Novel Rodent Arterivirus Detected in the Brazilian Amazon.

As part of a continuous effort to investigate the viral communities associated with wild mammals at the human-animal interface in an Amazonian metropolitan region, this study describes the detection of a novel rodent-borne arterivirus. A sample containing pooled organs of Oecomys paricola was submitted to RNA sequencing, and four sequences taxonomically assigned as related to the Arteriviridae family were recovered, corresponding to an almost complete genome of nearly 13 kb summed. In the phylogenetic analysis with the standard domains used for taxa demarcation in the family, the tentatively named Oecomys arterivirus 1 (OAV-1) was placed within the clade of rodent- and porcine-associated viruses, corresponding to the Variarterivirinae subfamily. The divergence analysis, based on the same amino acid alignment, corroborated the hypothesis that the virus may represent a new genus within the subfamily. These findings contribute to the expansion of the current knowledge about the diversity, host and geographical range of the viral family. Arterivirids are non-human pathogens and are usually species-specific, but the susceptibility of cell lines derived from different organisms should be conducted to confirm these statements for this proposed new genus in an initial attempt to assess its spillover potential.

Phylogenomic Characterization of Lopma Virus and Praja Virus, Two Novel Rodent-Borne Arteriviruses.

Recent years have witnessed the discovery of several new viruses belonging to the family Arteriviridae, expanding the known diversity and host range of this group of complex RNA viruses. Although the pathological relevance of these new viruses is not always clear, several well-studied members of the family Arteriviridae are known to be important animal pathogens. Here, we report the complete genome sequences of four new arterivirus variants, belonging to two putative novel species. These new arteriviruses were discovered in African rodents and were given the names Lopma virus and Praja virus. Their genomes follow the characteristic genome organization of all known arteriviruses, even though they are only distantly related to currently known rodent-borne arteriviruses. Phylogenetic analysis shows that Lopma virus clusters in the subfamily Variarterivirinae, while Praja virus clusters near members of the subfamily Heroarterivirinae: the yet undescribed forest pouched giant rat arterivirus and hedgehog arterivirus 1. A co-divergence analysis of rodent-borne arteriviruses confirms that they share similar phylogenetic patterns with their hosts, with only very few cases of host shifting events throughout their evolutionary history. Overall, the genomes described here and their unique clustering with other arteriviruses further illustrate the existence of multiple rodent-borne arterivirus lineages, expanding our knowledge of the evolutionary origin of these viruses.

ICTV Virus Taxonomy Profile: Arteriviridae 2021.

The family Arteriviridae comprises enveloped RNA viruses with a linear, positive-sense genome of approximately 12.7 to 15.7 kb. The spherical, pleomorphic virions have a median diameter of 50-74 nm and include eight to eleven viral proteins. Arteriviruses infect non-human mammals in a vector-independent manner. Infections are often persistent and can either be asymptomatic or produce overt disease. Some arteriviruses are important veterinary pathogens while others infect particular species of wild rodents or African non-human primates. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Arteriviridae, which is available at ictv.global/report/arteriviridae.

Transcriptome profiling analysis of lung tissue of Chinese soft-shell turtle infected by Trionyx sinensis Hemorrhagic Syndrome Virus.

Trionyx sinensis Hemorrhagic Syndrome Virus (TSHSV) is the firstly discovered aquatic arterivirus inducing high mortality of Trionyx sinensis. So far, the lack of genomic resources has hindered further research on revealing the immunological characteristics of T. sinensis in response to TSHSV. In the present study, we performed a transcriptome analysis from the lungs of T. sinensis challenged by TSHSV using Illumina-based RNA-Seq. The validity of transcriptomic data was confirmed with the gradual increase of TSHSV RNA copies detected in lung. A total of 103079339 clean reads were generated, and 58374764 unique mapped reads were analyzed. Assembly of the sequence data allowed identifying 16383 unigenes consisting of 36 significant differentially expressed genes (DEGs). These DEGs were categorized into 30 GO-enriched bioprocesses and 9 KEGG pathways. The combinational analysis of GO-enriched bioprocesses and KEGG pathways demonstrated that TSHSV modulated several immune genes of T. sinensis related to various biological processes, including virus recognition (RIG-I/MDA-5), immune initiation (IFIT-1 and IFIT-5), endocytosis (CUBN, ENPP2 and LRP2) and steroid metabolism (FCNIL and STAR). In summary, the finding of this study revealed several immune pathways and candidated genes involved in the immune response of T. sinensis against TSHSV-infection. These results will provide helpful information to investigate molecular mechanism of T. sinensis in response to TSHSV.

Arterivirus nsp4 Antagonizes Interferon Beta Production by Proteolytically Cleaving NEMO at Multiple Sites.

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae and pose major threats for the horse- and swine-breeding industries worldwide. A previous study suggested that PRRSV nsp4, a 3C-like protease, antagonizes interferon beta (IFN-ß) production by cleaving the NF-κB essential modulator (NEMO) at a single site, glutamate 349 (E349). Here, we demonstrated that EAV nsp4 also inhibited virus-induced IFN-ß production by targeting NEMO for proteolytic cleavage and that the scission occurred at four sites: E166, E171, glutamine 205 (Q205), and E349. Additionally, we found that, besides the previously reported cleavage site E349 in NEMO, scission by PRRSV nsp4 took place at two additional sites, E166 and E171. These results imply that while cleaving NEMO is a common strategy utilized by EAV and PRRSV nsp4 to antagonize IFN induction, EAV nsp4 adopts a more complex substrate recognition mechanism to target NEMO. By analyzing the abilities of the eight different NEMO fragments resulting from EAV or PRRSV nsp4 scission to induce IFN-ß production, we serendipitously found that a NEMO fragment (residues 1 to 349) could activate IFN-ß transcription more robustly than full-length NEMO, whereas all other NEMO cleavage products were abrogated for the IFN-ß-inducing capacity. Thus, NEMO cleavage at E349 alone may not be sufficient to completely inactivate the IFN response via this signaling adaptor. Altogether, our findings suggest that EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is critical for disarming the innate immune response for viral survival.IMPORTANCE The arterivirus nsp4-encoded 3C-like protease (3CLpro) plays an important role in virus replication and immune evasion, making it an attractive target for antiviral therapeutics. Previous work suggested that PRRSV nsp4 suppresses type I IFN production by cleaving NEMO at a single site. In contrast, the present study demonstrates that both EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is essential for disruption of type I IFN production. Moreover, we reveal that EAV nsp4 also cleaves NEMO at glutamine 205 (Q205), which is not targeted by PRRSV nsp4. Notably, targeting a glutamine in NEMO for cleavage has been observed only with picornavirus 3C proteases (3Cpro) and coronavirus 3CLpro In aggregate, our work expands knowledge of the innate immune evasion mechanisms associated with NEMO cleavage by arterivirus nsp4 and describes a novel substrate recognition characteristic of EAV nsp4.

Subclinical Infection of Macaques and Baboons with A Baboon Simarterivirus.

Simarteriviruses (Arteriviridae: Simarterivirinae) are commonly found at high titers in the blood of African monkeys but do not cause overt disease in these hosts. In contrast, simarteriviruses cause severe disease in Asian macaques upon accidental or experimental transmission. Here, we sought to better understand the host-dependent drivers of simarterivirus pathogenesis by infecting olive baboons (n = 4) and rhesus monkeys (n = 4) with the simarterivirus Southwest baboon virus 1 (SWBV-1). Surprisingly, none of the animals in our study showed signs of disease following SWBV-1 inoculation. Three animals (two rhesus monkeys and one olive baboon) became infected and sustained high levels of SWBV-1 viremia for the duration of the study. The course of SWBV-1 infection was highly predictable: plasma viremia peaked between 1 × 107 and 1 × 108 vRNA copies/mL at 3⁻10 days post-inoculation, which was followed by a relative nadir and then establishment of a stable set-point between 1 × 106 and 1 × 107 vRNA copies/mL for the remainder of the study (56 days). We characterized cellular and antibody responses to SWBV-1 infection in these animals, demonstrating that macaques and baboons mount similar responses to SWBV-1 infection, yet these responses are ineffective at clearing SWBV-1 infection. SWBV-1 sequencing revealed the accumulation of non-synonymous mutations in a region of the genome that corresponds to an immunodominant epitope in the simarterivirus major envelope glycoprotein GP5, which likely contribute to viral persistence by enabling escape from host antibodies.

Expanding the Arterivirus Host Spectrum: Olivier's Shrew Virus 1, A Novel Arterivirus Discovered in African Giant Shrews.

The family Arteriviridae harbors a rapidly expanding group of viruses known to infect a divergent group of mammals, including horses, pigs, possums, primates, and rodents. Hosts infected with arteriviruses present with a wide variety of (sub) clinical symptoms, depending on the virus causing the infection and the host being infected. In this study, we determined the complete genome sequences of three variants of a previously unknown virus found in Olivier's shrews (Crocidura olivieri guineensis) sampled in Guinea. On the nucleotide level, the three genomes of this new virus, named Olivier's shrew virus 1 (OSV-1), are 88-89% similar. The genome organization of OSV-1 is characteristic of all known arteriviruses, yet phylogenetic analysis groups OSV-1 separately from all currently established arterivirus lineages. Therefore, we postulate that OSV-1 represents a member of a novel arterivirus genus. The virus described here represents the first discovery of an arterivirus in members of the order Eulipotyphla, thereby greatly expanding the known host spectrum of arteriviruses.

Development of an indirect ELISA for detection of antibody to wobbly possum disease virus in archival sera of Australian brushtail possums (Trichosurus vulpecula) in New Zealand.

AIMS: To develop an indirect ELISA based on recombinant nucleocapsid (rN) protein of wobbly possum disease (WPD) virus for investigation of the presence of WPD virus in Australian brushtail possums (Trichosurus vulpecula) in New Zealand. METHODS: Pre- and post-infection sera (n=15 and 16, respectively) obtained from a previous experimental challenge study were used for ELISA development. Sera were characterised as positive or negative for antibody to WPD virus based on western-blot using WPD virus rN protein as antigen. An additional 215 archival serum samples, collected between 2000-2016 from five different regions of New Zealand, were also tested using the ELISA. Bayesian modelling of corrected optical density at 450 nm (OD450) results from the ELISA was used to obtain estimates of receiver operating characteristic (ROC) curves to establish cut-off values for the ELISA, and to estimate the prevalence of antibody to WPD virus. RESULTS: Western blot analysis showed 5/14 (36%) pre-infection sera and 11/11 (100%) post-infection sera from experimentally infected possums were positive for antibodies to WPD virus. Bayesian estimates of the ROC curves established cut-off values of OD450≥0.41 for samples positive, and OD450<0.28 for samples negative for antibody to WPD virus, for sera diluted 1:100 for the ELISA. Based on the model, the estimated proportion of samples with antibodies to WPD virus was 0.30 (95% probability interval=0.196-0.418). Of the 230 archival serum samples tested using the ELISA, 48 (20.9%) were positive for antibody to WPD virus, 155 (67.4%) were negative and 27 (11.7%) equivocal, using the established cut-off values. The proportion of samples positive for WPD virus antibody differed between geographical regions (p<0.001). CONCLUSION: The results suggested that WPD virus or a related virus has circulated among possums in New Zealand with differences in the proportion of antibody-positive samples from different geographical regions. Antibodies to WPD virus did not seem to protect possums from disease following experimental infection, as one third of possums from the previous challenge study showed evidence of pre-existing antibody at the time of challenge. These results provide further support for existence of different pathotypes of WPD virus, but the exact determinants of protection against WPD and epidemiology of infection in various regions of New Zealand remain to be established. CLINICAL RELEVANCE: Availability of the indirect ELISA for detection of WPD virus antibody will facilitate prospective epidemiological investigation of WPD virus circulation in wild possum populations in New Zealand.

The Histopathological Characteristics Caused by Trionyx sinensis Hemorrhagic Syndrome Virus (TSHSV) and Comparative Proteomic Analysis of Liver Tissue in TSHSV-Infected Chinese Soft-Shelled Turtles (Pelodiscus sinensis).

Trionyx sinensis hemorrhagic syndrome virus (TSHSV) is a pathogen that causes severe hemorrhagic syndrome and irreversible damage to different infected tissues of Pelodis cus sinensis, ending in the death of affected organisms. In the present study, the histopathological characteristics of TSHSV-infected P. sinensis were analyzed and compared by HE staining. Relative and absolute quantification (iTRAQ)-based proteomic analysis was employed to explore the molecular pathology of liver injury. Anatomical features indicated that TSHSV caused obvious congestion in the liver, kidney, intestine, and other tissues of P. sinensis. The typical clinical symptoms included hepatomegaly, fragility, spotty and severe congestion in liver tissue, and also obvious intestinal bleeding. The histopathological studies corroborated such lesions in the liver and kidney, etc. iTRAQ-based proteomic analysis revealed that there were 252 differentially expressed proteins in the liver tissue between healthy and infected P. sinensis, of which 118 proteins were upregulated and 134 proteins were downregulated. GO enrichment analysis and KEGG pathway analysis initially revealed the molecular mechanism of pathological changes in P. sinensis by TSHSV infection. The expression of some differentially expressed proteins was further confirmed by qRT-PCR. These results provided important information for the pathological diagnosis of TSHSV-caused disease, as well as the mechanism underlying TSHSV-caused disease.

Reorganization and expansion of the nidoviral family Arteriviridae.

The family Arteriviridae presently includes a single genus Arterivirus. This genus includes four species as the taxonomic homes for equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), porcine respiratory and reproductive syndrome virus (PRRSV), and simian hemorrhagic fever virus (SHFV), respectively. A revision of this classification is urgently needed to accommodate the recent description of eleven highly divergent simian arteriviruses in diverse African nonhuman primates, one novel arterivirus in an African forest giant pouched rat, and a novel arterivirus in common brushtails in New Zealand. In addition, the current arterivirus nomenclature is not in accordance with the most recent version of the International Code of Virus Classification and Nomenclature. Here we outline an updated, amended, and improved arterivirus taxonomy based on current data. Taxon-specific sequence cut-offs are established relying on a newly established open reading frame 1b phylogeny and pairwise sequence comparison (PASC) of coding-complete arterivirus genomes. As a result, the current genus Arterivirus is replaced by five genera: Equartevirus (for EAV), Rodartevirus (LDV + PRRSV), Simartevirus (SHFV + simian arteriviruses), Nesartevirus (for the arterivirus from forest giant pouched rats), and Dipartevirus (common brushtail arterivirus). The current species Porcine reproductive and respiratory syndrome virus is divided into two species to accommodate the clear divergence of the European and American "types" of PRRSV, both of which now receive virus status. The current species Simian hemorrhagic fever virus is divided into nine species to accommodate the twelve known simian arteriviruses. Non-Latinized binomial species names are introduced to replace all current species names to clearly differentiate them from virus names, which remain largely unchanged.

Two novel simian arteriviruses in captive and wild baboons (Papio spp.).

UNLABELLED: Since the 1960s, simian hemorrhagic fever virus (SHFV; Nidovirales, Arteriviridae) has caused highly fatal outbreaks of viral hemorrhagic fever in captive Asian macaque colonies. However, the source(s) of these outbreaks and the natural reservoir(s) of this virus remain obscure. Here we report the identification of two novel, highly divergent simian arteriviruses related to SHFV, Mikumi yellow baboon virus 1 (MYBV-1) and Southwest baboon virus 1 (SWBV-1), in wild and captive baboons, respectively, and demonstrate the recent transmission of SWBV-1 among captive baboons. These findings extend our knowledge of the genetic and geographic diversity of the simian arteriviruses, identify baboons as a natural host of these viruses, and provide further evidence that baboons may have played a role in previous outbreaks of simian hemorrhagic fever in macaques, as has long been suspected. This knowledge should aid in the prevention of disease outbreaks in captive macaques and supports the growing body of evidence that suggests that simian arterivirus infections are common in Old World monkeys of many different species throughout Africa. IMPORTANCE: Historically, the emergence of primate viruses both in humans and in other primate species has caused devastating outbreaks of disease. One strategy for preventing the emergence of novel primate pathogens is to identify microbes with the potential for cross-species transmission in their natural state within reservoir species from which they might emerge. Here, we detail the discovery and characterization of two related simian members of the Arteriviridae family that have a history of disease emergence and host switching. Our results expand the phylogenetic and geographic range of the simian arteriviruses and define baboons as a natural host for these viruses. Our findings also identify a potential threat to captive macaque colonies by showing that simian arteriviruses are actively circulating in captive baboons.

Biogenesis of non-structural protein 1 (nsp1) and nsp1-mediated type I interferon modulation in arteriviruses.

Type I interferons (IFNs-α/ß) play a key role for the antiviral state of host, and the porcine arterivirus; porcine reproductive and respiratory syndrome virus (PRRSV), has been shown to down-regulate the production of IFNs during infection. Non-structural protein (nsp) 1 of PRRSV has been identified as a viral IFN antagonist, and the nsp1α subunit of nsp1 has been shown to degrade the CREB-binding protein (CBP) and to inhibit the formation of enhanceosome thus resulting in the suppression of IFN production. The study was expanded to other member viruses in the family Arteriviridae: equine arteritis virus (EAV), murine lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV). While PRRSV-nsp1 and LDV-nsp1 were auto-cleaved to produce the nsp1α and nsp1ß subunits, EAV-nsp1 remained uncleaved. SHFV-nsp1 was initially predicted to be cleaved to generate three subunits (nsp1α, nsp1ß, and nsp1γ), but only two subunits were generated as SHFV-nsp1αß and SHFV-nsp1γ. The papain-like cysteine protease (PLP) 1α motif in nsp1α remained inactive for SHFV, and only the PLP1ß motif of nsp1ß was functional to generate SHFV-nsp1γ subunit. All subunits of arterivirus nsp1 were localized in the both nucleus and cytoplasm, but PRRSV-nsp1ß, LDV-nsp1ß, EAV-nsp1, and SHFV-nsp1γ were predominantly found in the nucleus. All subunits of arterivirus nsp1 contained the IFN suppressive activity and inhibited both interferon regulatory factor 3 (IRF3) and NF-κB mediated IFN promoter activities. Similar to PRRSV-nsp1α, CBP degradation was evident in cells expressing LDV-nsp1α and SHFV-nsp1γ, but no such degradation was observed for EAV-nsp1. Regardless of CBP degradation, all subunits of arterivirus nsp1 suppressed the IFN-sensitive response element (ISRE)-promoter activities. Our data show that the nsp1-mediated IFN modulation is a common strategy for all arteriviruses but their mechanism of action may differ from each other.

Clustering of and risk factors for the porcine high fever disease in a region of Vietnam.

Porcine high fever disease (PHFD) emerged in 2006 in China and spread to Vietnam. Little work has been carried out to investigate PHFD risk factors and space-time dynamics. To fill this gap, we investigated probable cases of PHFD at household level as the outcome. A study area, approximately 100 sq. km, was selected from a province of southern Vietnam that had reported the outbreak of PHFD in 2008. A survey was conducted in the study area to collect information about swine health problems during 2008. The questionnaire included three sections: general information, clinical signs of disease in pigs and production factors believed to be risk factors. Cases were defined at the household level and included interpretation of clinical signs in series. Logistic regression with a random intercept at the hamlet level was used to assess risk factors for PHFD at the household level. Spatial clustering was investigated using the D-function and a Cuzick-Edward's test. Spatial clusters were evaluated using a spatial relative risk surface and the spatial scan statistic using a Bernoulli model. Space-time clustering was explored using a space-time K-function and Knox's test. Space-time clusters were evaluated using a space-time permutation model in SaTScan. Of 955 households with questionnaire data, 33.4% were classified as cases. The statistical significance of space and space-time clustering differed between methods employed. The risk factors associated with occurrence of cases were higher numbers of sows and finishing pigs (log 2 transformed), receiving pigs from an external source and the interaction between using 'water green crop' (WGC) as pig feed and owning ducks with or without direct contact with pigs. The interaction between the presence of ducks and feeding WGC to pigs suggested the involvement of pathogens that might be present in water (environment) and could further replicate in or on ducks.

Intracellular Localization of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

Porcine reproductive and respiratory syndrome virus (PRRSV) is a small enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. It causes the porcine reproductive and respiratory syndrome in swine. The virus has 7 structural proteins: Of the seven, the N protein is the nucleocapsid that comprises a core of the virus particle. We have expressed the N protein of PRRSV PL97-1/LP1 strain using a heterologous gene expression vector derived from Sindbis virus, called pSinrep5. Immunofluorescence analysis showed that the N proteins were mainly found in the cytoplasm as well as in the nucleus of BHK-21 cells transfected with pSinrep5-N-derived RNA. Moreover, expression of the N protein did not change the incompetence of RNA replication of Mutant/nt14900 that lacks a 3' cis-acting replication element and the efficiency of RNA replication of Mutant/nt14800 that has a low level of RNA replication. Overall, our findings are consistent with previous results and help to understand a role of the N protein in PRRSV biology.

5' and 3' cis-Acting RNA Elements Required for RNA Replication of Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the genus Arterivirus in the family Arteriviridae, is the most important viral pathogens in swine industry worldwide. Here, we have investigated 5' and 3' cis-acting RNA elements required for PRRSV genome replication. Using the infectious PRRSV cDNA, we have manipulated the genomic RNA to generate mutant genomic RNAs, transfected these mutants into susceptible MARC-145 cells, and examined the competence of RNA replication. We found three genetic factors that were essential for viral replication. First, the cap structure present at the 5'-end of the genome was absolutely required for RNA replication. Secondly, polyadenylation of the genomic RNA at the 3'-end was also essential for RNA replication. Thirdly, approximately 100-nucleotide region just upstream of the N protein-coding region was crucial for genomic RNA replication. Taken together, our findings indicate that replication of PRRSV genomic RNA requires three important cis-acting RNA elements: 5' cap structure, 3' poly(A) motif, and an internal sequence of about 100 nucleotides. Further investigation is needed to elucidate the molecular mechanism(s) of how these elements act on PRRSV genome replication.

Crystal structure of the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona- and arteriviridae.

The causative agent of severe acute respiratory syndrome (SARS) is the SARS-associated coronavirus, SARS-CoV. The nucleocapsid (N) protein plays an essential role in SARS-CoV genome packaging and virion assembly. We have previously shown that SARS-CoV N protein forms a dimer in solution through its C-terminal domain. In this study, the crystal structure of the dimerization domain, consisting of residues 270-370, is determined to 1.75A resolution. The structure shows a dimer with extensive interactions between the two subunits, suggesting that the dimeric form of the N protein is the functional unit in vivo. Although lacking significant sequence similarity, the dimerization domain of SARS-CoV N protein has a fold similar to that of the nucleocapsid protein of the porcine reproductive and respiratory syndrome virus. This finding provides structural evidence of the evolutionary link between Coronaviridae and Arteriviridae, suggesting that the N proteins of both viruses have a common origin.

Site-directed mutagenesis of the Nidovirus replicative endoribonuclease NendoU exerts pleiotropic effects on the arterivirus life cycle.

The highly conserved NendoU replicative domain of nidoviruses (arteriviruses, coronaviruses, and roniviruses) belongs to a small protein family whose cellular branch is prototyped by XendoU, a Xenopus laevis endoribonuclease involved in nucleolar RNA processing. Recently, sequence-specific in vitro endoribonuclease activity was demonstrated for the NendoU-containing nonstructural protein (nsp) 15 of several coronaviruses. To investigate the biological role of this novel enzymatic activity, we have characterized a comprehensive set of arterivirus NendoU mutants. Deleting parts of the NendoU domain from nsp11 of equine arteritis virus was lethal. Site-directed mutagenesis of conserved residues exerted pleiotropic effects. In a first-cycle analysis, replacement of two conserved Asp residues in the C-terminal part of NendoU rendered viral RNA synthesis and virus production undetectable. In contrast, mutagenesis of other conserved residues, including two putative catalytic His residues that are absolutely conserved in NendoU and cellular homologs, produced viable mutants displaying reduced plaque sizes (20 to 80% reduction) and reduced yields of infectious progeny of up to 5 log units. A more detailed analysis of these mutants revealed a moderate reduction in RNA synthesis, with subgenomic RNA synthesis consistently being more strongly affected than genome replication. Our data suggest that the arterivirus nsp11 is a multifunctional protein with a key role in viral RNA synthesis and additional functions in the viral life cycle that are as yet poorly defined.