Differing Transcriptomic Responses in High Titer versus Low Titer Aedes aegypti Mosquitoes after Oral Infection with Sindbis Virus.

Oral infection of mosquitoes by arboviruses often results in a large degree of variation in the amount of infectious virus between individual mosquitoes, even when the mosquitoes are from inbred laboratory strains. This variability in arbovirus load has been shown to affect virus transmissibility. Previously, our group described population genetic and specific infectivity differences between the virus populations found in high and low titer Aedes aegypti mosquitoes that had been orally infected with Sindbis virus (SINV). In this study, we sought to investigate whether there were also differences in transcriptomic response between these high and low titer mosquitoes. Results from the transcriptomic data analysis showed that more genes involved in antiviral activity, endopeptidase activity, and methyltransferase activity were upregulated in low titer mosquitoes than in high titer mosquitoes, relative to blood-fed controls. Meanwhile, genes involved in ion transport, energy metabolism, acetylation, glycosylation, lipid metabolism, and transport tended to be upregulated in high titer mosquitoes more than in low titer mosquitoes, relative to blood-fed mosquitoes. Overall, genes involved in antiviral activities tended to be upregulated in low titer mosquitoes while genes involved in proviral activities were mostly upregulated in high titer mosquitoes. This study has identified a number of candidate mosquito genes that are putatively associated with SINV titer variability after oral infection of Ae. aegypti, and these can now be investigated in order to ascertain their roles in virus replication and their contributions to determining vector competence.

Oncolytic Activity of Sindbis Virus with the Help of GM-CSF in Hepatocellular Carcinoma.

Hepatocellular carcinoma is a refractory tumor with poor prognosis and high mortality. Many oncolytic viruses are currently being investigated for the treatment of hepatocellular carcinoma. Based on previous studies, we constructed a recombinant GM-CSF-carrying Sindbis virus, named SINV-GM-CSF, which contains a mutation (G to S) at amino acid 285 in the nsp1 protein of the viral vector. The potential of this mutated vector for liver cancer therapy was verified at the cellular level and in vivo, respectively, and the changes in the tumor microenvironment after treatment were also described. The results showed that the Sindbis virus could effectively infect hepatocellular carcinoma cell lines and induce cell death. Furthermore, the addition of GM-CSF enhanced the tumor-killing effect of the Sindbis virus and increased the number of immune cells in the intra-tumor microenvironment during the treatment. In particular, SINV-GM-CSF was able to efficiently kill tumors in a mouse tumor model of hepatocellular carcinoma by regulating the elevation of M1-type macrophages (which have a tumor-resistant ability) and the decrease in M2-type macrophages (which have a tumor-promoting capacity). Overall, SINV-GM-CSF is an attractive vector platform with clinical potential for use as a safe and effective oncolytic virus.

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.

Phenotypic and transcriptional changes in peripheral blood mononuclear cells during alphavirus encephalitis in mice.

Sindbis virus (SINV) infection of mice provides a model system for studying the pathogenesis of alphaviruses that infect the central nervous system (CNS) to cause encephalomyelitis. While studies of human viral infections typically focus on accessible cells from the blood, this compartment is rarely evaluated in mice. To bridge this gap, single-cell RNA sequencing (scRNAseq) was combined with flow cytometry to characterize the transcriptional and phenotypic changes of peripheral blood mononuclear cells (PBMCs) from SINV-infected mice. Twenty-one clusters were identified by scRNAseq at 7 days after infection, with a unique cluster and overall increase in naive B cells for infected mice. Uninfected mice had fewer immature T cells and CCR9+ CD4 T cells and a unique immature T cell cluster. Gene expression was most altered in the Ki67+ CD8 T cell cluster, with chemotaxis and proliferation-related genes upregulated. Global analysis indicated metabolic changes in myeloid cells and increased expression of Ccl5 by NK cells. Phenotypes of PBMCs and cells infiltrating the CNS were analyzed by flow cytometry over 14 days after infection. In PBMCs, CD8 and Th1 CD4 T cells increased in representation, while B cells showed a transient decrease at day 5 in total, Ly6a+, and naive cells, and an increase in activated B cells. In the brain, CD8 T cells increased for the first 7 days, while Th1 CD4 T cells and naive and Ly6a+ B cells continued to accumulate for 14 days. Therefore, dynamic immune cell changes can be identified in the blood as well as the CNS during viral encephalomyelitis. IMPORTANCE: The outcome of viral encephalomyelitis is dependent on the host immune response, with clearance and resolution of infection mediated by the adaptive immune response. These processes are frequently studied in mouse models of infection, where infected tissues are examined to understand the mechanisms of clearance and recovery. However, studies of human infection typically focus on the analysis of cells from the blood, a compartment rarely examined in mice, rather than inaccessible tissue. To close this gap, we used single-cell RNA sequencing and flow cytometry to profile the transcriptomic and phenotypic changes of peripheral blood mononuclear cells (PBMCs) before and after central nervous system (CNS) infection in mice. Changes to T and B cell gene expression and cell composition occurred in PBMC and during entry into the CNS, with CCL5 being a differentially expressed chemokine. Therefore, dynamic changes occur in the blood as well as the CNS during the response of mice to virus infection, which will inform the analysis of human studies.

Towards modelling tick-virus interactions using the weakly pathogenic Sindbis virus: Evidence that ticks are competent vectors.

Most tick-borne viruses (TBVs) are highly pathogenic and require high biosecurity, which severely limits their study. We found that Sindbis virus (SINV), predominantly transmitted by mosquitoes, can replicate in ticks and be subsequently transmitted, with the potential to serve as a model for studying tick-virus interactions. We found that both larval and nymphal stages of Rhipicephalus haemaphysaloides can be infected with SINV-wild-type (WT) when feeding on infected mice. SINV replicated in two species of ticks (R. haemaphysaloides and Hyalomma asiaticum) after infecting them by microinjection. Injection of ticks with SINV expressing enhanced Green Fluorescent Protein (eGFP) revealed that SINV-eGFP specifically aggregated in the tick midguts for replication. During blood-feeding, SINV-eGFP migrated from the midguts to the salivary glands and was transmitted to a new host. SINV infection caused changes in expression levels of tick genes related to immune responses, substance transport and metabolism, cell growth and death. SINV mainly induced autophagy during the early stage of infection; with increasing time of infection, the level of autophagy decreased, while the level of apoptosis increased. During the early stages of infection, the transcript levels of immune-related genes were significantly upregulated, and then decreased. In addition, SINV induced changes in the transcription levels of some functional genes that play important roles in the interactions between ticks and tick-borne pathogens. These results confirm that the SINV-based transmission model between ticks, viruses, and mammals can be widely used to unravel the interactions between ticks and viruses.

Extreme infectious titer variability in individual Aedes aegypti mosquitoes infected with Sindbis virus is associated with both differences in virus population structure and dramatic disparities in specific infectivity.

Variability in how individuals respond to pathogens is a hallmark of infectious disease, yet the basis for individual variation in host response is often poorly understood. The titer of infectious virus among individual mosquitoes infected with arboviruses is frequently observed to vary by several orders of magnitude in a single experiment, even when the mosquitoes are highly inbred. To better understand the basis for this titer variation, we sequenced populations of Sindbis virus (SINV) obtained from individual infected Aedes aegypti mosquitoes that, despite being from a highly inbred laboratory colony, differed in their titers of infectious virus by approximately 10,000-fold. We observed genetic differences between these virus populations that indicated the virus present in the midguts of low titer mosquitoes was less fit than that of high titer mosquitoes, possibly due to founder effects that occurred during midgut infection. Furthermore, we found dramatic differences in the specific infectivity or SI (the ratio of infectious units/viral genome equivalents) between these virus populations, with the SI of low titer mosquitoes being up to 10,000-fold lower than that of high titer mosquitoes. Despite having similar amounts of viral genomes, low titer mosquitoes appeared to contain less viral particles, suggesting that viral genomes were packaged into virions less efficiently than in high titer mosquitoes. Finally, antibiotic treatment, which has been shown to suppress mosquito antiviral immunity, caused an increase in SI. Our results indicate that the extreme variation that is observed in SINV infectious titer between individual Ae. aegypti mosquitoes is due to both genetic differences between virus populations and to differences in the proportion of genomes that are packaged into infectious particles.

A novel three-plasmid packaging system for chimeric SFV/SIN VRPs derived from Semliki Forest virus and Sindbis virus as a candidate gene delivery vector.

Semliki Forest virus (SFV) viral replicon particles (VRPs) have been frequently used in various animal models and clinical trials. Chimeric replicon particles offer different advantages because of their unique biological properties. We here constructed a novel three-plasmid packaging system for chimeric SFV/SIN VRPs. The capsid and envelope of SIN structural proteins were generated using two-helper plasmids separately, and the SFV replicon contained the SFV replicase gene, packaging signal of SIN, subgenomic promoter followed by the exogenous gene, and 3' UTR of SIN. The chimeric VRPs carried luciferase or eGFP as reporter genes. The fluorescence and electron microscopy results revealed that chimeric VRPs were successfully packaged. The yield of the purified chimeric VRPs was approximately 2.5 times that of the SFV VRPs (1.38 × 107 TU/ml vs. 5.41 × 106 TU/ml) (p < 0.01). Furthermore, chimeric VRPs could be stored stably at 4°C for at least 60 days. Animal experiments revealed that mice immunized with chimeric VRPs (luciferase) had stronger luciferase expression than those immunized with equivalent amount of SFV VRPs (luciferase) (p < 0.01), and successfully expressed luciferase for approximately 12 days. Additionally, the chimeric VRPs expressed the RBD of SARS-CoV-2 efficiently and induced robust RBD-specific antibody responses in mice. In conclusion, the chimeric VRPs constructed here met the requirements of a gene delivery tool for vaccine development and cancer therapy.

Utilization of nanopore direct RNA sequencing to analyze viral RNA modifications.

Modifications on viral RNAs (vRNAs), either genomic RNAs or RNA transcripts, have complex effects on the viral life cycle and cellular responses to viral infection. The advent of Oxford Nanopore Technologies Direct RNA Sequencing provides a new strategy for studying RNA modifications. To this end, multiple computational tools have been developed, but a systemic evaluation of their performance in mapping vRNA modifications is lacking. Here, 10 computational tools were tested using the Sindbis virus (SINV) RNAs isolated from infected mammalian (BHK-21) or mosquito (C6/36) cells, with in vitro-transcribed RNAs serving as modification-free control. Three single-mode approaches were shown to be inapplicable in the viral context, and three out of seven comparative methods required cutoff adjustments to reduce false-positive predictions. Utilizing optimized cutoffs, an integrated analysis of comparative tools suggested that the intersected predictions of Tombo_com and xPore were significantly enriched compared with the background. Consequently, a pipeline integrating Tombo_com and xPore was proposed for vRNA modification detection; the performance of which was supported by N6-methyladenosine prediction in severe acute respiratory syndrome coronavirus 2 RNAs using publicly available data. When applied to SINV RNAs, this pipeline revealed more intensive modifications in subgenomic RNAs than in genomic RNAs. Modified uridines were frequently identified, exhibiting substantive overlapping between vRNAs generated in different cell lines. On the other hand, the interpretation of other modifications remained unclear, underlining the limitations of the current computational tools despite their notable potential.IMPORTANCEComputational approaches utilizing Oxford Nanopore Technologies Direct RNA Sequencing data were almost exclusively designed to map eukaryotic epitranscriptomes. Therefore, extra caution must be exercised when using these tools to detect vRNA modifications, as in most cases, vRNA modification profiles should be regarded as unknown epitranscriptomes without prior knowledge. Here, we comprehensively evaluated the performance of 10 computational tools in detecting vRNA modification sites. All tested single-mode methods failed to differentiate native and in vitro-transcribed samples. Using optimized cutoff values, seven tested comparative tools generated very different predictions. An integrated analysis showed significant enrichment of Tombo_com and xPore predictions against the background. A pipeline for vRNA modification detection was proposed accordingly and applied to Sindbis virus RNAs. In conclusion, our study underscores the need for the careful application of computational tools to analyze viral epitranscriptomics. It also offers insights into alphaviral RNA modifications, although further validation is required.

Capsid protein mediated evasion of IRAK1-dependent signalling is essential to Sindbis virus neuroinvasion and virulence in mice.

ABSTRACTAlphaviruses are arthropod-borne, single-stranded positive-sense RNA viruses that are recognized as rapidly emerging pathogens. Despite being exquisitely sensitive to the effects of the innate immune response alphaviruses can readily replicate, disseminate, and induce pathogenesis in immunologically competent hosts. Nonetheless, how alphaviruses evade the induction of an innate immune response prior to viral gene expression, or in non-permissive infections, is unknown. Previously we reported the identification of a novel host/pathogen interaction between the viral Capsid (CP) protein and the host IRAK1 protein. The CP/IRAK1 interaction was determined to negatively impact IRAK1-dependent PAMP detection in vitro, however, the precise importance of the CP/IRAK1 interaction to alphaviral infection remained unknown. Here we detail the identification of the CP/IRAK1 interaction determinants of the Sindbis virus (SINV) CP protein and examine the importance of the interaction to alphaviral infection and pathogenesis in vivo using an interaction deficient mutant of the model neurotropic strain of SINV. Importantly, these interaction determinants are highly conserved across multiple Old-World alphaviruses, including Ross River virus (RRV), Mayaro virus (MAYV), Chikungunya virus (CHIKV), and Semliki Forest virus (SFV). In the absence of a functional CP/IRAK1 interaction, SINV replication is significantly restricted and fails to disseminate from the primary site of inoculation due to the induction of a robust type-I Interferon response. Altogether these data indicate that the evasion of IRAK1-dependent signalling is critical to overcoming the host innate immune response and the in vivo data presented here demonstrate the importance of the CP/IRAK1 interaction to neurovirulence and pathogenesis.

Host Derivation of Sindbis Virus Influences Mammalian Type I Interferon Response to Infection.

Arboviruses are defined by their ability to replicate in both mosquito vectors and mammalian hosts. There is good evidence that arboviruses "prime" their progeny for infection of the next host, such as via differential glycosylation of their outer glycoproteins or packaging of host ribosomal subunits. We and others have previously shown that mosquito-derived viruses more efficiently infect mammalian cells than mammalian-derived viruses. These observations are consistent with arboviruses acquiring host-specific adaptations, and we hypothesized that a virus derived from either the mammalian host or mosquito vector elicits different responses when infecting the mammalian host. Here, we perform an RNA-sequencing analysis of the transcriptional response of Human Embryonic Kidney 293 (HEK-293) cells to infection with either mosquito (Aedes albopictus, C7/10)- or mammalian (Baby Hamster Kidney, BHK-21)-derived Sindbis virus (SINV). We show that the C7/10-derived virus infection leads to a more robust transcriptional response in HEK-293s compared to infection with the BHK-derived virus. Surprisingly, despite more efficient infection, we found an increase in interferon-ß (IFN-ß) and interferon-stimulated gene (ISG) transcripts in response to the C7/10-derived virus infection versus the BHK-derived virus infection. However, translation of interferon-stimulated genes was lower in HEK-293s infected with the C7/10-derived virus, starkly contrasting with the transcriptional response. This inhibition of ISG translation is reflective of a more rapid overall shut-off of host cell translation following infection with the C7/10-derived virus. Finally, we show that the C7/10-derived virus infection of HEK-293 cells leads to elevated levels of phosphorylated eukaryotic translation elongation factor-2 (eEF2), identifying a potential mechanism leading to the more rapid shut-off of host translation. We postulate that the rapid shut-off of host translation in mammalian cells infected with the mosquito-derived virus acts to counter the IFN-ß-stimulated transcriptional response.

Alphavirus Evasion of Zinc Finger Antiviral Protein (ZAP) Correlates with CpG Suppression in a Specific Viral nsP2 Gene Sequence.

Certain re-emerging alphaviruses, such as chikungunya virus (CHIKV), cause serious disease and widespread epidemics. To develop virus-specific therapies, it is critical to understand the determinants of alphavirus pathogenesis and virulence. One major determinant is viral evasion of the host interferon response, which upregulates antiviral effectors, including zinc finger antiviral protein (ZAP). Here, we demonstrated that Old World alphaviruses show differential sensitivity to endogenous ZAP in 293T cells: Ross River virus (RRV) and Sindbis virus (SINV) are more sensitive to ZAP than o'nyong'nyong virus (ONNV) and CHIKV. We hypothesized that the more ZAP-resistant alphaviruses evade ZAP binding to their RNA. However, we did not find a correlation between ZAP sensitivity and binding to alphavirus genomic RNA. Using a chimeric virus, we found the ZAP sensitivity determinant lies mainly within the alphavirus non-structural protein (nsP) gene region. Surprisingly, we also did not find a correlation between alphavirus ZAP sensitivity and binding to nsP RNA, suggesting ZAP targeting of specific regions in the nsP RNA. Since ZAP can preferentially bind CpG dinucleotides in viral RNA, we identified three 500-bp sequences in the nsP region where CpG content correlates with ZAP sensitivity. Interestingly, ZAP binding to one of these sequences in the nsP2 gene correlated to sensitivity, and we confirmed that this binding is CpG-dependent. Our results demonstrate a potential strategy of alphavirus virulence by localized CpG suppression to evade ZAP recognition.

Alphavirus infection triggers antiviral RNAi immunity in mammals.

RNA interference (RNAi) is a well-established antiviral immunity. However, for mammalian somatic cells, antiviral RNAi becomes evident only when viral suppressors of RNAi (VSRs) are disabled by mutations or VSR-targeting drugs, thereby limiting its scope as a mammalian immunity. We find that a wild-type alphavirus, Semliki Forest virus (SFV), triggers the Dicer-dependent production of virus-derived small interfering RNAs (vsiRNAs) in both mammalian somatic cells and adult mice. These SFV-vsiRNAs are located at a particular region within the 5' terminus of the SFV genome, Argonaute loaded, and active in conferring effective anti-SFV activity. Sindbis virus, another alphavirus, also induces vsiRNA production in mammalian somatic cells. Moreover, treatment with enoxacin, an RNAi enhancer, inhibits SFV replication dependent on RNAi response in vitro and in vivo and protects mice from SFV-induced neuropathogenesis and lethality. These findings show that alphaviruses trigger the production of active vsiRNA in mammalian somatic cells, highlighting the functional importance and therapeutic potential of antiviral RNAi in mammals.

Proteomics-based determination of double-stranded RNA interactome reveals known and new factors involved in Sindbis virus infection.

Viruses are obligate intracellular parasites, which depend on the host cellular machineries to replicate their genome and complete their infectious cycle. Long double-stranded (ds)RNA is a common viral by-product originating during RNA virus replication and is universally sensed as a danger signal to trigger the antiviral response. As a result, viruses hide dsRNA intermediates into viral replication factories and have evolved strategies to hijack cellular proteins for their benefit. The characterization of the host factors associated with viral dsRNA and involved in viral replication remains a major challenge to develop new antiviral drugs against RNA viruses. Here, we performed anti-dsRNA immunoprecipitation followed by mass spectrometry analysis to fully characterize the dsRNA interactome in Sindbis virus (SINV) infected human cells. Among the identified proteins, we characterized SFPQ (splicing factor, proline-glutamine rich) as a new dsRNA-associated proviral factor upon SINV infection. We showed that SFPQ depletion reduces SINV infection in human HCT116 and SK-N-BE(2) cells, suggesting that SFPQ enhances viral production. We demonstrated that the cytoplasmic fraction of SFPQ partially colocalizes with dsRNA upon SINV infection. In agreement, we proved by RNA-IP that SFPQ can bind dsRNA and viral RNA. Furthermore, we showed that overexpression of a wild-type, but not an RNA binding mutant SFPQ, increased viral infection, suggesting that RNA binding is essential for its positive effect on the virus. Overall, this study provides the community with a compendium of dsRNA-associated factors during viral infection and identifies SFPQ as a new proviral dsRNA binding protein.

Dancing with the Devil: A Review of the Importance of Host RNA-Binding Proteins to Alphaviral RNAs during Infection.

Alphaviruses are arthropod-borne, single-stranded positive sense RNA viruses that rely on the engagement of host RNA-binding proteins to efficiently complete the viral lifecycle. Because of this reliance on host proteins, the identification of host/pathogen interactions and the subsequent characterization of their importance to viral infection has been an intensive area of study for several decades. Many of these host protein interaction studies have evaluated the Protein:Protein interactions of viral proteins during infection and a significant number of host proteins identified by these discovery efforts have been RNA Binding Proteins (RBPs). Considering this recognition, the field has shifted towards discovery efforts involving the direct identification of host factors that engage viral RNAs during infection using innovative discovery approaches. Collectively, these efforts have led to significant advancements in the understanding of alphaviral molecular biology; however, the precise extent and means by which many RBPs influence viral infection is unclear as their specific contributions to infection, as per any RNA:Protein interaction, have often been overlooked. The purpose of this review is to summarize the discovery of host/pathogen interactions during alphaviral infection with a specific emphasis on RBPs, to use new ontological analyses to reveal potential functional commonalities across alphaviral RBP interactants, and to identify host RBPs that have, and have yet to be, evaluated in their native context as RNA:Protein interactors.

Detection of West Nile Virus Lineage 2 in Eastern Romania and First Identification of Sindbis Virus RNA in Mosquitoes Analyzed using High-Throughput Microfluidic Real-Time PCR.

The impact of mosquito-borne diseases on human and veterinary health is being exacerbated by rapid environmental changes caused mainly by changing climatic patterns and globalization. To gain insight into mosquito-borne virus circulation from two counties in eastern and southeastern Romania, we have used a combination of sampling methods in natural, urban and peri-urban sites. The presence of 37 mosquito-borne viruses in 16,827 pooled mosquitoes was analyzed using a high-throughput microfluidic real-time PCR assay. West Nile virus (WNV) was detected in 10/365 pools of Culex pipiens (n = 8), Culex modestus (n = 1) and Aedes vexans (n = 1) from both studied counties. We also report the first molecular detection of Sindbis virus (SINV) RNA in the country in one pool of Culex modestus. WNV infection was confirmed by real-time RT-PCR (10/10) and virus isolation on Vero or C6/36 cells (four samples). For the SINV-positive pool, no cytopathic effectwas observed after infection of Vero or C6/36 cells, but no amplification was obtained in conventional SINV RT-PCR. Phylogenetic analysis of WNV partial NS5 sequences revealed that WNV lineage 2 of theCentral-Southeast European clade, has a wider circulation in Romania than previously known.

Genomic Analysis of Sindbis Virus Reveals Uncharacterized Diversity within the Australasian Region, and Support for Revised SINV Taxonomy.

Sindbis virus (SINV) is a widely dispersed mosquito-borne alphavirus. Reports of Sindbis disease are largely restricted to northern Europe and South Africa. SINV is frequently sampled in Australian mosquito-based arbovirus surveillance programs, but human disease has rarely been reported. Molecular epidemiological studies have characterized six SINV genotypes (G1-G6) based on E2 gene phylogenies, mostly comprising viruses derived from the African-European zoogeographical region and with limited representation of Australasian SINV. In this study, we conducted whole genome sequencing of 66 SINV isolates sampled between 1960 and 2014 from countries of the Australasian region: Australia, Malaysia, and Papua New Guinea. G2 viruses were the most frequently and widely sampled, with three distinct sub-lineages defined. No new G6 SINV were identified, confirming geographic restriction of these viruses to south-western Australia. Comparison with global SINV characterized large-scale nucleotide and amino acid sequence divergence between African-European G1 viruses and viruses that circulate in Australasia (G2 and G3) of up to 26.83% and 14.55%, respectively, divergence that is sufficient for G2/G3 species demarcation. We propose G2 and G3 are collectively a single distinct alphavirus species that we name Argyle virus, supported by the inapparent or mild disease phenotype and the higher evolutionary rate compared with G1. Similarly, we propose G6, with 24.7% and 12.61% nucleotide and amino acid sequence divergence, is a distinct alphavirus species that we name Thomson's Lake virus.

Host-Dependent Modifications of Packaged Alphavirus Genomic RNA Influence Virus Replication in Mammalian Cells.

Alphaviruses must interact efficiently with two distinct host environments in order to replicate and transmit between vertebrate and mosquito hosts. Some host-origin-dependent differences in virus particle composition that appear to facilitate the transmission cycle are known. However, the impact of host-mediated modification of packaged viral genomic RNA on subsequent infection has not been previously investigated. Here we show that in human (HEK-293) cells, mosquito-derived Sindbis virus (SINV) replicates and spreads faster, producing a more infectious virus than its mammalian-derived counterpart. This enhanced replication is neither a result of differences in the stability nor the production of the infecting genomic RNA. Nevertheless, purified genomic RNA from mosquito-derived SINV established infection in HEK-293 cells more efficiently than that of mammalian-derived SINV, indicating that the genomic RNA itself is different between the two producing hosts and this difference is a determinant of infection. In agreement with this idea, we show that mosquito-derived SINV genomic RNA is a more active template for translation than mammalian-derived SINV genomic RNA, and we attribute this difference to host-dependent changes in modification of packaged genomic RNA as determined by LC/MS-MS. Our data support the hypothesis that among other factors, the host-dependent modification profile of the packaged vRNA is likely to play an important role in the efficiency of SINV infection and replication in mammalian cells.

Requirement of a functional ion channel for Sindbis virus glycoprotein transport, CPV-II formation, and efficient virus budding.

Many viruses encode ion channel proteins that oligomerize to form hydrophilic pores in membranes of virus-infected cells and the viral membrane in some enveloped viruses. Alphavirus 6K, human immunodeficiency virus type 1 Vpu (HIV-Vpu), influenza A virus M2 (IAV-M2), and hepatitis C virus P7 (HCV-P7) are transmembrane ion channel proteins that play essential roles in virus assembly, budding, and entry. While the oligomeric structures and mechanisms of ion channel activity are well-established for M2 and P7, these remain unknown for 6K. Here we investigated the functional role of the ion channel activity of 6K in alphavirus assembly by utilizing a series of Sindbis virus (SINV) ion channel chimeras expressing the ion channel helix from Vpu or M2 or substituting the entire 6K protein with full-length P7, in cis. We demonstrate that the Vpu helix efficiently complements 6K, whereas M2 and P7 are less efficient. Our results indicate that while SINV is primarily insensitive to the M2 ion channel inhibitor amantadine, the Vpu inhibitor 5-N, N-Hexamethylene amiloride (HMA), significantly reduces SINV release, suggesting that the ion channel activity of 6K similar to Vpu, promotes virus budding. Using live-cell imaging of SINV with a miniSOG-tagged 6K and mCherry-tagged E2, we further demonstrate that 6K and E2 colocalize with the Golgi apparatus in the secretory pathway. To contextualize the localization of 6K in the Golgi, we analyzed cells infected with SINV and SINV-ion channel chimeras using transmission electron microscopy. Our results provide evidence for the first time for the functional role of 6K in type II cytopathic vacuoles (CPV-II) formation. We demonstrate that in the absence of 6K, CPV-II, which originates from the Golgi apparatus, is not detected in infected cells, with a concomitant reduction in the glycoprotein transport to the plasma membrane. Substituting a functional ion channel, M2 or Vpu localizing to Golgi, restores CPV-II production, whereas P7, retained in the ER, is inadequate to induce CPV-II formation. Altogether our results indicate that ion channel activity of 6K is required for the formation of CPV-II from the Golgi apparatus, promoting glycoprotein spike transport to the plasma membrane and efficient virus budding.

The VEGAS Platform Is Unsuitable for Mammalian Directed Evolution.

Directed evolution uses cycles of gene diversification and selection to generate proteins with novel properties. While traditionally directed evolution is performed in prokaryotic systems, recently a mammalian directed evolution system (viral evolution of genetically actuating sequences, or "VEGAS") has been described. Here we report that the VEGAS system has major limitations that preclude its use for directed evolution. The deconstructed Sindbis virus (SINV) genome that comprises the VEGAS system could no longer promote Sindbis structural gene (SSG)-dependent viral replication. Moreover, viral particles generated using the VEGAS system rapidly lost the target directed evolution transgene, and instead, "cheater" particles, primarily containing RNA encoding SINV structural components, arose. By sequencing, we found that this contamination came from RNA provided during initial SINV packaging, not RNA derived from the VEGAS system. Of note, both the structural RNA and target transgenes used in the VEGAS system contain viral packaging sequences. The impact of SINV "cheater" particles could be potentially overcome in the context of a robust VEGAS circuit, but since SSG complementation is also defective in the VEGAS system, selection for authentic evolution products is not currently possible. Similar results have been obtained in independent laboratories. Taken together, these results show that the VEGAS system does not work as described and, without significant redesign, cannot be used for mammalian directed evolution campaigns.

Vertical Transmission of Sindbis Virus in Culex Mosquitoes.

Vertical transmission (VT) is a phenomenon of vector-borne diseases where a pathogen is transferred from an infected arthropod mother to her offspring. For mosquito-borne flavi- and alphaviruses, VT is commonly viewed as rare; however, both field and experimental studies report on vertical transmission efficiency to a notably varying degree. It is likely that this reflects the different experimental methods used to test vertical transmission efficiency as well as differences between virus-vector combinations. There are very few investigations of the VT of an alphavirus in a Culex vector. Sindbis virus (SINV) is an arthritogenic alphavirus that utilizes Culex species as main vectors both in the summer transmission season and for its persistence over the winter period in northern latitudes. In this study, we investigated the vertical transmission of the SINV in Culex vectors, both in the field and in experimental settings. The detection of SINV RNA in field-collected egg rafts and emerging adults shows that vertical transmission takes place in the field. Experimentally infected females gave rise to adult offspring containing SINV RNA at emergence; however, three to four weeks after emergence none of the offspring contained SINV RNA. This study shows that vertical transmission may be connected to SINV's ability to persist throughout northern winters and also highlights many aspects of viral replication that need further study.