Chromosome level genome assembly and transcriptome analysis of E11 cells infected by tilapia lake virus.

The E11 cell line, derived from striped snakehead fish (Channa striata), possesses a distinctive feature: it is persistently infected with a C-type retrovirus. Notably, it exhibits high permissiveness to piscine nodavirus and the emerging tilapia lake virus (TiLV). Despite its popularity in TiLV research, the absence of genome assembly for the E11 cell line and Channa striata has constrained research on host-virus interactions. This study aimed to fill this gap by sequencing, assembling, and annotating the E11 cell line genome. Our efforts yielded a 600.5 Mb genome including 24 chromosomes with a BUSCO score of 98.8%. In addition, the complete proviral DNA sequence of snakehead retrovirus (SnRV) was identified in the E11 cell genome. Comparative genomic analysis between the E11 cell line and another snakehead species Channa argus revealed the loss of many immune-related gene families in the E11 cell genome, indicating a compromised immune response. We also conducted transcriptome analysis of mock- and TiLV-infected E11 cells, unveiling new perspectives on virus-virus and host-virus interactions. The TiLV infection suppressed the high expression of SnRV in E11 cells, and activated some other endogenous retroviruses. The protein-coding gene comparison revealed a pronounced up-regulation of genes involved in immune response, alongside a down-regulation of genes associated with specific metabolic processes. In summary, the genome assembly and annotation of the E11 cell line provide valuable resources to understand the SnRV and facilitate further studies on nodavirus and TiLV. The RNA-seq profiles shed light on the cellular mechanisms employed by fish cells in response to viral challenges, potentially guiding the development of therapeutic strategies against TiLV in aquaculture. This study also provides the first insights into the viral transcriptome profiles of endogenous SnRV and evading TiLV, enhancing our understanding of host-virus interactions in fish.

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.

Insect-Specific Chimeric Viruses Potentiated Antiviral Responses and Inhibited Pathogenic Alphavirus Growth in Mosquito Cells.

Most alphaviruses are transmitted by mosquito vectors and infect a wide range of vertebrate hosts, with a few exceptions. Eilat virus (EILV) in this genus is characterized by a host range restricted to mosquitoes. Its chimeric viruses have been developed as safe and effective vaccine candidates and diagnostic tools. Here, we investigated the interactions between these insect-specific viruses (ISVs) and mosquito cells, unveiling their potential roles in determining vector competence and arbovirus transmission. By RNA sequencing, we found that these ISVs profoundly modified host cell gene expression profiles. Two EILV-based chimeras, consisting of EILV's nonstructural genes and the structural genes of Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV), namely, EILV/CHIKV (E/C) and EILV/VEEV (E/V), induced more intensive transcriptome regulation than parental EILV and activated different antiviral mechanisms in host cells. We demonstrated that E/C robustly promoted antimicrobial peptide production and E/V strongly upregulated the RNA interference pathway components. This also highlighted the intrinsic divergences between CHIKV and VEEV, representatives of the Old World and New World alphaviruses. In contrast, EILV triggered a limited antiviral response. We further showed that initial chimera infections efficiently inhibited subsequent pathogenic alphavirus replication, especially in the case of E/V infection, which almost prevented VEEV and Sindbis virus (SINV) superinfections. Altogether our study provided valuable information on developing ISVs as biological control agents. IMPORTANCE Mosquito-borne alphaviruses can cause emerging and reemerging infectious diseases, posing a considerable threat to human and animal health worldwide. However, no specific antivirals or commercial vaccines are currently available. Therefore, it is vital to develop biological control measures to contain virus transmission. Insect-specific EILV and its chimeras are supposed to induce superinfection exclusion owing to the close phylogenetical relationship with pathogenic alphaviruses. These viruses might also, like bacterial symbionts, modulate mosquito hosts' vector competence for arboviruses. However, little is known about the responses of mosquitoes or mosquito cells to ISV infections. Here, we found that EILV barely elicited antiviral defenses in host cells, while its chimeras, namely, E/C and E/V, potentiated the responses via different mechanisms. Furthermore, we showed that initial chimera infections could largely inhibit subsequent pathogenic alphavirus infections. Taken together, our study proposed insect-specific chimeras as a promising candidate for developing biological control measures against pathogenic alphaviruses.

Complete genetic dissection and cell type-specific replication of old world alphaviruses, getah virus (GETV) and sagiyama virus (SAGV).

Getah virus (GETV), which was first isolated in Malaysia in 1955, and Sagiyama virus (SAGV), isolated in Japan in 1956, are members of the genus Alphavirus in the family Togaviridae. It is a consensus view that SAGV is a variant of GETV. In the present study, we determined the complete sequences of the prototype GETV MM2021 and SAGV M6-Mag132 genomic RNA extracted from plaque-purified viruses. The MM2021 genome was 11,692 nucleotides (nt) in length in the absence of 3' poly(A) tail, and the length of M6-Mag132 genome was 11,698 nt. Through sequence alignment of MM2021 and M6-Mag132, we located all the amino acid differences between these two strains, which were scattered in all the encoded proteins. Subsequently, we validated the close evolutionary relationship between GETV and SAGV by constructing phylogenetic trees based on either complete genomes or structural genomes. We eventually analyzed the growth kinetics of GETV and SAGV as well as other representative alphaviruses in various mammalian and insect cell lines. It was shown that human-oriented cell lines such as HEK-293T and Hela cells were relatively resistant to GETV and SAGV infection due to absence of proviral factors or species-specific barrier. On the other hand, both GETV and SAGV replicated efficiently in non-human cell lines. Our results provide essential genetic information for future epidemiological surveillance on Alphaviruses and lay the foundation for developing effective interventions against GETV and SAGV.

A Productive Expression Platform Derived from Host-Restricted Eilat Virus: Its Extensive Validation and Novel Strategy.

Most alphaviruses are transmitted by mosquitoes and infect a wide range of insects and vertebrates. However, Eilat virus (EILV) is defective for infecting vertebrate cells at multiple levels of the viral life cycle. This host-restriction property renders EILV an attractive expression platform since it is not infectious for vertebrates and therefore provides a highly advantageous safety profile. Here, we investigated the feasibility of versatile EILV-based expression vectors. By replacing the structural genes of EILV with those of other alphaviruses, we generated seven different chimeras. These chimeras were readily rescued in the original mosquito cells and were able to reach high titers, suggesting that EILV is capable of packaging the structural proteins of different lineages. We also explored the ability of EILV to express authentic antigens via double subgenomic (SG) RNA vectors. Four foreign genetic materials of varied length were introduced into the EILV genome, and the expressed heterologous genetic materials were readily detected in the infected cells. By inserting an additional SG promoter into the chimera genome containing the structural genes of Chikungunya virus (CHIKV), we developed a bivalent vaccine candidate against CHIKV and Zika virus. These data demonstrate the outstanding compatibility of the EILV genome. The produced recombinants can be applied to vaccine and diagnostic tool development, but more investigations are required.

Hypervariable Domain of Eastern Equine Encephalitis Virus nsP3 Redundantly Utilizes Multiple Cellular Proteins for Replication Complex Assembly.

Eastern equine encephalitis virus (EEEV) is a representative member of the New World alphaviruses. It is pathogenic for a variety of vertebrate hosts, in which EEEV induces a highly debilitating disease, and the outcomes are frequently lethal. Despite a significant public health threat, the molecular mechanism of EEEV replication and interaction with hosts is poorly understood. Our previously published data and those of other teams have demonstrated that hypervariable domains (HVDs) of the alphavirus nsP3 protein interact with virus-specific host factors and play critical roles in assembly of viral replication complexes (vRCs). The most abundantly represented HVD-binding proteins are the FXR and G3BP family members. FXR proteins drive the assembly of vRCs of Venezuelan equine encephalitis virus (VEEV), and G3BPs were shown to function in vRC assembly in the replication of chikungunya and Sindbis viruses. Our new study demonstrates that EEEV exhibits a unique level of redundancy in the use of host factors in RNA replication. EEEV efficiently utilizes both the VEEV-specific FXR protein family and the Old World alphavirus-specific G3BP protein family. A lack of interaction with either FXRs or G3BPs does not affect vRC formation; however, removal of EEEV's ability to interact with both protein families has a deleterious effect on virus growth. Other identified EEEV nsP3 HVD-interacting host proteins are also capable of supporting EEEV replication, albeit with a dramatically lower efficiency. The ability to use a wide range of host factors with redundant functions in vRC assembly and function provides a plausible explanation for the efficient replication of EEEV and may contribute to its highly pathogenic phenotype.IMPORTANCE Eastern equine encephalitis virus (EEEV) is one of the most pathogenic New World alphaviruses. Despite the continuous public health threat, to date, the molecular mechanisms of its very efficient replication and high virulence are not sufficiently understood. The results of this new study demonstrate that North American EEEV exhibits a high level of redundancy in using host factors in replication complex assembly and virus replication. The hypervariable domain of the EEEV nsP3 protein interacts with all of the members of the FXR and G3BP protein families, and only a lack of interaction with both protein families strongly affects virus replication rates. Other identified HVD-binding factors are also involved in EEEV replication, but their roles are not as critical as those of FXRs and G3BPs. The new data present a plausible explanation for the exceptionally high replication rates of EEEV and suggest a new means of its attenuation and new targets for screening of antiviral drugs.

New World and Old World Alphaviruses Have Evolved to Exploit Different Components of Stress Granules, FXR and G3BP Proteins, for Assembly of Viral Replication Complexes.

The positive-strand RNA viruses initiate their amplification in the cell from a single genome delivered by virion. This single RNA molecule needs to become involved in replication process before it is recognized and degraded by cellular machinery. In this study, we show that distantly related New World and Old World alphaviruses have independently evolved to utilize different cellular stress granule-related proteins for assembly of complexes, which recruit viral genomic RNA and facilitate formation of viral replication complexes (vRCs). Venezuelan equine encephalitis virus (VEEV) utilizes all members of the Fragile X syndrome (FXR) family, while chikungunya and Sindbis viruses exploit both members of the G3BP family. Despite being in different families, these proteins share common characteristics, which determine their role in alphavirus replication, namely, the abilities for RNA-binding and for self-assembly into large structures. Both FXR and G3BP proteins interact with virus-specific, repeating amino acid sequences located in the C-termini of hypervariable, intrinsically disordered domains (HVDs) of viral nonstructural protein nsP3. We demonstrate that these host factors orchestrate assembly of vRCs and play key roles in RNA and virus replication. Only knockout of all of the homologs results in either pronounced or complete inhibition of replication of different alphaviruses. The use of multiple homologous proteins with redundant functions mediates highly efficient recruitment of viral RNA into the replication process. This independently evolved acquisition of different families of cellular proteins by the disordered protein fragment to support alphavirus replication suggests that other RNA viruses may utilize a similar mechanism of host factor recruitment for vRC assembly. The use of different host factors by alphavirus species may be one of the important determinants of their pathogenesis.

The SD1 Subdomain of Venezuelan Equine Encephalitis Virus Capsid Protein Plays a Critical Role in Nucleocapsid and Particle Assembly.

UNLABELLED: Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, for which no safe and efficient vaccines or therapeutic means have been developed. Viral particle assembly and budding processes represent potential targets for therapeutic intervention. However, our understanding of the mechanistic process of VEEV assembly, RNA encapsidation, and the roles of different capsid-specific domains in these events remain to be described. The results of this new study demonstrate that the very amino-terminal VEEV capsid-specific subdomain SD1 is a critical player in the particle assembly process. It functions in a virus-specific mode, and its deletion, mutation, or replacement by the same subdomain derived from other alphaviruses has strong negative effects on infectious virus release. VEEV variants with mutated SD1 accumulate adaptive mutations in both SD1 and SD2, which result in a more efficiently replicating phenotype. Moreover, efficient nucleocapsid and particle assembly proceeds only when the two subdomains, SD1 and SD2, are derived from the same alphavirus. These two subdomains together appear to form the central core of VEEV nucleocapsids, and their interaction is one of the driving forces of virion assembly and budding. The similar domain structures of alphavirus capsid proteins suggest that this new knowledge can be applied to other alphaviruses. IMPORTANCE: Alphaviruses are a group of human and animal pathogens which cause periodic outbreaks of highly debilitating diseases. Despite significant progress made in understanding the overall structure of alphavirus and VEEV virions, and glycoprotein spikes in particular, the mechanistic process of nucleocapsid assembly, RNA encapsidation, and the roles of different capsid-specific domains in these processes remain to be described. Our new data demonstrate that the very amino-terminal subdomain of Venezuelan equine encephalitis virus capsid protein, SD1, plays a critical role in the nucleocapsid assembly. It functions synergistically with the following SD2 (helix I) and appears to form a core in the center of nucleocapsid. The core formation is one of the driving forces of alphavirus particle assembly.

Utilization of an Eilat Virus-Based Chimera for Serological Detection of Chikungunya Infection.

In December of 2013, chikungunya virus (CHIKV), an alphavirus in the family Togaviridae, was introduced to the island of Saint Martin in the Caribbean, resulting in the first autochthonous cases reported in the Americas. As of January 2015, local and imported CHIKV has been reported in 50 American countries with over 1.1 million suspected cases. CHIKV causes a severe arthralgic disease for which there are no approved vaccines or therapeutics. Furthermore, the lack of a commercially available, sensitive, and affordable diagnostic assay limits surveillance and control efforts. To address this issue, we utilized an insect-specific alphavirus, Eilat virus (EILV), to develop a diagnostic antigen that does not require biosafety containment facilities to produce. We demonstrated that EILV/CHIKV replicates to high titers in insect cells and can be applied directly in enzyme-linked immunosorbent assays without inactivation, resulting in highly sensitive detection of recent and past CHIKV infection, and outperforming traditional antigen preparations.

IFIT1 Differentially Interferes with Translation and Replication of Alphavirus Genomes and Promotes Induction of Type I Interferon.

Alphaviruses are a group of widely distributed human and animal pathogens. It is well established that their replication is sensitive to type I IFN treatment, but the mechanism of IFN inhibitory function remains poorly understood. Using a new experimental system, we demonstrate that in the presence of IFN-ß, activation of interferon-stimulated genes (ISGs) does not interfere with either attachment of alphavirus virions to the cells, or their entry and nucleocapsid disassembly. However, it strongly affects translation of the virion-delivered virus-specific RNAs. One of the ISG products, IFIT1 protein, plays a major role in this translation block, although an IFIT1-independent mechanism is also involved. The 5'UTRs of the alphavirus genomes were found to differ significantly in their ability to drive translation in the presence of increased concentration of IFIT1. Prior studies have shown that adaptation of naturally circulating alphaviruses to replication in tissue culture results in accumulation of mutations in the 5'UTR, which increase the efficiency of the promoter located in the 5'end of the genome. Here, we show that these mutations also decrease resistance of viral RNA to IFIT1-induced translation inhibition. In the presence of higher levels of IFIT1, alphaviruses with wt 5'UTRs became potent inducers of type I IFN, suggesting a new mechanism of type I IFN induction. We applied this knowledge of IFIT1 interaction with alphaviruses to develop new attenuated variants of Venezuelan equine encephalitis and chikungunya viruses that are more sensitive to the antiviral effects of IFIT1, and thus could serve as novel vaccine candidates.

Venezuelan equine encephalitis virus variants lacking transcription inhibitory functions demonstrate highly attenuated phenotype.

UNLABELLED: Alphaviruses represent a significant public health threat worldwide. They are transmitted by mosquitoes and cause a variety of human diseases ranging from severe meningoencephalitis to polyarthritis. To date, no efficient and safe vaccines have been developed against any alphavirus infection. However, in recent years, significant progress has been made in understanding the mechanism of alphavirus replication and virus-host interactions. These data have provided the possibility for the development of new rationally designed alphavirus vaccine candidates that combine efficient immunogenicity, high safety, and inability to revert to pathogenic phenotype. New attenuated variants of Venezuelan equine encephalitis virus (VEEV) designed in this study combine a variety of characteristics that independently contribute to a reduction in virulence. These constructs encode a noncytopathic VEEV capsid protein that is incapable of interfering with the innate immune response. The capsid-specific mutations strongly affect neurovirulence of the virus. In other constructs, they were combined with changes in control of capsid translation and an extensively mutated packaging signal. These modifications also affected the residual neurovirulence of the virus, but it remained immunogenic, and a single immunization protected mice against subsequent infection with epizootic VEEV. Similar approaches of attenuation can be applied to other encephalitogenic New World alphaviruses. IMPORTANCE: Venezuelan equine encephalitis virus (VEEV) is an important human and animal pathogen, which causes periodic outbreaks of highly debilitating disease. Despite a continuous public health threat, no safe and efficient vaccine candidates have been developed to date. In this study, we applied accumulated knowledge about the mechanism of VEEV replication, RNA packaging, and interaction with the host to design new VEEV vaccine candidates that demonstrate exceptionally high levels of safety due to a combination of extensive modifications in the viral genome. The introduced mutations did not affect RNA replication or structural protein synthesis but had deleterious effects on VEEV neuroinvasion and virulence. In spite of dramatically reduced virulence, the designed mutants remained highly immunogenic and protected mice against subsequent infection with epizootic VEEV. Similar methodologies can be applied for attenuation of other encephalitogenic New World alphaviruses.

[Effects of physics on development of optometry in the United States from the late 19th to the mid 20th century].

In this paper, it was studied how physics affected development of optometry in the United States, from aspects of formation and academization of optometry. It was also revealed that history of optometry was analogous to history of engineering. Optics in the 19th century was divided into electromagnetic study of light and visual optics. Development of the visual optics promoted professionalization of ophthalmology that had already started in the 18th century. The visual optics also stimulated formation of optometry and optometrists body in the late 19th century of the United States. The American optometrists body were originated from opticians who had studied visual optics. Publication of several English academic textbooks on visual optics induced appearance of educated opticians (and jewelers). They acquired a right to do the eye examination in the early 20th century after C. F. Prentice's trial in 1897, evolving into optometrists. The opticians could be considered as craftsmen, and they were divided into (dispensing) opticians and optometrists. Such history of American optometrists body is analogous to that of engineers body in the viewpoints of craftsmen origin and separation from craftsmen. Engineers were also originated from educated craftsmen, but were separated from craftsmen when engineering was built up. Education system and academization of optometry was strongly influenced by physics, too. When college education of optometry started at American universities, it was not belonged to medical school but to physics department. Physics and optics were of great importance in curriculum, and early faculty members were mostly physicists. Optometry was academized in the 1920s by the college education, standardization of curriculum, and formation of the American Academy of Optometry. This is also analogous to history of engineering, which was academized by natural sciences, especially by mathematics and physics. The reason why optometry was academized not by medicine but by physics is because ophthalmologists did not have conciliatory attitudes to optometry education. Optometry became independent of physics from the 1930s to the 1940s. Optometric researches concentrated on binocular vision that is not included to discipline of physics, and faculty members who majored in optometry increased, so that optometry departments and graduate schools were established around 1940. Such independence from natural sciences after academization also resembles history of engineering. On the contrary, history of optometry was different from history of ophthalmology in several aspects. Ophthalmology had already been formed in the 18th century before development of visual optics, and was not academized by visual optics. Ophthalmologists body were not originated from craftsmen, and were not separated from craftsmen. History of optometry in the United States from the late 19th to the mid 20th century is analogous to history of engineering rather than history of medicine, though optometry is a medical discipline.

Enhancement of protein expression by alphavirus replicons by designing self-replicating subgenomic RNAs.

Since the development of infectious cDNA clones of viral RNA genomes and the means of delivery of the in vitro-synthesized RNA into cells, alphaviruses have become an attractive system for expression of heterologous genetic information. Alphaviruses replicate exclusively in the cytoplasm, and their genetic material cannot recombine with cellular DNA. Alphavirus genome-based, self-replicating RNAs (replicons) are widely used vectors for expression of heterologous proteins. Their current design relies on replacement of structural genes, encoded by subgenomic RNAs (SG RNA), with heterologous sequences of interest. The SG RNA is transcribed from a promoter located in the alphavirus-specific RNA replication intermediate and is not further amplified. In this study, we have applied the accumulated knowledge of the mechanism of alphavirus replication and promoter structures, in particular, to increase the expression level of heterologous proteins from Venezuelan equine encephalitis virus (VEEV)-based replicons. During VEEV infection, replication enzymes are produced in excess to RNA replication intermediates, and a large fraction of them are not involved in RNA synthesis. The newly designed constructs encode SG RNAs, which are not only transcribed from the SG promoter, but are additionally amplified by the previously underused VEEV replication enzymes. These replicons produce SG RNAs and encoded proteins of interest 10- to 50-fold more efficiently than those using a traditional design. A modified replicon encoding West Nile virus (WNV) premembrane and envelope proteins efficiently produced subviral particles and, after a single immunization, elicited high titers of neutralizing antibodies, which protected mice from lethal challenge with WNV.

The amino-terminal domain of alphavirus capsid protein is dispensable for viral particle assembly but regulates RNA encapsidation through cooperative functions of its subdomains.

Venezuelan equine encephalitis virus (VEEV) is a pathogenic alphavirus, which circulates in the Central, South, and North Americas, including the United States, and represents a significant public health threat. In recent years, strong progress has been made in understanding the structure of VEEV virions, but the mechanism of their formation has yet to be investigated. In this study, we analyzed the functions of different capsid-specific domains and its amino-terminal subdomains in viral particle formation. Our data demonstrate that VEEV particles can be efficiently formed directly at the plasma membrane without cytoplasmic nucleocapsid preassembly. The entire amino-terminal domain of VEEV capsid protein was found to be dispensable for particle formation. VEEV variants encoding only the capsid's protease domain efficiently produce genome-free VEEV virus-like particles (VLPs), which are very similar in structure to the wild-type virions. The amino-terminal domain of the VEEV capsid protein contains at least four structurally and functionally distinct subdomains, which mediate RNA packaging and the specificity of packaging in particular. The most positively charged subdomain is a negative regulator of the nucleocapsid assembly. The three other subdomains are not required for genome-free VLP formation but are important regulators of RNA packaging. Our data suggest that the positively charged surface of the VEEV capsid-specific protease domain and the very amino-terminal subdomain are also involved in interaction with viral RNA and play important roles in RNA encapsidation. Finally, we show that VEEV variants with mutated capsid acquire compensatory mutations in either capsid or nsP2 genes.

Venezuelan equine encephalitis virus nsP2 protein regulates packaging of the viral genome into infectious virions.

Alphaviruses are one of the most geographically widespread and yet often neglected group of human and animal pathogens. They are capable of replicating in a wide variety of cells of both vertebrate and insect origin and are widely used for the expression of heterologous genetic information both in vivo and in vitro. In spite of their use in a range of research applications and their recognition as a public health threat, the biology of alphaviruses is insufficiently understood. In this study, we examined the evolution process of one of the alphaviruses, Venezuelan equine encephalitis virus (VEEV), to understand its adaptation mechanism to the inefficient packaging of the viral genome in response to serial mutations introduced into the capsid protein. The new data derived from this study suggest that strong alterations in the ability of capsid protein to package the viral genome leads to accumulation of adaptive mutations, not only in the capsid-specific helix I but also in the nonstructural protein nsP2. The nsP2-specific mutations were detected in the protease domain and in the amino terminus of the protein, which was previously proposed to function as a protease cofactor. These mutations increased infectious virus titers, demonstrated a strong positive impact on viral RNA replication, mediated the development of a more cytopathic phenotype, and made viruses capable of developing a spreading infection. The results suggest not only that packaging of the alphavirus genome is determined by the presence of packaging signals in the RNA and positively charged amino acids in the capsid protein but also that nsP2 is either directly or indirectly involved in the RNA encapsidation process.

Pseudoinfectious Venezuelan equine encephalitis virus: a new means of alphavirus attenuation.

Venezuelan equine encephalitis virus (VEEV) is a reemerging virus that causes a severe and often fatal disease in equids and humans. In spite of a continuous public health threat, to date, no vaccines or antiviral drugs have been developed for human use. Experimental vaccines demonstrate either poor efficiency or severe adverse effects. In this study, we developed a new strategy of alphavirus modification aimed at making these viruses capable of replication and efficient induction of the immune response without causing a progressive infection, which might lead to disease development. To achieve this, we developed a pseudoinfectious virus (PIV) version of VEEV. VEE PIV mimics natural viral infection in that it efficiently replicates its genome, expresses all of the viral structural proteins, and releases viral particles at levels similar to those found in wild-type VEEV-infected cells. However, the mutations introduced into the capsid protein make this protein almost incapable of packaging the PIV genome, and most of the released virions lack genetic material and do not produce a spreading infection. Thus, VEE PIV mimics viral infection in terms of antigen production but is safer due to its inability to incorporate the viral genome into released virions. These genome-free virions are referred to as virus-like particles (VLPs). Importantly, the capsid-specific mutations introduced make the PIV a very strong inducer of the innate immune response and add self-adjuvant characteristics to the designed virus. This unique strategy of virus modification can be applied for vaccine development against other alphaviruses.

Conservation of a packaging signal and the viral genome RNA packaging mechanism in alphavirus evolution.

Alphaviruses are a group of small, enveloped viruses which are widely distributed on all continents. In infected cells, alphaviruses display remarkable specificity in RNA packaging by encapsidating only their genomic RNA while avoiding packaging of the more abundant viral subgenomic (SG), cellular messenger and transfer RNAs into released virions. In this work, we demonstrate that in spite of evolution in geographically isolated areas and accumulation of considerable diversity in the nonstructural and structural genes, many alphaviruses belonging to different serocomplexes harbor RNA packaging signals (PSs) which contain the same structural and functional elements. Their characteristic features are as follows. (i) Sindbis, eastern, western, and Venezuelan equine encephalitis and most likely many other alphaviruses, except those belonging to the Semliki Forest virus (SFV) clade, have PSs which can be recognized by the capsid proteins of heterologous alphaviruses. (ii) The PS consists of 4 to 6 stem-loop RNA structures bearing conserved GGG sequences located at the base of the loop. These short motifs are integral elements of the PS and can function even in the artificially designed PS. (iii) Mutagenesis of the entire PS or simply the GGG sequences has strong negative effects on viral genome packaging and leads to release of viral particles containing mostly SG RNAs. (iv) Packaging of RNA appears to be determined to some extent by the number of GGG-containing stem-loops, and more than one stem-loop is required for efficient RNA encapsidation. (v) Viruses of the SFV clade are the exception to the general rule. They contain PSs in the nsP2 gene, but their capsid protein retains the ability to use the nsP1-specific PS of other alphaviruses. These new discoveries regarding alphavirus PS structure and function provide an opportunity for the development of virus variants, which are irreversibly attenuated in terms of production of infectious virus but release high levels of genome-free virions.

Chimeric Chikungunya viruses are nonpathogenic in highly sensitive mouse models but efficiently induce a protective immune response.

Chikungunya virus (CHIKV) is an important pathogen causing outbreaks of highly debilitating and often chronic, arthralgic human disease. We have designed chimeric alphaviruses encoding CHIKV-specific structural proteins but no structural or nonstructural proteins capable of interfering with development of cellular antiviral response. These chimeras demonstrate a highly attenuated phenotype in both immunocompetent and immunocompromised (A129) mice. However, after a single vaccination, they induced protective immune response against subsequent CHIKV challenge, characterized by high titers of neutralizing antibodies. The rational design of alphavirus genomes provides a strong basis for the development of new recombinant alphaviruses with irreversible, highly attenuated, cell type-restricted phenotypes.

Design of chimeric alphaviruses with a programmed, attenuated, cell type-restricted phenotype.

The Alphavirus genus in the Togaviridae family contains a number of human and animal pathogens. The importance of alphaviruses has been strongly underappreciated; however, epidemics of chikungunya virus (CHIKV), causing millions of cases of severe and often persistent arthritis in the Indian subcontinent, have raised their profile in recent years. In spite of a continuous public health threat, to date no licensed vaccines have been developed for alphavirus infections. In this study, we have applied an accumulated knowledge about the mechanism of alphavirus replication and protein function in virus-host interactions to introduce a new approach in designing attenuated alphaviruses. These variants were constructed from genes derived from different, geographically isolated viruses. The resulting viable variants encoded CHIKV envelope and, in contrast to naturally circulating viruses, lacked the important contributors to viral pathogenesis: genes encoding proteins functioning in inhibition of cellular transcription and downregulation of the cellular antiviral response. To make these viruses incapable of transmission by mosquito vectors and to differentially regulate expression of viral structural proteins, their replication was made dependent on the internal ribosome entry sites, derived from other positive-polarity RNA (RNA(+)) viruses. The rational design of the genomes was complemented by selection procedures, which adapted viruses to replication in tissue culture and produced variants which (i) demonstrated different levels of replication and production of the individual structural proteins, (ii) efficiently induced the antiviral response in infected cells, (iii) were incapable of replication in cells of mosquito origin, and (iv) efficiently replicated in Vero cells. This modular approach to genome design is applicable for the construction of other alphaviruses with a programmed, irreversibly attenuated phenotype.

Insertion and deletion in a non-essential region of the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome (PRRS) virus: effects on virulence and immunogenicity.

Developing a vaccine that can differentiate infected and vaccinated animals (DIVA) is a new challenge in the design of a vaccine for porcine reproductive and respiratory syndrome virus (PRRSV). Nonstructural protein 2 (nsp2) is the single largest viral product, and it has multiple roles in polypeptide processing and replication complex formation. Using reverse genetics and an infectious PRRSV cDNA clone, we constructed several deletion mutants in the non-essential region of nsp2. One mutant, which has a 131 amino acid deletion within a relatively conserved region of nsp2, was recovered and found to produce a viable virus. The deleted region was replaced with a peptide tag encoding eight amino acids. A recombinant virus containing the 131 amino acid deletion was found to produce normal virus yields in MARC-145 cells and porcine alveolar macrophages (PAM); however, gross and micro-histopathology showed that the virus was less virulent in pigs. The 131 amino acid peptide was expressed as a recombinant protein and used to coat enzyme-linked immunosorbent assay (ELISA) plates. This peptide was recognized by sera from pigs infected with wild-type virus, but not by sera from pigs infected with the deletion mutant. The results from this study show that nsp2 is an important target for the development of marker vaccines and for virus attenuation.