Lipid composition of an iridescent virus type 6 (CIV)

Summary The Chilo Iridescent Virus (CIV) is a lipid-containing virus propagatedin vitro inChoristoneura fumiferana cell cultures. We have analysed the individual lipids of the viral membrane which appeared interesting in their relative amounts and mainly in the high proportion of phosphatidylinositol. This fraction represented about 27 per cent of the phospholipid extract. The lipid composition of the viral membrane was unchanged whether the virus was propagatedin vivo in larvae orin vitro in invertebrate cell cultures and was clearly different from that of the hosts.With 1 Figure     

Further characterization of an alkaline protease activity associated with iridescent virus type 6

Summary Iridescent virus type 6 infectingGalleria mellonella larvae contained an associated alkaline protease activity which appeared to be tightly bound to the virions and essentially localized on the outside of the viral particle. Under alkaline conditions the virus was degraded by proteolytic cleavage of viral envelope proteins. Proteolytic activity was not present in virus propagated in a permissive insect cell line or when purified from isolated larval fat-bodies instead from whole larvae. The results suggest that the protease associated with Iridescent virus type 6 is of larval origin.With 4 Figures     

Structural and thermodynamic investigation of the Chilo iridescent virus (Iridovirus type 6)

Structural features and thermodynamic parameters of the complete Chilo iridescent virus (Iridovirus type 6) and its constituents, isolated from the larvae of Galleria mellonella, were evaluated by means of UV spectroscopy and microcalorimetry. It can be demonstrated that the viral DNA is attached to the coat protein in a chromatin-like fashion, which is preserved after disruption of the virus by low temperature or partial digestion of the coat protein with proteinase K. At elevated temperature however the viral DNA is denaturated irreversibly. The coat protein appears to remain in its native state during the DNA transition and its own thermal denaturation profile shows its independence from the DNA denaturation.     

Genetics of macrophage-controlled resistance to hepatitis induced by herpes simplex virus type 2 in mice.

The genetics of innate resistance of mice to hepatitis induced by herpes simplex virus type 2 (HSV-2) was analyzed by crossing resistant male GR to susceptible female BALB/c mice and backcrossing females of this F1 generation to susceptible male BALB/c mice. By scoring of macroscopic liver lesions and virus isolation studies from the liver 4 days after intraperitoneal inoculation of HSV-2, it appeared that the resistance was governed by one X-linked dominant gene or closely linked gene complex, as F1 female mice were resistant and F1 male mice were susceptible and the trait segregated in a ratio close to 1:1 in the backcross mating. A cellular expression in vitro of virus resistance was found in the macrophage population of the mice as measured by differences in the restriction of HSV-2 replication in macrophage cultures prepared from individual mice. In contrast to what was seen in macrophage cultures, virus replicated equally well in embryonic fibroblast cultures from susceptible and resistant strains of mice.     

Identification and characterization of the repetitive DNA element in the genome of insect iridescent virus type 6

The genome of the Chilo iridescent virus (CIV) was analyzed for existence of repetitive DNA sequences by DNA-DNA hybridization using a defined and complete gene library of the viral genome (209 kbp) and by heteroduplex mapping. These experiments revealed the presence of repetitive DNA elements in the CIV genome, which are located in the EcoRI fragment H and in the EcoRI DNA fragment C at the coordinates 0.535 to 0.548 (EcoRI/Pstl DNA fragment, 2.7 kbp) and 0.920 to 0.944 (PvuII CIV DNA fragment L, 5.1 kbp), respectively. The DNA nucleotide sequence (2708 bp) of the EcoRI/Pstl subfragment was determined. The comparative analysis of the DNA sequences of this particular region of the viral genome with the DNA sequences of the PvuII DNA fragment L (5064 bp) revealed the presence of several DNA sequences within the EcoRI/Pstl subfragment of the EcoRI CIV DNA fragment H which show homology to DNA sequences of the PvuII DNA fragment L. For example, a DNA element (box A, 91 bp) is located at nucleotide positions 1981 to 2072 of the EcoRI CIV DNA fragment H which are complementary (greater than 90%) to the nine regions of the PvuII DNA fragment L (L-boxes 1 to 9). Furthermore heteroduplex mapping revealed the existence of a stem-loop structure (stem, 65 +/- 10 bp and loop, 652 +/- 80 bp) at the genome coordinates 0.571 to 0.582 (2.5 kbp, HindIII/EcoRI subfragment of the EcoRI CIV DNA fragment H). This indicates that an inverted repeat sequence is located at this region of the viral genome. The DNA nucleotide sequence of this subfragment was determined (2555 bp) which confirmed the data obtained from electron microscopy. An inverted repeat DNA sequence located at nucleotide positions 304 and 1011 is able to form this type of stem-loop structure.     

Macrophages and age-dependent resistance to hepatitis induced by herpes simplex virus type 2 im mice.

An age-dependent increase in the resistance of BALB/c mice to induction of focal necrotic hepatitis by herpes simplex virus type 2 was demonstrated. In 3-week-old mice inoculated intraperitoneally with virus, numerous necrotic foci developed in the liver. As the mice matured, the number of lesions declined until the age of 8 weeks, when no further increase in resistance appeared. Corresponding to this, the virus titers of livers and spleens of 3-week-old mice were higher than in 8-week-old animals throughout the infection, and the infection was apparently terminated in these organs of the adult mice by day 5. In vitro infection of peritoneal macrophages from 3-week-old and 8-week-old mice showed that this age-related resistance was concomitant with an increased restriction of virus replication in peritoneal macrophages from adult mice. Since, furthermore, the resistance of adult mice could be abolished by intravenous inoculation of the macrophage-toxic agent silica before infection, and since adoptive transfer of 2 X 10(6) syngeneic macrophages from adult mice to young ones conferred to the latter a resistance comparable to that of the adult mice, it is concluded that macrophage maturation is responsible for the age-dependent resistance seen in this infection.     

Persistence of invertebrate iridescent virus 6 in tropical artificial aquatic environments

Summary. The rate of loss of activity of invertebrate iridescent virus 6 (IIV-6, family Iridoviridae) was determined in two artificial aquatic habitats in southern Mexico, using a sensitive insect bioassay technique. IIV-6 placed in trays of water in direct sunlight suffered rapid loss of activity (99.99% reduction) over a period of 36 h, during which water temperatures fluctuated between 24 and 41 °C. No significant deactivation occurred during the hours of darkness. In contrast, IIV-6 placed in trays of water in the shade lost 97% of original activity over a 60 h period, during which water temperatures fluctuated from 24 to 31 °C. Longitudinal analysis involving mixed effects models of time (shade) and cumulative exposure to ultraviolet radiation (sunlight) indicated that the rate of deactivation was best described by third order polynomial equations in both cases. We conclude that the likelihood of transmission of IIVs in aquatic habitats will be mediated by the intensity of UV radiation and water temperature.     

A comparison of techniques for detecting Invertebrate iridescent virus 6

The aim of this study was to compare the sensitivity and precision of various methods for the detection and quantification of Invertebrate iridescent virus 6 (IIV-6) (Iridoviridae) isolated from a the stem-boring moth Chilo suppressalis, and to apply these techniques to the detection of covert infections in the wax moth, Galleria mellonella. The relationship between the virus concentration and absorbance at 260 nm was linear over the range of 1.6 x 10(9)-5.6 x 10(10) particles/ml. TCID(50) assays using 12 different cell lines indicated that two Drosophila lines, DL2 and DR1, had the highest susceptibility whereas cell lines from Aedes albopictus and Plutella xylostella were four orders of magnitude less sensitive. TCID(50) values for IIV-6 in Spodoptera frugiperda Sf9 cells gave the particle-infectivity ratios of 15-64 virus particles/IU. An insect bioassay involved injecting doses of 1-100 IIV-6 particles into the third instar G. mellonella larvae. The prevalence of patent infection was 20-26% at a dose of 1 particle per larva rising to 86-92% at 10 particles and 100% at doses of 50 or 100 particles. Of the insects that survived to adulthood, between 5.8 and 75% caused patent infections when injected into G. mellonella larvae, indicating that they were covertly infected. A PCR technique resulted in 95% detection at 1000 virus particles per insect. Of the insects that proved positive for covert infection by insect bioassay, 41% also proved positive by PCR analysis. It is concluded that the G. mellonella bioassay is highly reliable for detection of doses of 10 particles or more and for determining the relative activity of IIV-6 preparations at doses as low as 1 particle per insect. PCR had a slightly lower sensitivity followed by the insect cell culture assay.     

Characterization of the third origin of DNA replication of the genome of insect iridescent virus type 6

The structure of the third origin of DNA replication (CIV-ori-M) of the genome (209 kbp) of Chilo iridescent virus (CIV) was determined by DNA nucleotide sequence analysis. The CIV-ori-M is located within the DNA sequences of theEcoRI CIV DNA fragment M (7 kbp; 0.310–0.345 viral map units) between the genome coordinates 0.310 (EcoRI site) and 0.317 (NcoI site). The DNA nucleotide sequence of theEcoRI/NcoI CIV DNA fragment (1601 bp) was determined for identifying the DNA sequence of the corresponding origin of DNA replication. The analysis of the DNA sequences of this region revealed the presence of a 12-mer inverted repeat at nucleotide positions 485–496 and 503–513 (485-AGATATTTGACT-496-TATGT-503-AGTCAAATATCT-513) that are able to form a hairpin-loop structure. A double-stranded DNA fragment was synthesized that corresponds to the nucleotide positions 485–513 that were cloned into the phages M13mp18 and M13mp19, and were screened for their ability to be amplified in CF-124 cell cultures infected with CIV. The successful amplification of the DNA sequence of the CIV-ori-M is strong evidence that this particular region of the CIV genome indeed serves as the origin of DNA replication.The analysis of the DNA sequence of CIV-ori-M in comparison to the DNA sequence of the two other characterized origins of DNA replication (CIV-ori-H and Y) of the CIV genome (9) was carried out, and the results are shown in Table 2. According to these data the DNA sequence homology between the DNA sequences of the CIV-ori-M and CIV-ori-Y, and between the CIV-ori-M or CIV-ori-H, was found to be 58% and 55%, respectively.     

Infectivity and pathogenesis of iridescent virus type 22 in various insect hosts

Summary This paper reports the results of a series of laboratory experiments to determine the infectivity and pathogenesis of iridescent virus type 22 (IV 22) for six species of mosquitoes, phlebotomine sand flies and triatomid bugs. Following inoculation, IV 22 replicated in all of the species tested, without producing noticeable mortality within a 14 day observation period. Examination of the infected insects by immunofluorescence demonstrated large amounts of viral antigen in many different organs. Electron microscopy done on infected mosquitoes (Aedes aegypti) showed large numbers of virus particles within cells of the fat body, muscle tracheal and midgut epithelium. Virus replication in the mosquitoes was confined to host cell cytoplasm and was similar to that described in the natural blackfly (Simulium) host. Transovarial transmission of IV 22 could not be demonstraed inA. aegypti, and only a small percentage of mosquito larvae could be infected orally. Results of these experiments are compatible with observations of other iridescent viruses; IV 22 is highly infectious for a wide range of insects when introduced into their hemolymph, but it is not very infectious per os. These characteristics would appear to limit its value as a potential biocontrol agent for Diptera.     

The replication and titration of iridescent virus type 22 in Spodoptera frugiperda cells

A plaque assay for iridescent virus type 22 (from Simulium sp.) using Spodoptera frugiperda cells has been devised, and the kinetics of growth of the virus in this cell line have been determined. The virus particle/p.f.u. ratio was 75 +/- 8, and the p.f.u./TCID50 ratio was 0.56 +/- 0.11.     

调节性T细胞在3型鼠肝炎病毒诱导的小鼠暴发型肝炎模型中作用的初步探讨

目的 检测调节性T细胞(Tr)在3型鼠肝炎病毒(MHV-3)诱导的小鼠暴发型肝炎模型中的比例变化及细胞因子表达,初步探讨Tr在该疾病模型中的作用.方法 通过腹腔注射MHV-3感染BALB/cJ小鼠诱导暴发型肝炎,观察小鼠的生存时间,检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)水平,利用苏木精-伊红(HE)染色法检测肝脏病理学改变,分离感染不同时间点外周血、脾脏以及肝脏中的淋巴细胞,利用流式细胞术来检测Tr的比例以及细胞因子IL-10表达水平.结果 BALB/cJ小鼠感染MHV-3后,全部在3～6d内死亡,血清ALT、AST水平随着感染时间延长逐渐升高,HE染色显示肝脏组织炎症及坏死程度逐渐加重,流式细胞术检测发现随着感染时间延长,小鼠肝脏中的Tr的比例明显升高.同时肝脏Tr分泌细胞因子1L-10的比例以及肝脏组织IL-10的mRNA水平逐渐升高.结论 MHV-3诱导的小鼠暴发型肝炎模型中Tr在肝脏中的比例和功能显著升高,这种代偿性升高提示Tr可能发挥调节机体过度免疫反应的功能.     

Role of activated macrophages in resistance of congenitally athymic nude mice to hepatitis induced by herpes simplex virus type 2.

Congenitally athymic nude (nu/nu) mice of a BALB/c genetic background were found considerably more resistant to the induction of focal necrotic hepatitis by herpes simplex virus type 2 (HSV-2) tha, were phenotypically normal littermates (nu/+) or BALB/c mice. The augmented resistance was age dependent, as it was only manifested in mice from 4 to 5 weeks of age. Studies of the course of infection showed that nude mice were able to restrain virus multiplication in the liver far better than normal mice in the early phase of infection. However, they seemed inferior to normal mice in eliminating the infectious process. In vitro investigation of peritoneal macrophages revealed that macrophages from 6-week-old nude mice exhibited accelerated spreading and were three times as restrictive in the replication of HSV-2 as macrophages from normal mice. However, no difference was found in the efficiency of adsorption/phagocytosis between macrophages from nude and normal mice. The increased resistance of nude mice could be abolished by blockade of the microphage function of the mice by silica. Nude mice reconstituted at birth with thymus cells were just as susceptible to infection as normal mice. These data suggest that the increased resistance of nude mice to HSV-2 hepatitis is due to the presence of nonspecifically activated macrophages before infection.     

Dhori virus induced lesions in mice

Dhori and Thogoto viruses are till now the only recognized tick-borne orthomyxoviruses. Like Thogoto virus, also Dhori is highly hepatotropic for laboratory mice; the lesions in several organs resemble those described for influenza virus.     

Identification and mapping of origins of DNA replication within the DNA sequences of the genome of insect iridescent virus type 6

The origins of DNA replication of the genome (209 kbp) of Chilo iridescent virus (CIV), which is circularly permuted and terminally redundant, were identified. The defined genomic library of CIV, which represents 100% of DNA sequences of the viral genome (e.g., all 32EcoRI CIV DNA fragments), was used for transfection ofChoristoneura fumiferana insect cell cultures (CF-124) that were previously infected with CIV. The plasmid rescue experiments were carried out to select those recombinant plasmids that were amplified during viral replication in CIV-infected cell cultures. It was found that six recombinant plasmids harboring theEcoRI DNA fragments C [13.5 kbp, 0.909-0.974 map units (m.u.)], H (9.8 kbp, 0.535–0.582 m.u.), M (7.25 kbp, 0.310–0.345 m.u.), O (6.5 kbp, 0.196–0.228 m.u.), Q (5.9 kbp, 0.603–0.631 m.u.), and Y (2.0 kbp, 0.381–0.391 m.u.) were able to be amplified under the conditions used. This indicates that the CIV genome possesses six DNA replication origins. Subclones of theEcoRI CIV DNA fragments C and H were screened under the same conditions. It was found that DNA sequences within theEcoRI DNA fragments C and H at the genome coordinates 0.924–0.930 and 0.535–0.548, respectively, contain origins of viral DNA replication. The DNA nucleotide sequences of theEcoRI CIV DNA fragment Y (1986 bp) were determined for identifying the DNA sequence of the corresponding origin of DNA replication. The computer-aided analysis revealed the presence of a 15-mer inverted repeat at nucleotide positions 661–675 and 677–691 (661-TAAATTTAATGAGAA-G-TTCTCATTAAATTTA-692). The analysis of the DNA sequence of theEcoRI DNA fragment H corresponding to the particular region at the genome coordinates 0.535–0.548 (1) showed that this region contains a 16-mer inverted repeat at the nucleotide positions 1315 and 1332 (1315-TAAATTTTAATGGTTA-A-TAACCATTAAAATTTA-1347), which is very similar to the inverted repetition found within theEcoRI DNA fragment Y. The successful recognition and amplification of the single-stranded synthetic DNA sequences of both strands of CIV-ori-Y (nucleotide position 661–691) using phage M13 system in CIV-infected cells is strong evidence that the CIV-ori-Y is bidirectionally active, and this DNA sequence is considered to be the origin of DNA replication within theEcoRI CIV DNA fragment Y.     

Lethal synergism induced in mice by influenza type A virus and type Ia group B streptococci.

Intranasal inoculation of CD-1 or BALB/c mice with low doses of influenza A/PR8/34 (HON1) virus followed 48 h later by intranasal inoculation of low doses of type Ia group B streptococci effected a lethal synergism. At a constant input dose of virus, a direct relationship between input dose of bacteria and percent mortality was observed; the converse was also true. An inverse relationship between input dose of group B streptococci, but not input dose of virus, and mean time to death was observed in CD-1 but not in BALB/c mice. The kinetics of influenza A/PR8/34 virus and group B streptococcal replication in singly and dually infected BALB/c mice was determined by assaying samples from the lungs, liver, spleen, and blood for viable group B streptococci and infectious influenza A/PR8/34 virus. No significant difference in virus replication in the lung was observed between singly and dually infected mice. Extrapulmonary dissemination of virus was not observed. Concurrent virus infection effected a 10,000- to 100,000-fold increase in the levels of type Ia group B streptococci in the lung. Potentiation of group B streptococcal infection of the lung was not associated with bacteremia or infection of the liver or spleen, a finding contrary to previous observations of fulminant septicemia after intranasal inoculation of mice with input doses of group B streptococci less than one-tenth of the pulmonary levels observed in the present study.     

Comparative study of different methods to genotype hepatitis C virus type 6 variants

Hepatitis C virus (HCV) genotype 6a is found frequently in Southeast Asia. In Thailand, however, genotype 6 variants may exist which posses a genotype 1 like sequence in the 5' non-coding region. In order to genotype correctly these viruses, four different methods; INNO-LiPA assay, two RFLP assays on the core region (using different restriction enzymes) and phylogenetic analysis of the core sequences were compared. Samples from 17 chronic HCV patients from the Netherlands and Thailand and 18 anti-HCV positive blood donors recruited from Thailand were tested. The INNO-LiPA could not distinguish genotype 6 variants. The RFLP methods used could not, or only in combination with 5'NCR genotyping methods, identify type 6 variants. In conclusion, for identification of type 6 variants at least two different regions of the HCV genome have to be analyzed (both 5'NCR and core).     

Amanita virosa induced toxic hepatitis: report of three cases

We report here three cases of Amanita virosa induced toxic hepatitis. Two of the three cases recovered but the other died 10 days after mushroom ingestion. Since the mortality of Amanita mushroom induced toxic hepatitis is very high, prompt diagnosis and aggressive therapeutic measures should be initiated as soon as possible. Our cases showed that the initial serum aminotransferase levels might not predict the clinical outcome of the patient, but that the prothrombin time (PT) seemed to be a more useful prognostic marker. Close monitoring of aminotransferase levels and PT as well as appropriate therapy are recommended. All three cases showed signs of proteinuria and we were able to characterize mixed tubular and glomerular type proteinuria at 3 or 4 days after ingestion in two cases. Among the previously reported Korean cases of suspected Amanita induced toxic hepatitis, most species could not be identified except for four cases of Amanita virosa. No cases of Amanita phalloides induced toxic hepatitis have been identified in Korea so far.