Molecular characterization of the SP3a gene, a negative regulator of viral infection in the orange-spotted grouper, Epinephelus coioides.

SP3 (specificity protein 3) is a transcription factor characterized by three conserved Cys2His2 zinc finger motifs that exert a transregulatory effect by binding to GC boxes, either upregulating or downregulating multiple genes or by co-regulating gene expression in coordination with other proteins. SP3 potentially regulates a series of processes, such as the cell cycle, growth, metabolic pathways, and apoptosis, and plays an important role in antiviral effect. The function of sp3 in fish is poorly understood. In this study, the Sp3a open reading frame was cloned from the orange-spotted grouper, Epinephelus coioides. The full-length open reading frame of Sp3a was 2034 bp, encoding 677 amino acids, with a predicted molecular weight of 72.34 kDa and an isoelectric point of 5.05. Phylogenetically, Sp3a in Epinephelus coioides was the most closely related to Sp3a in the Malabar grouper, Epinephelus malabaricus. RT-qPCR revealed ubiquitous expression of Sp3a in all examined grouper tissues, with no significant differences in expression levels among tissues. A eukaryotic expression vector, pEGFP-Sp3a, was constructed and transfected into grouper spleen (GS) cells. Subcellular localization of Sp3a was observed using an inverted fluorescence microscope. When Spa3 was overexpressed in GS cells, the expression of orange-spotted grouper nerve necrosis virus (RGNNV) genes (CP and RdRp) decreased significantly, indicating that Sp3a significantly inhibited RGNNV replication. siRNA inhibition of Sp3a accelerated the intracellular replication of RGNNV, implying the antiviral effect of Sp3a. Conclusively, our findings contribute to further research on the antiviral capabilities of Sp3a in grouper and other fish. Therefore, our research has potential implications on the development of the aquaculture industry.

3D Spheroid and Organoid Models to Study Neuroinfection of RNA Viruses.

Three-dimensional culture models of the brain enable the study of neuroinfection in the context of a complex interconnected cell matrix. Depending on the differentiation status of the neural cells, two models exist: 3D spheroids also called neurospheres and cerebral organoids. Here, we describe the preparation of 3D spheroids and cerebral organoids and give an outlook on their usage to study Rift Valley fever virus and other neurotropic viruses.

Fish HERC7: Phylogeny, Characterization, and Potential Implications for Antiviral Immunity in European Sea Bass.

E3 ubiquitin ligases, key components of the ubiquitin proteasome system, orchestrate protein degradation through ubiquitylation and profoundly impact cellular biology. Small HERC E3 ligases (HERC3-6) have diverse functions in mammals, including roles in spermatogenesis, protein degradation, and immunity. Until now, only mammals' HERC3, HERC5, and HERC6 are known to participate in immune responses, with major involvement in the antiviral response. Interestingly, an exclusive HERC7 has been characterized in fish showing great molecular conservation and antiviral roles. Thus, this study identifies and characterizes the herc7 gene in the European sea bass teleost. The European sea bass herc7 gene and the putative protein show good conservation of the promoter binding sites for interferons and the RCC1 and HECT domains characteristic of HERC proteins, respectively. The phylogenetic analysis shows a unique cluster with the fish-exclusive HERC7 orthologues. During ontogeny, the herc7 gene is expressed from 3 days post-fertilization onwards, being constitutively and widely distributed in adult tissues. In vitro, stimulated leucocytes up-regulate the herc7 gene in response to mitogens and viruses, pointing to a role in the immune response. Furthermore, sea bass herc7 expression is related to the interferon response intensity and viral load in different tissues upon in vivo infection with red-grouper betanodavirus (RGNNV), suggesting the potential involvement of fish HERC7 in ISGylation-based antiviral activity, similarly to mammalian HERC5. This study broadens the understanding of small HERC proteins in fish species and highlights HERC7 as a potential contributor to the immune response in European sea bass, with implications for antiviral defense mechanisms. Future research is needed to unravel the precise actions and functions of HERC7 in teleost fish immunity, providing insights into direct antiviral activity and viral evasion.

Natural variation in infection specificity of Caenorhabditis briggsae isolates by two RNA viruses.

Antagonistic relationships such as host-virus interactions potentially lead to rapid evolution and specificity in interactions. The Orsay virus is so far the only horizontal virus naturally infecting the nematode C. elegans. In contrast, several related RNA viruses infect its congener C. briggsae, including Santeuil (SANTV) and Le Blanc (LEBV) viruses. Here we focus on the host's intraspecific variation in sensitivity to these two intestinal viruses. Many temperate-origin C. briggsae strains, including JU1264 and JU1498, are sensitive to both, while many tropical strains, such as AF16, are resistant to both. Interestingly, some C. briggsae strains exhibit a specific resistance, such as the HK104 strain, specifically resistant to LEBV. The viral sensitivity pattern matches the strains' geographic and genomic relationships. The heavily infected strains mount a seemingly normal small RNA response that is insufficient to suppress viral infection, while the resistant strains show no small RNA response, suggesting an early block in viral entry or replication. We use a genetic approach from the host side to map genomic regions participating in viral resistance polymorphisms. Using Advanced Intercrossed Recombinant Inbred Lines (RILs) between virus-resistant AF16 and SANTV-sensitive HK104, we detect Quantitative Trait Loci (QTLs) on chromosomes IV and III. Building RILs between virus-sensitive JU1498 and LEBV-resistant HK104 followed by bulk segregant analysis, we identify a chromosome II QTL. In both cases, further introgressions of the regions confirmed the QTLs. This diversity provides an avenue for studying virus entry, replication, and exit mechanisms, as well as host-virus specificity and the host response to a specific virus infection.

Selection of nonstandard viral genomes during the evolution of RNA viruses: A virus survival strategy or a pesky inconvenience?

RNA viruses are some of the most successful biological entities due their ability to adapt and evolve. Despite their small genome and parasitic nature, RNA viruses have evolved many mechanisms to ensure their survival and maintenance in the host population. We propose that one of these mechanisms of survival is the generation of nonstandard viral genomes (nsVGs) that accumulate during viral replication. NsVGs are often considered to be accidental defective byproducts of the RNA virus replication, but their ubiquity and the plethora of roles they have during infection indicate that they are an integral part of the virus life cycle. Here we review the different types of nsVGs and discuss how their multiple roles during infection could be beneficial for RNA viruses to be maintained in nature. By shifting our perspectives on what makes a virus successful, we posit that nsVG generation is a conserved phenomenon that arose during RNA virus evolution as an essential component of a healthy virus community.

Pathogenicity of two different genotypes avian hepatitis E strains in laying hens and silkie fowl.

To determine the pathogenicity of two different genotypes of avian hepatitis E strains in two species of birds, a total of thirty healthy 12-week-old birds were used. After inoculation, fecal virus shedding, viremia, seroconversion, serum alanine aminotransferase (ALT) increases and liver lesions were evaluated. The results revealed that CHN-GS-aHEV and CaHEV could both infect Hy-Line hens and silkie fowls, respectively. Compared to the original avian HEV strain, the cross-infected virus exhibited a delay of 2 weeks and 1 week in emerged seroconversion, viremia, fecal virus shedding, and increased ALT level, and also showed mild liver lesions. These findings suggested that CHN-GS-aHEV may have circulated in chickens. Overall, these two different genotypes of avian HEV showed some variant pathogenicity in different bird species. This study provides valuable data for further analysis of the epidemic conditions of two avian HEVs in Hy-Line hens and silkie fowls.

Alpha-synuclein and RNA viruses: Exploring the neuronal nexus.

Alpha-synuclein (α-syn), known for its pivotal role in Parkinson's disease, has recently emerged as a significant player in neurotropic RNA virus infections. Upregulation of α-syn in various viral infections has been found to impact neuroprotective functions by regulating neurotransmitter synthesis, vesicle trafficking, and synaptic vesicle recycling. This review focuses on the multifaceted role of α-syn in controlling viral replication by modulating chemoattractant properties towards microglial cells, virus-induced ER stress signaling, anti-oxidative proteins expression. Furthermore, the text underlines the α-syn-mediated regulation of interferon-stimulated genes. The review may help suggest potential therapeutic avenues for mitigating the impact of RNA viruses on the central nervous system by exploiting α-syn neuroprotective biology.

M1-type polarized macrophage contributes to brain damage through CXCR3.2/CXCL11 pathways after RGNNV infection in grouper.

The vertebrate central nervous system (CNS) is the most complex system of the body. The CNS, especially the brain, is generally regarded as immune-privileged. However, the specialized immune strategies in the brain and how immune cells, specifically macrophages in the brain, respond to virus invasion remain poorly understood. Therefore, this study aimed to examine the potential immune response of macrophages in the brain of orange-spotted groupers (Epinephelus coioides) following red-spotted grouper nervous necrosis virus (RGNNV) infection. We observed that RGNNV induced macrophages to produce an inflammatory response in the brain of orange-spotted grouper, and the macrophages exhibited M1-type polarization after RGNNV infection. In addition, we found RGNNV-induced macrophage M1 polarization via the CXCR3.2- CXCL11 pathway. Furthermore, we observed that RGNNV triggered M1 polarization in macrophages, resulting in substantial proinflammatory cytokine production and subsequent damage to brain tissue. These findings reveal a unique mechanism for brain macrophage polarization, emphasizing their role in contributing to nervous tissue damage following viral infection in the CNS.

T-cells and CD45-cells discovery in the central nervous system of healthy and nodavirus-infected teleost fish Dicentrarchuslabrax.

To achieve insights in antiviral immune defense of the central nervous system (CNS), we investigated T cells and CD45 cells in the marine fish model Dicentrarchus labrax infected with the CNS-tropic virus betanodavirus. By employing markers for pan-T cells (mAb DLT15) and CD45-cells (mAb DLT22) in immunofluorescence (IIF) of leukocytes from brain, we obtained 3,7 ± 2.3 % of T cells and 7.3 ± 3.2 % of CD45+ cells. Both IIF and immunoelectron microscopy confirmed a leukocyte/glial morphology for the immunoreactive cells. Quantitative immunohistochemistry (qIHC) of brain/eye sections showed 1.9 ± 0.8 % of T+ cells and 2 ± 0.9 % of CD45+ cells in the brain, and 3.6 ± 1.9 % and 4.1 ± 2.2 % in the eye, respectively. After in vivo RGNNV infection the number of T cells/CD45+ leukocytes in the brain increased to 8.3 ± 2.1 % and 11.6 ± 4.4 % (by IIF), and 26.1 ± 3.4 % and 45.6 ± 5.9 % (by qIHC), respectively. In the eye we counted after infection 8.5 ± 4.4 % of T cells and 10.2 ± 5.8 % of CD45 cells. Gene transcription analysis of brain mRNA revealed a strong increase of gene transcripts coding for: antiviral proteins Mx and ISG-12; T-cell related CD3ε/δ, TcRß, CD4, CD8α, CD45; and for immuno-modulatory cytokines TNFα, IL-2, IL-10. A RAG-1 gene product was also present and upregulated, suggesting somatic recombination in the fish brain. Similar transcription data were obtained in the eye, albeit with differences. Our findings provide first evidence for a recruitment and involvement of T cells and CD45+ leukocytes in the fish eye-brain axis during antiviral responses and suggest similarities in the CNS immune defense across evolutionary distant vertebrates.

TRIM25 predominately associates with anti-viral stress granules.

Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25's ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.

Covert RNA viruses in medflies differ in their mode of transmission and tissue tropism.

Numerous studies have demonstrated the presence of covert viral infections in insects. These infections can be transmitted in insect populations via two main routes: vertical from parents to offspring, or horizontal between nonrelated individuals. Thirteen covert RNA viruses have been described in the Mediterranean fruit fly (medfly). Some of these viruses are established in different laboratory-reared and wild medfly populations, although variations in the viral repertoire and viral levels have been observed at different time points. To better understand these viral dynamics, we characterized the prevalence and levels of covert RNA viruses in two medfly strains, assessed the route of transmission of these viruses, and explored their distribution in medfly adult tissues. Altogether, our results indicated that the different RNA viruses found in medflies vary in their preferred route of transmission. Two iflaviruses and a narnavirus are predominantly transmitted through vertical transmission via the female, while a nodavirus and a nora virus exhibited a preference for horizontal transmission. Overall, our results give valuable insights into the viral tropism and transmission of RNA viruses in the medfly, contributing to the understanding of viral dynamics in insect populations. IMPORTANCE: The presence of RNA viruses in insects has been extensively covered. However, the study of host-virus interaction has focused on viruses that cause detrimental effects to the host. In this manuscript, we uncovered which tissues are infected with covert RNA viruses in the agricultural pest Ceratitis capitata, and which is the preferred transmission route of these viruses. Our results showed that vertical and horizontal transmission can occur simultaneously, although each virus is transmitted more efficiently following one of these routes. Additionally, our results indicated an association between the tropism of the RNA virus and the preferred route of transmission. Overall, these results set the basis for understanding how viruses are established and maintained in medfly populations.

Favipiravir Treatment Prolongs Survival in a Lethal BALB/c Mouse Model of Ebinur Lake Virus Infection.

Orthobunyavirus is the largest and most diverse genus in the family Peribunyaviridae. Orthobunyaviruses are widely distributed globally and pose threats to human and animal health. Ebinur Lake virus (EBIV) is a newly classified Orthobunyavirus detected in China, Russia, and Kenya. This study explored the antiviral effects of two broad-spectrum antiviral drugs, favipiravir and ribavirin, in a BALB/c mouse model. Favipiravir significantly improved the clinical symptoms of infected mice, reduced viral titer and RNA copies in serum, and extended overall survival. The median survival times of mice in the vehicle- and favipiravir-treated groups were 5 and 7 days, respectively. Favipiravir significantly reduced virus titers 10- to 100-fold in sera at all three time points compared to vehicle-treated mice. And favipiravir treatment effectively reduced the virus copies by approximately 10-fold across the three time points, relative to vehicle-treated mice. The findings expand the antiviral spectrum of favipiravir for orthobunyaviruses in vivo.

Rapid detection of goose megrivirus using TaqMan real-time PCR technology.

The aim of this study was to develop an efficient and accurate platform for the detection of the newly identified goose megrivirus (GoMV). To achieve this goal, we developed a TaqMan real-time PCR technology for the rapid detection and identification of GoMV. Our data showed that the established TaqMan real-time PCR assay had high sensitivity, with the lowest detection limit of 67.3 copies/µL. No positive signal can be observed from other goose origin viruses (including AIV, GPV, GoCV, GHPyV, and GoAstV), with strong specificity. The coefficients of variation of repeated intragroup and intergroup tests were all less than 1.5%, with excellent repeatability. Clinical sample investigation data from domestic Minbei White geese firstly provided evidence that GoMV can be transmitted both horizontally and vertically. In conclusion, since the TaqMan real-time PCR method has high sensitivity, specificity, and reproducibility, it can be a useful candidate tool for GoMV epidemiological investigation.

Establishment and validation of a 2D primary gill cell culture of the sevenband grouper (Hyporthodus septemfasciatus).

A 2D primary gill cell culture system of the sevenband grouper (Hyporthodus septemfasciatus) was established to validate the pathogenesis of nervous necrosis virus (NNV) as observed in previous studies. This system, developed using the double-seeded insert (DSI) technique, yielded confluent cell layers. Upon challenge with NNV in a setup containing both autoclaved salt water and L15 media in the apical compartment, viral replication akin to that anticipated based on previous studies was observed. Consequently, we advocate for the utilization of primary gill cell culture as a viable alternative to conventional methodologies for investigating host pathogen interactions.

Bipartite viral RNA genome heterodimerization influences genome packaging and virion thermostability.

Multi-segmented viruses often multimerize their genomic segments to ensure efficient and stoichiometric packaging of the correct genetic cargo. In the bipartite Nodaviridae family, genome heterodimerization is also observed and conserved among different species. However, the nucleotide composition and biological function for this heterodimer remain unclear. Using Flock House virus as a model system, we developed a next-generation sequencing approach ("XL-ClickSeq") to probe heterodimer site sequences. We identified an intermolecular base-pairing site which contributed to heterodimerization in both wild-type and defective virus particles. Mutagenic disruption of this heterodimer site exhibited significant deficiencies in genome packaging and encapsidation specificity to viral genomic RNAs. Furthermore, the disruption of this intermolecular interaction directly impacts the thermostability of the mature virions. These results demonstrate that the intermolecular RNA-RNA interactions within the encapsidated genome of an RNA virus have an important role on virus particle integrity and thus may impact its transmission to a new host.IMPORTANCEFlock House virus is a member of Nodaviridae family of viruses, which provides a well-studied model virus for non-enveloped RNA virus assembly, cell entry, and replication. The Flock House virus genome consists of two separate RNA molecules, which can form a heterodimer upon heating of virus particles. Although similar RNA dimerization is utilized by other viruses (such as retroviruses) as a packaging mechanism and is conserved among Nodaviruses, the role of heterodimerization in the Nodavirus replication cycle is unclear. In this research, we identified the RNA sequences contributing to Flock House virus genome heterodimerization and discovered that such RNA-RNA interaction plays an essential role in virus packaging efficiency and particle integrity. This provides significant insight into how the interaction of packaged viral RNA may have a broader impact on the structural and functional properties of virus particles.

Zebrafish TMEM47 is an effective blocker of IFN production during RNA and DNA virus infection.

IMPORTANCE: Mitochondrial antiviral signaling protein (MAVS) and stimulator of interferon (IFN) genes (STING) are key adaptor proteins required for innate immune responses to RNA and DNA virus infection. Here, we show that zebrafish transmembrane protein 47 (TMEM47) plays a critical role in regulating MAVS- and STING-triggered IFN production in a negative feedback manner. TMEM47 interacted with MAVS and STING for autophagic degradation, and ATG5 was essential for this process. These findings suggest the inhibitory function of TMEM47 on MAVS- and STING-mediated signaling responses during RNA and DNA virus infection.

The alg-1 Gene Is Necessary for Orsay Virus Replication in Caenorhabditis elegans.

The establishment of the Orsay virus-Caenorhabditis elegans infection model has enabled the identification of host factors essential for virus infection. Argonautes are RNA interacting proteins evolutionary conserved in the three domains of life that are key components of small RNA pathways. C. elegans encodes 27 argonautes or argonaute-like proteins. Here, we determined that mutation of the argonaute-like gene 1, alg-1, results in a greater than 10,000-fold reduction in Orsay viral RNA levels, which could be rescued by ectopic expression of alg-1. Mutation in ain-1, a known interactor of ALG-1 and component of the RNA-induced silencing complex, also resulted in a significant reduction in Orsay virus levels. Viral RNA replication from an endogenous transgene replicon system was impaired by the lack of ALG-1, suggesting that ALG-1 plays a role during the replication stage of the virus life cycle. Orsay virus RNA levels were unaffected by mutations in the ALG-1 RNase H-like motif that ablate the slicer activity of ALG-1. These findings demonstrate a novel function of ALG-1 in promoting Orsay virus replication in C. elegans. IMPORTANCE All viruses are obligate intracellular parasites that recruit the cellular machinery of the host they infect to support their own proliferation. We used Caenorhabditis elegans and its only known infecting virus, Orsay virus, to identify host proteins relevant for virus infection. We determined that ALG-1, a protein previously known to be important in influencing worm life span and the expression levels of thousands of genes, is required for Orsay virus infection of C. elegans. This is a new function attributed to ALG-1 that was not recognized before. In humans, it has been shown that AGO2, a close relative protein to ALG-1, is essential for hepatitis C virus replication. This demonstrates that through evolution from worms to humans, some proteins have maintained similar functions, and consequently, this suggests that studying virus infection in a simple worm model has the potential to provide novel insights into strategies used by viruses to proliferate.

Combining Phage Display Technology with In Silico-Designed Epitope Vaccine to Elicit Robust Antibody Responses against Emerging Pathogen Tilapia Lake Virus.

Antigen epitope identification is a critical step in the vaccine development process and is a momentous cornerstone for the development of safe and efficient epitope vaccines. In particular, vaccine design is difficult when the function of the protein encoded by the pathogen is unknown. The genome of Tilapia lake virus (TiLV), an emerging virus from fish, encodes protein functions that have not been elucidated, resulting in a lag and uncertainty in vaccine development. Here, we propose a feasible strategy for emerging viral disease epitope vaccine development using TiLV. We determined the targets of specific antibodies in serum from a TiLV survivor by panning a Ph.D.-12 phage library, and we identified a mimotope, TYTTRMHITLPI, referred to as Pep3, which provided protection against TiLV after prime-boost vaccination; its immune protection rate was 57.6%. Based on amino acid sequence alignment and structure analysis of the target protein from TiLV, we further identified a protective antigenic site (399TYTTRNEDFLPT410) which is located on TiLV segment 1 (S1). The epitope vaccine with keyhole limpet hemocyanin (KLH-S1399-410) corresponding to the mimotope induced the tilapia to produce a durable and effective antibody response after immunization, and the antibody depletion test confirmed that the specific antibody against S1399-410 was necessary to neutralize TiLV. Surprisingly, the challenge studies in tilapia demonstrated that the epitope vaccine elicited a robust protective response against TiLV challenge, and the survival rate reached 81.8%. In conclusion, this study revealed a concept for screening antigen epitopes of emerging viral diseases, providing promising approaches for development and evaluation of protective epitope vaccines against viral diseases. IMPORTANCE Antigen epitope determination is an important cornerstone for developing efficient vaccines. In this study, we attempted to explore a novel approach for epitope discovery of TiLV, which is a new virus in fish. We investigated the immunogenicity and protective efficacy of all antigenic sites (mimotopes) identified in serum of primary TiLV survivors by using a Ph.D.-12 phage library. We also recognized and identified the natural epitope of TiLV by bioinformatics, evaluated the immunogenicity and protective effect of this antigenic site by immunization, and revealed 2 amino acid residues that play important roles in this epitope. Both Pep3 and S1399-410 (a natural epitope identified by Pep3) elicited antibody titers in tilapia, but S1399-410 was more prominent. Antibody depletion studies showed that anti-S1399-410-specific antibodies were essential for neutralizing TiLV. Our study demonstrated a model for combining experimental and computational screens to identify antigen epitopes, which is attractive for epitope-based vaccine development.

Clonal replacement sustains long-lived germinal centers primed by respiratory viruses.

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naive-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures.

Multiple Viral Protein Genome-Linked Proteins Compensate for Viral Translation in a Positive-Sense Single-Stranded RNA Virus Infection.

Viral protein genome-linked (VPg) protein plays an essential role in protein-primed replication of plus-stranded RNA viruses. VPg is covalently linked to the 5' end of the viral RNA genome via a phosphodiester bond typically at a conserved amino acid. Whereas most viruses have a single VPg, some viruses have multiple VPgs that are proposed to have redundant yet undefined roles in viral replication. Here, we use cricket paralysis virus (CrPV), a dicistrovirus that has four nonidentical copies of VPg, as a model to characterize the role of VPg copies in infection. Dicistroviruses contain two main open reading frames (ORFs) that are driven by distinct internal ribosome entry sites (IRESs). We systematically generated single and combinatorial deletions and mutations of VPg1 to VPg4 within the CrPV infectious clone and monitored viral yield in Drosophila S2 cells. Deletion of one to three VPg copies progressively decreased viral yield and delayed viral replication, suggesting a threshold number of VPgs for productive infection. Mass spectrometry analysis of CrPV VPg-linked RNAs revealed viral RNA linkage to either a serine or threonine in VPg, mutations of which in all VPgs attenuated infection. Mutating serine 4 in a single VPg abolished viral infection, indicating a dominant negative effect. Using viral minigenome reporters that monitor dicistrovirus 5' untranslated (UTR) and IRES translation revealed a relationship between VPg copy number and the ratio of distinct IRES translation activities. We uncovered a novel viral strategy whereby VPg copies in dicistrovirus genomes compensate for the relative IRES translation efficiencies to promote infection. IMPORTANCE Genetic duplication is exceedingly rare in small RNA viral genomes, as there is selective pressure to prevent RNA genomes from expanding. However, some small RNA viruses encode multiple copies of a viral protein, most notably an unusual viral protein that is linked to the viral RNA genome. Here, we investigate a family of viruses that contains multiple viral protein genome-linked proteins and reveal a novel viral strategy whereby viral protein copy number counterbalances differences in viral protein synthesis mechanisms.