brazilian Amazon. Arthropods belonging to the family Culicidae were collected within a forest fragment located in the Environmental Protection Area of the metropolitan region of Belem. Subsequently, these specimens were meticulously identified to the species level. Afterward, the collected batches were macerated, and the resulting supernatant was then inoculated into C6/36 and Vero cell cultures to facilitate viral isolation. The presence of arboviruses within the inoculated cell cultures was determined through indirect immunofluorescence analysis. Furthermore, positive supernatant samples underwent nucleotide sequencing to precisely identify the viral strains present. Notably, a batch containing Culex (Melanoconion) mosquitoes was identified to be positive for the genus Alphavirus via indirect immunofluorescence. This study is the first report on insect-specific alphavirus isolation in Brazil and the first-ever description of Agua Salud alphavirus isolation within Amazon Forest remnants.

Packaging of alphavirus-based self-amplifying mRNA yields replication-competent virus through a mechanism of aberrant homologous RNA recombination.

Messenger (m)RNA has taken center stage in vaccine development, gene therapy, and cancer immunotherapy. A next-generation of mRNA is the self-amplifying (sa)mRNA, which induces broad and long-lasting immunity at a lower dose which provides better clinical outcomes in conjunction with fewer adverse effects. SamRNA, also known as "replicon" RNA, encodes the replication machinery of an alphavirus together with an antigen. Efficient delivery of replicon RNA to target tissues can be accomplished by packaging the replicon RNA in virus-like replicon particles (VRPs) via co-transfection of producer cells with defective helper RNA(s) encoding the alphavirus structural proteins. During the manufacture of VRPs, however, there is a potential risk of RNA recombination, which may lead to the formation of replication-competent virus (RCV). To investigate the factors influencing the unwanted RCV formation, we evaluated how sequence homology orchestrates alphavirus RNA recombination. Several combinations of complementing alphavirus replicon and helper RNAs varying in length of sequences overlap were co-transfected in mammalian cells. The culture fluid was serially passaged to detect RCV. Nanopore sequencing of cells after the first passage in combination with amplicon-based Sanger sequencing of RCV in the culture fluid after four passages led to the detection of RNA recombination. RCV was generated between replicon and helper RNAs with sequence homology in either the non-structural or structural genes, whereas RNAs without overlapping gene regions did not generate RCV. Remarkably, no sequence overlap was detected at the recombination junction sites in the RCV genome, suggesting a mechanism of "aberrant homologous RNA recombination." Accordingly, we conclude that the alphavirus RNA recombination process leading to the formation of RCV is homology-assisted and can be prevented by avoiding sequence homology between replicon and helper RNAs.IMPORTANCEThere is a growing interest in the use of self-amplifying (sa)mRNA vectors for next-generation vaccine development, gene therapy, and cancer immunotherapy. The delivery of samRNA in the form of virus-like replicon particles (VRPs) enables efficient delivery of samRNA to target tissue. The production of these VRPs, however, suffers from contamination with replication-competent virus (RCV) that is thought to arise from recombination events between samRNA and helper RNAs for VRP packaging. The presence of RCV in samRNA in the clinical product is undesirable as alphaviruses may cause serious disease in humans. However, the underlying recombination mechanism leading to RCV is currently unknown. In our work, we demonstrate a detailed evaluation of the recombination sites, which indicates that RCV is formed through an unusual mechanism of "aberrant homologous RNA recombination." The results are useful for researchers in the field of RNA vaccine manufacture and delivery.

Stress granule formation helps to mitigate neurodegeneration.

Cellular stress pathways that inhibit translation initiation lead to transient formation of cytoplasmic RNA/protein complexes known as stress granules. Many of the proteins found within stress granules and the dynamics of stress granule formation and dissolution are implicated in neurodegenerative disease. Whether stress granule formation is protective or harmful in neurodegenerative conditions is not known. To address this, we took advantage of the alphavirus protein nsP3, which selectively binds dimers of the central stress granule nucleator protein G3BP and markedly reduces stress granule formation without directly impacting the protein translational inhibitory pathways that trigger stress granule formation. In Drosophila and rodent neurons, reducing stress granule formation with nsP3 had modest impacts on lifespan even in the setting of serial stress pathway induction. In contrast, reducing stress granule formation in models of ataxia, amyotrophic lateral sclerosis and frontotemporal dementia largely exacerbated disease phenotypes. These data support a model whereby stress granules mitigate, rather than promote, neurodegenerative cascades.

Mayaro Virus: An Emerging Alphavirus in the Americas.

Mayaro virus (MAYV) is an arbovirus first isolated in Trinidad and Tobago in 1954. MAYV is the causative agent of Mayaro fever, which is characterised by high fever, maculopapular rash, myalgia and arthralgia. The potential for chronic arthralgia is of particular clinical concern. Currently, MAYV outbreaks are restricted to South and Central America, with some cases reported in Africa as well as several imported cases in Europe. However, in recent years, MAYV has become a growing global concern due to its potential to emerge into urban transmission cycles. Challenges faced with diagnostics, as well as a lack of specific antivirals or licensed vaccines further exacerbate the potential global health threat posed by MAYV. In this review, we discuss this emerging arboviral threat with a particular focus on the current treatment and vaccine development efforts. Overall, MAYV remains a neglected arbovirus due to its limited area of transmission. However, with the potential of its urbanisation and expanding circulation, the threat MAYV poses to global health cannot be overlooked. Further research into the improvement of current diagnostics, as well as the development of efficacious antivirals and vaccines will be crucial to help prevent and manage potential MAYV outbreaks.

Positive selection analyses identify a single WWE domain residue that shapes ZAP into a more potent restriction factor against alphaviruses.

The host interferon pathway upregulates intrinsic restriction factors in response to viral infection. Many of them block a diverse range of viruses, suggesting that their antiviral functions might have been shaped by multiple viral families during evolution. Host-virus conflicts have led to the rapid adaptation of host and viral proteins at their interaction hotspots. Hence, we can use evolutionary genetic analyses to elucidate antiviral mechanisms and domain functions of restriction factors. Zinc finger antiviral protein (ZAP) is a restriction factor against RNA viruses such as alphaviruses, in addition to other RNA, retro-, and DNA viruses, yet its precise antiviral mechanism is not fully characterized. Previously, an analysis of 13 primate ZAP orthologs identified three positively selected residues in the poly(ADP-ribose) polymerase-like domain. However, selective pressure from ancient alphaviruses and others likely drove ZAP adaptation in a wider representation of mammals. We performed positive selection analyses in 261 mammalian ZAP using more robust methods with complementary strengths and identified seven positively selected sites in all domains of the protein. We generated ZAP inducible cell lines in which the positively selected residues of ZAP are mutated and tested their effects on alphavirus replication and known ZAP activities. Interestingly, the mutant in the second WWE domain of ZAP (N658A) is dramatically better than wild-type ZAP at blocking replication of Sindbis virus and other ZAP-sensitive alphaviruses due to enhanced viral translation inhibition. The N658A mutant is adjacent to the previously reported poly(ADP-ribose) (PAR) binding pocket, but surprisingly has reduced binding to PAR. In summary, the second WWE domain is critical for engineering a more potent ZAP and fluctuations in PAR binding modulate ZAP antiviral activity. Our study has the potential to unravel the role of ADP-ribosylation in the host innate immune defense and viral evolutionary strategies that antagonize this post-translational modification.

Unraveling the complex interplay: immunopathology and immune evasion strategies of alphaviruses with emphasis on neurological implications.

Arthritogenic alphaviruses pose a significant public health concern due to their ability to cause joint inflammation, with emerging evidence of potential neurological consequences. In this review, we examine the immunopathology and immune evasion strategies employed by these viruses, highlighting their complex mechanisms of pathogenesis and neurological implications. We delve into how these viruses manipulate host immune responses, modulate inflammatory pathways, and potentially establish persistent infections. Further, we explore their ability to breach the blood-brain barrier, triggering neurological complications, and how co-infections exacerbate neurological outcomes. This review synthesizes current research to provide a comprehensive overview of the immunopathological mechanisms driving arthritogenic alphavirus infections and their impact on neurological health. By highlighting knowledge gaps, it underscores the need for research to unravel the complexities of virus-host interactions. This deeper understanding is crucial for developing targeted therapies to address both joint and neurological manifestations of these infections.

Mosquitoes as Vectors of Mycobacterium ulcerans Based on Analysis of Notifications of Alphavirus Infection and Buruli Ulcer, Victoria, Australia.

Alphavirus infections are transmitted by mosquitoes, but the mode of transmission for Mycobacterium ulcerans, which causes Buruli ulcer, is contested. Using notification data for Victoria, Australia, during 2017-2022, adjusted for incubation period, we show close alignment between alphavirus and Buruli ulcer seasons, supporting the hypothesis of mosquito transmission of M. ulcerans.

A getah virus-like-particle vaccine provides complete protection from viremia and arthritis in wild-type mice.

Getah virus (GETV) is an emerging mosquito-borne virus with economic impact on the livestock industry in East Asia. In this study, we successfully produced GETV virus-like particles (VLPs) in insect cells using the baculovirus expression vector system. We show that the GETV envelope glycoproteins were successfully expressed at the surface of the insect cell and were glycosylated. VLPs were isolated from the culture fluid as enveloped particles of 60-80 nm in diameter. Two 1 µg vaccinations with this GETV VLP vaccine, without adjuvant, generated neutralizing antibody responses and protected wild-type C57/BL6 mice against GETV viremia and arthritic disease. The GETV VLP vaccine may find application as a horse and/or pig vaccine in the future.

A recombinant Getah Virus expressing a GFP gene for rapid neutralization testing and antiviral drug screening assay.

Getah virus (GETV) is a re-emerging mosquito-borne RNA virus that induces fever, hind limb edema, swollen submandibular lymph nodes, and urticaria in horses. In pigs, the virus often results in stillbirths among pregnant sows, and neurological symptoms leading to death in piglets. Currently, there are no specific treatments or drugs available for GETV infection. The use of reporter viruses to monitor viral replication and spread in real-time within infected cells and animals provides a powerful tool for targeting antiviral drugs throughout the viral life cycle. Their fluorescence-tracked characteristics greatly facilitate virus neutralization tests (VNTs). In this study, we engineered two recombinant viruses by inserting different reporter protein genes at the 3' end of the structural protein gene, an unreported location that can accommodate exogenous genes. The rGEEiLOV and rGEEGFP viruses demonstrated genetic stability for at least five passages and replicated at a rate similar to that of the parental virus in BHK-21 cells. The rGEEGFP virus facilitated viral neutralization testing. Additionally, we used the reporter virus rGEEGFP to confirm ivermectin, a broad-spectrum antiparasitic agent, as a potential inhibitor of GETV in vitro. Ivermectin appears to inhibit the early replication stages of the virus and can block cell-to-cell viral transmission. In conclusion, rGEEGFP holds significant potential for antiviral screening to identify specific inhibitors against GETV and for use in viral neutralization tests.

An improved alphaviruses-specific RT-qPCR facilitates monitoring and prevention of alphaviruses.

Molecular surveillance is vital for monitoring arboviruses, often employing genus-specific quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Despite this, an overlooked chikungunya fever outbreak occurred in Yunnan province, China, in 2019 and false negatives are commonly encountered during alphaviruses screening practice, highlighting the need for improved detection methods. In this study, we developed an improved alphaviruses-specific RT-qPCR capable of detecting chikungunya virus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, Sindbis virus, Mayaro virus, and Ross River virus with high sensitivity and specificity. The assay identified three chikungunya virus-positive cases out of 188 sera retrospectively. Later genetic characterization suggested that imported cases from neighboring countries may be responsible for the neglected chikungunya fever outbreak of 2019 in Yunnan. Our findings underscore the value of improved alphaviruses-specific RT-qPCR in bolstering alphaviruses surveillance and informing preventive strategies.

Comparative pathogenesis of three Mayaro virus genotypes in the cynomolgus macaque.

Mayaro virus (MAYV), a mosquito-borne alphavirus, is considered an emerging threat to public health with epidemic potential. Phylogenetic studies show the existence of three MAYV genotypes. In this study, we provide a preliminary analysis of the pathogenesis of all three MAYV genotypes in cynomolgus macaques (Macaca facicularis, Mauritian origin). Significant MAYV-specific RNAemia and viremia were detected during acute infection in animals challenged intravenously with the three MAYV genotypes, and strong neutralizing antibody responses were observed. MAYV RNA was detected at high levels in lymphoid tissues, joint muscle and synovia over 1 month after infection, suggesting that this model could serve as a promising tool in studying MAYV-induced chronic arthralgia, which can persist for years. Significant leucopenia was observed across all MAYV genotypes, peaking with RNAemia. Notable differences in the severity of acute RNAemia and composition of cytokine responses were observed among the three MAYV genotypes. Our model showed no outward signs of clinical disease, but several major endpoints for future MAYV pathology and intervention studies are described. Disruptions to normal blood cell counts and cytokine responses were markedly distinct from those observed in macaque models of CHIKV infection, underlining the importance of developing non-human primate models specific to MAYV infection.

Displaying alphavirus physicochemical consensus antigens on immunogenic liposomes enhances antibody elicitation in mice.

Cobalt-porphyrin phospholipid displays recombinant protein antigens on liposome surfaces via antigen polyhistidine-tag (His-tag), and when combined with monophosphorylated lipid A and QS-21 yields the "CPQ" vaccine adjuvant system. In this proof of principle study, CPQ was used to generate vaccine prototypes that elicited antibodies for two different alphaviruses (AV). Mice were immunized with computationally designed, His-tagged, physicochemical property consensus (PCPcon) protein antigens representing the variable B-domain of the envelope protein 2 (E2) from the serotype specific Venezuelan Equine Encephalitis Virus (VEEVcon) or a broad-spectrum AV-antigen termed EVCcon. The CPQ adjuvant enhanced the antigenicity of both proteins without eliciting detectable anti-His-tag antibodies. Antibodies elicited from mice immunized with antigens admixed with CPQ showed orders-of-magnitude higher levels of antigen-specific IgG compared to alternative control adjuvants. The ELISA results correlated with antiviral activity against VEEV strain TC83 and more weakly to Chikungunya virus 118/25. Thus, display of E.coli-produced His-tagged E2 protein segments on the surface of immunogenic liposomes elicits high levels of antigen-specific and AV neutralizing antibodies in mice with vaccination, while facilitating vaccine preparation and providing dose-sparing potential.

Exploring Barmah Forest virus pathogenesis: molecular tools to investigate non-structural protein 3 nuclear localization and viral genomic determinants of replication.

Barmah Forest virus (BFV) is a mosquito-borne virus that causes arthralgia with accompanying rash, fever, and myalgia in humans. The virus is mainly found in Australia and has caused outbreaks associated with significant health concerns. As the sole representative of the Barmah Forest complex within the genus Alphavirus, BFV is not closely related genetically to other alphaviruses. Notably, basic knowledge of BFV molecular virology has not been well studied due to a lack of critical investigative tools such as an infectious clone. Here we describe the construction of an infectious BFV cDNA clone based on Genbank sequence and demonstrate that the clone-derived virus has in vitro and in vivo properties similar to naturally occurring virus, BFV field isolate 2193 (BFV2193-FI). A substitution in nsP4, V1911D, which was identified in the Genbank reference sequence, was found to inhibit virus rescue and replication. T1325P substitution in nsP2 selected during virus passaging was shown to be an adaptive mutation, compensating for the inhibitory effect of nsP4-V1911D. The two mutations were associated with changes in viral non-structural polyprotein processing and type I interferon (IFN) induction. Interestingly, a nuclear localization signal, active in mammalian but not mosquito cells, was identified in nsP3. A point mutation abolishing nsP3 nuclear localization attenuated BFV replication. This effect was more prominent in the presence of type I interferon signaling, suggesting nsP3 nuclear localization might be associated with IFN antagonism. Furthermore, abolishing nsP3 nuclear localization reduced virus replication in mice but did not significantly affect disease.IMPORTANCEBarmah Forest virus (BFV) is Australia's second most prevalent arbovirus, with approximately 1,000 cases reported annually. The clinical symptoms of BFV infection include rash, polyarthritis, arthralgia, and myalgia. As BFV is not closely related to other pathogenic alphaviruses or well-studied model viruses, our understanding of its molecular virology and mechanisms of pathogenesis is limited. There is also a lack of molecular tools essential for corresponding studies. Here we describe the construction of an infectious clone of BFV, variants harboring point mutations, and sequences encoding marker protein. In infected mammalian cells, nsP3 of BFV was located in the nuclei. This finding extends our understanding of the diverse mechanisms used by alphavirus replicase proteins to interact with host cells. Our novel observations highlight the complex synergy through which the viral replication machinery evolves to correct mutation errors within the viral genome.

Inhibitors of the Structural and Nonstructural Proteins of Alphaviruses.

The Alphavirus genus includes viruses that cause encephalitis due to neuroinvasion and viruses that cause arthritis due to acute and chronic inflammation. There is no approved therapeutic for alphavirus infections, but significant efforts are ongoing, more so in recent years, to develop vaccines and therapeutics for alphavirus infections. This review article highlights some of the major advances made so far to identify small molecules that can selectively target the structural and the nonstructural proteins in alphaviruses with the expectation that persistent investigation of an increasingly expanding chemical space through a variety of structure-based design and high-throughput screening strategies will yield candidate drugs for clinical studies. While most of the works discussed are still in the early discovery to lead optimization stages, promising avenues remain for drug development against this family of viruses.

Safety concern of recombination between self-amplifying mRNA vaccines and viruses is mitigated in vivo.

Self-amplifying mRNA (SAM) vaccines can be rapidly deployed in the event of disease outbreaks. A legitimate safety concern is the potential for recombination between alphavirus-based SAM vaccines and circulating viruses. This theoretical risk needs to be assessed in the regulatory process for SAM vaccine approval. Herein, we undertake extensive in vitro and in vivo assessments to explore recombination between SAM vaccine and a wide selection of alphaviruses and a coronavirus. SAM vaccines were found to effectively limit alphavirus co-infection through superinfection exclusion, although some co-replication was still possible. Using sensitive cell-based assays, replication-competent alphavirus chimeras were generated in vitro as a result of rare, but reproducible, RNA recombination events. The chimeras displayed no increased fitness in cell culture. Viable alphavirus chimeras were not detected in vivo in C57BL/6J, Rag1-/- and Ifnar-/- mice, in which high levels of SAM vaccine and alphavirus co-replicated in the same tissue. Furthermore, recombination between a SAM-spike vaccine and a swine coronavirus was not observed. In conclusion we state that although the ability of SAM vaccines to recombine with alphaviruses might be viewed as an environmental safety concern, several key factors substantially mitigate against in vivo emergence of chimeric viruses from SAM vaccine recipients.