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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.

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.

Type I/III interferons provide powerful and universal innate intracellular defense mechanisms against viruses. Among the antiviral effectors induced, Mx proteins of some species appear as key components of defense against influenza A viruses. It is expected that such an antiviral protein must display a platform dedicated to the recognition of said viruses. In an attempt to identify such platform in human MxA, an evolution-guided approach capitalizing on the antagonistic arms race between MxA and its viral targets and the genomic signature it left on primate genomes revealed that the surface-exposed so-called "loop L4", which protrudes from the compact structure of the MxA stalk, is a hotspot of recurrent positive selection. Since MxA is archetypic of Mx1 proteins in general, we reasoned that the L4 loop also functions as a recognition platform for influenza viruses in the Mx1 proteins of other species that had been exposed to the virus for ever. In this study, the anti-influenza activity of 5 distinct mammalian Mx1 proteins was measured by comparing the number of viral nucleoprotein-positive cells 7 h after infection in a sample of 100,000 cells expected to contain both Mx1-positive and Mx1-negative cell subpopulations. The systematic depletion (

Myxovirus resistance proteins represent a family of interferon-induced restriction factors of the innate and adaptive immune system. Human MxB acts as a novel restriction factor with antiviral activity against a range of HIV-1 and other retroviruses mainly by inhibiting the uncoating process after reverse transcription but prior to integration. Based on published data and conservation analysis, we propose a novel hypothesis, in which MxB dimers form higher order oligomers that restrict retroviral replication by binding to the viral capsid. Insights into the mechanistic basis of structural and functional characteristics of MxB will greatly advance our understanding of MxB.

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 &micro, g/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.

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. IMPORTANCE Bats are a natural reservoir for various viruses that rarely cause clinical symptoms in bats but are dangerous zoonotic pathogens, like Ebola or rabies virus. It has been hypothesized that the interferon system might play a key role in controlling viral replication in bats. We speculate that the interferon-induced Mx proteins might be key antiviral factors of bats and have coevolved with bat-borne viruses. This study evaluated for the first time a large set of bat Mx1 proteins spanning three major bat families for their antiviral potential, including activity against Ebola virus and bat influenza A-like virus, and we describe here their phylogenetic relationship, revealing patterns of positive selection that suggest a coevolution with viral pathogens. By understanding the molecular mechanisms of the innate resistance of bats against viral diseases, we might gain important insights into how to prevent and fight human zoonotic infections caused by bat-borne viruses.

非病原性アクアビルナウイルス(ABV)を予め接種され、その 7 日後にベータノダウイルス(NNV)で攻撃されたマハタは NNV 感染に対して強い抵抗性を示した。他方、ヒラメではNNVに対する感受性が低いため死亡率からABVの効果をみることはできなかったが、脳および腎臓におけるNNVの消長をみると、両魚種とも ABV 処理魚において明らかにウイルス感染力価の低下が認められた。この防御は、ABVにより誘導されるインターフェロン関連タンパク質に因ると考えられた。, Experimental dual-infections with a non-lethal aquabirnavirus (ABV) and a lethal betanodavirus (redspotted grouper nervous necrosis virus: RGNNV) were carried out in Japanese flounder Paralichthys olivaceus and sevenband grouper Epinephelus septemfasciatus. In the dual-infection group, ABV was intramuscularly (IM) injected into fish seven days before the IM-injection with RGNNV. In the experiments with flounder, a high expression of an Mx gene, a molecular marker for type I interferon(s) (IFM) production, occurred in the head kidneys and brains at Day 7 post-ABV injection. Although-no mortality was found not only in the dual-infected group but also in the single infection group with RGNNV (control group), the infective titers of RGNNV in the tissues of the dual-infected group were significantly lower at any sampling times than those in the control group. In the experiments with grouper, the preceding ABV infection resulted in complete protection against RGNNV infection. The infective titers of RGNNV in the tissues were also lower in the dual-infected group than in the control group throughout the experiments, and finally the virus disappeared from the head kidneys and brains of the dual-infected group at Day 14 and Day 56 postinjections, respectively. These results suggest that an ABV-induced IFN(s) effectively suppresses the progression of secondary betanodavirus infection.

We have recently identified the two major determinants of the glycoprotein G of the viral hemorrhagic septicaemia rhabdovirus (gpG(VHSV)), peptides p31 and p33 implicated in triggering the host type I IFN antiviral response associated to these rhabdoviral antigens. With the aim to investigate the properties of these viral glycoprotein regions as DNA molecular adjuvants, their corresponding cDNA sequences were cloned into a plasmid (pMCV1.4) flanked by the signal peptide and transmembrane sequences of gpG(VHSV). In addition, a plasmid construct encoding both sequences p31 and p33 (pMCV1.4-p31 + p33) was also designed. In vitro transitory cell transfection assays showed that these VHSV gpG regions were able to induce the expression of type I IFN stimulated genes as well as to confer resistance to the infection with a different fish rhabdovirus, the spring viremia of carp virus (SVCV). In vivo, zebrafish intramuscular injection of only 1 mu g of the construct pMCV1.4-p31 + p33 conferred fish protection against SVCV lethal challenge up to 45 days post-immunization. Moreover, pMCV1.4-p31 + p33 construct was assayed for molecular adjuvant city's for a DNA vaccine against SVCV based in the surface antigen of this virus (pAE6-G(SVCV)). The results showed that the co-injection of the SVCV DNA vaccine and the molecular adjuvant allowed (i) a ten-fold reduction in the dose of pAE6-GSVCV without compromising its efficacy (ii) an increase in the duration of protection, and (iii) an increase in the survival rate. To our knowledge, this is the first report in which specific IFN-inducing regions from a viral gpG are used to design more-efficient and cost-effective viral vaccines, as well as to improve our knowledge on how to stimulate the innate immune system Refereed/Peer-reviewed

Type I interferons (IFN-α/β) provide powerful and universal innate intracellular defense mechanisms against viruses. Among the antiviral effectors induced by IFN-α/β, Mx proteins of some species appear as key components of defense against influenza A viruses. The body of work published to date suggests that to exert anti-influenza activity, an Mx protein should possess a GTP-binding site, structural bases allowing multimerisation, and a specific C-terminal GTPase effector domain (GED). Both the human MxA and bovine Mx1 proteins meet these minimal requirements, but the bovine protein is more active against influenza viruses. Here, we measured the anti-influenza activity exerted by 2 human/bovine chimeric Mx proteins. We show that substituting the bovine GED for the human one in human MxA does not affect the magnitude of anti-influenza activity. Strikingly, however, substituting the human GED for the bovine one in bovine Mx1 yields a chimeric protein with a much higher anti-influenza activity than the human protein. We conclude, in contradiction to the hypothesis currently in vogue in the literature, that the GED is not the sole determinant controlling the magnitude of the anti-influenza activity exercised by an Mx protein that can bind GTP and multimerise. Our results suggest that 1 or several motifs that remain to be discovered, located N-terminally with regard to the GED, may interact with a viral component or a cellular factor so as to alter the viral cycle. Identifying, in the N-terminal portion of bovine Mx1, the motif(s) responsible for its higher anti-influenza activity could contribute to the development of new anti-influenza molecules.

Anti-IFNbeta antibodies are related to IFNbeta bioavailability loss in multiple sclerosis. We investigated the reliability of radioimmunoprecipitation and cytopathic assays in detecting binding (BAbs) and neutralizing (NAbs) antibodies and the correlation of these antibodies to MxA mRNA production. Eleven percent of IFNbeta-treated patients showed a lack of MxA induction, with an inverse correlation between MxA mRNA and the presence of BAbs and NAbs. Some patients had contemporary MxA induction in the presence of high NAb titres, thus calling into question the reliability of cytopathic assay. Since anti-IFNbeta antibodies well correlated with MxA induction loss, MxA assay is an appropriate test to determine IFNbeta bioavailability.

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 1 log10 or 3 log 10 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 (1 log10 loss of infective titer) and IHNV (2 log10 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., We thank Mercedes Sánchez Carmona for her excellent technical assistance. This study was supported by Grant AGL2001-1256 from the Ministerio de Ciencia y Tecnología (Spain).

Hepatitis C virus (HCV), especially the genotype 1, is naturally resistant to the antiviral effects of interferon-alpha (IFN-alpha). Expression of the whole HCV genome and the NS5A protein has been suggested to interfere with the antiviral activity of IFN-alpha. Here we have analyzed the effect of individual or various combinations of HCV proteins on IFN-alpha-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-alpha 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-gamma (10 IU/ml) partially restored the antiviral activity of IFN-alpha. 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-alpha-induced antiviral activity. Our results indicate that the expression of structural proteins of HCV may impair the antiviral activity of IFNs.

Serum neopterin (Np), beta 2-microglobulin (beta 2-M), and 2',5'-adenylate (2',5'A) levels and intracellular 2',5'A and human Mx (Hu-Mx) protein synthesis were measured in 20-24 chronic myeloid leukemia patients before and during 1 year of IFN-alpha treatment and in a further 8-9 patients before and at the end of the first and second treatment weeks only. Univariate analysis showed that IFN-alpha increased Np and 2',5'A serum levels and intracellular concentrations of 2',5'A and Hu-Mx significantly from the end of the first week to month 12 of therapy. The biologic marker profiles were similar in cytogenetic responders and nonresponders, as well as in patients treated with IFN-alpha early (12 months from diagnosis) or late (after12 months standard chemotherapy). Further, there were no differences in the short-term (first 14 days) or long-term (during 12 month therapy) induction of the biologic markers irrespective of whether IFN-alpha 2a or IFN-alpha 2b was given. Because multivariate analysis revealed no significant interactions between cytogenetic response, time to treatment, and type of IFN-alpha used, increments in intracellular 2',5'A and Hu-Mx protein were similar at all study times for all factor combinations tested. Np levels varied significantly only during the first 14 therapy days; changes in serum 2',5'A were never statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

The clinical tolerance and biological properties of 6 x 10(6) IU of Chinese hamster glycosylated recombinant interferon-beta (rHuIFN-beta) and natural IFN-beta (Frone) given i.v. were compared in 12 healthy volunteers in a randomized cross-over, double-blind trial. All subjects received a single injection of each type of IFN-beta. Both were well tolerated and provoked similar changes in clinical indices. Serum neopterin (Np) values increased significantly from the 24th to 72nd h post-injection of rHuIFN-beta and Frone. beta 2-Microglobulin (beta 2-M) serum levels were statistically above baseline 24-96 h after rHuIFN-beta, and from the 24th to the 120th h with Frone. Both IFNs provoked a rise in intracellular 2',5'-adenylate (2-5A) levels from the 10th to the 48th h, as well as in Hu-Mx synthesis, which was significant from the 10th to the 96th h. Serum levels of 2-5A, interleukin-1 alpha (IL-1 alpha), and interleukin-1 beta (IL-1 beta) remained unchanged. There were no statistical differences in the changes provoked by the two differently derived IFN-beta in any of the biological parameters studied. Overall, the results of this study indicate that rHuIFN-beta and Frone have similar pharmacodynamics.

In mouse cells carrying the dominant influenza resistance allele Mx+ (but not in Mx− cells) interferon-α/β (IFN) induces an efficient antiviral state against influenza viruses and, concomitantly, the synthesis of a 75,000-Da protein (protein Mx). Here, indirect immunofluorescence using monoclonal antibodies was used to demonstrate that protein Mx accumulates in the nucleus of IFN-treated Mx+ cells, suggesting a nuclear site of action. Protein Mx is present in the nucleus of untreated influenza virus-resistant macrophages freshly explanted from the peritoneal cavity of Mx+ mice but is lost with time in culture when peritoneal macrophages become permissive for influenza virus.