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.

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.

A novel quasi-viral agent, MaTu, is a two-component system.

MaTu is a quasi-viral agent presumably derived from a human mammary tumor. In some respects it resembles classical viruses and in some the "slow viruses," and in others it is different from both. Using monoclonal antibodies (Mabs), we showed that it is a two-component system. One part of the complex, MX, is exogenous; it is manifested by a protein, p58X, which is a cytoplasmic antigen and it reacts with some natural sera of man and of various animals. The other component, MN, is endogenous to human cells. This is manifested by a twin protein(s), p54/58N, localized on the cell surface and in the nucleus. This protein is absent in rapidly growing, sparse cultures of HeLa, but it is inducible either by keeping the cells in dense cultures or, more efficiently, by infecting them with MX. Both inducing factors are synergistic. Only p54/58N is associated with virions of vesicular stomatitis virus (VSV), reproduced in MaTu-infected HeLa. This suggests that MN is responsible for complementation of VSV mutants and for the formation of the VSV (MaTu) pseudotype. Both p54/58N peptides are glycosylated and they form oligomers linked by disulfidic bonds; p58X is not glycosylated and it does not form S-S-linked oligomers.

Isolation of cytopathic small round viruses with BS-C-1 cells from patients with gastroenteritis.

Fecal extracts from 12 subjects in outbreaks of oyster-associated nonbacterial gastroenteritis were inoculated with BS-C-1 cells for isolation of the causative viruses. Cytopathic agents were isolated from 3 patients. No cross-neutralizing reactions were observed between the isolates and prototypes of human enteroviruses. The isolates were approximately 30 nm in diameter and had a distinct ultrastructure resembling that of astroviruses. Four polypeptide bands with molecular sizes of 42, 28, 27, and 22 kDa were seen on SDS-PAGE analyses. Seroconversion against the isolate was observed in 18 (31.6%) of 57 patients involved in five of seven outbreaks examined by neutralization test. A protein band characteristically reactive with the paired serum samples was detectable at 42 kDa by immunoblot assay. These results suggested that some small round viruses resembling astroviruses might show cytopathic effect in BS-C-1 cells and may be associated with an oyster-related gastroenteritis.

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.

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.

[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.

Foodborne Snow Mountain agent gastroenteritis with secondary person-to-person spread in a retirement community.

A variety of small round-structured viruses are being recognized with increasing frequency as a cause of gastroenteritis in the community, but have rarely been reported to cause outbreaks in hospitals or extended-care facilities. From March 20 through April 15, 1988, an outbreak of gastroenteritis occurred in a retirement facility in the San Francisco Bay area. Illness was characterized by diarrhea, nausea, and vomiting; two residents died. Attack rates were 46% (155 of 336) in residents and 37% (28 of 75) in employees. During the initial outbreak period, illness among residents was associated with two shrimp meals served in the facility dining hall (odds ratio = 6.7). Person-to-person transmission probably occurred: The risk of becoming ill one or two days after a roommate became ill was significantly greater than that of becoming ill at other times during the outbreak (risk ratio = 6.5). Microbiologic examinations for bacterial and parasitic enteric pathogens were negative; however, 27-nm viral particles were detected by immune electron microscopy and by blocking enzyme immunoassay to Snow Mountain agent in stools obtained at the onset of illness from one of six ill residents. Seroconversion (greater than fourfold antibody rise) to Snow Mountain agent was detected in acute- and convalescent-phase serum specimens from five of six ill residents as measured by enzyme immunoassay, but not for Norwalk agent as measured by radioimmunoassay. This report of an outbreak of Snow Mountain agent gastroenteritis in an extended-care facility documents that these difficult-to-identify 27-nm viruses can cause outbreaks in inpatient settings.

Diagnosis of astrovirus gastroenteritis by antigen detection with monoclonal antibodies.

An enzyme-linked immunosorbent assay (ELISA), based on monoclonal antibodies to the astrovirus group antigen, was designed for the detection of astroviruses in stools of patients with gastroenteritis. Compared to immune electron microscopy used as the standard test, the sensitivity of the astrovirus ELISA was 91% (31/34) and the specificity was 96% (54/56). All five of the known astrovirus serotypes could be detected in 16 samples on which serotyping was done. In tests on 155 stools containing other enteric viruses, including adenoviruses, rotaviruses, caliciviruses, Hawaii virus, Snow Mountain virus, and Norwalk virus (30, 20, 70, 24, 4, and 7 samples, respectively), only 3 were positive in the astrovirus ELISA. The combined specificity for all astrovirus immune electron microscopy-negative samples was 98% (206/211). The results demonstrate that the new ELISA provides a sensitive and specific means for the diagnosis of astrovirus gastroenteritis.

Immunological characterization of the Marin County strain of astrovirus.

Marin County virus (MCV) was isolated from a stool suspension and serially propagated in human embryonic kidney cell cultures. MCV particles in stool and cell-propagated virus stocks showed reactivity by immune electron microscopy (IEM) with rabbit antiserum to astrovirus type 5. MCV antigen was also detected in two MCV stool samples by enzyme immunoassay (EIA) with an astrovirus group-specific monoclonal antibody. Acute and convalescent sera from 3 of 3 MCV-infected patients showed seroconversion to cell-propagated MCV by EIA. Immunofluorescence of MCV propagated in cell culture showed positive reactivity with an astrovirus group specific monoclonal antibody and astrovirus type 5 antiserum, with some cross-reactivity with astrovirus type 1. Similar results were obtained with the prototype strain of astrovirus type 5. However, in plaque-reduction assays, both the prototype astrovirus type 5 and MCV were neutralized by type 5 antiserum only. We conclude that MCV can be serially propagated by techniques used for previously described astroviruses and is serotypically an astrovirus type 5.

Western blot (immunoblot) assay of small, round-structured virus associated with an acute gastroenteritis outbreak in Tokyo.

Small, round-structured virus (SRSV) was detected in a stool specimen of a patient during an acute gastroenteritis outbreak in Tokyo and was tentatively named SRSV-9. SRSV-9 was purified by sucrose velocity gradient centrifugation after CsCl density gradient centrifugation. The buoyant density of SRSV-9 appeared to be 1.36 g/ml in CsCl. A Western blot (immunoblot) assay using the biotin-avidin system revealed that SRSV-9 was antigenically related to the Hawaii agent but distinct from the Norwalk agent and contained a single major structural protein with a molecular size of 63.0 +/- 0.6 kilodaltons. The prevalence of SRSV-9 infection in Tokyo was surveyed by the Western blot antibody assay by using a crude virus preparation as the antigen. Seroconversion was observed in 56.5% of the patients involved in the outbreaks from which SRSV was detected by electron microscopy.

Antigenic characterization of cell-cultivated astrovirus serotypes and development of astrovirus-specific monoclonal antibodies.

Cultivation of human astroviruses in human embryonic kidney or LLCMK2 cell cultures was corroborated for four of the five serotypes originally reported (types 1, 2, 4, and 5). By using type-specific rabbit antisera and immunofluorescence of virus-infected cells, we readily distinguished between serotypes of astrovirus; however, these serotypes showed a high degree of cross-reactivity by enzyme-linked immunoassay, a result indicating the presence of a group antigen. We prepared monoclonal antibodies to astrovirus type 2 antigen and selected them on the basis of group antigen reactivity. The antibodies were reactive with the four astrovirus serotypes that we could cultivate, as well as with the Marin County strain of astrovirus. A previously reported cell-cultivated astrovirus type 3 also reacted with the monoclonal antibodies. These monoclonal antibodies, and the finding of group reactivity among the human astroviruses, should facilitate studies on the importance of these viruses as agents of viral gastroenteritis.

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.

Snow Mountain agent gastroenteritis from clams.

A 1983 investigation of two clambake-related gastroenteritis outbreaks in Rochester, New York, showed that 84 (43%) of 196 persons interviewed had an acute illness characterized by watery diarrhea, vomiting, and abdominal cramps. None of the ill persons were hospitalized or had complications. Illness was associated with eating raw (p = 0.002) or baked (p less than 0.01) hard-shell clams, with the risk of illness increasing with the total number of clams consumed (p less than 0.01). The median incubation period and duration of illness were 36 and 44 hours, respectively. Stool samples obtained 2-4 days after onset of illness were negative for commonly recognized bacterial and viral pathogens. However, of 31 persons whose stools were tested, the stool of only one ill person was positive by enzyme-linked immunosorbent assay for the Snow Mountain agent, one of the Norwalk-like viruses. Paired serum specimens from six (67%) of nine ill and two (29%) of seven well persons showed a fourfold or greater rise in antibody titer to Snow Mountain agent. Persons who ate clams were more likely to seroconvert to Snow Mountain agent (eight of 12) than were those who did not eat clams (zero of four) (p = 0.04). The clams were harvested off the coast of southern Massachusetts in late October, when harvest waters were documented to be contaminated by untreated municipal sewage. This report describes the first documented outbreak of shellfish-associated gastroenteritis attributed to Snow Mountain agent of which we are aware.

Preliminary characterisation of torovirus-like particles of humans: comparison with Berne virus of horses and Breda virus of calves.

Pleomorphic virus-like particles have been observed by electron microscopy in the faeces of children and adults with diarrhoea. Some of these particles were approximately 100 nm in diameter and had a "fringe" of closely applied peplomers approximately 10 nm long; they closely resembled Berne virus of horses and Breda virus of calves, the two representatives of a newly proposed family called the Toroviridae. In one sample a toroidal nucleoprotein-like structure was observed within the particles. For two samples a buoyant density of 1.14 g/ml was determined by centrifugation through a sucrose density gradient. One sample possessed a haemagglutinin for rat erythrocytes. The serological relationship between these different viruses was observed by immune electron microscopy, haemagglutination inhibition, and serum neutralisation. The role of these virus-like particles as candidate pathogens of humans is discussed.

Enzyme-linked immunosorbent assays for Snow Mountain and Norwalk agents of viral gastroenteritis.

Enzyme-linked immunosorbent assays (ELISAs) for antigen detection and blocking ELISAs for serum antibody rises were developed for the Snow Mountain and Norwalk agents of viral gastroenteritis. The ELISAs were as sensitive as the existing radioimmunoassays and were specific for the Snow Mountain or Norwalk agent. The blocking ELISAs detected the same number of significant rises in antibodies to these agents as did the existing blocking radioimmunoassays.