Two Conserved Amino Acids within the NSs of Severe Fever with Thrombocytopenia Syndrome Phlebovirus Are Essential for Anti-interferon Activity.

The nonstructural protein (NSs) of severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) sequesters TANK-binding kinase 1 (TBK1) into NSs-induced cytoplasmic structures to inhibit the phosphorylation and nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3) and subsequent interferon beta (IFN-ß) production. Although the C-terminal region of SFTSV NSs (NSs66-249) has been linked to the formation of NSs-induced cytoplasmic structures and inhibition of host IFN-ß responses, the role of the N-terminal region in antagonizing host antiviral responses remains to be defined. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the SFTSV and heartland virus (HRTV) NSs are essential for suppression of IRF3 phosphorylation and IFN-ß mRNA expression following infection with SFTSV or recombinant influenza virus lacking the NS1 gene. Surprisingly, formation of SFTSV/HRTV NSs-induced cytoplasmic structures is not essential for inhibition of host antiviral responses. Rather, an association between SFTSV/HRTV NSs and TBK1 is required for suppression of mitochondrial antiviral signaling protein (MAVS)-mediated activation of IFN-ß promoter activity. Although SFTSV NSs did not prevent the ubiquitination of TBK1, it associates with TBK1 through its N-terminal kinase domain (residues 1 to 307) to block the autophosphorylation of TBK1. Furthermore, we found that both wild-type NSs and the 21/23A mutant (NSs in which residues at positions 21 and 23 were replaced with alanine) of SFTSV suppressed NLRP3 inflammasome-dependent interleukin-1ß (IL-1ß) secretion, suggesting that the importance of these residues is restricted to TBK1-dependent IFN signaling. Together, our findings strongly implicate the two conserved amino acids at positions 21 and 23 of SFTSV/HRTV NSs in the inhibition of host interferon responses.IMPORTANCE Recognition of viruses by host innate immune systems plays a critical role not only in providing resistance to viral infection but also in the initiation of antigen-specific adaptive immune responses against viruses. Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infectious disease caused by the SFTS phlebovirus (SFTSV), a highly pathogenic tick-borne phlebovirus. The 294-amino-acid nonstructural protein (NSs) of SFTSV associates with TANK-binding kinase 1 (TBK1), a key regulator of host innate antiviral immunity, to inhibit interferon beta (IFN-ß) production and enhance viral replication. Here, we demonstrate that two conserved amino acids at positions 21 and 23 in the NSs of SFTSV and heartland virus, another tick-borne phlebovirus, are essential for association with TBK1 and suppression of IFN-ß production. Our results provide important insight into the molecular mechanisms by which SFTSV NSs helps to counteract host antiviral strategies.

Susceptibility of Four Abalone Species, Haliotis gigantea, Haliotis discus discus, Haliotis discus hannai and Haliotis diversicolor, to Abalone asfa-like Virus.

Abalone amyotrophia is a viral disease that causes mass mortality of juvenile Haliotis discus and H. madaka. Although the cause of this disease has yet to be identified, we had previously postulated a novel virus with partial genome sequence similarity to that of African swine fever virus is the causative agent and proposed abalone asfa-like virus (AbALV) as a provisional name. In this study, three species of juvenile abalone (H. gigantea, H. discus discus, and H. diversicolor) and four species of adult abalone (the above three species plus H. discus hannai) were experimentally infected, and their susceptibility to AbALV was investigated by recording mortality, quantitatively determining viral load by PCR, and conducting immunohistological studies. In the infection test using 7-month-old animals, H. gigantea, which was previously reported to be insusceptible to the disease, showed multiplication of the virus to the same extent as in H. discus discus, resulting in mass mortality. H. discus discus at 7 months old showed abnormal cell masses, notches in the edge of the shell and brown pigmentation inside of the shell, which are histopathological and external features of this disease, while H. gigantea did not show any of these characteristics despite suffering high mortality. Adult abalones had low mortality and viral replication in all species; however, all three species, except H. diversicolor, became carriers of the virus. In immunohistological observations, cells positive for viral antigens were detected predominantly in the gills of juvenile H. discus discus and H. gigantea, and mass mortality was observed in these species. In H. diversicolor, neither juvenile nor adult mortality from infection occurred, and the AbALV genome was not increased by experimental infection through cohabitation or injection. Our results suggest that H. gigantea, H. discus discus and H. discus hannai are susceptible to AbALV, while H. diversicolor is not. These results confirmed that AbALV is the etiological agent of abalone amyotrophia.

Immunopathogenesis of acute central nervous system disease produced by lymphocytic choriomeningitis virus. II. Adoptive immunization of virus carriers.

Lymphocytic choriomeningitis (LCM) virus carriers were established by intracerebral inoculation of adult BALB/c mice followed by a single dose of cyclophosphamide (CY) (150 mg/kg) 3 days after infection, and by intracerebral injection within 24 hr of birth. These carriers were then adoptively immunized with spleen cells or serum from immune or normal BALB/c donors. Transfer of immune spleen cells into drug-induced carriers consistently resulted in acutely fatal choriomeningitis, histologically strikingly similar to classical LCM. Normal spleen cells or immune serum failed to produce either central nervous system (CNS) pathology or illness with any regularity. In addition, focal necrosis of the cerebellum was seen after adoptive immunization of drug-induced carriers but only when mice received cells at least 3 wk after inoculation, which is probably explained by the gradual spread of infection from membranes to the neural parenchyma during the first month after establishment of the carrier state in adult mice. Immune spleen cells, when transferred to neonatal carriers, led to a decrease in virus titers in blood and brains and to development of antibody without acute CNS disease. It appears that the production of fatal choriomeningitis after LCM infection is determined in part by the distribution of viral antigen, and this is markedly different in neonatal and drug-induced carriers at the time of cell transfer. Another factor of potential importance is the much higher level of circulating viral antigen in the plasma of neonatal than in that of drug-induced LCM carriers. Classical LCM disease can only be transferred by immune lymphoid cells and not by antiserum. Furthermore, little or no complement-fixing (CF) antibody was found in the plasma of mice dying of acute choroiditis. These observations strongly suggest that acute choroiditis is dependent upon the cell-mediated immune response.

Immunopathogenesis of acute central nervous system disease produced by lymphocytic choriomeningitis virus. I. Cyclophosphamide-mediated induction by the virus-carrier state in adult mice.

A single dose of 150 mg/g of cyclophosphamide (CY), given 3 days after intracerebral (i.c.) inoculation of lymphocytic choriomeningitis (LCM) virus, protected over 90% of adult BALB/c mice against acutely fatal choriomeningitis. Surviving mice became persistently infected carriers, with high virus titers in blood and brain. Immunofluorescent examination of the brain showed that in CY-induced carriers infection was initially confined to the choroid plexus, ependyma, and leptomeninges, but over the next 30 days gradually spread to the neural parenchyma, most notably to the molecular layer of the cerebellum. By contrast, LCM virus-carrier mice produced by neonatal virus injection and examined as adults, showed a much less marked infection of choroid plexus and much more widespread infection of parenchyma, with a different distribution among brain nuclei, including heavy infection of the Purkinje cells of the cerebellum.

Detection of serum antibodies to Borna disease virus in patients with psychiatric disorders.

Borna disease virus causes a rare meningoencephalitis in horses and sheep and has been shown to produce behavioral effects in some species. The possibility that the Borna virus is associated with mental disorders in humans was evaluated by examining serum samples from 979 psychiatric patients and 200 normal volunteers for the presence of Borna virus-specific antibodies. Antibodies were detected by the indirect immunofluorescence focus assay. Antibodies to the virus were demonstrated in 16 of the patients but none of the normal volunteers. The patients with the positive serum samples were characterized by having histories of affective disorders, particularly of a cyclic nature. Further studies are needed to define the possible involvement of Borna virus in human psychiatric disturbances.

Isolation of Aleutian mink disease virus by affinity chromatography.

Affinity chromatography was used to isolate the Aleutian disease virus of mink. Dissociation of the immunoadsorbent-virus complex with 0.75 molar sodium chloride and then with a glycine-hydrochloride gradient released infective particles resembling picornaviruses. The elution profile suggests that two different types of virus-antibody complexes are formed, one dissociated by sodium chloride and another that requires glycine-hydrochloride in addition to sodium chloride for release of virus.

Borna disease virus. A possible etiologic factor in human affective disorders?

Borna disease virus is a unique neurotropic agent that appears to have a predilection for the limbic area of the brain. In some animal species, it can produce a behavioral syndrome characterized by aggressive and passive phases. This syndrome has suggested an analogy to certain human affective disorders. In this preliminary study, we examined the possible involvement of Borna disease virus in the etiology of human mood disorders by assaying for virus-specific antibodies in 265 patients with unipolar or bipolar depression and 105 normal, healthy volunteers. Twelve patients (4.5%) and none of the healthy controls demonstrated this antibody in their serum samples. It will be necessary to replicate and extend these intriguing preliminary results to determine if Borna disease virus is possibly involved in the pathogenesis of affective disorders in humans.

Antigenic relationship between human and simian rotaviruses.

The simian rotavirus, SA 11, and the murine rotavirus, EDIM, were investigated for antigenic relatedness to the human rotavirus, by immunoelectron-microscopy. These studies led to the recognition of two types of rotavirus antibody. One agglutinated "rough" virus particles only and was group-reactive; it appears to be widely distributed in various animal species, including human infants. The second antibody agglutinated "smooth" virus particles and was more species-specific, demonstrating only a one-way cross-reaction between the simian and human viruses; it was found only in convalescent-phase human sera and in hyperimmune rabbit sera and is probably protective. The simian rotavirus is easy to propagate in primary cell culture and in cell lines and should prove useful for serodiagnosis of human gastroenteritis. It may be a candidate for immunoprophylaxis.

Rocket line immunoelectrophoresis: an improved assay for simultaneous quantification of a mink parvovirus (Aleutian disease virus) antigen and antibody.

A rocket line immunoelectrophoretic assay (RLIE) was developed for the simultaneous quantification of viral antigens and antiviral antibodies of the important mink parvovirus, Aleutian disease virus (ADV). The sensitivity of the RLIE assay was found to be 5 log2 higher than that of the counter current immunoelectrophoresis which is the assay routinely used for diagnostic purposes.

The development and evaluation of radioimmune assays for the detection of immune globulins M and G against astrovirus.

The development and evaluation of radioimmune assays for the detection of IgM and IgG antibodies to astrovirus are described. The test was shown to be sensitive and specific, and suitable for screening large numbers of sera. The use of the assays has established that astrovirus type 1 is prevalent in the United Kingdom and that not only infants but also schoolchildren and elderly patients are affected. Further evidence is given to support the view that Marin County Agent is antigenically related to astrovirus type 1.

Immune complexes in Aleutian disease: demonstration of antibody on isolated virus.

The specific binding of Staphylococcal protein A for mammalian immunoglobulin G was used to demonstrate IgG associated with Aleutian disease virus (ADV) when isolated from infected mink tissues. Protein A specifically bound to mink serum Ig with no reaction with other serum or tissue proteins. Protein A labeled with 131Iodine reacted with crude virus preparations but not with virus that had been purified by freon extraction to the point where it became reactive with antibody by counterimmunoelectrophoresis. Binding to purified ADV was restored when the purified virus was first reacted with antibody. Results of urea treatment indicated this as an alternative method for isolation of ADV free from antibody.

Application of control measures against viral haemorrhagic disease of rabbits in the Czech and Slovak Federal Republic.

The first outbreaks of viral haemorrhagic disease (VHD) of rabbits were reported from eastern Slovakia in 1987. In 1988, the infection spread throughout the Czech and Slovak Federal Republic. Electron microscopy was used by the Veterinary Research Institute in Brno to diagnose the disease during the early stage of infection. At present, the regional laboratories of the veterinary investigation services use the haemagglutination and the direct immunofluorescence tests as the principal methods to demonstrate the causal agent. Indirect immunofluorescence and immunoperoxidase techniques have been developed to demonstrate VHD virus, while the enzyme-linked immunosorbent assay (ELISA) has been used to detect antibodies. Diagnostic kits, allowing a wide use of these methods, are now available commercially. Two types of inactivate vaccines were developed and produced in 1988 and 1989. VHD is controlled by vaccination of exposed rabbit colonies. This is accompanied by other preventive and protective measures, directed by district veterinary officers following instructions from federal authorities.

Protides of the Mustelidae: immunoresponse of mustelids to Aleutian mink disease virus.

Members of North American Mustelidae were tested for their response to inoculation with 10(6) infective doses of Aleutian disease virus. In subfamily Mustelinae, 3 species in the genus Mustela (M vision, M erminea, and M putorius) and 2 species in genus Martes (Ma pennanti and Ma americana) responded immunologically with some features resembling Aleutian disease in mink. In subfamily Mephitinae, only Mephitis mephitis responded, and others of the subfamily did not, nor did members of subfamilies Melinae and Lutrinae. The responses observed ranged from development of detectable antibody levels determined by counterimmunoelectrophoresis to histopathologic changes typical of Aleutian disease.

Isolation of virus and antibody containing immune complexes from mink with Aleutian disease by affinity chromatography of equine complement clq.

Affinity chromatography on immobilized equine complement Clq was used for the isolation of complement-binding immune complexes in sera of mink infected with Aleutian disease virus. Immune complexes were isolated and quantitated from 4 of 5 infected mink, as early as 2 weeks after infection and before hypergammaglobulinemia had appeared. The quantity of immunoglobulin G in these immune complexes ranged from 180 to 370 micrograms/ml serum. There were no Clq-binding immune complexes found in mink which were negative for Aleutian disease antibody. Using 125I-labeled BSA-anti-BSA complexes, we demonstrated that the affinity columns bound selectively immune complexes which had formed in antibody excess, whereas immune complexes in antigen excess were not bound. By neutralization of sensitized virus with anti-mink IgG serum, non Clq-binding immune complexes were also detected, which indicates that circulating immune complexes in persistently infected mink are heterogeneous as far as their reactivity with equine Clq is concerned.

Comparative studies on three isolates of Breda virus of calves.

Three isolates of Breda virus of calves were compared morphologically and antigenically. The isolates demonstrated similar morphology and shared common antigens, as determined by enzyme-linked immunosorbent assay and immunoelectron microscopy. On the basis of results of the hemagglutination-inhibition test, enzyme-linked immunosorbent assay, and immunoelectron microscopy, the 3 isolates were further subdivided into 2 serotypes: serotype 1 (Breda virus 1) represented by the Iowa isolate 1; and serotype 2 (Breda virus 2), by the Ohio isolate and the Iowa isolate 2. The 3 isolates caused diarrhea in gnotobiotic calves.

Norwalk agent-like particles associated with gastroenteritis in human beings.

Currently, 6 agents of viral gastroenteritis with properties similar to the Norwalk agent have been described. These are the Hawaii, Montgomery County, W, Ditchling, and Cockle agents, as well as the Norwalk agent. These agents are 25 to 27 nm in diameter, are nonenveloped, and have a buoyant density of 1.38 to 1.40 g/cc in CsCl. Multiple antigenic types exist among these agents. These particles have been detected by immune electron microscopy in feces of acutely ill patients. None has been successfully cultivated in vitro, and suitable animal models of disease do not exist. Studies in volunteers indicate that acute infection is associated with a reversible histopathologic change in the jejunal mucosa and with transient malabsorption. Pathogenesis of these changes remains unknown. Other similar agents will likely emerge from ongoing studies.

Mystery swine disease in The Netherlands: the isolation of Lelystad virus.

In early 1991, the Dutch pig-industry was struck by the so-called mystery swine disease. Large-scale laboratory investigations were undertaken to search for the etiological agent. We focused on isolating viruses and mycoplasmas, and we tested paired sera of affected sows for antibodies against ten known pig viruses. The mycoplasmas M. hyosynoviae, M. hyopneumoniae, and Acholeplasma laidlawii, and the viruses encephalomyocarditis virus and porcine enterovirus types 2 and 7 were isolated from individual pigs. An unknown agent, however, was isolated from 16 of 20 piglets and from 41 of 63 sows. This agent was characterised as a virus and designated Lelystad virus. No relationship between this virus and other viruses has yet been established. Of 165 sows reportedly afflicted by the disease, 123 (75 per cent) seroconverted to Lelystad virus, whereas less than 10 per cent seroconverted to any of the other virus isolates or to the known viral pathogens. Antibodies directed against Lelystad virus were also found in pigs with mystery swine disease in England, Germany, and in the United States. We conclude that infection with Lelystad virus is the likely cause of mystery swine disease.

Isolation of a virus strain from Argas persicus ticks.

A previously undescribed virus was isolated from Argas persicus ticks collected on sentinel chicken in western Slovakia. The strain was lethal for suckling mice only after intracerebral inoculation. No symptoms were induced in adult mice. The virus strain was insensitive to sodium deoxycholate and resistant to ether treatment. An antigen prepared from the virus did not agglutinate goose or human O erythrocytes. The single strain obtained in 1976 appeared to be untrelated to a large number of known arboviruses when tested by the complement-fixation reaction.

[Intravital laboratory diagnosis of human subacute spongioid encephalopathies (Creutzfeldt-Jakob syndrome and amyotrophic leukospongiosis)]. / Prizhiznennaia laboratornaia diagnostika podostrykh spongioznykh éntsefalopatiicheloveka (bolezni Kreittsfel'da-Jakoba i amiotroficheskogo leikospongioza).

Methods aimed at the detection of causative agents in the CSF and peripheral blood lymphocytes are recommended for the use in intravital laboratory diagnosis of slow infections of the central nervous system. The results obtained enable recommending the biotest on guinea-pigs or indication of the causative agent of amyotrophic leukospongiosis (AL) in cell culture coupled with the punctate immunoenzyme assay for the diagnosis of AL. As to the diagnosis of Creutzfeldt-Jacob disease, it is suggested that the biotest on guinea-pigs and the punctate immunoenzyme assay may be used.