Large Open Reading Frame Research Articles

Characterization of three mycoviruses co-infecting the plant pathogenic fungus Sclerotinia nivalis

Two dsRNAs of approximately 6.0-kb and 3.0-kb in length were detected in strain SsSn-1 of Sclerotinia nivalis. Genomic analysis showed that the 6.0-kb dsRNA was a victorivirus, named as Sclerotinia nivalis victorivirus 1 (SnVV1). The genome of SnVV1 is 5162bp in length containing two large open reading frames (ORFs), ORF1 and ORF2. ORF1 was deduced to encode a coat protein (CP) showing homology to CPs of viruses belonging to the family Totiviridae. The stop codon of ORF1 overlaps the start codon of ORF2 in the tetranucleotide sequence AUGA. ORF2 was predicted to encode for a RNA-dependent RNA polymerase (RdRp) that was very similar to the RdRps of victoriviruses. The 3.0-kb dsRNA was consisted of two species of mitoviruses, named as Sclerotinia nivalis mitovirus 1/SsSn-1 (SnMV1/SsSn-1) and Sclerotinia sclerotiorum mitovirus 3/SsSn-1 (SsMV3/SsSn-1). The genomes of SnMV1/SsSn-1 and SsMV3/SsSn-1 were 2720nt and 2583nt in length, respectively. Both mitoviruses were AU-rich and deduced to contain a major large ORF encoding a mitoviral RdRp with the fungal mitochondrial codon usages. SnMV1/SsSn-1 was most closely related to Sclerotinia sclerotiorum mitovirus 4 (SsMV4/NZ1) and shared 76.5% and 80.1% identity with SsMV4/NZ1 for nucleotide and RdRp sequences, respectively. In addition, the nucleotide and RdRp sequences of SsMV3/SsSn-1 were 90.6% and 95.9% identical to the nucleotide and RdRp sequences of SsMV3/NZ1, respectively. Considering their nucleotide and RdRp sequence identities with other mitoviruses, SnMV1/SsSn-1 may belong to the genus Mitovirus, whereas SsMV3/SsSn-1 is possibly a strain of SsMV3. Both SnMV1/SsSn-1 and SsMV3/SsSn-1 were transmitted to a recipient virus-free colony faster than was SnVV1 through hyphal anastomosis. Co-infection by these mycoviruses had no apparent effects on growth and pathogenicity of S. nivalis.

Nonstructural protein (NS1) of human parvovirus B19 stimulates host innate immunity and blunts the exogenous type I interferon signaling in vitro

B19 virus is a non-enveloped DNA virus and belongs to the family of parvoviridae. There are two large open reading frames (ORFs), nonstructural protein (NS1) and two capsid proteins (VP1 and VP2). Host innate immune responses form the first line of defense against many pathogen invasion. How B19 virus, especially its encoded viral proteins interacts with host innate immune system remains unknown. In this study we aim to investigate the effect of NS1 on the host innate immune response and exogenous type I IFN signaling. Here we found that the type I IFN can be stimulated by NS1. Interestingly, NS1 also plays an important role in inhibiting the exogenous type I IFN signaling at p-STAT1, ISRE and ISGs levels. We concluded that NS1 may play pivotal role in evading the host immune surveillance. Our data shed novel light on the pathogenesis of B19 viral infection and virus evasion strategies.

A survey of endogenous retrovirus (ERV) sequences in the vicinity of multiple sclerosis (MS)-associated single nucleotide polymorphisms (SNPs).

Although multiple sclerosis (MS) is one of the most common central nervous system diseases in young adults, little is known about its etiology. Several human endogenous retroviruses (ERVs) are considered to play a role in MS. We are interested in which ERVs can be identified in the vicinity of MS associated genetic marker to find potential initiators of MS. We analysed the chromosomal regions surrounding 58 single nucleotide polymorphisms (SNPs) that are associated with MS identified in one of the last major genome wide association studies. We scanned these regions for putative endogenous retrovirus sequences with large open reading frames (ORFs). We observed that more retrovirus-related putative ORFs exist in the relatively close vicinity of SNP marker indices in multiple sclerosis compared to control SNPs. We found very high homologies to HERV-K, HCML-ARV, XMRV, Galidia ERV, HERV-H/env62 and XMRV-like mouse endogenous retrovirus mERV-XL. The associated genes (CYP27B1, CD6, CD58, MPV17L2, IL12RB1, CXCR5, PTGER4, TAGAP, TYK2, ICAM3, CD86, GALC, GPR65 as well as the HLA DRB1*1501) are mainly involved in the immune system, but also in vitamin D regulation. The most frequently detected ERV sequences are related to the multiple sclerosis-associated retrovirus, the human immunodeficiency virus 1, HERV-K, and the Simian foamy virus. Our data shows that there is a relation between MS associated SNPs and the number of retroviral elements compared to control. Our data identifies new ERV sequences that have not been associated with MS, so far.

Identification of helitron sequences in the coral genome and implications for host defense.

Abstract Helitrons are mobile elements found in the genomes of numerous plant and animal species. Helitrons can mobilize flanking sequences when they transpose, and so have the potential to duplicate genes and remodel genomes. The stony coral Acropora has an expanded immune gene repertoire in comparison to the closely-related anemone species Nematostella, especially in the number of TLR and IL-1R genes. Mechanisms behind the expansion are not currently known. Although helitron sequences have been identified in Nematostella, no helitrons have been identified in Acropora. BLAST searches against the Acropora genome using Nematostella amino acid sequences as queries revealed seven candidate Acropora helitrons. Six of the seven Acropora candidates had large open reading frames in the forward direction. One candidate element was split between two adjacent reading frames. Conserved domain searches were performed within the open reading frames using the databases at NCBI. As expected, Helitron N-like and replicative protein A (RPA) domains were shared between Acropora and Nematostella elements. UPGMA trees were constructed using highly conserved regions, and one candidate Acropora element clustered more closely with the Nematostella reference helitron than with other Acropora sequences. Interestingly, P-loop domains were common to cnidarian helitrons and a subset of plant helitron reference sequences containing plant disease resistance gene motifs in regions overlapping with a nucleotide-binding domain critical for resistance gene function. Together, these studies will help to determine if helitrons played a role in the expansion of the Acropora immune gene repertoire.

Ctenopharyngodon idella NF-κB subunit p65 modulates the transcription of IκBα in CIK cells.

NF-κB is an important transcription factor for regulating the multiple inflammatory and immune related gene transcription. It can bind with the nuclear factor κB site within the promoter of target genes to regulate their transcriptions. p65, the all-important subunit of NF-κB, is ubiquitously expressed in cells. In the present study, we cloned and identified the p65 subunit from grass carp (Ctenopharyngodon idella) (named Cip65) by homologous cloning and RACE technique. The full length of Cip65 cDNA is 2481bp along with 9bp 5' UTR, 639bp 3' UTR and the largest open reading frame (1833bp) encoding a polypeptide of 610 amino acids with a well conserved Rel-homology domain (RHD) in N-terminal and a putative transcription activation domain (TAD) in C-terminal. Cip65 gathers with other teleost p65 proteins to form a fish-specific clade clearly distinct from those of mammalian and amphibian counterparts on the phylogenetic tree. In CIK (C.idellus kidney) cells, the expression of Cip65 was significantly up-regulated under the stimulation with Poly I:C. As one member of the NF-κB inhibitor protein (IκB) family, IκBα can dominate the activity of NF-κB by interacting with it. To study the molecular mechanisms of negative feedback loop of NF-κB signaling in fish, we cloned grass carp IκBα (CiIκBα) promoter sequence. CiIκBα promoter is 414bp in length containing two RelA binding sites and a putative atypical TATA-box. Meanwhile, Cip65 and its mutant proteins including C-terminus deletion mutant of Cip65 (Cip65-ΔC) and N-terminus deletion mutant of Cip65 (Cip65-ΔN) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. Invitro, Cip65 rather than Cip65-ΔC and Cip65-ΔN showed high affinity with CiIκBα promoter sequence by gel mobility shift assays. Invivo, the cotransfection of pcDNA3.1-Cip65 (or pcDNA3.1-Cip65-ΔC, pcDNA3.1-Cip65-ΔN respectively) with pGL3-CiIκBα and pRL-TK renilla luciferase plasmid into CIK cells showed that pcDNA3.1-Cip65 rather than pcDNA3.1-Cip65-ΔC and pcDNA3.1-Cip65-ΔN, can increase the luciferase activity. Taken together, these results suggested that Cip65 can regulate the expression of CiIκBα and works as a negative feedback loop in NF-κB pathway.

Crystal structure of the 3C protease from Southern African Territories type 2 foot-and-mouth disease virus.

The replication of foot-and-mouth disease virus (FMDV) is dependent on the virus-encoded 3C protease (3Cpro). As in other picornaviruses, 3Cpro performs most of the proteolytic processing of the polyprotein expressed from the large open reading frame in the RNA genome of the virus. Previous work revealed that the 3Cpro from serotype A—one of the seven serotypes of FMDV—adopts a trypsin-like fold. On the basis of capsid sequence comparisons the FMDV serotypes are grouped into two phylogenetic clusters, with O, A, C, and Asia 1 in one, and the three Southern African Territories serotypes, (SAT-1, SAT-2 and SAT-3) in another, a grouping pattern that is broadly, but not rigidly, reflected in 3Cpro amino acid sequences. We report here the cloning, expression and purification of 3C proteases from four SAT serotype viruses (SAT2/GHA/8/91, SAT1/NIG/5/81, SAT1/UGA/1/97, and SAT2/ZIM/7/83) and the crystal structure at 3.2 Å resolution of 3Cpro from SAT2/GHA/8/91.

Identification of a naturally occurring Bean common mosaic virus recombinant isolate infecting azuki bean.

The near complete nucleotide sequence of Bean common mosaic virus (BCMV) infecting azuki bean from China was determined to be 10047 nucleotides in length excluding the 3′-terminal poly (A) sequence. The isolate was designated as BCMV-Az and caused stunting, mosaic and crumpling on leaves of azuki plants. It contains a large open reading frame (ORF) flanked by a 131 nt 5′-untranslated region (UTR) and a 253 nt 3′-UTR and the putative polyprotein encoded by this large ORF is comprised of 3220 amino acid residues. Phylogenetic analysis showed that BCMV-Az clustered with strains isolated from different hosts and geographic origins, suggesting that it is a novel strain. Recombination analyses indicated that it was a recombinant originating from isolates R (AJ312437) and US10 (KF919299).

The P1N-PISPO trans-Frame Gene of Sweet Potato Feathery Mottle Potyvirus Is Produced during Virus Infection and Functions as an RNA Silencing Suppressor.

The positive-sense RNA genome of Sweet potato feathery mottle virus (SPFMV) (genus Potyvirus, family Potyviridae) contains a large open reading frame (ORF) of 3,494 codons translatable as a polyprotein and two embedded shorter ORFs in the -1 frame: PISPO, of 230 codons, and PIPO, of 66 codons, located in the P1 and P3 regions, respectively. PISPO is specific to some sweet potato-infecting potyviruses, while PIPO is present in all potyvirids. In SPFMV these two extra ORFs are preceded by conserved G2A6 motifs. We have shown recently that a polymerase slippage mechanism at these sites could produce transcripts bringing these ORFs in frame with the upstream polyprotein, thus leading to P1N-PISPO and P3N-PIPO products (B. Rodamilans, A. Valli, A. Mingot, D. San Leon, D. B. Baulcombe, J. J. Lopez-Moya, and J.A. Garcia, J Virol 89:6965-6967, 2015, doi:10.1128/JVI.00337-15). Here, we demonstrate by liquid chromatography coupled to mass spectrometry that both P1 and P1N-PISPO are produced during viral infection and coexist in SPFMV-infected Ipomoea batatas plants. Interestingly, transient expression of SPFMV gene products coagroinfiltrated with a reporter gene in Nicotiana benthamiana revealed that P1N-PISPO acts as an RNA silencing suppressor, a role normally associated with HCPro in other potyviruses. Moreover, mutation of WG/GW motifs present in P1N-PISPO abolished its silencing suppression activity, suggesting that the function might require interaction with Argonaute components of the silencing machinery, as was shown for other viral suppressors. Altogether, our results reveal a further layer of complexity of the RNA silencing suppression activity within the Potyviridae family. Gene products of potyviruses include P1, HCPro, P3, 6K1, CI, 6K2, VPg/NIaPro, NIb, and CP, all derived from the proteolytic processing of a large polyprotein, and an additional P3N-PIPO product, with the PIPO segment encoded in a different frame within the P3 cistron. In sweet potato feathery mottle virus (SPFMV), another out-of-frame element (PISPO) was predicted within the P1 region. We have shown recently that a polymerase slippage mechanism can generate the transcript variants with extra nucleotides that could be translated into P1N-PISPO and P3N-PIPO. Now, we demonstrate by mass spectrometry analysis that P1N-PISPO is indeed produced in SPFMV-infected plants, in addition to P1. Interestingly, while in other potyviruses the suppressor of RNA silencing is HCPro, we show here that P1N-PISPO exhibited this activity in SPFMV, revealing how the complexity of the gene content could contribute to supply this essential function in members of the Potyviridae family.

The first complete genome sequence of iris severe mosaic virus.

The first complete genome sequence of ISMV was determined by deep sequencing of a small RNA library constructed from ISMV-infected samples and rapid amplification of cDNA ends (RACE) PCR. The ISMV genome consists of 10,403 nucleotides excluding the poly(A) tail and contains a large open reading frame encoding a polyprotein of 3316 amino acids. Putative proteolytic cleavage sites were identified by BLAST analysis. The ISMV polyprotein showed highest amino acid sequence identity to that encoded by onion yellow dwarf virus. Phylogenetic analysis of the polyprotein amino acid sequence confirmed that ISMV forms a cluster with shallot yellow stripe virus, Cyrtanthus elatus virus A, narcissus degeneration virus and onion yellow dwarf virus. These results confirm that ISMV is a distinct member of the genus Potyvirus.

Genomic characterization and phylogenetic analysis of Chinese sacbrood virus isolated from Loess Plateau, China.

The complete genomic RNA of the Chinese sacbrood virus (CSBV) strain, which infects the honeybees in the Loess plateau, was sequenced and analyzed. The CSBV-SX strain contains 8705 nucleotides, which includes a single large open reading frame (99-8681 nucleotides) encoding 2860 amino acids. A novel efficient identification method was used to investigate the samples infected by CSBV. The putative amino acid sequence alignment analysis showed that, except for some normal well characterized domains such as RNA helicase, RNA protease, and RNA-dependent RNA polymerase domains, a calicivirus coat protein domain was identified at amino acids 493-564. Phylogenetic analysis indicated that CSBV-SX was closely related to CSBV-BJ, and this result was supported by nucleotide multiple sequence alignment and protein multiple sequence alignment analysis results. These differences in the CSBV-SX strain may be related to virus adaptation to the xerothermic, low relative humidity, and strong ultraviolet radiation conditions in the Loess Plateau.

Novel mitoviruses in Rhizoctonia solani AG-3PT infecting potato

Double-stranded RNA (dsRNA) elements are ubiquitous in Rhizoctonia solani. Total dsRNA was randomly amplified from a R. solani isolate (RS002) belonging to anastomosis group-3PT (AG-3PT), associated with black scurf in potato. Assembly of resulting cDNA sequences identified a nearly complete genome of a novel virus related to the genus Mitovirus (family Narnaviridae), herein named Rhizoctonia mitovirus 1 RS002 (RMV-1-RS002). The 2797 nucleotide partial genome of RMV-1-RS002 is A-U rich (59.06 %), and can be folded into stable stem-loop structures at 5′ and 3′ ends. Universal and mold mitochondrial codon usages revealed a large open reading frame in the genome, putatively encoding an 826 amino acid polypeptide, which has conserved motifs for mitoviral RNA-dependent RNA polymerase. The full length putative polypeptide shared 25.6 % sequence identity with the corresponding region of Tuber excavatum mitovirus (TeMV). The partial genome of a second mitovirus (proposed name Rhizoctonia mitovirus 2 RS002 (RMV-2-RS002)) was also amplified from RS002. A nearly identical copy of RMV-1-RS002 was detected in two additional AG-3PT isolates. These data indicate that multiple mitoviruses can exist in a single isolate of R. solani AG-3PT, and that mitoviruses such as RMV-1-RS002 are probably widespread in this pathogen. The roles of mitoviruses in the biology of R. solani AG-3PT remain unknown.

Characterization of a novel Sclerotinia sclerotiorum RNA virus as the prototype of a new proposed family within the order Tymovirales

Recent studies have shown that Sclerotinia sclerotiorum, an important plant pathogen fungus, harbors diverse mycoviruses. A new mycovirus, tentatively named as Sclerotinia sclerotiorum deltaflexivirus 1 (SsDFV1), was isolated from a S. sclerotiorum strain AX19 containing multiple dsRNA elements. The complete genome of SsDFV1 was shown to be 8178 nucleotides long excluding the poly (A) tail. SsDFV1 has a large putative open reading frame (ORF1) and three smaller ORFs (2–4). ORF1 encodes a putative methyltransferase-helicase-RdRp polyprotein of 2075 amino acids. ORFs (2–4) encode three putative small hypothetical proteins (<40kDa) with unknown biological functions. No evidence for a coat protein encoded by SsDFV1 was obtained. Multiple alignment suggested that three conserved domains, RdRp, methyltransferase, and helicase, from SsDFV1 have lower identity (approximately 25%) with all the reported viruses of four approved families, Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae and Tymoviridae in the order Tymovirales. Moreover, a phylogenetic tree also suggested that the SsDFV1 could not be phylogenetically placed in any of the approved families, and forms a separated cluster distinct from other known viruses. Therefore, these combined results suggest that SsDFV1 could represent a new positive-sense single-stranded RNA virus with some unique molecular features, and we propose to create a tentative family Deltaflexiviridae that accommodates SsDFV1.

Fish IRF3 up-regulates the transcriptional level of IRF1, IRF2, IRF3 and IRF7 in CIK cells.

Interferon Regulatory Factors (IRFs) belong to a family of transcription factor involved in transcriptional regulation of type I IFN and IFN-stimulated genes (ISG) in cells. In the present study, an IRF3 full-length cDNA (termed CiIRF3, JX999055) and its promoter sequence were cloned by homology cloning strategy and genome walking from grass carp (Ctenopharyngodon idella). The full-length cDNA sequence of CiIRF3 is comprised of a 5'UTR (195 bp), a 3'UTR (269 bp) and a largest open reading frame (ORF) of 1377 bp encoding a polypeptide of 458 amino acids. CiIRF3 has a conservative DNA-binding domain (DBD) at N-terminal and a relatively conserved interferon regulatory factors association domain (IAD). Phylogenetic tree analysis indicated that CiIRF3 gathers together with other IRF-3 from higher vertebrates in the same branch. The promoter sequence of CiIRF3 (596 bp) consists of three IRF-E, a C/EBP beta, a NF-kappa B and a TATA-BOX. CiIRF3 was constitutively expressed at low level in different grass carp tissues but was rapidly up-regulated with Poly I:C stimulation. To study the molecular mechanism of CiIRF3 regulating the transcription of IRFs, CiIRF3 was expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. Gel mobility shift assays revealed the affinity of CiIRF3 protein with promoters of CiIRF1, CiIRF2, CiIRF3 and CiIRF7 respectively. Then, CIK cells were co-transfected with pcDNA3.1-CiIRF3, pGL3-promotor (pGL3-CiIRF1, pGL3-CiIRF2, pGL3-CiIRF3, pGL3-CiIRF7) and luciferase reporter vector respectively. The cotransfection experiment showed that CiIRF3 increased the promoter activity of CiIRF1, CiIRF2, CiIRF3 and CiIRF7. Furthermore, overexpression of CiIRF3 in CIK cells also up-regulated the expressions of CiIRF1, CiIRF2, CiIRF3 and CiIRF7. So, CiIRF3 can improve the transcriptional level of CiIRF1, CiIRF2, CiIRF3 and CiIRF7.

Identification and Genome Characterization of the First Sicinivirus Isolate from Chickens in Mainland China by Using Viral Metagenomics.

Unlike traditional virus isolation and sequencing approaches, sequence-independent amplification based viral metagenomics technique allows one to discover unexpected or novel viruses efficiently while bypassing culturing step. Here we report the discovery of the first Sicinivirus isolate (designated as strain JSY) of picornaviruses from commercial layer chickens in mainland China by using a viral metagenomics technique. This Sicinivirus isolate, which contains a whole genome of 9,797 nucleotides (nt) excluding the poly(A) tail, possesses one of the largest picornavirus genome so far reported, but only shares 88.83% and 82.78% of amino acid sequence identity to that of ChPV1 100C (KF979332) and Sicinivirus 1 strain UCC001 (NC_023861), respectively. The complete 939 nt 5′UTR of the isolate strain contains at least twelve stem-loop domains (A–L), representing the highest set of loops reported within Sicinivirus genus. The conserved 'barbell-like' structure was also present in the 272 nt 3′UTR of the isolate as that in the 3′ UTR of Sicinivirus 1 strain UCC001. The 8,586 nt large open reading frame encodes a 2,862 amino acids polyprotein precursor. Moreover, Sicinivirus infection might be widely present in commercial chicken farms in Yancheng region of the Jiangsu Province as evidenced by all the tested stool samples from three different farms being positive (17/17) for Sicinivirus detection. This is the first report on identification of Sicinivirus in commercial layer chickens with a severe clinical disease in mainland China, however, further studies are needed to evaluate the pathogenic potential of this picornavirus in chickens.

Complete genome sequence of a novel endornavirus isolated from hot pepper.

The complete genome of a putative new endornavirus infecting hot peppers (Capsicum annuum) was determined to be 14,729 nt in size, including 12 cytosines at the 3' end. The hot pepper-infecting virus has the highest nucleotide sequence similarity (94% query cover and 72% identity) to bell pepper endornavirus (BPEV) isolated from the cultivar Yolo Wonder in the USA (GenBank accession no. JN019858). The putative single, large open reading frame encodes a 4,884-amino-acid-long polyprotein that contains four putative functional domains: a viral methyltransferase, a viral RNA helicase, a glycosyltransferase, and an RNA-dependent RNA polymerase. A phylogenetic tree based on whole polyprotein sequences confirmed the close evolutionary relationship of the studied endornavirus to BPEV. The hot pepper-infecting virus also has a nick at nt position 975. Taken together, these results suggest that this virus belongs to a new species in the genus Endornavirus (family Endornaviridae), for which the name hot pepper endornavirus (HPEV) is proposed.

Rice necrosis mosaic virus, a fungal transmitted Bymovirus: complete nucleotide sequence of the genomic RNAs and subgrouping of bymoviruses

The complete nucleotide sequence of Rice necrosis mosaic virus (RNMV) RNA1 was determined to be 7178 nt long with one large open reading frame, potentially encoding a polyprotein of 258 kDa with the features of a typical bymovirus. The nucleotide sequence showed 56 % identity with that of Barley mild mosaic virus (BaMMV) and 46 % with those of Oat mosaic virus, Barley yellow mosaic virus and Wheat yellow mosaic virus. RNA2 was 3579 nt long, encoding a 110-kDa polyprotein, and showed 42 and 35/26 % identity with BaMMV and others, respectively. Phylogenetic analysis showed that bymoviruses were classified into two subgroups.

Out of the Reservoir: Phenotypic and Genotypic Characterization of a Novel Cowpox Virus Isolated from a Common Vole.

The incidence of human cowpox virus (CPXV) infections has increased significantly in recent years. Serological surveys have suggested wild rodents as the main CPXV reservoir. We characterized a CPXV isolated during a large-scale screening from a feral common vole. A comparison of the full-length DNA sequence of this CPXV strain with a highly virulent pet rat CPXV isolate showed a sequence identity of 96%, including a large additional open reading frame (ORF) of about 6,000 nucleotides which is absent in the reference CPXV strain Brighton Red. Electron microscopy analysis demonstrated that the vole isolate, in contrast to the rat strain, forms A-type inclusion (ATI) bodies with incorporated virions, consistent with the presence of complete ati and p4c genes. Experimental infections showed that the vole CPXV strain caused only mild clinical symptoms in its natural host, while all rats developed severe respiratory symptoms followed by a systemic rash. In contrast, common voles infected with a high dose of the rat CPXV showed severe signs of respiratory disease but no skin lesions, whereas infection with a low dose led to virus excretion with only mild clinical signs. We concluded that the common vole is susceptible to infection with different CPXV strains. The spectrum ranges from well-adapted viruses causing limited clinical symptoms to highly virulent strains causing severe respiratory symptoms. In addition, the low pathogenicity of the vole isolate in its eponymous host suggests a role of common voles as a major CPXV reservoir, and future research will focus on the correlation between viral genotype and phenotype/pathotype in accidental and reservoir species. We report on the first detection and isolation of CPXV from a putative reservoir host, which enables comparative analyses to understand the infection cycle of these zoonotic orthopox viruses and the relevant genes involved. In vitro studies, including whole-genome sequencing as well as in vivo experiments using the Wistar rat model and the vole reservoir host allowed us to establish links between genomic sequences and the in vivo properties (virulence) of the novel vole isolate in comparison to those of a recent zoonotic CPXV isolated from pet rats in 2009. Furthermore, the role of genes present only in a reservoir isolate can now be further analyzed. These studies therefore allow unique insights and conclusions about the role of the rodent reservoir in CPXV epidemiology and transmission and about the zoonotic threat that these viruses represent.

Torque teno virus: a ubiquitous virus.

The TTV genome can be divided into an untranslatedregion (UTR) of 1.2kb and a potential coding region of 2.6kb.The UTR is relatively conserved, suggesting that it playsan important regulatory role in viral replication. The cod-ing region contains two large open reading frames: ORF1 andORF2. Several other open reading frames have been described,and the peptides that they encode differ in length for differentisolates.

MOLECULAR ANALYSIS OF THE COMPLETE GENOME SEQUENCE OF A TOBACCO ETCH VIRUS ISOLATE FROM CHINA

An isolate of Tobacco etch virus (TEV-Shaanxi) induces systemic mosaic and necrotic lines or etching in N. Tabacum and is transmitted by M. persicae more efficiently than A. gossypii. The complete genome of TEV-Shaanxi consists of 9,494 nucleotides, excluding the 3’poly (A) tail. It contains a single large open reading frame (ORF) coding for a polyprotein of 3,054 amino acids, with an AUG start codon and a UGA stop codon. The polyprotein encodes 10 proteins (P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, Nib and CP), whose putative cleavage sites were identified by comparison with sequences of other known potyviruses. The identities of the 10 proteins of TEV-Shaanxi with other members of the genus Potyvirus were 30.5-62.7% and 29.4-68.8% at the nucleotide and amino acid levels, respectively. A short internal open reading frame, PIPO, was also highly conserved. TEV isolates whose sequences are available cluster into two groups which correspond with their host of origin.

The polymerase slips and PIPO exists.

Plant viruses that contain plus‐sensed single‐stranded RNA genomes are highly abundant in nature. As the equivalents of large mRNAs, these viral genomes utilize a wide variety of gene expression strategies for the production of their encoded proteins. The potyviruses, which are among the most agriculturally important members in this category, contain a single large open reading frame (ORF) coding for a polyprotein that is processed into functional units. For many years, the products derived from the full‐length polyprotein were thought to be the only functional viral proteins. However, this notion was dispelled when an additional essential viral ORF, PIPO, was discovered encoded in an alternative reading frame. Since then, the PIPO protein—P3N‐PIPO, which mediates virus movement in plants—has been intensively studied, but its mode of expression remained elusive until now. Two articles, one in this issue of EMBO Reports , now report that slippage of the viral polymerase during viral genome replication is responsible for shifting PIPO into a translated reading frame, thereby allowing for production of P3N‐PIPO [1], [2]. This mechanism of gene expression represents a novel strategy for plant viruses.