A Novel Picornavirus Discovered in White Leg Shrimp Penaeus vannamei.

Global shrimp farming is increasingly threatened by various emerging viruses. In the present study, a novel picornavirus, Penaeus vannamei picornavirus (PvPV), was discovered in moribund White leg shrimp (Penaeus vannamei) collected from farm ponds in China in 2015. Similar to most picornaviruses, PvPV is non-enveloped RNA virus, with a particle diameter of approximately 30 nm. The sequence of the positive single-stranded RNA genome with a length of 10,550 nts was characterized by using genome sequencing and reverse transcription PCR. The existence of PvPV related proteins was further proved by confirmation of viral amino acid sequences, using mass spectrometry analysis. Phylogenetic analysis based on the full-length genomic sequence revealed that PvPV was more closely related to the Wenzhou shrimp virus 8 than to any other dicistroviruses in the order Picornavirales. Genomic sequence conservative domain prediction analysis showed that the PvPV genome encoded a large tegument protein UL36, which was unique among the known dicistroviruses and different from other dicistroviruses. According to these molecular features, we proposed that PvPV is a new species in the family Dicistroviridae. This study reported the first whole-genome sequence of a novel and distinct picornavirus in crustaceans, PvPV, and suggests that further studies of PvPV would be helpful in understanding its evolution and potential pathogenicity, as well as in developing diagnostic techniques.

Structure of Nora virus at 2.7 Å resolution and implications for receptor binding, capsid stability and taxonomy.

Nora virus, a virus of Drosophila, encapsidates one of the largest single-stranded RNA virus genomes known. Its taxonomic affinity is uncertain as it has a picornavirus-like cassette of enzymes for virus replication, but the capsid structure was at the time for genome publication unknown. By solving the structure of the virus, and through sequence comparison, we clear up this taxonomic ambiguity in the invertebrate RNA virosphere. Despite the lack of detectable similarity in the amino acid sequences, the 2.7 Å resolution cryoEM map showed Nora virus to have T = 1 symmetry with the characteristic capsid protein ß-barrels found in all the viruses in the Picornavirales order. Strikingly, α-helical bundles formed from the extended C-termini of capsid protein VP4B and VP4C protrude from the capsid surface. They are similar to signalling molecule folds and implicated in virus entry. Unlike other viruses of Picornavirales, no intra-pentamer stabilizing annulus was seen, instead the intra-pentamer stability comes from the interaction of VP4C and VP4B N-termini. Finally, intertwining of the N-termini of two-fold symmetry-related VP4A capsid proteins and RNA, provides inter-pentamer stability. Based on its distinct structural elements and the genetic distance to other picorna-like viruses we propose that Nora virus, and a small group of related viruses, should have its own family within the order Picornavirales.

Capsid Structure of a Marine Algal Virus of the Order Picornavirales.

The order Picornavirales includes viruses that infect different kinds of eukaryotes and that share similar properties. The capsid proteins (CPs) of viruses in the order that infect unicellular organisms, such as algae, presumably possess certain characteristics that have changed little over the course of evolution, and thus these viruses may resemble the Picornavirales ancestor in some respects. Herein, we present the capsid structure of Chaetoceros tenuissimus RNA virus type II (CtenRNAV-II) determined using cryo-electron microscopy at a resolution of 3.1 Å, the first alga virus belonging to the family Marnaviridae of the order Picornavirales A structural comparison to related invertebrate and vertebrate viruses revealed a unique surface loop of the major CP VP1 that had not been observed previously, and further, revealed that another VP1 loop obscures the so-called canyon, which is a host-receptor binding site for many of the mammalian Picornavirales viruses. VP2 has an N-terminal tail, which has previously been reported as a primordial feature of Picornavirales viruses. The above-mentioned and other critical structural features provide new insights on three long-standing theories about Picornavirales: (i) the canyon hypothesis, (ii) the primordial VP2 domain swap, and (iii) the hypothesis that alga Picornavirales viruses could share characteristics with the Picornavirales ancestor.IMPORTANCE Identifying the acquired structural traits in virus capsids is important for elucidating what functions are essential among viruses that infect different hosts. The Picornavirales viruses infect a broad spectrum of hosts, ranging from unicellular algae to insects and mammals and include many human pathogens. Those viruses that infect unicellular protists, such as algae, are likely to have undergone fewer structural changes during the course of evolution compared to those viruses that infect multicellular eukaryotes and thus still share some characteristics with the Picornavirales ancestor. This article describes the first atomic capsid structure of an alga Marnavirus, CtenRNAV-II. A comparison to capsid structures of the related invertebrate and vertebrate viruses identified a number of structural traits that have been functionally acquired or lost during the course of evolution. These observations provide new insights on past theories on the viability and evolution of Picornavirales viruses.

Cryo-EM Studies of Virus-Antibody Immune Complexes.

Antibodies play critical roles in neutralizing viral infections and are increasingly used as therapeutic drugs and diagnostic tools. Structural studies on virus-antibody immune complexes are important for better understanding the molecular mechanisms of antibody-mediated neutralization and also provide valuable information for structure-based vaccine design. Cryo-electron microscopy (cryo-EM) has recently matured as a powerful structural technique for studying bio-macromolecular complexes. When combined with X-ray crystallography, cryo-EM provides a routine approach for structurally characterizing the immune complexes formed between icosahedral viruses and their antibodies. In this review, recent advances in the structural understanding of virus-antibody interactions are outlined for whole virions with icosahedral T = pseudo 3 (picornaviruses) and T = 3 (flaviviruses) architectures, focusing on the dynamic nature of viral shells in different functional states. Glycoprotein complexes from pleomorphic enveloped viruses are also discussed as immune complex antigens. Improving our understanding of viral epitope structures using virus-based platforms would provide a fundamental road map for future vaccine development.

Cryo-Electron Microscopy Structure of Seneca Valley Virus Procapsid.

Seneca Valley virus (SVV), like some other members of the Picornaviridae, forms naturally occurring empty capsids, known as procapsids. Procapsids have the same antigenicity as full virions, so they present an interesting possibility for the formation of stable virus-like particles. Interestingly, although SVV is a livestock pathogen, it has also been found to preferentially infect tumor cells and is being explored for use as a therapeutic agent in the treatment of small-cell lung cancers. Here we used cryo-electron microscopy to investigate the procapsid structure and describe the transition of capsid protein VP0 to the cleaved forms of VP4 and VP2. We show that the SVV receptor binds the procapsid, as evidence of its native antigenicity. In comparing the procapsid structure to that of the full virion, we also show that a cage of RNA serves to stabilize the inside surface of the virus, thereby making it more acid stable.IMPORTANCE Viruses are extensively studied to help us understand infection and disease. One of the by-products of some virus infections are the naturally occurring empty virus capsids (containing no genome), termed procapsids, whose function remains unclear. Here we investigate the structure and formation of the procapsids of Seneca Valley virus, to better understand how they form, what causes them to form, how they behave, and how we can make use of them. One potential benefit of this work is the modification of the procapsid to develop it for targeted in vivo delivery of therapeutics or to make a stable vaccine against SVV, which could be of great interest to the agricultural industry.

Cryo-EM study of slow bee paralysis virus at low pH reveals iflavirus genome release mechanism.

Viruses from the family Iflaviridae are insect pathogens. Many of them, including slow bee paralysis virus (SBPV), cause lethal diseases in honeybees and bumblebees, resulting in agricultural losses. Iflaviruses have nonenveloped icosahedral virions containing single-stranded RNA genomes. However, their genome release mechanism is unknown. Here, we show that low pH promotes SBPV genome release, indicating that the virus may use endosomes to enter host cells. We used cryo-EM to study a heterogeneous population of SBPV virions at pH 5.5. We determined the structures of SBPV particles before and after genome release to resolutions of 3.3 and 3.4 Å, respectively. The capsids of SBPV virions in low pH are not expanded. Thus, SBPV does not appear to form "altered" particles with pores in their capsids before genome release, as is the case in many related picornaviruses. The egress of the genome from SBPV virions is associated with a loss of interpentamer contacts mediated by N-terminal arms of VP2 capsid proteins, which result in the expansion of the capsid. Pores that are 7 Å in diameter form around icosahedral threefold symmetry axes. We speculate that they serve as channels for the genome release. Our findings provide an atomic-level characterization of the genome release mechanism of iflaviruses.

Honey Bee Deformed Wing Virus Structures Reveal that Conformational Changes Accompany Genome Release.

The picornavirus-like deformed wing virus (DWV) has been directly linked to colony collapse; however, little is known about the mechanisms of host attachment or entry for DWV or its molecular and structural details. Here we report the three-dimensional (3-D) structures of DWV capsids isolated from infected honey bees, including the immature procapsid, the genome-filled virion, the putative entry intermediate (A-particle), and the empty capsid that remains after genome release. The capsids are decorated by large spikes around the 5-fold vertices. The 5-fold spikes had an open flower-like conformation for the procapsid and genome-filled capsids, whereas the putative A-particle and empty capsids that had released the genome had a closed tube-like spike conformation. Between the two conformations, the spikes undergo a significant hinge-like movement that we predicted using a Robetta model of the structure comprising the spike. We conclude that the spike structures likely serve a function during host entry, changing conformation to release the genome, and that the genome may escape from a 5-fold vertex to initiate infection. Finally, the structures illustrate that, similarly to picornaviruses, DWV forms alternate particle conformations implicated in assembly, host attachment, and RNA release. IMPORTANCE: Honey bees are critical for global agriculture, but dramatic losses of entire hives have been reported in numerous countries since 2006. Deformed wing virus (DWV) and infestation with the ectoparasitic mite Varroa destructor have been linked to colony collapse disorder. DWV was purified from infected adult worker bees to pursue biochemical and structural studies that allowed the first glimpse into the conformational changes that may be required during transmission and genome release for DWV.

The novel asymmetric entry intermediate of a picornavirus captured with nanodiscs.

Many nonenveloped viruses engage host receptors that initiate capsid conformational changes necessary for genome release. Structural studies on the mechanisms of picornavirus entry have relied on in vitro approaches of virus incubated at high temperatures or with excess receptor molecules to trigger the entry intermediate or A-particle. We have induced the coxsackievirus B3 entry intermediate by triggering the virus with full-length receptors embedded in lipid bilayer nanodiscs. These asymmetrically formed A-particles were reconstructed using cryo-electron microscopy and a direct electron detector. These first high-resolution structures of a picornavirus entry intermediate captured at a membrane with and without imposing icosahedral symmetry (3.9 and 7.8 Å, respectively) revealed a novel A-particle that is markedly different from the classical A-particles. The asymmetric receptor binding triggers minimal global capsid expansion but marked local conformational changes at the site of receptor interaction. In addition, viral proteins extrude from the capsid only at the site of extensive protein remodeling adjacent to the nanodisc. Thus, the binding of the receptor triggers formation of a unique site in preparation for genome release.

Extended phylogenetic analysis of a new Israeli isolate of Brevicoryne brassicae virus (BrBV-IL) suggests taxonomic revision of the genus Iflavirus.

BACKGROUND: Brevicoryne brassicae virus (BrBV) is a positive-strand genomic RNA virus which is unassigned tentative member of the genus Iflavirus. BrBv was first identified and characterized in the late 90's in the cabbage aphid in the United Kingdom (UK) (J Gen Virol 88:2590-2595, 2007) and was fully sequenced, using random amplification of encapsidated RNA. No other reports have been published demonstrating detection of this virus outside the UK. FINDINGS: A new isolate of the cabbage aphid virus Brevicoryne brassicae virus was identified from Brevicoryne brassicae aphids growing on wild mustard plants (Sinapis arvensis) in northern Israel. The virus genome was partially assembled from purified siRNA using the Illumina MiSeq Sequencing System with limited success. Combining classical viral RNA purification and RT-PCR amplification followed by traditional Sanger sequencing enabled obtaining the complete genomic sequence. The Israeli strain of BrBV shared 95 % nucleotide sequence identity with the BrBV found in the United Kingdom. CONCLUSIONS: The detection of BrBV in Israel indicates a broader geographical distribution of the virus".

Complete genome sequence and structural characterization of a novel iflavirus isolated from Opsiphanes invirae (Lepidoptera: Nymphalidae).

Opsiphanes invirae (Lepidopera: Nymphalidae) is a common pest of the African oil palm tree (Elaeis guineensis) in Brazil. Dead larvae were collected in canopy of oil palm trees cultivated in the amazon region (Para State) and analyzed for viral infection. Electron microscopy of caterpillar extracts showed an icosahedral picorna-like virus particle with 30nm in diameter. Total RNA extracted from partially purified virus particles was sequenced. A contig of 10,083 nucleotides (nt) was identified and showed to encode one single predicted polyprotein with 3185 amino acid residues. Phylogenetic analysis showed that the new virus was closely related to another lepidopteran infective virus Spodoptera exigua iflavirus 1(SeIV-1), with 35% amino acid pairwise identity. The novel virus fulfils all ICTV requirements for a new iflavirus species and was named Opsiphanes invirae Iflavirus 1 (OilV-1).

Enterovirus replication: go with the (counter)flow.

All (+)RNA viruses replicate on distinct membranous domains; however, how they induce and maintain their unique lipid composition is largely unknown. Two recent studies reveal that enteroviruses harness the PI4P-cholestrol exchange cycle driven by OSBP1 protein and PI4 kinase(s), and that blocking the dynamic lipid flow inhibits virus replication.

Picornavirus morphogenesis.

The Picornaviridae represent a large family of small plus-strand RNA viruses that cause a bewildering array of important human and animal diseases. Morphogenesis is the least-understood step in the life cycle of these viruses, and this process is difficult to study because encapsidation is tightly coupled to genome translation and RNA replication. Although the basic steps of assembly have been known for some time, very few details are available about the mechanism and factors that regulate this process. Most of the information available has been derived from studies of enteroviruses, in particular poliovirus, where recent evidence has shown that, surprisingly, the specificity of encapsidation is governed by a viral protein-protein interaction that does not involve an RNA packaging signal. In this review, we make an attempt to summarize what is currently known about the following topics: (i) encapsidation intermediates, (ii) the specificity of encapsidation (iii), viral and cellular factors that are required for encapsidation, (iv) inhibitors of encapsidation, and (v) a model of enterovirus encapsidation. Finally, we compare some features of picornavirus morphogenesis with those of other plus-strand RNA viruses.

Membranous replication factories induced by plus-strand RNA viruses.

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two "morphotypes" of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments.

Picornavirus entry.

The essential event in picornavirus entry is the delivery of the RNA genome to the cytoplasm of a target cell, where replication occurs. In the past several years progress has been made in understanding the structural changes in the virion important for uncoating and RNA release. In addition, for several viruses the endocytic mechanisms responsible for internalization have been identified, as have the cellular sites at which uncoating occurs. It has become clear that entry is not a passive process, and that viruses initiate specific signals required for entry. And we have begun to recognize that for a given virus, there may be multiple routes of entry, depending on the particular target cell and the receptors available on that cell.

TaqMan MGB probe fluorescence real-time quantitative PCR for rapid detection of Chinese Sacbrood virus.

Sacbrood virus (SBV) is a picorna-like virus that affects honey bees (Apis mellifera) and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV) in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB) probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.

Picornaviruses.

The picornavirus family consists of a large number of small RNA viruses, many of which are significant pathogens of humans and livestock. They are amongst the simplest of vertebrate viruses comprising a single stranded positive sense RNA genome within a T = 1 (quasi T = 3) icosahedral protein capsid of approximately 30 nm diameter. The structures of a number of picornaviruses have been determined at close to atomic resolution by X-ray crystallography. The structures of cell entry intermediate particles and complexes of virus particles with receptor molecules or antibodies have also been obtained by X-ray crystallography or at a lower resolution by cryo-electron microscopy. Many of the receptors used by different picornaviruses have been identified, and it is becoming increasingly apparent that many use co-receptors and alternative receptors to bind to and infect cells. However, the mechanisms by which these viruses release their genomes and transport them across a cellular membrane to gain access to the cytoplasm are still poorly understood. Indeed, detailed studies of cell entry mechanisms have been made only on a few members of the family, and it is yet to be established how broadly the results of these are applicable across the full spectrum of picornaviruses. Working models of the cell entry process are being developed for the best studied picornaviruses, the enteroviruses. These viruses maintain particle integrity throughout the infection process and function as genome delivery modules. However, there is currently no model to explain how viruses such as cardio- and aphthoviruses that appear to simply dissociate into subunits during uncoating deliver their genomes into the cytoplasm.

The three-dimensional structure of Infectious flacherie virus capsid determined by cryo-electron microscopy.

Cryo-electron microscopy and image reconstruction were used to determine the three-dimensional structure of Infectious flacherie virus (IFV). 5047 particles were selected for the final reconstruction. The FSC curve showed that the resolution of this capsid structure was 18 A. The structure is a psuedo T=3 (P=3) icosahedral capsid with a diameter of 302.4 A and a single shell thickness of 15 A. The density map showed that IFV has a smooth surface without any prominent protrude or depression. Comparison of the IFV structure with those of the insect picorna-like virus-Cricket paralysis virus (CrPV)and human picornavirus-Human rhinovirus 14 (HRV 14) revealed that the IFV structure resembles the CrPV structure. The "Rossmann canyon" is absent in both IFV and CrPV particles. The polypeptide topology of IFV VP2, IFV VP3 was predicted and the subunit location at the capsid surface was further analyzed.

Characterization of structural proteins of Solenopsis invicta virus 1.

Purification of Solenopsis invicta virus 1 (SINV-1) from its host, S. invicta, and subsequent examination by electron microscopy revealed a homogeneous fraction of spherical particles with a diameter of 30-35 nm. Quantitative PCR with SINV-1-specific oligonucleotide primers verified that this fraction contained high copy numbers of the SINV-1 genome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the SINV-1 purified fraction revealed three major and one minor protein bands. The protein bands were labeled VP1 (40.8+/-1.4 kDa), VP2 (35.7+/-2.8 kDa), VP3 (25.2+/-1.8 kDa), and VP4 (22.2+/-2.5 kDa) based on mass. N-terminal sequence was acquired successfully for VP1, VP2, and VP3, but not VP4, and delineated each capsid protein within the 3'-proximal open reading frame of SINV-1. Positional organization of the viral proteins within the SINV-1 structural polyprotein was consistent with dicistroviruses (when based on sequence similarity). Blastp analysis of SINV-1 VP1, VP2, and VP3 revealed significant identity with corresponding structural capsid proteins of positive-strand RNA viruses, particularly acute bee paralysis virus (ABPV), Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV). Amino acid residues about the scissile bonds for VP1 and VP3 were consistent with dicistroviruses and insect-infecting picorna-like viruses. N-terminal sequencing of VP2 also established that translation initiation of the SINV-1 structural polyprotein was mediated by an internal ribosomal entry site and is AUG-independent.

AC-ELISA and RT-PCR assays for the diagnosis of triatoma virus (TrV) in triatomines (Hemiptera: Reduviidae) species.

Triatoma virus (TrV) is the only entomopathogenic virus found in triatomines. TrV replicates in cells of the midgut epithelium of triatomines, causing a high mortality rate and delayed development of the infected insect. In this work, we report an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of TrV infection. For antiserum production, rabbits and hens where inoculated with purified TrV. Antiserum reactivity was checked by immunodiffusion, and its specificity was confirmed by western blot and AC-ELISA. Totally 90 fecal samples from T. infestans were analysed. AC-ELISA and RT-PCR results correlated well with transmission electron microscopy (EM) observations, which are considered the gold standard, with Kappa values of 0.73 for AC-ELISA and 0.93 for RT-PCR when compared with EM. Applications and complementary uses of the two techniques reported in this work are discussed.