云南辛德毕斯病毒的生物学性状研究

本文对云南首次分离到的Sindbis病毒进行了滤过试验,耐酸耐醚试验、致细胞病变、动物敏感性试验、血压 凝特性、空斑和毒力等试验研究,结果符合披膜病毒科的病毒特性。交 因抑试验和免疫荧光试验,以及空斑减小中和试验进一步证实为甲病毒属的Sindbis病毒,其空斑纯化株的生物学特性也与原株相符,纯化株的制备为该株病毒分子生物学研究的准确性和一致性提供了条件。云南Sindbis病毒的首次分离具有重要的流行病学意义,其生物学特性研究结果对我省该病的诊断和防治具有重要的指导意义。     

Normal rainbow trout serum (RTS)-resistant variants of the infectious pancreatic necrosis virus (IPNV)-Jasper differ with respect to inhibition by RTS, serotype and cDNA sequence

In order to determine if the infectious pancreatic necrosis virus isolate IPNV-Jasper (Ja-ATCC) is homogeneous or heterogeneous with respect to inhibition by normal rainbow trout serum (RTS), 50 clones were tested for sensitivity to RTS. The initial isolate was very sensitive to RTS, losing from 10(4) to 10(8) 50 % tissue culture infection dose (TCID50) ml(-1) with a 1:100 dilution of RTS. The sensitivity of the clones ranged from highly sensitive to completely resistant (0 to 10(8) TCID50 ml(-1) reduction). Eight percent of clones (4/50) were very sensitive to RTS (Ja-S) and 84% of clones (42/50) showed a mid-range of sensitivity to RTS. The final 8 % of clones (4/50) were resistant to RTS (Ja-R). Enzyme immunodot assay revealed that Ja-S clones showed a monoclonal reaction identical to the parents, Ja-ATCC; however, Ja-R clones differed by several epitopes from the parental strain. Analysis of Ja-S and Ja-R revealed that there were significant differences in their nucleic acid sequences for the capsid protein VP2. These 2 strains shared 80.7 and 86.5% identity in nucleic acid and in amino acid sequences, respectively. Ja-S had 99.7 and 91.0 % identity in nucleic acid sequences, and 99.5 and 95.9 % in amino acid sequences with Ja-ATCC and Jasper-Dobos (Ja-D), respectively, while Ja-R showed 80.6 and 79.8 % identity in nucleic acid sequences and 86.5 and 87.0 % in amino acid sequences with Ja-ATCC and Ja-D, respectively. In conclusion, the Ja-ATCC population was heterogeneous in terms of RTS sensitivity, serotype and cDNA sequences from the VP2 coding region.     

Release of fatty acids from virus glycoproteins by hydroxylamine

The fatty acids bound to the glycoproteins of Sindbis and vesicular stomatitis viruses can be released by treating the protein with 1 M hydroxylamine at pH 8.0, but the rates of release vary greatly among the three proteins. The most labile fatty acyl bonds were in the Sindbis virus PE2/E2 proteins and the most stable were in the E1 protein. Some of the fatty acids in Sindbis virus glycoproteins were reduced to the alcohol after treatment with sodium borohydride, indicating that protein-bound fatty acids could be in thiolester linkage. Sindbis virus PE2/E2 has several cysteine residues near the carboxy terminus, a region of the protein postulated to be localized on the inside (cytoplasmic face) of the bilayer, and protease digestion of microsomal membranes containing E2 protein removed a small portion of this cytoplasmic tail as well as significant amounts of the fatty acid. For the vesicular stomatitis virus G protein, the sensitivity of fatty acid hydrolysis appeared to depend on the conformation of the protein and a significant fraction of G protein was converted to a disulfide-linked dimer by hydroxylamine. These data implicate cysteinyl groups on these proteins as sites involved in fatty acid acylation.     

Luciferase imaging of a neurotropic viral infection in intact animals

The identification of viral determinants of virulence and host determinants of susceptibility to virus-induced disease is essential for understanding the pathogenesis of infection. Obtaining this information requires infecting large numbers of animals to assay amounts of virus in a variety of organs and to observe the onset and progression of disease. As an alternative approach, we have used a murine model of viral encephalitis and an in vivo imaging system that can detect light generated by luciferase to monitor over time the extent and location of virus replication in intact, living mice. Sindbis virus causes encephalomyelitis in mice, and the outcome of infection is determined both by the strain of virus used for infection and by the strain of mouse infected. The mode of entry into the nervous system is not known. Virulent and avirulent strains of Sindbis virus were engineered to express firefly luciferase, and the Xenogen IVIS system was used to monitor the location and extent of virus replication in susceptible and resistant mice. The amount of light generated directly reflected the amount of infectious virus in the brain. This system could distinguish virulent and avirulent strains of virus and susceptible and resistant strains of mice and suggested that virus entry into the nervous system could occur by retrograde axonal transport either from neurons innervating the initial site of replication or from the olfactory epithelium after viremic spread.     

Molecular basis of Sindbis virus neurovirulence in mice.

We examined a variety of strains of Sindbis virus for the genetic changes responsible for differences in neurovirulence in mice. SV1A (a low passage of the AR339 strain of Sindbis virus), a neuroadapted Sindbis virus (NSV), and two laboratory strains of Sindbis virus (HRSP and Toto1101) were examined. NSV causes severe encephalomyelitis with hind-limb paralysis and high mortality after intracerebral inoculation in weanling mice. In contrast, SV1A causes only mild, nonfatal disease in weanling mice; however, in suckling mice, SV1A causes a fatal encephalomyelitis after either intracerebral or subcutaneous inoculation. The two laboratory strains used have a greatly reduced neurovirulence for suckling mice and are avirulent for weanling mice. The nucleotide sequences and encoded amino acid sequences of the structural glycoproteins of these four strains were compared. Hybrid genomes were constructed by replacing restriction fragments in a full-length cDNA clone of Sindbis virus, from which infectious RNA can be transcribed in vitro, with fragments from cDNA clones of the various strains. These recombinant viruses allowed us to test the importance of each amino acid difference between the various strains for neurovirulence in weanling and suckling mice. Glycoproteins E2 and E1 were of paramount importance for neurovirulence in adult mice. Recombinant viruses containing the nonstructural protein region and the capsid protein region from an avirulent strain and the E1 and E2 glycoprotein regions from NSV were virulent, although they were less virulent than NSV. Furthermore, changes in either E2 (His-55 in NSV to Gln in SV1A) or E1 (Ala-72 in NSV to Val in SV1A and Asp-313 in NSV to Gly in SV1A) reduced virulence. For virulence in suckling mice, we found that a number of changes in E2 and E1 can lead to decreased virulence and that in fact, a gradient of virulence exists.     

一株辛德毕斯病毒的基因组序列测定及分析

目的：对引进的一株辛德毕斯病毒的基因组序列进行测定,阐明其与已报道毒株序列的关系。方法：对辛德毕斯病毒基因组编码区进行分段RT-PCR扩增,对非编码区采用RACE法进行扩增,将扩增产物直接进行测序,应用DNAStar软件将测序结果拼接得到基因组序列,采用MEGA3.1软件对9株辛德毕斯病毒基因组序列进行系统进化发生树的构建。结果与结论：此株辛德毕斯病毒基因组共11663nt,编码3745个氨基酸残基,其中5＇端的2/3基因组编码4种非结构蛋白NSp1、NSp2、NSp3和NSp4,3＇端的1/3基因组编码5种结构蛋白E1、E2、E3、6K和C;结构基因和非结构基因之间有48nt的连接区为非翻译区;病毒基因组5＇末端和3＇末端分别有59、318nt的非编码区;序列同源性分析结果表明,此株病毒与S.A.AR86株的同源性最高,两者核苷酸序列的同源性为99.7%,氨基酸序列的同源性为99.6%,而与本室保存的另一辛德毕斯病毒MEI株的遗传进化关系稍远,系统进化发生树处于不同分支上。     

Molecular analysis of neurovirulent strains of Sindbis virus that evolve during persistent infection of scid mice.

To understand the role of tissue-specific adaptation and antibody-induced selectional pressures in the evolution of neurovirulent viruses, we analyzed three strains of Sindbis virus isolated from the brains of persistently infected scid mice and four strains of Sindbis virus isolated from the brains of scid mice with viral reactivation following immune serum treatment. For each viral isolate, we tested neurovirulence in weanling BALB/c mice and sequenced regions of the E2 and E1 envelope glycoprotein genes that are known to contain important determinants of Sindbis virus neurovirulence. One strain isolated from a persistently infected scid mouse and two strains isolated from scid mice with viral reactivation were neurovirulent, resulting in mortality in 80 to 100% of weanling BALB/c mice. All three neurovirulent strains contained an A-->U change at nucleotide 8795, which predicts a Gln-->His substitution at E2 amino acid position 55. No nucleotide changes were detected in the other sequenced regions of the E2 and E1 envelope glycoprotein genes or in the avirulent isolates. Our findings indicate that tissue-specific adaptations, rather than antibody-induced selectional pressures, are a critical determinant of the evolution of neurovirulent strains of Sindbis virus and provide evidence that E2 His-55 is an important neuroadaptive mutation that confers neurovirulence properties on Sindbis virus.     

Comparative Toxicity of Three Organic Acids to Freshwater Organisms and Their Impact on Aquatic Ecosystems

The comparative toxicity of lactic acid, acetic acid, and benzoic acid to tilapia (Oreochromis mossambicus), cladoceran crustacea (Moina micrura), and oligochaete worm (Branchiura sowerbyi) were determined using static bioassay tests. Worms were found most sensitive to all the acids whereas the cladoceran was found most resistant to lactic acid and the fish most resistant to acetic acid and benzoic acid. The 96h LC₅₀ values of lactic acid, acetic acid, and benzoic acid, were, respectively, 257.73, 272.87, and 276.74 mg L^?1 for O. mossambicus; 329.12, 163.72, and 71.65 mg L^?1 for M. micrura and 50.82, 14.90, and 39.47 mg L^?1 for B. sowerbyi. Tilapia lost appetite at sub-lethal concentrations as low as 2.18 mg L^?1 lactic acid, 1.26 mg L^?1 acetic acid, and 13.84 mg L^{? 1} of benzoic acid. Growth and reproduction of the fish were affected following 90-day chronic exposure to sub-lethal concentrations of the acids. Minimum effective concentration of the acids that significantly reduced food conversion efficiency (FCE), percent increase of weight, specific growth rate, yield and fecundity of the fish were 2.18, 1.47, and 3.95 mg · L^?1 of lactic acid, acetic acid, and benzoic acid, respectively. Effects of acetic acid and benzoic acid on FCE, weight increase, and yield were not significantly different from each other whereas lactic acid produced different effects from acetic acid as well as benzoic acid. Mean values of dissolved oxygen, primary productivity, and plankton populations of the test medium significantly reduced from control at 16.94 mg L^?1 lactic acid, 16.79 mg L^?1 acetic acid, and 13.84 mg L^?1 benzoic acid.     

云南辛德毕斯病毒的生物学性状研究

本文对云南首次分离到的Sindbis病毒进行了滤过试验、耐酸耐醚试验、致细胞病变、动物敏感性试验、血凝特性、空斑和毒力等试验研究,结果符合披膜病毒科的病毒特性。交叉血抑试验和免疫荧光试验,以及空斑减少中和试验进一步证实为甲病毒属的Sindbis病毒。其空斑纯化株的生物学特性也与原株相符,纯化株的制备为该株病毒分子生物学研究的准确性和一致性提供了条件。云南Sindbis病毒的首次分离具有重要的流行病学意义,其生物学特性研究结果对我省该病的诊断和防治具有重要的指导意义。     

Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.     

Aeromonas, a possible etiological agent in the formation of secondary tumors of crown gall

Abstract From a secondary tumor in a bean stem we have isolated a Gram-negative bacteria, named by us T.2. These bean stems had crown gall tumors induced by the ATV strain of Agrobacterium tumefaciens . This bacterium was classified as belonging to the genus Aeromonas and possesses the capacity of inducing overgrowths in plants, synthesizing indole acetic acid (IAA). The codified phenotypic characteristics of bacterium T.2. via the Ti-plasmid of A. tumefaciens , such as opine utilization and sensitivity to agrocin 84, have been studied. Neither octopine nor nopaline is utilized by T.2. and it is resistant to agrocin 84, whereas the strain ATV of A. tumefaciens utilizes nopaline, and is sensitive to agrocin 84.     

Nucleocapsid-glycoprotein interactions required for assembly of alphaviruses.

We have studied interactions between nucleocapsids and glycoproteins required for budding of alphaviruses, using Ross River virus-Sindbis virus chimeras in which the nucleocapsid protein is derived from one virus and the envelope glycoproteins are derived from the second virus. A virus containing the Ross River virus genome in which the capsid protein had been replaced with that from Sindbis virus was almost nonviable. Nucleocapsids formed in normal numbers in the infected cell, but very little virus was released from the cell. There are 11 amino acid differences between Ross River virus and Sindbis virus in their 33-residue E2 cytoplasmic domains. Site-specific mutagenesis was used to change 9 of these 11 amino acids in the chimera from the Ross River virus to the Sindbis virus sequence in an attempt to adapt the E2 of the chimera to the nucleocapsid. The resulting mutant chimera grew 4 orders of magnitude better than the parental chimeric virus. This finding provides direct evidence for a sequence-specific interaction between the nucleocapsid and the E2 cytoplasmic domain during virus budding. The mutated chimeric virus readily gave rise to large-plaque variants that grew almost as well as Ross River virus, suggesting that additional single amino acid substitutions in the structural proteins can further enhance the interactions between the disparate capsid and the glycoproteins. Unexpectedly, change of E2 residue 394 from lysine (Ross River virus) to glutamic acid (Sindbis virus) was deleterious for the chimera, suggesting that in addition to its role in nucleocapsid-E2 interactions, the N-terminal part of the E2 cytoplasmic domain may be involved in glycoprotein-glycoprotein interactions required to assemble the glycoprotein spikes. The reciprocal chimera, Sindbis virus containing the Ross River virus capsid, also grew poorly. Suppressor mutations arose readily in this chimera, producing a virus that grew moderately well and that formed larger plaques.     

Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence.

Sindbis virus induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells in vivo may contribute to the resulting disease and mortality. To investigate the role of apoptosis in Sindbis virus pathogenesis, infected mouse brains were assayed by the in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique and for DNA ladder formation. Infection with recombinant Sindbis virus strain 633 resulted in widespread apoptosis in newborn mouse brains and spinal cords, but few apoptotic cells were observed following infection of 2-week-old animals. This finding correlates with the age-dependent mortality observed in mice. The more neurovirulent virus TE, which differs from 633 by a single amino acid in the E2 glycoprotein, induced significant apoptosis in brains and spinal cords of 2-week-old animals, consistent with its ability to cause fatal disease in older animals. Double-labeling experiments demonstrated that the apoptotic cells were also infected with Sindbis virus. Thus, Sindbis virus-induced apoptosis appears to be a result of virus infection and is likely to reflect pathogenic mechanisms for other viruses.     

Host modification of Sindbis virus sialic acid content influences alternative complement pathway activation and virus clearance

Previous studies have shown that Sindbis virus, an enveloped alphavirus of the togavirus group, activates the alternative complement pathway in the absence of detectable antiviral immunoglobulin. The present studies examined the role of the host-determined sialic acid content of Sindbis virus on activation of the alternative complement pathway. Purified Sindbis virus grown in baby hamster kidney (BHK-SV) and in mosquito (MOSQ-SV) cells yielded virus with 10.2 and less than 2.0 nmol sialic acid/mg viral protein, respectively. Sindbis virus deficient in sialic acid (2.0 nmol sialic/mg) was also produced by treating the BHK-SV with neuraminidase (NANase-SV). When MOSQ-SV or NANase-SV was incubated in either C4DGPS or C2DHS, each consumed significantly more C3 than did BHK-SV, indicating that the ability of Sindbis virus to activate the alternative pathway is inversely related to its sialic acid content. Studies in vivo showed that virus deficient in sialic acid (MOSQ-SV) was cleared from the blood of mice much more efficiently than was virus rich in sialic acid (BHK-SV), after i.v. inoculation. Furthermore, when animals were depleted of C3 through C9 by cobra venom factor (CoVF) treatment, no differences in the clearance of high and low sialic acid-containing viruses were observed. Thus both the activation in vitro and complement-dependent clearance in vivo are significantly affected by the host-determined sialic acid content of Sindbis virus.     

Abortive infection with Sindbis virus of a Chinese hamster ovary cell mutant defective in phosphatidylserine and phosphatidylethanolamine biosynthesis

The effects of phosphatidylserine starvation on the infection with Sindbis virus (an enveloped RNA virus) have been investigated in a Chinese hamster ovary (CHO) cell mutant (strain PSA-3) which requires exogenously added phosphatidylserine for cell growth because it lacks the ability to synthesize this phospholipid. When PSA-3 cells were grown in the absence of phosphatidylserine, the cellular contents of phosphatidylserine and also phosphatidylethanolamine produced through decarboxylation of phosphatidylserine decreased. Sindbis virus production in the mutant cells decreased immediately upon phosphatidylserine deprivation as did the contents of phosphatidylserine and phosphatidylethanolamine, whereas the cell growth, viability, and syntheses of protein, DNA and RNA remained normal for approx. 40 h phosphatidylserine starvation. Although PSA-3 cells grown without phosphatidylserine for 24 h were able to bind and internalize Sindbis virus almost normally, viral RNA synthesis was greatly reduced in the cells, suggesting that nucleocapsids of internalized Sindbis virus are not normally released into the cytoplasm. Unlike mammalian cell mutants defective in endosomal acidification, PSA-3 cells grown without phosphatidylserine were not resistant to diphtheria toxin. Furthermore, the yield of virions and viral RNA synthesis in PSA-3 cells were not completely restored on brief exposure of the cells to low pH medium following virus adsorption, which is known to induce artificial fusion of the viral envelope with the plasma membrane of normal host cells and then injection of viral nucleocapsids into the cytoplasm. Our data demonstrate the requirement of membrane phospholipids, such as phosphatidylserine and/or phosphatidylethanolamine, in CHO cells for Sindbis virus infection, and we discuss their possible roles.     

Zygosaccharomyces kombuchaensis: the physiology of a new species related to the spoilage yeasts Zygosaccharomyces lentus and Zygosaccharomyces bailii

Zygosaccharomyces kombuchaensis was recently discovered in the 'tea fungus' used to make fermented tea. Z. kombuchaensis was shown by ribosomal DNA sequencing to be a novel species, and a close relative of Zygosaccharomyces lentus, from which it could not be distinguished by conventional physiological tests. Z. lentus was originally established as a new taxon by growth at 4 degrees C, sensitivity for heat and oxidative stress, and lack of growth in aerobic shaken culture at temperatures above 25 degrees C. Subsequent analysis of Z. kombuchaensis reveals that this species shares these unusual characteristics, confirming its close genealogical relationship to Z. lentus. Detailed physiological data from a number of Z. kombuchaensis and Z. lentus strains clearly demonstrate that these two species can in fact be distinguished from one another based on their differing resistance/sensitivity to the food preservatives benzoic acid and sorbic acid. The spoilage yeasts Zygosaccharomyces bailii and Z. lentus are resistant to both acetic acid and sorbic acid, whereas Z. kombuchaensis is resistant to acetic acid but sensitive to sorbic acid. This would indicate that Z. kombuchaensis strains lack the mechanism for resistance to sorbic acid, but possess the means of resistance to acetic acid. This observation would therefore suggest that these two resistance mechanisms are different, and that in all probability acetic and sorbic acids inhibit yeast growth by different modes of action. Z. kombuchaensis strains were also sensitive to benzoic acid, again suggesting inhibition dissimilar from that to acetic acid.     

Heterologous interference in Aedes albopictus cells infected with alphaviruses.

Maximum amounts of 42S and 26S single-stranded viral RNA and viral structural proteins were synthesized in Aedes albopictus cells at 24 h after Sindbis virus infection. Thereafter, viral RNA and protein syntheses were inhibited. By 3 days postinfection, only small quantities of 42S RNA and no detectable 26S RNA or structural proteins were synthesized in infected cells. Superinfection of A. albopictus cells 3 days after Sindbis virus infection with Sindbis, Semliki Forest, Una, or Chikungunya alphavirus did not lead to the synthesis of intracellular 26S viral RNA. In contrast, infection with snowshoe hare virus, a bunyavirus, induced the synthesis of snowshoe hare virus RNA in both A. Ablpictus cells 3 days after Sindbis virus infection and previously uninfected mosquito cells. These results suggested that at 3 days after infection with Sindbis virus, mosquito cells restricted the replication of both homologous and heterologous alphaviruses but remained susceptible to infection with a bunyavirus. In superinfection experiments the the alphaviruses were differentiated on the basis of plaque morphology and the electrophoretic mobility of their intracellular 26S viral RNA species. Thus, it was shown that within 1 h after infection with eigher Sindbis or Chikungunya virus, A. albopictus cells were resistant to superinfection with Sindbis, Chikungunya, Una, and Semliki Forest viruses. Infected cultures were resistant to superinfection with the homologous virus indefinitely, but maximum resistance to superinfection with heterologous alphaviruses lasted for approximately 8 days. After that time, infected cultures supported the replication of heterologous alphaviruses to the same extent as did persistently infected cultures established months previously. However, the titer of heterologous alphavirus produced after superinfection of persistently infected cultures was 10- to 50-fold less than that produced by an equal number of previously uninfected A. albopictus cells. Only a small proportion (8 to 10%) of the cells in a persistently infected culture was capable of supporting the replication of a heterologous alphavirus.