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Mx proteins are key factors of the innate intracellular defense mechanisms that act against viruses induced by type I/III interferons. The family Peribunyaviridae includes many viruses of veterinary importance, either because infection results in clinical disease or because animals serve as reservoirs for arthropod vectors. According to the evolutionary arms race hypothesis, evolutionary pressures should have led to the selection of the most appropriate Mx1 antiviral isoforms to resist these infections. Although human, mouse, bat, rat, and cotton rat Mx isoforms have been shown to inhibit different members of the Peribunyaviridae, the possible antiviral function of the Mx isoforms from domestic animals against bunyaviral infections has, to our knowledge, never been studied. Herein, we investigated the anti-Schmallenberg virus activity of bovine, canine, equine, and porcine Mx1 proteins. We concluded that Mx1 has a strong, dose-dependent anti-Schmallenberg activity in these four mammalian species. [ABSTRACT FROM AUTHOR]

Mx proteins are key factors of the innate intracellular defense mechanisms that act against viruses induced by type I/III interferons. The family Peribunyaviridae includes many viruses of veterinary importance, either because infection results in clinical disease or because animals serve as reservoirs for arthropod vectors. According to the evolutionary arms race hypothesis, evolutionary pressures should have led to the selection of the most appropriate Mx1 antiviral isoforms to resist these infections. Although human, mouse, bat, rat, and cotton rat Mx isoforms have been shown to inhibit different members of the Peribunyaviridae, the possible antiviral function of the Mx isoforms from domestic animals against bunyaviral infections has, to our knowledge, never been studied. Herein, we investigated the anti-Schmallenberg virus activity of bovine, canine, equine, and porcine Mx1 proteins. We concluded that Mx1 has a strong, dose-dependent anti-Schmallenberg activity in these four mammalian species.

Myxovirus resistance (Mx) proteins are dynamin-like GTPases that are inducible by interferons (IFNs) following virus infections. Most studies investigating Mx proteins have focused on their activity against influenza A viruses (IAV), although emerging evidence suggests that some Mx proteins may exhibit broader antiviral activity. Herein, we demonstrate that in addition to IAV, overexpression of mouse Mx1 (mMx1), but not mMx2, resulted in potent inhibition of growth of the human alphaherpesviruses herpes simplex virus 1 (HSV-1) and HSV-2, whereas neither inhibited the mouse betaherpesvirus murine cytomegalovirus (MCMV) in vitro. IFN induction of a functional endogenous mMx1 in primary mouse fibroblasts ex vivo was also associated with inhibition of HSV-1 growth. Using an in vitro overexpression approach, we demonstrate that mutations that result in redistribution of mMx1 from the nucleus to the cytoplasm or in loss of its combined GTP binding and GTPase activity also abrogated its ability to inhibit HSV-1 growth. Overexpressed mMx1 did not inhibit early HSV-1 gene expression but was shown to inhibit both replication of the HSV-1 genome as well as subsequent late gene expression. In a mouse model of cutaneous HSV-1 infection, mice expressing a functional endogenous mMx1 showed significant reductions in the severity of skin lesions as well as reduced HSV-1 titers in both the skin and dorsal root ganglia (DRG). Together, these data demonstrate that mMx1 mediates potent antiviral activity against human alphaherpesviruses by blocking replication of the viral genome and subsequent steps in virus replication. Moreover, endogenous mMx1 potently inhibited pathogenesis in the zosteriform mouse model of HSV-1 infection. IMPORTANCE While a number of studies have demonstrated that human Mx proteins can inhibit particular herpesviruses in vitro, we are the first to report the antiviral activity of mouse Mx1 (mMx1) against alphaherpesviruses both in vitro and in vivo. We demonstrate that both overexpressed mMx1 and endogenous mMx1 potently restrict HSV-1 growth in vitro. mMx1-mediated inhibition of HSV-1 was not associated with inhibition of virus entry and/or import of the viral genome into the nucleus, but rather with inhibition of HSV-1 genomic replication as well as subsequent late gene expression. Therefore, inhibition of human alphaherpesviruses by mMx1 occurs by a mechanism that is distinct from that reported for human Mx proteins against herpesviruses. Importantly, we also provide evidence that expression of a functional endogenous mMx1 can limit HSV-1 pathogenesis in a mouse model of infection. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 4 (IRF4), in conjunction with thermogenic regulation, is a negative regulator of immune responses. Therefore, we examined whether temperature changes regulated the antiviral response of IRF4 in nervous necrosis virus (NNV)-infected orange-spotted groupers. We found that osgIRF4 mRNA expression was responsive to poly I:C stimulation and NNV infection. In vitro overexpression of osgIRF4 caused a marked decrease in the promoter activity of the antiviral protein Mx1, and magnified NNV replication. Notably, we showed that the IAD domain of osgIRF4 exerted a dominant inhibitory effect on the Mx1 promoter. Furthermore, on exposure to high temperatures, the action of osgIRF4 was dependent on heat shock factor 1 (HSF1) expression. Additionally, small interfering RNA knockdown of HSF1 abrogated high temperature-mediated osgIRF4 activity. These findings suggest that osgIRF4 is an essential negative regulator of innate antiviral immunity and enhances viral replication during heat stress in the orange-spotted grouper. • Orange-spotted grouper IRF4 (osgIRF4) enhanced nervous necrosis virus infection. • OsgIRF4 suppressed the expression of Mx1 through the IAD domain. • The antiviral response of osgIRF4 is regulated by HSF1 during heat stress. [ABSTRACT FROM AUTHOR]

We compared the Mx expression and anti-viral function and the 3D structure of Mx protein in four species: chicken (Gallus gallus), whooper swan (Cygnus cygnus), jungle crow (Corvus macrorhynchos), and rock dove (Columba livia). We observed different mortalities associated with highly pathogenic avian influenza virus (HPAIV) infection to understand the relationship between Mx function as an immune response factor and HPAIV proliferation in bird cells. Different levels of Mx were observed among the different bird species after virus infection. Strong Mx expression was confirmed in the rock dove and whooper swan 6 hr after viral infection. The lowest virus copy numbers were observed in rock dove. The virus infectivity was significantly reduced in the BALB/3T3 cells expressing rock dove and jungle crow Mx. These results suggested that high Mx expression and significant Mx-induced anti-viral effects might result in the rock dove primary cells having the lowest virus copy number. Comparison of the expected 3D structure of Mx protein in all four bird species demonstrated that the structure of loop L4 varied among the investigated species. It was reported that differences in amino acid sequence in loop L4 affect antiviral activity in human and mouse cells, and a significant anti-viral effect was observed in the rock dove Mx. Thus, the amino acid sequence of loop L4 in rock dove might represent relatively high anti-viral activity. [ABSTRACT FROM AUTHOR]

Myxovirus resistance (Mx) proteins represents the subclass of the dynamin superfamily of large Guanosine triphosphates (GTPases), play esential role in intracellular vesicle trafficking, endocytosis, organelle homeostasis and mitochondria distribution. These proteins are key players of the vertebrate immune system, induced by type-I and type-III interferons (IFN) of infected host and inhibit viral replication by sequestering its nucleoprotein. In the present study, we report the sequencing and characterization of Cirrhinus mrigala Mx protein (CmMx) for the first time and observed its constitutive expression in different tissues for a period of fourteen days. The synthetic peptide, LSGVALPRGTGI, was dissolved in PBS and injected into a rabbit and the antibody raised against CmMx was used to study the level of its expression. The full length of the CmMx cDNA is 2244 bp with a molecular mass of 70.9 kDa and a predicted isoelectric point of 8.25. The 627 amino acids polypeptide formed of three main functional domains: N-terminal GTPase domain (GD), a middle domain (MD) and GTPase effector domain (GED) with carboxy terminal leucine zipper motif. The 3D models of CmMx protein was modeled based on available close structural homologs and further validated through molecular dynamics (MD) simulations. MD study revealed the importance of G-domain responsible for recognition of GTP, which perfectly corroborate with earlier studies. MM/PBSA binding free energy analysis displayed that van der Waals and electrostatic energy were the key driving force behind molecular recognition of GTP by CmMx protein. The results from this study will illuminate more lights into the ongoing research on myxovirus resistance protein and its role in inhibition of viral replication in other eukaryotic system as well. [ABSTRACT FROM AUTHOR]

Feline parvovirus (FPV) causes severe gastroenteritis and leukopenia in cats; the outcome is poor. Information regarding specific treatments is lacking. Class A CpG oligodeoxynucleotides (CpG-A) are short single-stranded DNAs, stimulating type I interferon production. In cats, CpG-A induced an antiviral response in vivo and inhibited FPV replication in vitro. The aim was to prospectively investigate the effects of CpG-A on survival, clinical score, hematological findings, antiviral response (cytokines), viremia, and fecal shedding (real-time qPCR) in cats naturally infected with FPV. Forty-two FPV-infected cats were randomized to receive 100 µg/kg of CpG-A (n = 22) or placebo (n = 20) subcutaneously, on admission and after 48 h. Blood and fecal samples were collected on admission, after 1, 3, and 7 days. All 22 cats showed short duration pain during CpG-A injections. The survival rate, clinical score, leukocyte and erythrocyte counts, viremia, and fecal shedding at any time-point did not differ between cats treated with CpG-A (50%) and placebo (40%). Antiviral myxovirus resistance (Mx) gene transcription increased in both groups from day 1 to 3 (p = 0.005). Antibodies against FPV on admission were associated with survival in cats (p = 0.002). In conclusion, CpG-A treatment did not improve the outcome in cats with FPV infection. FPV infection produced an antiviral response.

Viral attack within host cells triggers the production of type I interferons and leads to the induction of interferon stimulated genes (ISGs). One of the ISG Mx, encodes type I interferon inducible GTPase that is responsible for the establishment of an anti-viral state within cells. Intriguingly, several isoforms of Mx have been reported in fish, but the structural analysis of fish Mx proteins remains unexplored. For the first time, we have identified and unraveled the molecular structure of Mx protein from Indian snow trout, Schizothorax richardsonii (Gray) a Coldwater fish that inhabits the water bodies in the sub-Himalayan region. The snow trout Mx coding region consists of 2518 nucleotides with an open reading frame (ORF) of 1854 nucleotides. It codes for a polypeptide of 617 amino acids with a predicted molecular weight of 70 kDa. In silico analysis of snow trout Mx protein revealed signature of dynamin family (LPRGTGIVTR) along with a tripartite GTP-binding domain (GDQSSGKS, DLPG, and TKPD). Homology modelling established that the Mx protein is an elongated structure with a G domain, bundle signaling element (BSE) and a GTPase effector domain (GED). Moreover, the GED of Mx contains two highly conserved leucine zippers at the COOH-terminal of the protein suggesting its structural similarity with human homologues. However, snow trout Mx lacks the essential features of its mammalian homologues questioning its functional characteristics. Further, a ligand binding site in the said protein has also been predicted adjacent to the GTPase switch within the G domain. [ABSTRACT FROM AUTHOR]

Heart diseases caused by viruses are major causes of Atlantic salmon aquaculture loss. Two Atlantic salmon cardiovascular cell lines, an endothelial cell line (ASHe) from the heart and a fibroblast cell line (BAASf) from the bulbus arteriosus, were evaluated for their response to four fish viruses, CSV, IPNV, VHSV IVa and VHSV IVb, and the innate immune agonist, double-stranded RNA mimic poly IC. All four viruses caused cytopathic effects in ASHe and BAASf. However, ASHe was more susceptible to all four viruses than BAASf. When comparing between the viruses, ASHe cells were found to be moderately susceptible to CSV and VHSV IVb, but highly susceptible to IPNV and VHSV IVa induced cell death. All four viruses were capable of propagating in the ASHe cell line, leading to increases in virus titre over time. In BAASf, CSV and IPNV produced more than one log increase in titre from initial infection, but VHSV IVb and IVa did not. When looking at the antiviral response of both cell lines, Mx proteins were induced in ASHe and BAASf by poly IC. All four viruses induced Mx proteins in BAASf, while only CSV and VHSV IVb induced Mx proteins in ASHe. IPNV and VHSV IVa suppressed Mx proteins expression in ASHe. Pretreatment of ASHe with poly IC to allow for Mx proteins accumulation protected the culture from subsequent infections with IPNV and VHSV IVa, resulting in delayed cell death, reduced virus titres and reduced viral proteins expression. These data suggest that endothelial cells potentially can serve as points of infections for viruses in the heart and that two of the four viruses, IPNV and VHSV IVa, have mechanisms to avoid or downregulate antiviral responses in ASHe cells. Furthermore, the high susceptibility of the ASHe cell line to IPNV and VHSV IVa can make it a useful tool for studying antiviral compounds against these viruses and for general detection of fish viruses. [ABSTRACT FROM AUTHOR]

Bats serve as a reservoir for various, often zoonotic viruses, including significant human pathogens such as Ebola and influenza viruses. However, for unknown reasons, viral infections rarely cause clinical symptoms in bats. A tight control of viral replication by the host innate immune defense might contribute to this phenomenon. Transcriptomic studies revealed the presence of the interferon-induced antiviral myxovirus resistance (Mx) proteins in bats, but detailed functional aspects have not been assessed. To provide evidence that bat Mx proteins might act as key factors to control viral replication we cloned Mx1 cDNAs from three bat families, Pteropodidae, Phyllostomidae, and Vespertilionidae. Phylogenetically these bat Mx1 genes cluster closely with their human ortholog MxA. Using transfected cell cultures, minireplicon systems, virus-like particles, and virus infections, we determined the antiviral potential of the bat Mx1 proteins. Bat Mx1 significantly reduced the polymerase activity of viruses circulating in bats, including Ebola and influenza A-like viruses. The related Thogoto virus, however, which is not known to infect bats, was not inhibited by bat Mx1. Further, we provide evidence for positive selection in bat Mx1 genes that might explain species-specific antiviral activities of these proteins. Together, our data suggest a role for Mx1 in controlling these viruses in their bat hosts. [ABSTRACT FROM AUTHOR]

[Objectives]The purpose of this research was to investigate the antiviral activity of Mx proteins from four species(porcine, human, mouse and bovine)against vesicular stomatitis virus(VSV), and to reveal the conservatism of antiviral activities of cytoplasmic Mx proteins. [Methods]Specific primers were designed to amplify full-length genes of Mx proteins from four species by PCR and then eukaryotic expressing plasmids were constructed. After PK-15 cells overexpressing the recombinant plasmids, Western blot and indirect immunofluorescence assay(IFA)was used to detect the expressing activity and distribution of Mx proteins from four species in PK-15 cells. PK-15 cells overexpressing Mx proteins were infected with VSV for 8 or 16 h and harvested. The replication of VSV was analyzed using RT-qPCR, Western blot, IFA and plaque reduction experiment respectively. [Results]The full-length genes of Mx proteins from four species were successfully cloned and expressed, furthermore, the size of Mx proteins was correct. Although porcine Mx1 protein and mouse Mx2 protein were distributed in the cytoplasm in the form of rods, the porcine Mx2 protein, human MxA protein and bovine Mx1 protein were dispersed in the cytoplasm in the form of granules, all Mx proteins from four species can significantly inhibit the proliferation of VSV by inhibiting the transcription of VSV-G mRNA, reducing the synthesis of VSV-G protein and reducing the titer of progeny virus. [Conclusions]All Mx proteins from four species can significantly inhibit the proliferation of VSV in PK-15 cells. [ABSTRACT FROM AUTHOR]

The understanding of virus–host interactions relies on the knowledge of the regulatory mechanisms of the type I interferon (IFN I)-stimulated genes (ISGs). Among ISGs, those coding Mx proteins play a main role due to their direct antiviral activity. The study of these genes in gilthead seabream is interesting, since this species displays high natural resistance to viral diseases, being asymptomatic carrier of infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV). Gilthead seabream has three Mx genes ( Mx1 , Mx2 , and Mx3 ), encoding proteins with a wide spectrum of antiviral activity. The structure of the three promoters (pMx1, pMx2 and pMx3) has been previously disclosed, and their response to poly I:C in RTG-2 cells characterized. To further analyze these promoters, their response to two viral infections has been evaluated in the present study. For that purpose, RTG-2 cells transiently transfected with the luciferase gene under the control of each promoter were inoculated with either IPNV or VHSV at two different doses. The highest and lowest fold induction values were recorded for pMx2 and pMx3, respectively. The promoter induction was always stronger after VHSV inoculation than in IPNV-inoculated cells. In addition, the higher dose of VHSV tested induced higher response of the three promoters, whereas in IPNV-infected cells the highest induction was recorded after inoculation with the lower viral dose. To further study the response of the Mx2 promoter, RTG-2 cells stably transfected with the luciferase gene under the control of pMx2 were stimulated with poly I:C and subsequently infected with IPNV or VHSV. Interestingly, IPNV infection inhibited the induction caused by poly I:C, suggesting an antagonistic activity of IPNV on Mx2 transcription. In contrast, VHSV infection did not alter the response triggered by poly I:C. These results highlight the specific regulation that controls the activity of each promoter, and support the existence of complex interactions between host cells, specific Mx promoters, and viruses, which are responsible for the final outcome of a viral infection. [ABSTRACT FROM AUTHOR]

Due to their direct antiviral activity, Mx proteins play a main role in the response mediated by type I interferon against viral infections. The study on gilthead seabream Mx proteins is especially interesting, since this species is unusually resistant to viral diseases, being asymptomatic carrier of several viruses pathogenic to other fish species. Gilthead seabream has three Mx proteins (Mx1, Mx2 and Mx3) that, separately, display antiviral activity against a wide range of viruses, showing interesting differences in their antiviral specificities. In this work, the possible synergy between the three Mx isoforms has been studied using in vitro systems consisting of CHSE-214 cells stably expressing two or the three gilthead seabream Mx proteins. The antiviral activity of these Mx combinations has been tested against the Infectious Pancreatic Necrosis Virus (IPNV), the Viral Haemorrhagic Septicaemia Virus (VHSV), the European Sheatfish Virus (ESV) and the Lymphocystis Disease Virus (LCDV). A synergistic effect of the Mx proteins was only detected against ESV, no synergy was observed against LCDV, and a negative interference was detected against the two RNA viruses tested, IPNV and VHSV, as viral replication was higher in cells expressing certain Mx combinations than in cells expressing these proteins separately. These results suggest a functional interaction between gilthead seabream Mx isoforms, which results in a higher or lower antiviral activity depending on the virus tested, thus supporting the idea of complex virus-host interactions and finely tuned mechanisms controlling the antiviral activity of Mx proteins. [ABSTRACT FROM AUTHOR]

Abstract: Mx proteins are main effectors of the antiviral innate immune response mediated by type I interferon (IFN I). Actually, diverse Mx proteins from fish proved highly active against fish viruses, standing out among them the Mx1, Mx2 and Mx3 from gilthead seabream (Sparus aurata), a species exhibiting a natural resistance to viral diseases. In this study, the structure and functional activity of their corresponding promoters (pMx1, pMx2 and pMx3) have been assessed. The three promoters present an identical 3′ region of 157 bp, exhibiting a single canonical interferon-stimulated response element (ISRE), which is indispensible for the poli:IC induction of pMx1 and pMx3, while not for that of pMx2. In the remaining part of the three promoters other regulatory motifs were identified, as gamma IFN activated sites in variable number (1, 4 and 2 in pMx1, pMx2 and pMx3, respectively), as well as several independent GAAA elements or ISRE core sequences (13, 15 and 12 in pMx1, pMx2 and pMx3, respectively). The structural dissimilarities shown by the three promoters parallels with the differences observed in their response profiles, in terms of the time course of the induction, and basal and induced expression levels of each promoter. Altogether, these findings indicate that the expression of Mx1, Mx2 and Mx3 genes from the gilthead seabream might be specifically regulated, in accordance with the functional role of each Mx protein in the successful antiviral response shown by this species. [Copyright &y& Elsevier]

Abstract: Salmonid alphavirus (SAV) infection in cultured salmonids causes severe economic losses across Europe. Immune protection and antiviral mechanisms of the host against SAV are poorly characterised in vivo. Analysis of immune gene expression in head kidney of Atlantic salmon (Salmo salar L.) experimentally infected with SAV 1, using a quantitative reverse transcription polymerase chain reaction (qRT-PCR), revealed rapid induction of interferon I (INF-I), interferon II (INF-II) and INF-I associated Mx genes in SAV 1 infected fish compared to control fish injected with tissue culture supernatant. Mx protein was found to be highly expressed in the heart and mucosal membranes of infected fish by immunohistochemistry (IHC). Interestingly, the pathological changes that were observed in the target tissues of the virus became visible some time after peak expression of genes associated with the INF-I-pathway in head kidney tissue. These findings suggest that a non-specific antiviral immune response is rapidly induced during the early stages of SAV infection in salmon. [Copyright &y& Elsevier]

Abstract: Mx proteins are key components of the antiviral state triggered by interferon type I in response to viral infections. In this study, two different Mx genes have been identified in European sea bass (Dicentrarchus labrax), and their sequences were cloned and characterized. MxA cDNA consists of 1881bp coding for a putative 626 aminoacids protein, while MxB cDNA has 1920bp and results in a protein with 639 residues. Their corresponding genomic sequences contain 3538bp and 5326bp, respectively, and both present 12 exons and 11 introns. The expression patterns of the two Mx genes after an in vivo challenge with the viral nervous necrosis virus (VNNV), a serious pathogen in farmed European sea bass, have been characterized by real-time PCR. The results showed interesting differences in the transcription profile of both Mx, thus suggesting a differential role for each Mx isoform in the immune response of European sea bass to VNNV, and most likely in the general viral response of this species. [Copyright &y& Elsevier]

Interferons play a key role in fish resistance to viral infections by inducing the expression of antiviral proteins, such as Mx. The aim of the present study was to test the antiviral activity of the Senegalese sole Mx protein (SsMx) against RNA and DNA viruses pathogenic to fish, i.e. the infectious pancreatic necrosis virus (IPNV, dsRNA), the viral haemorrhagic septicaemia virus (VHSV, ssRNA), and the European sheatfish virus (ESV, dsDNA), using a CHSE-214 cell clone expressing this antiviral protein. A strong inhibition of IPNV and VHSV replication was recorded in SsMx-expressing cells, as has been shown by the virus yield reduction and the decrease in the synthesis of the viral RNA encoding the polyprotein (for IPNV) and the nucleoprotein (for VHSV). The titres of these viruses replicating on SsMx-expressing cells were 100 times lower than those recorded on non-transfected cells. In contrast, SsMx did not inhibit ESV replication since no significant differences were observed regarding the virus yield or the major capsid protein gene transcription in transfected and non-transfected cells. [ABSTRACT FROM AUTHOR]

Abstract: Mx is a structural protein, induced by type I interferon (IFN), with direct antiviral properties. In fish the inherent contribution of Mx protein to viral protection is unknown. The transgenic Chinook salmon embryonic (CHSE)-TOF cell line was genetically modified to express the rainbow trout Mx (rbtMx1) protein under the control of the tetracycline derivative, doxycycline (DOX). Two clones CHSE-TOF-MX8 and CHSE-TOF-MX10 were isolated and characterised by qPCR. The level of resistance to Infectious Pancreatic Necrosis Virus (IPNV), Salmon Alphavirus (SAV), Infectious Haematopoietic Necrosis Virus (IHNV) and Epizootic Haematopoietic Necrosis Virus (EHNV) of the CHSE-TOF, CHSE-TOF-MX8 and CHSE-TOF-MX10 cell lines cultivated with and without DOX was measured. A novel method was established to measure accurately the level of sensitivity of any given viral isolate to Mx protein. IPNV and SAV viruses were highly sensitive to the presence of rbtMx1 in the cells whereas IHNV and EHNV showed partial resistance suggesting contrasting viral evasion strategies between these categories of viruses. [Copyright &y& Elsevier]

Abstract: Type I interferons are secreted by infected cells and promote an antiviral state in neighbouring cells by the induction of numerous genes, some of which present antiviral activity, as the Mx proteins. In this study, three different Mx cDNAs (Mx1, Mx2 and Mx3) from gilthead seabream (Sparus aurata), the most important fish species in Southern European aquaculture, have been cloned and characterized. A Southern blot assay revealed the existence of three Mx loci, thus the three Mx isoforms correspond to three different genes that seem to have a common origin. The genomic sequences of Mx1, Mx2 and Mx3 have been completely obtained, and consist on 11 introns and 12 exons in a full length of 5971bp, 7391bp and 6938bp, respectively. As a first approach to the functional meaning of these three genes, their response to the viral nervous necrosis virus (VNNV) infection was tested. Important differences in terms of tissue, time course and level of induction were found between them, thus suggesting a differential functional role for each isoform, which can represent a key point in the natural resistance of this fish species, that has been repeatedly reported as an asymptomatic carrier of VNNV. [Copyright &y& Elsevier]

Abstract: Nervous necrosis virus (NNV) belongs to the betanodavirus of the Nodaviridae family. It is the causative agent of viral nervous necrosis (VNN) disease, and has inflicted devastating damage on the world-wide aquaculture industry. The fish that survived after the outbreak of VNN become persistently NNV-infected carriers. NNV-persistent infection has been demonstrated in a barramundi brain (BB) cell line, and it involves the type I interferon (IFN) response with the expression of Mx gene. However, little of the defense mechanism in fish cells against NNV infection is understood. In this study, the anti-NNV mechanism of barramundi Mx protein (BMx) was elucidated in cBB cells which were derived from BB cell line after serial treatments by NNV-specific antiserum and then became an NNV-free cell line. After NNV infection of cBB cells, the level of viral RNA-dependent RNA polymerase (RdRp) increased with time over a period of 24 h post-infection (hpi), but decreased when the BMx expression increased 48 and 72 hpi. When the expression of BMx was down-regulated by BMx-specific siRNA, the expression levels of viral RNA, proteins and progeny viral titers were restored. The BMx was found to colocalize with viral RdRp at the perinuclear area 24 hpi and coprecipitate with viral RdRp, indicating that they could bind with each other. Viral RdRp was also revealed to colocalize with lysosomes 48 hpi as the NNV RdRp level started to decline. Therefore, it is suggested that BMx inhibited the viral RNA synthesis by interaction with viral RdRp, and redistributed RdRp to perinuclear area for degradation. [Copyright &y& Elsevier]

Summary: Expression of interferon-α (IFN-α) and Mx protein in the livers of pigs experimentally infected with swine hepatitis E virus (HEV) was examined immunohistochemically. Five infected and five non-infected pigs were killed at 3, 7, 14, 21, 29 and 50 days post-inoculation (dpi). Tissues were collected from each pig at necropsy. Multifocal lymphoplasmacytic hepatitis (P =0.042) and focal hepatocellular necrosis (P =0.037) were significantly more frequent in the swine HEV-infected pigs than in the non-infected control pigs. Immunohistochemical analysis detected expression of IFN-α and Mx protein by macrophages/Kupffer cells, lymphocytes and hepatocytes in the livers of infected pigs. [Copyright &y& Elsevier]

Biliary atresia (BA) is the most frequent indication for paediatric liver transplantation. We tested the hypothesis of a viral aetiology of this disease by screening liver samples of a large number of BA patients for the common human hepatotropic viruses. Moreover, we correlated our findings to the expression of Mx protein, which has been shown to be significantly up-regulated during viral infections. Seventy-four liver biopsies (taken during Kasai portoenterostomy) were tested by polymerase chain reaction (PCR) for DNA viruses (herpes simplex virus [HSV], Epstein-Barr virus [EBV], varicella zoster virus [VZV], cytomegalovirus [CMV], adenovirus, parvovirus B19 and polyoma BK) and RNA viruses (enteroviruses, rotavirus and reovirus 3). Mx protein expression was assessed by immunohistochemistry. Virus DNA/RNA was found in less than half of the biopsies (8/74 CMV, 1/74 adenovirus; 21/64 reovirus, 1/64 enterovirus). A limited number presented with double infection. Patients that had detectable viral RNA/DNA in their liver biopsies were significantly older than virus-free patients (P = 0.037). The majority (54/59) of the liver biopsies showed expression of Mx proteins in hepatocytes, bile ducts and epithelium. Our data suggest that the known hepatotropic viruses do not play a major role in the aetiology and progression of BA. Their incidence appears to be, rather, a secondary phenomenon. Nonetheless, the inflammatory response in the livers of BA patients mimics that observed during viral infections. [ABSTRACT FROM AUTHOR]

Abstract: The expression of Mx transcripts and Mx protein was monitored at weekly intervals for 7weeks, by qRT-PCR and immunohistochemistry, in the kidney, liver, gill and blood of Atlantic salmon parr following injection of poly I:C. Elevated levels of Mx transcripts compared to PBS injected control fish were found in the tissues at week 1. Background levels were then found up to week 7, with the exception of week 4 when high levels were again found in poly I:C injected fish as well as control fish. Immunostaining for Mx protein in the kidney, liver and gill of poly I:C injected fish was higher than in control fish from weeks 1–4, but little staining was found in the tissues of both poly I:C treated and control fish thereafter. Blood monocytes stained consistently in all fish, suggesting that this leucocyte type constitutively expressed Mx protein. From weeks 2–4, lymphocytes of both groups consistently stained for Mx protein but the consistency decreased at weeks 5–7. Staining of neutrophils was also inconsistent. Western blots of plasma showed an immunoreactive band of 76kDa typical of salmon Mx protein. Semi-quantitative measurements of dot blots showed poly I:C injected fish to have higher levels of plasma Mx protein than controls on weeks 1–4 with very low levels on weeks 5–7. The results indicate that following induction of an interferon response with poly I:C, Atlantic salmon parr maintain elevated levels of Mx protein in tissues, leucocytes and blood plasma for about 4weeks. Production of Mx protein by blood monocytes appears to be constitutive, though production by lymphocytes and neutrophils was less consistent. [Copyright &y& Elsevier]

Abstract: Transfection with synthesized virus-specific small interfering RNAs (siRNAs) efficiently inhibits viral replication in viral-infected fish cell lines, implying the involvement of RNA interference (RNAi)-related pathways in the antiviral response of fish cells. Here, we demonstrate that plasmid expressing virus-encoded pre-microRNAs (pre-miRNAs) can also inhibit viral replication through these pathways. By incorporating sequences encoding miRNAs specific to major capsid protein (MCP) gene of red sea bream iridovirus (RSIV) and a miRNA specific to hirame rhabdovirus (HIRRV) genome into a murine miR-155 pre-miRNA backbone, we were able to intracellularly express viral pre-miRNAs (miR-MCPs and miR-HIRRV) in a fish cell line. The miR-MCPs and miR-HIRRV, delivered as pre-miRNA precursors in transfected cells, inhibited viral replication when these cells were infected with the target virus. Although this may suggest sequence-specific interference, inhibitory effect on viral replication was also observed in cells transfected with a plasmid expressing pre-miRNA targeting β-galactosidase gene (miR-LacZ) that served as a specificity control. Expression of pre-miRNAs was found to activate interferon (IFN)-related pathways, correlating with upregulation of the antiviral IFN-induced Mx protein. The antiviral effects of viral-miRNAs observed here were partly the result of the antiviral miRNA-related pathways and partly the result of the antiviral IFN-related pathways. We propose that engineered virus-encoded pre-miRNA can engage not only RNAi-related pathways but also IFN-related pathways to induce potent antiviral responses in fish cells. [Copyright &y& Elsevier]

Abstract: A polyclonal rabbit antiserum directed against the conserved region of the Atlantic salmon antiviral Mx1 protein was used to detect the putative Atlantic cod Mx protein using Western and dot blotting. A doublet band at about 75kDa and 65kDa was detected by Western blotting in kidney and spleen extracts of cod 3 and 4 days after i.p. injection with poly I:C but not in control fish injected with PBS. In blood leucocyte lysates, similar immunostaining could also be detected in Atlantic cod weakly after injection with PBS and more intensely after injection with poly I:C, suggesting some constitutive expression of Mx protein by leucocytes. Dot blot analysis showed that the Mx protein level was significantly higher in spleen, kidney, liver and gill of cod at least up to 4 days after injection with poly I:C when compared with the PBS-injected controls. [Copyright &y& Elsevier]

Summary: Over-expression of grouper Mx negatively regulated nodavirus activity through direct interaction, likely via the binding and perturbation of the intracellular localization of nodavirus coat protein. Deletion analysis of grouper Mx indicated that the coat protein binds to the effector domain of Mx. The presence of grouper Mx in a poly [I:C] interferon system inhibited nodavirus infection, demonstrating that grouper Mx over-expression has an inhibitory effect on both coat protein and RNA-dependent RNA polymerase of nodavirus antigens, which results in reduced viral yields. We conclude that grouper Mx has a key role in cellular resistance to nodavirus infection. [Copyright &y& Elsevier]

Abstract: Betanodaviruses and megalocytiviruses are the causative agents of viral nervous necrosis and iridoviral disease, respectively, among marine species farmed in Taiwan. Because there are few cell lines susceptible to these viruses, we wished to identify additional lines with which to study viral pathology and epidemiology. Thus, we established two clonal cell lines designated GBC1 and GBC4 from the brain of an orange-spotted grouper, Epinephelus coioides. Both cell lines grew well in Leibovitz''s L-15 medium supplemented with 5% (GBC1) or 10% (GBC4) fetal bovine serum at temperatures between 20 °C and 35 °C. Cytokeratin immunofluorescence staining revealed that both cell lines were of epithelial origin. GBC4 cells expressed glial fibril acidic protein suggesting, that they are astroglial lineage cells. The modal diploid chromosome number was 44 and 48 for GBC1 and GBC4, respectively. GBC1 cells were highly susceptible to grouper nerve necrosis virus (GNNV) and yielded titers of 1010 TCID50 ml−1 but were non-susceptible to the giant seaperch iridovirus (GSIV). By contrast, GBC4 cells were susceptible to GSIV with titers approaching 109 TCID50 ml−1 whereas GNNV infection only yielded titers of 106 TCID50 ml−1. GNNV propagated in GBC1 across a wide range of temperatures (15–37 °C) whereas in GBC4, GSIV propagated over 15–30 °C. Induction of the Mx protein upon GNNV infection occurred in GBC4 but not in GBC1, suggesting that the Mx protein inhibits virus production. Our results suggest that these two cell lines provide a valuable tool for the isolation and investigation of betanodavirus and megalocytivirus. [Copyright &y& Elsevier]

Abstract: Type I (α/β) interferons (IFNs) are a family of cytokines that stimulate the expression of numerous proteins that mediate an antiviral state in uninfected cells. Two Atlantic salmon (Salmo salar) IFN-α (SasaIFN-α1 & 2) genes have previously been cloned and both were found to contain a putative N-linked glycosylation site. Recombinant SasaIFN-α1 (rSasaIFN-α1) produced in eukaryotic systems has repeatedly been shown to confer antiviral properties. However, different IFN-α subtypes may exhibit differential antiviral activities and be subject to glycosylation. To evaluate the potential therapeutic impact of a rSasaIFN-α, the mature form of the SasaIFN-α2 protein was produced in a high-level Escherichia coli expression system. Expression of the rSasaIFN-α2 was detected by SDS-PAGE and Western blot, and its identity was confirmed by mass spectrometry. In the homologous Chinook salmon embryonic (CHSE-214) cell line, the rSasaIFN-α2 incited early expression of the IFN-induced Mx protein and exhibited high antiviral activity of about 2.8×106 Umg−1 against infectious pancreatic necrosis virus (IPNV). Conversely, antiviral protection by rSasaIFN-α2 was not observed in a heterologous Japanese flounder embryo (HINAE) cell line. Hence, a biologically active form of rSasaIFN-α2 was successfully produced using a glycosylation-deficient prokaryotic system and purified to homogeneity, suggesting that glycosylation is not required for its antiviral activity. [Copyright &y& Elsevier]

Abstract: Senegalese sole, Solea senegalensis, is a flat fish of growing interest in European aquaculture. In its culture viral infections are constant threats, thus understanding antiviral defences is a key factor for a successful industry. Mx proteins are IFN-induced proteins widespread in eukaryotes; however, their antiviral activity is unclear and the results variable among species. Therefore assessment of the putative Mx antiviral activity in each species is of interest. Our group has recently cloned the Senegalese sole Mx (SsMx) cDNA and in this study its antiviral activity was assessed by infecting CHSE-214 cells expressing recombinant SsMx, with sole aquabirnavirus. The antiviral activity against this pathogen was demonstrated by reduction in induced cytopathic effects, reduction in virus yield and decrease in viral transcripts. These findings contribute to our understanding of fish antiviral mechanisms and open the possibility of using this protein as a tool for fighting viral infections in aquaculture. [Copyright &y& Elsevier]

Abstract: The transcription of Mx mRNA after poly I:C induction and sole aquabirnavirus infection has been analysed in SAF-1 and TV-1 cells (derived from gilt-head seabream and turbot, respectively). Both cell lines were stimulated with 10μgml−1 poly I:C and Mx mRNA was analysed by a specific RT-PCR at several times post-induction. The results showed a high level of Mx expression from 12 to 120h after induction in SAF-1 cells, whereas in TV-1 cells Mx mRNA was only detected at 12 and 24h. The treatment with different concentrations of poly I:C showed that TV-1 cells are less sensitive to this inductor than the SAF-1 cell line. The antiviral activity derived from poly I:C induction has been clearly demonstrated against sole aquabirnavirus on both cell lines. The inoculation of sole aquabirnavirus resulted in the Mx mRNA transcription at 48, 72, and 96h post-infection (p.i.) in SAF-1 cells. On the contrary, inoculated TV-1 cells only showed a faint Mx mRNA band at 24 and 48h p.i. This study has established different patterns of Mx expression in both cells under study as a consequence of the poly I:C induction and sole aquabirnavirus infection, and it shows that gilt-head seabream and turbot Mx inhibit sole aquabirnavirus replication. [Copyright &y& Elsevier]

Abstract: A rabbit antiserum was produced from a 12-amino acid long peptide common to the 3 known isoforms of Atlantic salmon Mx proteins. The antibody stained ASK-1 cells 48h after stimulation with poly I:C. In Western blots of these cells, the antibody stained a doublet with MW about 75kDa and another band at about 65kDa, typical of the MW of Atlantic salmon Mx. Western blots of kidney from IPNV-injected salmon showed a similar staining pattern. In immunohistochemistry, the antibody stained the gill, kidney and liver tissue of a fish infected with IPNV by cohabitation. These tissues also expressed high levels of interferon (IFN) and Mx transcripts as determined by real-time qRT-PCR. Normal healthy salmon post-smolts sampled at 4–8 weeks after transfer to sea water had very low-level expression of IFN and Mx transcripts. However, at 4 and 5 weeks after sea water transfer the gill, kidney and liver of these fish stained strongly for Mx protein. Thereafter, immunostaining of Mx markedly diminished in all tissues, persisting weakly in the gill. It has been reported that Atlantic salmon smolts constitutively express IFN and Mx transcripts around the time of smolting. Presumably the Mx protein detected in the tissues for about 6 weeks after transfer to sea water resulted from such a transcriptional event. As Mx is known to provide protection against IPNV infections it is tempting to associate the duration of persistence of Mx protein with the outbreaks of IPN-related mortalities in post-smolts, 6–8 weeks after transfer to sea water. [Copyright &y& Elsevier]

Abstract: Double-stranded RNA and type I interferon-like activity induce an antiviral state in vertebrate cells and in several fish cell lines by increasing the expression of proteins that inhibit virus replication. We compared the protection induced by the polyinosinic:polycytidylic acid (poly I:C) or poly I:C plus transfection agents against the infectious pancreatic necrosis virus (IPNV) and the infectious hematopoietic necrosis virus (IHNV) in BF-2 cells, with that induced in RTG-2, CHSE-214, or SAF cells. In addition, we examined the reduction in the infective titers of these viruses and the correlation with Mx protein expression as IFN marker. Furthermore, the suitability of BF-2 cells for the evaluation and optimization of immune responses in an IPNV-IHNV co-infection was assessed. The results demonstrated strong anti-IPNV and anti-IHNV activity (around 90% of infected cells surviving) in BF-2 cells transfected with poly I:C, in which a loss of 1log10 or 3log10 of the IPNV or IHNV infective titers, respectively, was observed. No antiviral activity was evident in the cells incubated with poly I:C alone. The protection recorded in the co-infection experiments was comparable with those of the single infections. The SAF cell line exhibited the lowest antiviral capacity (45%), which was also increased after transfection with poly I:C. In addition, medium from transfected BF-2 provided protection against IPNV (1log10 loss of infective titer) and IHNV (2log10 loss of infective titer) in new monolayers, indicating that these cells secreted the factors that induce antiviral activity. A correlation between antiviral activity and Mx protein expression was observed in all the cells. These results indicate that poly I:C transfection could improve IFN-like production in these cell lines. However, the antiviral effectiveness of poly I:C differed between cell lines. On the basis of our findings, we conclude that the BF-2 cell line is a useful model in which to study the role of IFN-induced cytokines in resistance against single or double infections with salmonid fish viruses. [Copyright &y& Elsevier]

Abstract: Interferons (IFNs) are secreted proteins, which induce vertebrate cells into an antiviral state. In mammals, three families of IFNs (type I IFN, type II IFN and IFN-λ) can be distinguished on the basis of gene structure, protein structure and functional properties. Type I IFNs, which include IFN-α and IFN-β, are encoded by intron lacking genes and have a major role in the first line of defense against viruses. The human IFN-λs have similar biological properties as type I IFNs, but are encoded by intron containing genes. Type II IFN is identical to IFN-γ, which is produced by T helper 1 cells in response to mitogens and antigens and has a key role in adaptive cell mediated immunity. IFNs, which show structural and functional properties similar to mammalian type I IFNs, have recently been cloned from Atlantic salmon, channel catfish, pufferfish, and zebrafish. Teleost fish appear to have at least two type I IFN genes. Phylogenetic sequence analysis shows that the fish type I IFNs form a group separated from the avian type I IFNs and the mammalian IFN-α, -β and -λ groups. Interestingly, the fish IFNs possess the same exon/intron structure as the IFN-λs, but show most sequence similarity to IFN-α. Recently, IFN-γ genes have also been cloned from several fish species and shown to have the same exon/intron structure as mammalian IFN-γ genes. The antiviral effect of mammalian type I IFN is exerted through binding to the IFN-α/β-receptor, which triggers signal transduction through the JAK–STAT signal transduction pathway resulting in expression of Mx and other antiviral proteins. Putative IFN receptor genes have been identified in pufferfish. Several interferon regulatory factors and members of the JAK–STAT pathway have also been identified in various fish species. Moreover, Mx and several other interferon stimulated genes have been cloned and studied in fish. Furthermore, antiviral activity of Mx protein from Atlantic salmon and Japanese flounder has recently been demonstrated. [Copyright &y& Elsevier]

Abstract: Molecular cloning and nucleotide sequencing of cDNA encoding an orange-spotted grouper (Epinephelus coioides) homolog of Mx (“OsgMx”) was conducted and its possible role in fish immunity was analysed. Similar to mammalian Mx, the OsgMx are members of a family of interferon-inducible genes that are expressed by cells in response to nodavirus and iridovirus naturally-infected. Expression of OsgMx mRNA was noticeably upregulated in all tissues by nodavirus naturally-infected grouper. The transcription of OsgMx gene increased 6 h after intramuscular injection of nodavirus experimentally-infected fish and peaked at 72 h in their brains. Analysis of the 5′-flanking sequence of the gene shows that as in pufferfish and zebrafish, the OsgMx promoter contains two potential interferon-stimulated response element (ISRE) responsible for the induction of interferon-inducer polyinosinic-polycytidylic acid (Poly[I:C]). Transient transfection of grouper cells in gfp-reporter gene assays shows that the activation of the grouper Mx promoter fragment by Poly[I:C] is sufficient to allow the expression of green fluorescent protein (GFP). These results may provide a possible regulated pathway against nodavirus. [Copyright &y& Elsevier]

Abstract: Mx proteins are interferon-inducible GTPases that possess antiviral properties in vertebrates. Japanese flounder (Paralichthys olivaceus) Mx protein has previously been shown to possess some antiviral activity against rhabdoviruses. A polyclonal antibody was generated against a purified peptide fragment of Japanese flounder Mx protein that had been produced in an Escherichia coli expression system. The PAb detected the approximately 71kDa Mx protein from Japanese flounder (hirame) natural embryo (HINAE) cells that had been cultured with poly I:C, an interferon inducer, but not in unstimulated cells. The polyclonal antibody did not cross react with Mx protein from carp epithelial, grouper fin and zebrafish embryo cell lines that had been similarly induced or transfected with poly I:C. By immunofluorescence cytochemistry, Japanese flounder Mx protein was localized to the cell cytoplasm. Hirame rhabdovirus stimulated expression of Mx protein in the infected and surrounding HINAE cells. Within virus-infected cells, there was some indication of Mx protein colocalizing with viral proteins. Poly I:C stimulation of HINAE cells induced an early increase in Mx protein mRNA transcripts, but maximum Mx mRNA transcript and protein expression was reached after 48h. Both Mx mRNA transcripts and protein levels were maintained till at least 72h. [Copyright &y& Elsevier]

Mx proteins are antiviral GTPases that are induced by type I interferons in vertebrates. An Atlantic halibut Mx cDNA (HHMx) was recently cloned. In this work, a polyclonal antiserum against HHMx protein was generated that detected a 71 kDa protein in the nuclei of Chinook salmon embryo cells transfected with the HHMx cDNA. Mx protein expression in organs of halibut was studied by immunoblot analysis after injection with the double-stranded RNA poly I:C or infectious pancreatic necrosis virus. Poly I:C stimulated increased Mx protein expression in liver, kidney, heart, spleen, gills and intestine. The Mx protein level in liver reached a maximum after 3 days and remained elevated for 14 days after treatment. IPNV infection resulted in increased Mx protein in liver from 4 to at least 35 days. Immunocytochemical detection of Mx proteins in blood smears from poly I:C treated halibut indicated that a cytoplasmic Mx form might exist in this species. Detection of Mx proteins in blood leukocytes could thus work as an early non-lethal test for viral infections. [Copyright &y& Elsevier]

The resistance of Japanese flounder (Paralichthys olivaceus Temminck et Schlegel) against a viral haemorrhagic septicaemia virus (VHSV) challenge induced by a preceding non-lethal aquabirnavirus (ABV) challenge was investigated through experimental dual-infections with different intervals between the two challenges. The non-specific protection conferred by the primary ABV infection against the secondary VHSV infection commenced at Day 3 and persisted up to Day 14 but vanished at Day 21 post-ABV challenge. The in vitro assay using HINAE (hirame natural embryo) cells demonstrated anti-VHSV activity in the serum of ABV-challenged flounder from Day 1 to Day 14 but not at Day 21 post-ABV challenge. A high expression of a Mx gene, a molecular marker of type I interferon(s) (IFN) occurred in the head kidneys of ABV-challenged flounder from Day 1 to Day 7. These results suggest that the non-specific protection against the secondary VHSV infection in flounder was due to IFN(s) induced by the primary ABV infection. [Copyright &y& Elsevier]

An attempt was made to determine whether amino acid variation at position 631 in the chicken Mx protein definitely influences antiviral specificity, using an artificial mutation technique by which a single amino acid was reciprocally substituted between Ser (AGT) and Asn (AAT) at position 631 of the negative and positive chicken Mx, respectively. Using permanently transfected 3T3 cell lines, the antiviral potential of chicken Mx against vesicular stomatitis virus infection was analysed. The results indicated that the phenotype of antiviral activity depends on the amino acid difference at position 631; that is, the genotype coding Asn at position 631 corresponds to the positive antiviral phenotype, and the genotype coding Ser corresponds to the negative phenotype. The present study has confirmed that the antiviral specificity of chicken Mx protein is determined by an amino acid substitution at the carboxy terminus. [ABSTRACT FROM AUTHOR]

Mx proteins are induced by type I interferons (IFN α and β) in mice and humans and inhibit the replication of orthomyxoviruses and some other single-stranded RNA viruses. Recently, Mx genes have been cloned from Atlantic salmon. Mx transcripts were shown to be induced in head-kidney, liver and gills of the fish by the synthetic double-stranded RNA polyinosinic polycytidylic acid (poly I:C). In the present work we have studied expression of Mx protein in organs of Atlantic salmon treated with poly I:C. A quantitative immunoblot method was established to monitor expression of Mx protein and to compare relative amounts of Mx protein in different organs. Treatment of Atlantic salmon with poly I:C increased the relative amount of Mx protein in liver, stomach, hindgut, head-kidney and spleen. In gills the levels of Mx protein were similar in control fish and poly I:C treated fish. Immunohistochemistry of tissue sections from liver, head-kidney and gills from poly I:C treated fish was in accordance with the immunoblotting data and showed staining for Mx protein in several different cell types. Classification of infectious salmon anaemia virus as an orthomyxovirus makes it a putative target for Atlantic salmon Mx protein. Atlantic salmon treated with poly I:C showed reduced cumulative mortality compared to the control fish when challenged with infectious salmon anaemia virus (ISAV) by intraperitoneal injection. This demonstrates that poly I:C has some protective effect against ISAV in vivo. [Copyright &y& Elsevier]

Hepatitis C virus (HCV), especially the genotype 1, is naturally resistant to the antiviral effects of interferon-alpha (IFN-α). Expression of the whole HCV genome and the NS5A protein has been suggested to interfere with the antiviral activity of IFN-α. Here we have analyzed the effect of individual or various combinations of HCV proteins on IFN-α-mediated antiviral effect against vesicular stomatitis virus (VSV). When the structural proteins (core-E1-E2) of HCV genotype 1 were expressed in human osteosarcoma cells in a tetracycline-regulated manner, partial VSV resistance to IFN-α was established. This was seen as an enhancement of both viral protein synthesis and production of infectious virus. Priming of core-E1-E2-expressing cells with low doses of IFN-γ (10 IU/ml) partially restored the antiviral activity of IFN-α. The core (high-level expression) and NS4B protein expression also showed some rescue of VSV replication. In this model cell system NS3A–NS4A complex and NS5A showed no inhibition of IFN-α-induced antiviral activity. Our results indicate that the expression of structural proteins of HCV may impair the antiviral activity of IFNs. [Copyright &y& Elsevier]

Myxovirus resistance (Mx) proteins are antiviral GTPases induced by type I interferons (IFNs). In chickens, a single Mx protein variant, S631N, has been suggested to possess antiviral activity. However, the impact of this variant on chicken Mx (chMx) protein structure and conformation has not been investigated. Hence, in this study, we applied computational methods such as molecular modeling, molecular dynamic simulation, inter domain motion and residue networks to examine the structure and dynamic behavior of wild-type and mutant chMx. At first, we built 3-dimensional structural models for both wild-type and mutant chMx proteins, which revealed that the structural organization of chMx was similar to that of human Mx proteins. Subsequently, molecular dynamics simulations revealed that angle variation around the hinge1 region led to the different stalk domain conformations between the wild-type and mutant chMx proteins. Domain motion analysis further suggested that the conformational differences in the loop region surrounded by the mutant residue may lead to an inclined stalk domain conformation in the mutant compared to the wild-type protein. In addition, we performed betweenness centrality analysis from residue interaction networks, to identify the crucial residues for intramolecular signal flow in chMx. The results of this study provided information on the differences in structure and dynamics between wild-type and mutant chMx, which may aid in understanding the structural features of the S631N mutant, that may be associated with chMx protein antiviral activity.Communicated by Ramaswamy H. Sarma.