Development of a new cell system for the infectivity assay of dengue viruses: plaque formation and virus growth of prototype and wild-type dengue virus strains in a newly established cell line, GK

A newly established cell line, GK, derived from the kidney tissue of Mongolian gerbils, produced plaques by infection of prototype and wild-type dengue virus strains. Both prototype and wild strains of type 2 virus grew in GK cells and formed plaques at 35.5 C and at 31 C, while types 1, 3, and 4 wild strains grew and formed plaques only at 31 C. In GK cells, plaque formation and the growth of dengue viruses depended on the high (35.5 C) and low (31 C) incubation temperatures. Virus yields in GK cells of all the 14 dengue virus strains tested, including four prototype and ten wild-type viruses, were 5 to 1,000-times lower than those in C6/36 cells. After five serial passages in GK cells, types 2, 3, and 4 prototype viruses and type 2 wild strain increased virus yields, and one strain of prototype virus and one strain of wild-type virus decreased mouse neurovirulence.     

Isolation and preliminary characterisation of twenty-five temperature-sensitive mutants of mouse cytomegalovirus

Abstract To study the pathogenicity of mouse cytomegalovirus (MCMV) and to identify virulence determinants, we have isolated and phenotypically characterised 25 temperature-sensitive ( ts ) mutants. Six of these ( tsm 9, tsm 13, tsm 20, tsm 22, tsm 28 and tsm 30) failed to replicate in mice and were avirulent. Five mutants ( tsm 14, tsm 18, tsm 19, tsm 25 and tsm27 ) were to similar virulence to the parenthal wild-type ( wt ) virus, five ( tsm 7, tsm 15, tsm 24, tsm31 ) were 12–100 fold less virulent, five ( tsm 8, tsm 12, tsm 16, tsm 23 and tsm 29) were 150–1500 fold less virulent and four ( tsm 10, tsm 11, tsm 17 and tsm 21) were between 2,000 and 85,000 fold less virulent than wt . One mutant ( tsm 28) did not plaque or replicate at 39°C while 5 other mutants ( tsm 7, tsm 9, tsm 23, tsm 24 and tsm 27) also failed to plaque at 39°C but only failed to replicate or replicated poorly at 40°C. A further two mutants ( tsm 10 and tsm 13) were able to plaque and replicate at 39°C but not 40°C. Six other mutants ( tsm 14, tsm 15, tsm 16, tsm21 , tsm 22 and tsm 30) failed to form plaques at 40°C and were severely restricted in their replication at 40°C. The remaining 11 mutants exhibited varying degrees of restriction in ability to plaque and/or replicate at non-permissive temperatures. These 25 mutants, together with 6 isolated previously, comprise at least 24 complementation groups.     

A型流感病毒持续感染细胞系来源的IVpi-189病毒生物学性状的初步研究

为明确E61-24-P15 A型重组流感病毒的第189代传代子病毒(IVpi-189)是否具备流感病毒温度敏感减毒活疫苗候选株的特点,将IVpi-189病毒感染MDCK细胞,并于不同培养温度条件下培养,观察其致细胞病变效应,病毒合成、释放情况,以及不同温度条件下病毒存活时间。结果显示32℃培养温度下,IVpi-189病毒具有等同于亲代野生病毒株的诱导细胞病变能力,而当培养温度上调至38℃,IVpi-189病毒致细胞病变效果出现缓慢且程度明显减轻。空斑形成单位实验发现IVpi-189病毒在38℃培养条件下增殖能力明显下降,其原因与病毒灭活速度及子病毒释放无关,但与感染细胞病毒合成能力下降有关。上述实验结果初步证实流感病毒持续感染细胞系来源的IVpi-189病毒具有温度敏感减毒活疫苗的生物学特性,在许可培养温度条件下具有良好的增殖能力,而在非许可培养温度下,病毒增殖活性受到明显抑制。本研究为流感病毒减毒活疫苗的开发研制提供实验佐证。     

Impact of mutations in nucleoprotein on replication of influenza virus A/Hong Kong/1/68/162/35 reassortants at different temperatures

The nucleoprotein (NP) of influenza virus is a multifunctional RNA binding protein. The role of NP in the adaptation of influenza viruses to a host has been experimentally proved. Ambiguous data are available on the role of nucleoprotein in the attenuation of influenza A viruses, which is characterized by ability to replicate at low temperature (26°C) and inability to replicate at high temperature (39°C). Influenza virus donor strain A/Hong Kong/1/68/162/35 (H3N2), adapted to growth at low temperature, differs from the wild type virus by 14 amino acid mutations in the internal and non-structural proteins. Two mutations occurred in the NP: Gly102Arg and Glu292Gly. We have obtained viruses with point reverse-mutations in these positions and compared their replication at different temperatures by measuring infectious activity in chicken embryos. It has been shown that reverse mutation Gly292Glu in the NP reduced virus ability to replicate at low temperature, the introduction of the second reverse mutation Arg102Gly completely abolished virus cold adaptation.     

Antibody-resistant spread of vesicular stomatitis virus infection in cell lines of epithelial origin.

In MDCK cells, vesicular stomatitis virus (VSV) buds exclusively from the basolateral plasma membranes beneath tight junctions, whereas influenza virus forms only at the free apical surface. Anti-VSV antiserum did not prevent the formation of plaques on MDCK cell monolayers infected with VSV, whereas plaque formation in BHK-21 cells was completely inhibited by such antiserum. Under similar conditions, homologous antiserum completely prevented plaque formation by influenza virus on MDCK cells. In several other epithelioid cell lines, VSV also formed plaques in the presence of specific antiserum. These results suggest that VSV receptors are present on basolateral membranes in the cells studied and that junctional complexes present between cells may exclude antibody from intercellular spaces and thus permit the lateral spread of virus infection in the presence of neutralizing antibody.     

The growth of simian virus 40 (SV40) host range/adenovirus helper function mutants in an African green monkey cell line that constitutively expresses the SV40 agnoprotein.

The simian virus 40 T-antigen carboxy-terminal mutants, dlA2459 and dlA2475, are cell line and temperature dependent for growth and plaque formation in monkey kidney cells. Although these mutants did form plaques on BSC-1 cells at 37 degrees C, they were about fivefold less efficient for plaque formation than wild-type simian virus 40. These mutants did not grow in CV-1 cells and did not synthesize agnoprotein in those cells. CV-1 cells which constitutively express the agnoprotein were permissive for mutant plaque formation. However, late mRNAs, virion proteins, and progeny virion yields did not accumulate to wild-type levels during mutant infection of the agnoprotein-producing cells.     

Construction and Preliminary Characterization of a Library of “Lethal” Preterminal Protein Mutant Adenoviruses

Adenoviruses containing lethal in-frame insertion mutant alleles of the preterminal protein (pTP) gene were constructed with cell lines that express pTP. Thirty in-frame insertion mutant alleles, including 26 alleles previously characterized as lethal and 4 newly constructed mutant alleles, were introduced into the viral chromosome in place of the wild-type pTP gene. The viruses were tested for ability to form plaques at 37 degrees C in HeLa-pTP cells and at 32 degrees C and 39.5 degrees C in HeLa cells. Two of the newly constructed viruses exhibited temperature sensitivity for plaque formation, one virus did not form plaques in the absence of complementation, seven additional mutants exhibited a greater than 10-fold reduction in plaque formation in the absence of complementation, and another eight mutants exhibited stronger phenotypes than did previously characterized in-frame insertion mutants in the plaque assay. These mutant viruses offer promise for analysis of pTP functions.     

Isolation and genetic characterization of temperature-sensitive mutants of vaccinia virus WR.

One hundred temperature-sensitive mutants of vaccinia virus WR were isolated from virus that had been mutagenized with 5-bromodeoxyuridine or N-methyl-N'-nitro-N-nitrosoguanidine. A rapid screening procedure based on the ability of vaccinia virus to form plaques under liquid overlay medium was used to identify potential mutants among randomly picked plaque isolates or plaques preselected for their small size after temperature shift-up. The preselection technique resulted in a sixfold increase in the number of successful mutant isolations relative to the number of plaques picked. All of the mutants had efficiencies of plating at 39.5 degrees C relative to that at 33 degrees C of 10(-4) or less, and 33 of 40 produced 10% or less of the amount of virus at the nonpermissive temperature (39.5 degrees C) relative to that at the permissive temperature (33 degrees C). Experiments with the fluorescent DNA binding dye Hoechst 33258 demonstrated that 6 of the 100 mutants failed to form characteristic cytoplasmic DNA factories at 39.5 degrees C. To facilitate the functional grouping of such a large number of mutants, a rapid infectious center assay was developed. Thirty of the mutants were assigned to 16 or 17 complementation-recombination groups by using this assay. Recombination experiments have allowed the construction of a genetic map representing 22 mutants in 12 of these groups.     

Characteristics of Anabaena variabilis influencing plaque formation by cyanophage N-1.

Phage N-1 grown in Anabaena strain 7120 [N-1 . 7120] forms plaques on A. variabilis about 10(-7) to 10(-6) as efficiently as on Anabaena 7120. By manipulating different characteristics of the interaction between phage and host, it was possible to increase the relative efficiency of plaque formation to 0.38. Growth of A. variabilis at 40 degrees C for at least three generations resulted in an increase in the rate of phage adsorption and a 10-fold increase in the efficiency of plaque formation. The efficiency of plaque formation was further increased about 42-fold, with little or no further increase in rate of adsorption, in a variant strain. A. variabilis strain FD, isolated from a culture of A. variabilis which had grown for more than 30 generations at 40 degrees C. The low relative efficiency of plaque formation by N-1 . 7120 on A. variabilis could be partially accounted for if A. variabilis contains a deoxyribonucleic acid restriction endonuclease which is absent from Anabaena 7120. Indirect evidence for such an endonuclease included the following: (i) phage N-1 grown in A. variabilis (N-1 . Av) had approximately a 7 X 10(3)-fold higher relative efficiency of plaque formation on A. variabilis than had N-1 . 7120; and (ii) the efficiency of plaque formation by N-1 . 7120 on A. variabilis strain FD was increased by up to 146-fold after heating the latter organism at 51 degrees C.     

Isolation, Complementation, and Initial Characterization of Temperature-Sensitive Mutants of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus

Sixteen temperature-sensitive mutants of Autographa californica nuclear polyhedrosis virus were isolated. Several interesting phenotypes were observed. A large proportion of the mutants were unable to form polyhedral occlusion bodies (polyhedra) at the nonpermissive temperature (32.5°C). At 32.5°C, one mutant formed plaques in which the cells lacked polyhedra. Another mutant type was defective in the production of progeny extracellular nonoccluded virus and produced a “plaque” consisting of only a single cell containing polyhedra at 32.5°C. One mutant was defective in plaque formation, progeny nonoccluded virus formation, and polyhedra formation at 32.5°C. Several mutants produced nonoccluded virus but failed to produce plaques or polyhedra at 32.5°C. Other phenotypes were also distinguished. Complementation analyses, performed by either measuring the increase in extracellular nonoccluded virus formation or by observing polyhedra formation in mixed infections at 32.5°C, indicated the presence of 15 complementation groups. A high frequency of recombination was observed. Four of the mutants were found to be host dependent in their temperature sensitivity for polyhedra formation.     

Separation of Plaque-type Variants of Encephalomyocarditis Virus by Chromatography on Calcium Phosphate

Encephalomyocarditis (EMC) virus (K-2 strain) was separated into two distinct peaks of optical density by chromatography on calcium phosphate, eluting at 0.17 and 0.31 m, respectively. The virus in these peaks could not be distinguished by infectivity (plaque-forming units), hemagglutinin activity, electron microscopic appearance, sedimentation coefficient, or base ratios. However, variants producing large plaques predominated in the first peak eluted, whereas variants producing small plaques were concentrated in the second peak. When plaque-purified large and small plaque variants were chromatographed individually, each gave a single peak eluting at 0.16 and at 0.43 m, respectively. However, one small plaque type isolated was eluted in the 0.17 m region expected for large plaque variants, and not with the bulk of the small plaque variants. There were indications that EMC virus (K-2 strain) could be separated into more than two peaks by use of selected elution gradients. The possibility that there may be a relationship between plaque size and point of elution from calcium phosphate is discussed.     

Features of mutated changes of genomic RNA of cold-adapted and hr-variants of influenza group A virus, detected by RNA:RNA hybridization]

The presence of mutations in the majority of the genes of cold-adapted strains A/Leningrad/134/17/57 (H2N2), A/Leningrad/134/47/57 (H2N2) and A/PR/8/59/1 (H1N1) of influenza A virus has been demonstrated by the RNA-RNA hybridization with the subsequent electrophoresis of double-stranded RNA in 7.5% polyacrylamide gel. The strains were cultivated 17, 47 and 59 passages in the chicken embryos at 25 degrees C. In the genomes of variants passaged in chicken embryos at optimal temperature of incubation 36 degrees C (hr-variants) the used technique permits identification of a single mutant gene. The obtained data suppose the attenuation of cold-adapted vaccine strains of influenza A virus and their high genetic stability to be a result of selection of the variants obtaining multiple mutations in the genome during passaging of the virions at cold temperature. The attenuation of hr-variants is defined by 1-2 mutations (first of all in HA-gene) that makes understandable their inability to serve as donors for recombinant live influenza vaccines construction.     

Susceptibility of influenza viruses to interferon and to poly(I) . Poly(C) determined by the plaque reduction method

Susceptibility of eight strains of influenza A and B viruses to interferon and to poly(I) . poly(C) were determined by the plaque reduction method. All strains tested were slightly less susceptible than vesicular stomatitis virus (VSV) in an established line of canine kidney (MDCK) cells. The 50% plaque depression doses (PD50) of poly(I) . poly(C) for influenza A and B viruses were as high as 3.0- to 4.5-fold and 6- to 18-fold that for VSV, respectively. The amounts of interferon required to inhibit plaque formation of influenza A and B viruses by 50% were 3.0-6.2 and 7.3-15.2 units/ml, respectively. The ratio of PD50 of poly(I) . poly(C) for each strain of influenza viruses tested to that for VSV in chick embryo cells was almost the same as in MDCK cells. Furthermore, in chick embryo cells, the strains of influenza virus tested were demonstrated to be much more susceptible to poly(I) . poly(C) than both Newcastle disease virus and vaccinia virus. It is suggested that influenza viruses may be relatively susceptible to interferon and to poly(I) . poly(C).     

Evaluation of Culture Media for the Isolation of Salmonellae from Feces

Serial passage of the 64-2389 strain of type 3 parainfluenza virus in cercopithecus monkey kidney tissue cultures at low temperatures resulted in the selection of a variant which had a higher efficiency of plaque formation at 25 C than the parent line grown at 37 C. The cold variant, unlike the parent strain, plaqued readily at 25 C, and at 37 C it produced significantly larger plaques. Virus titers of the cold variant in hamster lungs were significantly lower and this was probably caused by the stimulation of interferon by the cold variant during the early phase of the infection. The cold variant, like the virus grown at 37 C, also induced the synthesis of interferon late in the infection. Hamsters responded to the intranasal inoculation of each virus line by the development of hemagglutinating-inhibiting antibodies in the sera.     

Ultraviolet enhanced reactivation of a human virus: effect of delayed infection

The ability of UV-irradiated herpes simplex virus to form plaques was examined in monolayers of CV-1 monkey kidney cells preexposed to UV radiation at different intervals before virus assay. From analysis of UV reactivation (Weigle reactivation) curves it was found that as the interval between cell UV irradiation (0-20 J/m2) and initiation of the virus assay was increased over a period of five days, (1) the capacity of the cells to support unirradiated virus plaque formation, which was decreased immediately following UV exposure to the monolayers, increased and returned to approximately normal levels within five days, and (2) at five days an exponential increase was observed in the relative plaque formation of irradiated virus as a function of UV fluence to the monolayers. For high UV fluence (20 J/m2) to the cells, the relative plaque formation by the UV-irradiated virus at five days was about 10-fold higher than that obtained from assay on unirradiated cells. This enhancement in plaque formation is interpreted as a delayed expression of Weigle reactivation. The amount of enhancement resulting from this delayed reactivation was several fold greater than that produced by the Weigle reactivation which occurred when irradiated herpes virus was assayed immediately following cell irradiation.     

Isolation of Two Plaque Variants from the Adenovirus Type 2-Simian Virus 40 Hybrid Population Which Differ in Their Efficiency in Yielding Simian Virus 40

Studies on the adenovirus type 2-simian virus 40 (SV40) hybrid population demonstrated two genetically stable variants within this population, which were isolated by plaquing in African green monkey kidney cells. These variants were similar in that each induced SV40 T antigen in human embryonic kidney cells and contained similar concentrations of nonhybrid adenovirus type 2 virions and adenovirus-encapsidated particles containing the infectious SV40 genome. These variants differed markedly, however, in their ability to produce SV40 viral antigen in human embryonic kidney cells and the efficiency with which they produce SV40 plaques in monkey cell monolayers. It is postulated that the differences in SV40-yielding efficiency between these variants lie in the nature of the recombinant deoxyribonucleic acid composing the genome of the hybrid particles.     

Markers Differentiating Type 3 Parainfluenza Virus Grown at 25 and 37 C

Serial passage of the 64-2389 strain of type 3 parainfluenza virus in cercopithecus monkey kidney tissue cultures at low temperatures resulted in the selection of a variant which had a higher efficiency of plaque formation at 25 C than the parent line grown at 37 C. The cold variant, unlike the parent strain, plaqued readily at 25 C, and at 37 C it produced significantly larger plaques. Virus titers of the cold variant in hamster lungs were significantly lower and this was probably caused by the stimulation of interferon by the cold variant during the early phase of the infection. The cold variant, like the virus grown at 37 C, also induced the synthesis of interferon late in the infection. Hamsters responded to the intranasal inoculation of each virus line by the development of hemagglutinating-inhibiting antibodies in the sera.     

Avian Influenza Virus Glycoproteins Restrict Virus Replication and Spread through Human Airway Epithelium at Temperatures of the Proximal Airways

Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37°C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32°C). These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40°C), rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32°C and 37°C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32°C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA) and neuraminidase (NA) from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and suggests that adaptation of avian influenza viruses to efficient infection at 32°C may represent a critical evolutionary step enabling human-to-human transmission.     

Contrasting Effects of Polycations on Plaquing Efficiency of Encephalomyocarditis Virus Variants

Polycation treatment of L cell monolayers affected plaquing efficiency of both the r(+) and r variants of the encephalomyocarditis virus. Plaque formation by r(+) variant was decreased markedly by three structurally different types of synthetic basic polymers, diethylaminoethyl dextran, hexadimethrene (polybrene), and basic polyamino acids. In contrast, these same substances increased substantially the number of plaques formed by the r variant. The effect on the two variants was observed when polycations were applied to the cells before or simultaneously with the introduction of virus. The molar concentration and size of the polymer proved important. Thus, basic polyamino acids of low molecular weight were significantly more inhibitory for the r(+) variant than were those of high molecular weight. On the other hand, plaquing efficiency of the r variant was increased by relatively large polyamino acids, but not by polymers of small size. Basic polyamino acids inhibited r(+) plaque formation to a greater degree at low than at high pH values. However, plaquing efficiency of the r variant in polycation-treated cultures was not affected by changes in pH. Basic polymers appear to bind to cell membranes and affect either attachment or uptake of the viruses. The evidence suggests that the substances influence by different mechanisms the interaction of the r(+) and r variants with cells.     

Genetic analysis of the yellow fever virus NS1 protein: identification of a temperature-sensitive mutation which blocks RNA accumulation.

The flavivirus NS1 protein is a highly conserved nonstructural glycoprotein that is capable of eliciting protective immunity. NS1 homodimers are secreted from virus-infected mammalian cells, but the protein is also present at the plasma membrane and in the lumen of intracellular vesicles. Based on these properties, it has been speculated that NS1 may function in virus maturation or release. To gain further insight into NS1 function, we used clustered charged-amino-acid-to-alanine mutagenesis to create 28 clustered substitutions in the NS1 protein of yellow fever virus. To screen for conditional mutations, full-length RNAs containing each mutation were assayed for plaque formation at 32 and 39 degrees C after RNA transfection. We found that 9 mutations were lethal, 18 allowed plaque formation at both temperatures, and 1, ts25, was strongly heat sensitive and was unable to form plaques at 39 degrees C. Lethal mutations clustered in the amino-terminal half of NS1, whereas those leading to impaired replication relative to the parent were distributed throughout the protein. High-multiplicity infections at 39 degrees C demonstrated that ts25 was defective for RNA accumulation, leading to depressed viral protein synthesis and delayed virus production. Although ts25 secreted less NS1 than did the parent, temperature shift experiments failed to demonstrate any temperature-dependent differences in polyprotein processing, NS1 stability and secretion, or release of infectious virus. The ts lesion of ts25 was shown to be due to a single alanine substitution for Arg-299, a residue which is conserved among flaviviruses. These results argue that NS1 plays an essential but as yet undefined role in flavivirus RNA amplification.