Minding the message: tactics controlling RNA decay, modification, and translation in virus-infected cells.

With their categorical requirement for host ribosomes to translate mRNA, viruses provide a wealth of genetically tractable models to investigate how gene expression is remodeled post-transcriptionally by infection-triggered biological stress. By co-opting and subverting cellular pathways that control mRNA decay, modification, and translation, the global landscape of post-transcriptional processes is swiftly reshaped by virus-encoded factors. Concurrent host cell-intrinsic countermeasures likewise conscript post-transcriptional strategies to mobilize critical innate immune defenses. Here we review strategies and mechanisms that control mRNA decay, modification, and translation in animal virus-infected cells. Besides settling infection outcomes, post-transcriptional gene regulation in virus-infected cells epitomizes fundamental physiological stress responses in health and disease.

The roles and mechanisms of endoplasmic reticulum stress-mediated autophagy in animal viral infections.

The endoplasmic reticulum (ER) is a unique organelle responsible for protein synthesis and processing, lipid synthesis in eukaryotic cells, and the replication of many animal viruses is closely related to ER. A considerable number of viral proteins are synthesised during viral infection, resulting in the accumulation of unfolded and misfolded proteins in ER, which in turn induces endoplasmic reticulum stress (ERS). ERS further drives three signalling pathways (PERK, IRE1, and ATF6) of the cellular unfolded protein response (UPR) to respond to the ERS. In numerous studies, ERS has been shown to mediate autophagy, a highly conserved cellular degradation mechanism to maintain cellular homeostasis in eukaryotic cells, through the UPR to restore ER homeostasis. ERS-mediated autophagy is closely linked to the occurrence and development of numerous viral diseases in animals. Host cells can inhibit viral replication by regulating ERS-mediated autophagy, restoring the ER's normal physiological process. Conversely, many viruses have evolved strategies to exploit ERS-mediated autophagy to achieve immune escape. These strategies include the regulation of PERK-eIF2α-Beclin1, PERK-eIF2α-ATF4-ATG12, IRE1α-JNK-Beclin1, and other signalling pathways, which provide favourable conditions for the replication of animal viruses in host cells. The ERS-mediated autophagy pathway has become a hot topic in animal virological research. This article reviews the most recent research regarding the regulatory functions of ERS-mediated autophagy pathways in animal viral infections, emphasising the underlying mechanisms in the context of different viral infections. Furthermore, it considers the future direction and challenges in the development of ERS-mediated autophagy targeting strategies for combating animal viral diseases, which will contribute to unveiling their pathogenic mechanism from a new perspective and provide a scientific reference for the discovery and development of new antiviral drugs and preventive strategies.

Current research on viral proteins that interact with fibrillarin.

The nucleolus is a multifunctional nuclear domain primarily dedicated to ribosome biogenesis. Certain viruses developed strategies to manipulate host nucleolar proteins to facilitate their replication by modulating ribosomal RNA (rRNA) processing. This association interferes with nucleolar functions resulting in overactivation or arrest of ribosome biogenesis, induction or inhibition of apoptosis, and affecting stress response. The nucleolar protein fibrillarin (FBL) is an important target of some plant and animal viruses. FBL is an essential and highly conserved S-adenosyl methionine (SAM) dependent methyltransferase, capable of rRNA degradation by its intrinsically disordered region (IDR), the glycine/arginine-rich (GAR) domain. It forms a ribonucleoprotein complex that directs 2'-O-methylations in more than 100 sites of pre-rRNAs. It is involved in multiple cellular processes, including initiation of transcription, oncogenesis, and apoptosis, among others. The interaction with animal viruses, including human viruses, triggered its redistribution to the nucleoplasm and cytoplasm, interfering with its role in pre-rRNA processing. Viral-encoded proteins with IDRs as nucleocapsids, matrix, Tat protein, and even a viral snoRNA, can associate with FBL, forcing the nucleolar protein to undergo atypical functions. Here we review the molecular mechanisms employed by animal and human viruses to usurp FBL functions and the effect on cellular processes, particularly in ribosome biogenesis.

Detection and molecular characterisation of bovine Enterovirus in Brazil: four decades since the first report.

It is suggested that bovine enteroviruses (BEV) are involved in the aetiology of enteric infections, respiratory disease, reproductive disorders and infertility. In this study, bovine faecal samples collected in different Brazilian states were subjected to RNA extraction, reverse transcription-polymerase chain reaction analysis and partial sequencing of the 5'-terminal portion of BEV. One hundred and three samples were tested with an overall positivity of 14.5%. Phylogenetic analysis clustered these BEV Brazilian samples into the Enterovirus F clade. Our results bring an important update of the virus presence in Brazil and contribute to a better understanding of the distribution and characterisation of BEV in cattle.

The measles virus nucleocapsid protein tail domain is dispensable for viral polymerase recruitment and activity.

Paramyxovirus genomes are ribonucleoprotein (RNP) complexes consisting of nucleoprotein (N)-encapsidated viral RNA. Measles virus (MeV) N features an amino-terminal RNA-binding core and a 125-residue tail domain, of which only the last 75 residues are considered fully mobile on the nucleocapsid surface. A molecular recognition element (MoRE) domain mediates binding of the viral phosphoprotein (P). This P N-tail interaction is considered instrumental for recruiting the polymerase complex to the template. We have engineered MeV N variants with tail truncations progressively eliminating the MoRE domain and upstream tail sections. Confirming previous reports, RNPs with N truncations lacking the carboxyl-terminal 43-residues harboring the MoRE domain cannot serve as polymerase template. Remarkably, further removal of all tail residues predicted to be surface-exposed significantly restores RNP bioactivity. Insertion of structurally dominant tags into the central N-tail section reduces bioactivity, but the negative regulatory effect of exposed N-tail stems is sequence-independent. Bioactive nucleocapsids lacking exposed N-tail sections are unable to sustain virus replication, because of weakened interaction of the advancing polymerase complex with the template. Deletion of the N-MoRE-binding domain in P abrogates polymerase recruitment to standard nucleocapsids, but polymerase activity is partially restored when N-tail truncated RNPs serve as template. Revising central elements of the current replication model, these data reveal that MeV polymerase is capable of productively docking directly to the nucleocapsid core. Dispensable for polymerase recruitment, N-MoRE binding to P-tail stabilizes the advancing polymerase-RNP complex and may rearrange unstructured central tail sections to facilitate polymerase access to the template.

Antiviral drugs in animal-derived matrices: A review.

The ban of antiviral drugs in food-producing animals in several parts of the world, latest by Commission Delegated Regulation (EU) 2022/1644, has increased the need for food control laboratories to develop analytical methods and perform official controls. However, little is known about antiviral drugs, their use, and its analysis in food-producing animals in the EU. This review aims to provide insights into relevant viruses, antiviral drugs, and animal-derived matrices for analytical method development and monitoring purposes to implement in food control laboratories. For years, animal viruses, such as African swine fever and avian influenza, have caused many outbreaks. Besides, they led to large economic losses due to the applied control measures and a lack of available treatments. Considering these losses and the known effectiveness of authorized human antiviral drugs in different organisms, medicines such as amantadine in Chinese poultry have been misused. Various analytical methods, including screening assays and sensors (published 2016-2023), and mass spectrometry methods (published 2012-2023) have been outlined in this review for the monitoring of antiviral drugs in animal-derived matrices. However, pharmacokinetics information on metabolite formation and distribution of these substances in different animal-derived matrices is incomplete. Additionally, apart from a few countries, there is a lack of available data on the potential misuse of different antiviral drugs in animal-derived matrices. Although a handful of important antiviral drugs, such as influenza, broad-spectrum, antiretroviral, and herpes drugs, and animal-derived matrices, such as chicken muscle, are identified, the priority of the scope should be further specified by closing the aforementioned gaps.

Breaking Barriers: Animal viruses as oncolytic and immunotherapeutic agents for human cancers.

Oncolytic viruses, defined as viruses capable of lysing cancer cells, emerged as a groundbreaking class of therapeutic entities poised to revolutionize cancer treatment. Their mode of action encompasses both direct tumor cell lysis and the indirect enhancement of anti-tumor immune responses. Notably, four leading contenders in this domain, Rigvir® in Latvia, T-VEC in the United States, H101 in China and Teserpaturev (DELYTACT®) in Japan, have earned approval for treating metastatic melanoma (Rigvir and T-VEC), nasopharyngeal carcinoma and malignant glioma, respectively. Despite these notable advancements, the integration of oncolytic viruses into cancer therapy encounters several challenges. Foremost among these hurdles is the considerable variability observed in clinical responses to oncolytic virus interventions. Moreover, the adaptive immune system may inadvertently target the oncolytic viruses themselves, diverting immune resources away from tumor antigens and undermining therapeutic efficacy. Another significant limitation arises from the presence of preexisting immunity against oncolytic viruses in certain patient populations, hampering treatment outcomes. To circumvent this obstacle, researchers are investigating the utilization of animal viruses, for which humans lack preexisting immunity, as a compelling alternative to human-derived counterparts. In our comprehensive review, we delve into the intricate nuances of oncolytic virotherapy, elucidating the multifaceted mechanisms through which these viruses exert their anti-cancer effects. Furthermore, we provide a thorough examination of animal-derived oncolytic viruses, highlighting their respective strengths and limitations. Lastly, we explore the promising potential of leveraging animal viruses as potent oncolytic agents, offering new avenues for enhancing the efficacy and reach of human cancer therapeutics.

Risk assessment of SARS-CoV-2 replicating and evolving in animals.

The retransmissions of SARS-CoV-2 from several mammals - primarily mink and white-tailed deer - to humans have raised concerns for the emergence of a new animal-derived SARS-CoV-2 variant to worsen the pandemic. Here, we discuss animal species that are susceptible to natural or experimental infection with SARS-CoV-2 and can transmit the virus to mates or humans. We describe cutting-edge techniques to assess the impact of a mutation in the viral spike (S) protein on its receptor and on antibody binding. Our review of spike sequences of animal-derived viruses identified nine unique amino acid exchanges in the receptor-binding domain (RBD) that are not present in any variant of concern (VOC). These mutations are present in SARS-CoV-2 found in companion animals such as dogs and cats, and they exhibit a higher frequency in SARS-CoV-2 found in mink and white-tailed deer, suggesting that sustained transmissions may contribute to maintaining novel mutations. Four of these exchanges, such as Leu452Met, could undermine acquired immune protection in humans while maintaining high affinity for the human angiotensin-converting enzyme 2 (ACE2) receptor. Finally, we discuss important avenues of future research into animal-derived viruses with public health risks.

Viruses and Cajal Bodies: A Critical Cellular Target in Virus Infection?

Nuclear bodies (NBs) are dynamic structures present in eukaryotic cell nuclei. They are not bounded by membranes and are often considered biomolecular condensates, defined structurally and functionally by the localisation of core components. Nuclear architecture can be reorganised during normal cellular processes such as the cell cycle as well as in response to cellular stress. Many plant and animal viruses target their proteins to NBs, in some cases triggering their structural disruption and redistribution. Although not all such interactions have been well characterised, subversion of NBs and their functions may form a key part of the life cycle of eukaryotic viruses that require the nucleus for their replication. This review will focus on Cajal bodies (CBs) and the viruses that target them. Since CBs are dynamic structures, other NBs (principally nucleoli and promyelocytic leukaemia, PML and bodies), whose components interact with CBs, will also be considered. As well as providing important insights into key virus-host cell interactions, studies on Cajal and associated NBs may identify novel cellular targets for development of antiviral compounds.

Environmental Factors and Their Threshold Affecting the Survival of Five Aquatic Animal Viruses in Different Animal Cells.

Aquatic animal viruses infect and transmit in aquatic environments, causing serious harm to the aquaculture industry and a variety of wild aquatic animals. How are they affected by environmental factors and do they represent potential threat to mammalian heath or not? Here, the effects of environmental factors (ultraviolet radiation (UV), temperature, pH, and drying) and their threshold on five epidemic aquatic animal viruses infecting amphibians and bony fish, including Rana grylio virus (RGV), Andrias davidianus ranavirus (ADRV), Grass carp reovirus (GCRV), Paralichthys olivaceus rhabdovirus (PORV), and Scophthalmus maximus rhabdovirus (SMRV), were measured and compared in a fish cell line. The examination of virus titers after different treatment in fish cells showed that the two iridoviruses, RGV and ADRV, had a higher tolerance to all of the environmental factors, such as they only had a decay rate of 22-36% when incubated at 37 °C for 7 days. However, the rhabdovirus SMRV was sensitive to all of the factors, with a decay rate of more than 80% in most of the treatments; even a complete inactivation (100%) can be observed after drying treatment. To address the potential threat to mammals, infectivity and limitation factors of the five viruses in Baby hamster kidney fibroblast cells (BHK-21) were tested, which showed that three of the five viruses can replicate at a low temperature, but a high temperature strongly inhibited their infection and none of them could replicate at 37 °C. This study clarified the sensitivity or tolerance of several different types of aquatic animal viruses to the main environmental factors in the aquatic environment and proved that the viruses cannot replicate in mammalian cells at normal physiological temperature.

Universal Virucidal Activity of Calcium Bicarbonate Mesoscopic Crystals That Provides an Effective and Biosafe Disinfectant.

We investigated the virucidal effects in solution of a new type of disinfectant, calcium bicarbonate mesoscopic crystals, designated CAC-717, against various types of virus. CAC-717 in solution is alkaline (pH 12.4) and has a self-electromotive force that generates pulsed electrical fields. Upon application to human skin, the pH of the solution becomes 8.4. CAC-717 contains no harmful chemicals and is thus non-irritating and harmless to humans and animals. Its virucidal effects were tested against six types of animal virus: enveloped double-strand (ds)-DNA viruses, non-enveloped ds-DNA viruses, non-enveloped single strand (ss)-DNA viruses, enveloped ss-RNA viruses, non-enveloped ss-RNA viruses, and non-enveloped ds-RNA viruses. The treatment resulted in a reduction in viral titer of at least 3.00 log10 to 6.38 log10. Fetal bovine serum was added as a representative organic substance. When its concentration was ≥20%, the virucidal effect of CAC-717 was reduced. Real-time PCR revealed that CAC-717 did not reduce the quantity of genomic DNA of most of the DNA viruses, but it greatly reduced that of the genomic RNA of most of the RNA viruses. CAC-717 may therefore be a useful biosafe disinfectant for use against a broad range of viruses.

Review of antiviral peptides for use against zoonotic and selected non-zoonotic viruses.

Viruses remain one of the leading causes of animal and human disease. Some animal viral infections spread sporadically to human populations, posing a serious health risk. Particularly the emerging viral zoonotic diseases such as the novel, zoonotic coronavirus represent an actual challenge for the scientific and medical community. Besides human health risks, some animal viral infections, although still not zoonotic, represent important economic loses to the livestock industry. Viral infections pose a genuine concern for which there has been an increasing interest for new antiviral molecules. Among these novel compounds, antiviral peptides have been proposed as promising therapeutic options, not only for the growing body of evidence showing hopeful results but also due to the many adverse effects of chemical-based drugs. Here we review the current progress, key targets and considerations for the development of antiviral peptides (AVPs). The review summarizes the state of the art of the AVPs tested in zoonotic (coronaviruses, Rift Valley fever viruses, Eastern Equine Encephalitis Virus, Dengue and Junín virus) and also non-zoonotic farm animal viruses (avian and cattle viruses). Their molecular target, amino acid sequence and mechanism of action are summarized and reviewed. Antiviral peptides are currently on the cutting edge since they have been reported to display anti-coronavirus activity. Particularly, the review will discuss the specific mode of action of AVPs that specifically inhibit the fusion of viral and host-cell membranes for SARS-CoV-2, showing in detail some important features of the fusion inhibiting peptides that target the spike protein of these risky viruses.

Intestinal Viral Loads and Inactivation Kinetics of Livestock Viruses Relevant for Natural Casing Production: a Systematic Review and Meta-Analysis.

Animal intestines are the source of edible sausage casings, which are traded worldwide and may come from areas where notifiable infectious animal diseases are prevalent. To estimate the risks of virus contamination, knowledge about the quantity of virus and decimal reduction values of the standard preservation method by salting is of great importance. A literature search, based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, was performed in search engine CAB Abstracts to determine the viral load of 14 relevant animal viruses in natural casings or intestines. Only a very limited number of scientific publications per virus were found and viral loads in the intestines varied from high for ASFV (five publications), BVDV (3), CSFV (6), PPRV (3), RPV(2) and TGEV (3) to moderate for PEDV (2) and SVDV (3), low for HEV (2) and FMDV (5), very low for VESV (1) and negative for PrV (2) and VSV (1). PRRSV was found in intestines, however, viral titers were not published. Three viruses (BVDV, CSFV and PPRV) with high viral loads were selected to search for their inactivation kinetics. For casings, no inactivation data were found, however, thermal inactivation data of these viruses were available, but differed in quantity, quality and matrices. In conclusion, important data gaps still exist when it comes to the quantitative inactivation of viruses in sausage casings or livestock intestines.

A Fluorescent Real-Time Plaque Assay Enables Single-Cell Analysis of Virus-Induced Cytopathic Effect by Live-Cell Imaging.

Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to countable plaques that are visualized with standard techniques such as crystal violet, neutral red, or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluorescent dyes with differential cell permeability to visualize them by live-cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applications in virology.

Serological relationship between a novel ovine pestivirus and classical swine fever virus.

The genus Pestivirus comprises globally distributed members of the family Flaviviridae, which cause severe losses in livestock. The most common species of the genus are bovine viral diarrhoea virus type 1 (BVDV-1) and type 2 (BVDV-2), classical swine fever virus (CSFV) and border disease virus (BDV). Recently, a novel ovine pestivirus was repeatedly detected in aborted lamb foetuses on a farm located in the Brescia Province (Italy). Complete genome characterization of this isolate showed that it was highly divergent from known pestivirus species and that it was genetically closely related to CSFV. The aim of this study was to determine the serological relatedness between the identified novel pestivirus and BVDV, BDV and CSFV selected strains for which homologous serum was available, by antigenic characterization performed using cross-neutralization assays. The serological relatedness was expressed as the coefficient of antigenic similarity (R). Both field and specific antisera raised against the ovine pestivirus neutralized the CSFV reference strain Diepholz with titres significantly higher than those specific for the BDV and BVDV strains. Furthermore, the calculated R values clearly indicated that the novel ovine pestivirus is antigenically more related to CSFV than to ruminant pestiviruses, in agreement with the results of the genomic analysis. This would have severe consequences on CSFV serology in the event of a switch to porcine hosts with implications for CSFV surveillance and porcine health management.

Comparative Metagenomics of Palearctic and Neotropical Avian Cloacal Viromes Reveal Geographic Bias in Virus Discovery.

Our understanding about viruses carried by wild animals is still scarce. The viral diversity of wildlife may be best described with discovery-driven approaches to the study of viral diversity that broaden research efforts towards non-canonical hosts and remote geographic regions. Birds have been key organisms in the transmission of viruses causing important diseases, and wild birds are threatened by viral spillovers associated with human activities. However, our knowledge of the avian virome may be biased towards poultry and highly pathogenic diseases. We describe and compare the fecal virome of two passerine-dominated bird assemblages sampled in a remote Neotropical rainforest in French Guiana (Nouragues Natural Reserve) and a Mediterranean forest in central Spain (La Herrería). We used metagenomic data to quantify the degree of functional and genetic novelty of viruses recovered by examining if the similarity of the contigs we obtained to reference sequences differed between both locations. In general, contigs from Nouragues were significantly less similar to viruses in databases than contigs from La Herrería using Blastn but not for Blastx, suggesting that pristine regions harbor a yet unknown viral diversity with genetically more singular viruses than more studied areas. Additionally, we describe putative novel viruses of the families Picornaviridae, Reoviridae and Hepeviridae. These results highlight the importance of wild animals and remote regions as sources of novel viruses that substantially broaden the current knowledge of the global diversity of viruses.

Tenth Scientific Biennial Meeting of the Australasian Virology Society-AVS10 2019.

The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2-5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on "Successfully transitioning from post-doc to lab head", winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia.

Adenoviruses across the animal kingdom: a walk in the zoo.

Adenoviruses (AdVs) infect representatives of numerous species from almost every major vertebrate class, albeit their incidence shows great variability. AdVs infecting birds, reptiles, and bats are the most common and diverse, whereas only one AdV has been so far isolated both from fish and amphibians. The family Adenoviridae is divided into five genera, each corresponding to an independent evolutionary lineage that supposedly coevolved with its respective vertebrate hosts. Members of genera Mastadenovirus and Aviadenovirus seem to infect exclusively mammals and birds, respectively. The genus Ichtadenovirus includes the single known AdV from fish. The majority of AdVs in the genus Atadenovirus originated from squamate reptiles (lizards and snakes), but also certain mammalian and avian AdVs are classified within this genus. The genus Siadenovirus contains the only AdV isolated from frog, along with numerous avian AdVs. In turtles, members of a sixth AdV lineage have been discovered, pending official recognition as an independent genus. The most likely scenario for AdV evolution includes long-term cospeciation with the hosts, as well as occasional switches between closely or, rarely, more distantly related hosts.

Virus Entry: Looking Back and Moving Forward.

Research over a period of more than half a century has provided a reasonably accurate picture of mechanisms involved in animal virus entry into their host cells. Successive steps in entry include binding to receptors, endocytosis, passage through one or more membranes, targeting to specific sites within the cell, and uncoating of the genome. For some viruses, the molecular interactions are known in great detail. However, as more viruses are analyzed, and as the focus shifts from tissue culture to in vivo experiments, it is evident that viruses display considerable redundancy and flexibility in receptor usage, endocytic mechanism, location of penetration, and uncoating mechanism. For many viruses, the picture is still elusive because the interactions that they engage in rely on sophisticated adaptation to complex cellular functions and defense mechanisms.

Assessment of air sampling methods and size distribution of virus-laden aerosols in outbreaks in swine and poultry farms.

Swine and poultry viruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and highly pathogenic avian influenza virus (HPAIV), are economically important pathogens that can spread via aerosols. The reliability of methods for quantifying particle-associated viruses as well as the size distribution of aerosolized particles bearing these viruses under field conditions are not well documented. We compared the performance of 2 size-differentiating air samplers in disease outbreaks that occurred in swine and poultry facilities. Both air samplers allowed quantification of particles by size, and measured concentrations of PRRSV, PEDV, and HPAIV stratified by particle size both within and outside swine and poultry facilities. All 3 viruses were detectable in association with aerosolized particles. Proportions of positive sampling events were 69% for PEDV, 61% for HPAIV, and 8% for PRRSV. The highest virus concentrations were found with PEDV, followed by HPAIV and PRRSV. Both air collectors performed equally for the detection of total virus concentration. For all 3 viruses, higher numbers of RNA copies were associated with larger particles; however, a bimodal distribution of particles was observed in the case of PEDV and HPAIV.