The shrinkage and swelling of the Particle of minute-body-forming proteid,including virus proteid,due to the addition of sugars or of NaCl

Summary The minute-body-forming proteid particles (protoplasm-lipoproteid particles) including vaccinia virus proteid particles are exerted on some influences in the degree of its swelling through the addition of sugars (glucose and lactose). The swelling of vaccinia particles isolated from cattle's skin tissues is enhanced, while, in the case of vaccinia isolated from rabbit's testicles and the proteid particles from rabbit's brain, it causes shrinkage.Such an influence appears to be unrelated to the penetration or non-penetration of the sugars into the particles, because NaCl which seems to penetrate freely into the particle exerts almost a similar influence upon the degree of its swelling.The decrease of sugar-insoluble space of the particle with the increase of the concentration of sugars added, is not due to the shrinkage of the particle, but probably to the penetration of sugars into the particle.     

The water content of the particle of vaccinia virus proteid

Summary When vaccinia virus proteid particles agglutinated in a weakly acid solution were centrifuged by an ordinary centrifuge for a long period of time, and the water contents of the precipitate thus sedimented were measured successively after every period of centrifuging, then it was found that the water content of the sediment diminished rapidly at the beginning of the centrifuging, but that at later periods of sedimentation the diminution rate became low and constant, decreasing rectilinearly.If we assume that the decrease in the water content with a constant rate is due to the squeezing out the water contained in the particle itself and that the rapid decrease at the beginning of the centrifugation with an inconstant rate is due to the pressing out of the water interposing among proteid particles, the water content of the particle can be estimated by measuring the content at the time when the decreasing rate becomes constant. From this point of view, the proteid particles are estimated as containing about 90% water. In the same way coli-phage proteid particles are found to have a similar water content. Sugar-insoluble space of the proteid particle was calculated by measuring the quantity of sugar added to the suspension of elementary bodies which were centrifuged off after the addition of the sugar; the water content was estimated from this sugar-insoluble space to be likewise about 90%, although the space varies over a wide range with the concentration of sugar added.Also from the viewpoint of the filtrability of the particles it is reasonable to consider that they strongly swells on absorbing water of about 10 times as great as its mass.These conclusions can be applied also generally to minute-bodyforming proteid (protoplasm-lipoproteid particles) having no virus activity.I should like to express my sincere thanks to Prof. Dr.M. Y. Dzen, the former director of the Shanghai Hygienic Institute, for his generous imparting of a great quantity of vaccine pulp having a strong activity.     

Replication of human adenoviruses in guinea pig embryo cells: an ultrastructural study

Summary Guinea pig embryo (GPE) cells showed different degrees of susceptibility to human adenovirus types as determined by virus infectivity assay and electron microscopic examination. Adenovirus 2 and 5 induced extensive cellular changes and produced high titers of infectious virus in GPE cells as in human cells. Mature progeny virus and protein crystals were observed in both cell types. Adenovirus 7 induced some cellular changes in GPE cells but only a small number of cells yielded progeny virus as determined by electron microscopy. Adenovirus 3, 8 and 31 induced some cellular changes but no progeny virus was found under electron microscopic examination. Characteristic fibers were observed in nuclei of adenovirus 31 infected cells. The ability of human adenovirus 2 and 5 to replicate in GPE cells is an example of an unusual cross-species biological property of certain adenovirus types. This property may be useful as a biological marker for these virus types.With 8 Figures     

Study on West Nile virus persistence in monkeys

Experiments in M. rhesus showed persistence to be a typical property of West Nile virus. This property was exhibited by strains belonging to different antigenic types, and varying in virulence and in the isolation area (U.S.S.R., Uganda, India). The duration of persistence was at least 5 1/2 months in asymptomatic infection and in convalescence after encephalitis or a febrile disease. The virus isolated within the first 2 weeks after inoculation of monkeys has the standard properties. The virus persisting for 2 months retains its cytopathic and antigenic activity, however, is non-pathogenic for white mice. After 5 1/2 months of persistence the virus has no neurovirulence or cytopathic properties but is capable of infecting the susceptible cells and induces in them the synthesis of virus-specific antigen detectable by immunofluorescence. The persisting virus has been isolated by cocultivation of trypsinized monkey organ cells and cells of the indicator culture. This virus was located mostly in the cerebellum, cerebral subcortical ganglia, lymph nodes, and kidneys. The monkeys experiencing encephalitis, febrile, or asymptomatic infection showed in morphological examinations a subacute inflammatory-degenerative process in the central nervous system. The results suggest that West Nile virus, one of the most widely spread arboviruses in Africa, Asia, and Europe, may be implicated in the etiology of subacute diseases of the CNS.     

Further studies on the mechanism of centrifugal enhancement of cytomegalovirus infectivity

The phenomenon of centrifugal enhancement of infectivity, previously shown to be an intrinsic property of murine and human cytomegalovirus (CMV) was studied further in an attempt to understand the underlying mechanism of action and to try to influence the phenomenon by means of extrinsic factors. The sand-rat herpes virus also displayed centrifugal enhancement in rat, mouse, and human cells. Further investigations were performed with murine CMV. The property of enhancement was not restricted to a specific phase of cell cycle. A variety of factors, known to influence cellular physiology, did not significantly affect the ratio of centrifugal infectivity to standard infectivity i.e., the c/s ratio. However, it was necessary to centrifuge the infected cultures within one hour of the establishment of infection, since centrifugation after this delay failed to yield the enhancement; but there was no partial effect; the cultures either showed complete centrifugal enhancement or no such effect. Thus, it appears that some herpes viruses, during their initial encounter with susceptible cells, can be forced into preferential productive infection by the process of centrifugation, but after a lapse of an hour or more, centrifugation can no longer do this and most of the infected cells fail to replicate virus. Consequently we continue to recommend low speed centrifugation (1000 x g, for 30 min) in order to enhance CMV detection and to optimize infectivity assays.     

Host cell-dependent lateral mobility of viral glycoproteins

The lateral mobility of viral envelope proteins on the plasma membranes of infected cells is an important factor in both virus assembly and pathogenesis. The envelope glycoproteins of measles and human parainfluenza virus are mobile on the surfaces of infected HeLa cells and undergo lateral redistribution in the presence of specific antibody, forming unipolar caps. In contrast, no such redistribution was observed with influenza virus hemagglutinin (HA) or vesicular stomatitis virus (VSV) G glycoproteins on infected HeLa cell surfaces. However, the HA and G glycoproteins were both found to be mobile in the plasma membrane of CV-1 cells, or human or murine peritoneal macrophages. These results indicate that host cell-dependent as well as virus-specific factors are involved in determining viral glycoprotein mobility. No significant differences in the patterns of synthesis of influenza or VSV viral proteins were found in the various cell types examined. The HA and G proteins, when expressed from vaccinia virus recombinants, were each found to be immobile in HeLa cells and mobile in CV-1 cells, thus indicating that the host cell-dependent differences in mobility are an intrinsic property of each viral glycoprotein molecule and not the result of interaction with other viral components. It is suggested that the association of viral glycoproteins with either the cytoskeleton or membrane-associated cellular proteins may be related to the observed differences in lateral mobility.     

Restriction of Moloney murine leukemia virus replication in Moloney murine sarcoma virus-infected cells

MuSV349 is a TB-cell line which produces infectious MuSV with little or no MuLV detectable by the XC assay. The apparent restriction of MuLV replication in MuSV349 cells was investigated. A replication-competent helper virus, with protein composition nearly identical to that of Mo-MuLV was isolated from the viruses produced by MuSV349 cells. This helper MuLV after it was separated from MuSV and upon infection of TB cells produced viral titer similar to that of Mo-MuLV-infected TB cells indicating that its replication might have been restricted in the MuSV349 cells. To find out whether the suppression of the helper virus replication is due to the genetic peculiarities of the virus or MuSV349 cells, the relative amounts of MuLV and MuSV produced by several distinct clonal MuSV isolates (derived from a common progenitor) upon superinfection with Mo-MuLV were determined. The results of these experiments showed that while both SV7 and SV15F on coinfection with Mo-MuLV produced MuSV in excess over MuLV; and ts110 and tsSV13 on coinfection with Mo-MuLV produced MuLV in excess over MuSV. Since the same Mo-MuLV is used in these experiments and since upon transfer to a different cell, SV7, SV15F, and ts110 retain the property to restrict or not restrict MuLV replication it appears that the above property is determined by the genetics of the MuSV.     

The cytotoxic effect of fowlpox virus on primary human amniotic cell cultures

Summary This report describes a cytotoxic effect of fowlpox virus on human amnion cells. Changes began after 48 hours and were complete within a week following inoculation. The infective property and the cytotoxic property were compared. Suspensions which were cytotoxic nearly always contained at least 10³ PFU/ml. Titers of both properties fell to undetectable levels during 30 days in human amnion cell culture. Titers of both properties were reduced by centrifugation, filtration, heat, ultraviolet light, acid, chloroform, and ether. Exposure to ultraviolet light reduced infectivity more rapidly than cytotoxicity. Treatment with ultrasound regularly increased cytotoxicity titer; infectivity increased to a much smaller and less predictable degree. Cytotoxicity-neutralizing serum antibodies developed in guinea pigs inoculated with fowipox virus. Cytotoxicity was identical in normal and IDUR-treated human amnion cells, but no plaques appeared in IDUR-treated chick embryo cells. The data suggest a mechanism of cytotoxicity that requires contact between virus and cell that does not require assembly of infectious viral particles.This investigation was supported by research grant AI-01023 and by training grant AI-177 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service.     

Characteristics of the Interaction in Vitro Between Cytotoxic Thymus-Derived Lymphocytes and Target Monolayers Infected with Lymphocytic Choriomeningitis Virus

Cell-mediated lysis of target monolayers intccted with lymphocytic choriomeningitis virus has previously been shown to be a property of specifically sensitized thymus-derived lymphocytes (T cells). The cytotoxic potential of individual effector lymphocytes is maximally expressed when infected monolayers are confluent and overlaying immune spleen cells cover less than the total target area. The data presented support the following interpretations [l] Cell-mediated injury depends on close association between sensitized T cells and infected L cells. [2] Any attachment made is readily reversible. [3] Effector lymphocytes may damage more than one target cell, operating, perhaps, in a rather circumscribed area. [4] Cell death is a 'one hit' phenomenon. [5] Cytolysis is a property of individual T cells, there being no obvious requirement for co-operation between different populations of spleen cells, [6] Substances secreted into the medium at large are neither directly cytotoxic nor capable of sensitizing normal spleen cells. [7] Specific lysis occurs at temperatures ranging from 27°C to 40°C, being maximal at the higher temperature     

Effects of lysine to arginine mutations in HIV-1 Vif on its expression and viral infectivity

We previously demonstrated that the expression in cells of human immunodeficiency virus type 1 (HIV-1) Vif is maintained at low level by proteasome-degradation. We examined the contribution of 16 lysines present in Vif (NL432 clone), which is composed of 192 amino acids (aa), to its expression within cells and to viral infectivity for non-permissive cells. To this end, various lysine-arginine mutations were introduced into wild-type (wt) Vif, and the mutational effects were monitored by transfection experiments. When all the lysines were changed to arginines, the mutant Vif was expressed in cells at much higher level than wt and was much more stable. Both N-terminal (aa nos. 34 and 36) and C-terminal (aa nos. 179 and 181) lysines were found to be almost sufficient for wt property. Different from this observation, one of the lysines at aa nos. 22 and 26 was demonstrated to be essential for the virus to grow in non-permissive cells. Our results showed that there is no clear co-relationship between the expression level of HIV-1 Vif and viral infectivity.     

Differential budding efficiencies of human T-cell leukemia virus type I (HTLV-I) Gag and Gag-Pro polyproteins from insect and mammalian cells

In this study, we examined the ability of human T-cell leukemia virus type I (HTLV-I) Gag and Gag-Pro to assemble immature virus-like particles (VLPs) and bud from insect and mammalian cells. Transmission electron microscopy of insect cells infected with a recombinant baculovirus carrying the entire gag gene revealed that Pr53(Gag) is targeted to the plasma membrane, where it extensively accumulates and forms electron-dense evaginations. However, no particles could be detected either inside the cells or in the culture supernatants. With the Gag-Pro-expressing construct, we observed HTLV-I-specific cytoplasmic proteolysis of the Gag precursor, but again no particle released in the culture supernatants. Transmission electron microscopic analysis of insect cells expressing Gag-Pro polyprotein revealed large vacuoles in the cytoplasm and no budding particles at the plasma membrane. In contrast, human immunodeficiency virus type 1 Gag polyprotein expressed in insect cells is able to release VLPs. These data showed that unlike other retroviruses, Pr53(Gag) is unable to be released as immature VLPs from insect cells. To determine whether the block in particle budding and release is due to an intrinsic property of Pr53(Gag) or the absence of essential cellular factors in insect cells, we expressed Gag and Gag-Pro polyproteins in human 293 cells. The results indicate that Pr53(Gag) and p24 capsid are released within particles into the culture supernatants of human 293 cells. We found that the myristylation of the N-terminal glycine residue is essential for Gag release. Altogether, these results strongly suggest that the proper assembly of HTLV-I particles is dependent on mammalian host cell factors.     

Cytomegalovirus infectivity: analysis of the phenomenon of centrifugal enhancement of infectivity.

The phenomenon of centrifugal enhancement of infectivity was examined in some detial for the murine cytomegalovirus (MCV). Centrifugal enhancement (meaning that application of virus to monolayer cultures with the aid of a centrifugal field resulted in a 20- to 80-fold increase in infectious centers), was observed in mouse embryo cultures from three different strains of mice, in 3T3 cells, and in rat kidney cells. Three different strains of MCV and 20 plaque-purified strains of MCV all showed the property, as did the one strain of human cytomegalovirus examined. In contrast, herpes simplex virus type 1 and type 2 did not. Centrifugal enhancement could not be explained by increased penetration of MCV or its DNA into cells and their nuclei. Other experiments involving PFU-dose response, uv-inactivation kinetics, electron microscopy, and a variety of labeling regimes with thymidine and uridine isotopes, ruled out the presence of interfering microorganisms. It is concluded, therefore, that the property of centrifugal enhancement is an inherent property of MCV particles, and furthermore, it is suggested that some cytomegaloviruses may have a tendency to enter into a nonreplicating state in homologous fibroblasts.     

Evidence for helper independent murine sarcoma virus. I. Segregation of replication-defective and transformation-defective viruses

In the conventional focus assay for murine sarcoma virus (Hartley and Rowe, 1966), the formation of a focus involves repeated rounds of infection, and, as is shown in this report, the possibility of alterations in the genome of the virus is thereby increased. Multiple rounds of infection were avoided by infecting cells in suspension, plating them sparsely, and allowing them to grow into colonies. XC cells were added to detect which colonies were producing leukemia virus. When cells were infected with the Moloney sarcoma-leukemia virus (M-MuSV(MuLV)), four types of colonies were seen: (1) morphologically normal with syncytia (XC⁺) or (2) without syncytia (XC^?), (3) morphologically transformed with no syncytia, (4) transformed with syncytia. The proportions infected by MuSV (transformed cells) or by MuLV (XC⁺) conformed to Poisson's distribution, and this allowed the calculation of the titers of MuSV and MuLV. Clones of chronically infected cells could readily be isolated.A clone of transformed cells called G8 was derived from JLS-V9 cells infected with M-MuSV(MuLV). The cells produced no MuLV detectable by cocultivation with XC cells, but they did produce sarcoma virus detected by the production of sarcomas in mice and morphological transformation of several lines of mouse cells in culture. The virus had a density of 1.16 g/cm³.The kinetics of focus formation were one-hit when assayed by the conventional assay. Virus picked from most ($\text{32}\text{38}$) of these foci consisted of a mixture of sarcoma virus and leukemia virus but some ($\text{4}\text{38}$) foci were found that produced sarcoma virus alone (presumably “competent” sarcoma virus, i.e., helper-independent). The presumed “competent” sarcoma virus was carried through 4 successive passages and each time, most of the foci were found to contain both MuSV and MuLV, but some produced MuSV only. In contrast, the original, chronically infected G8 cells did not release detectable MuLV through more than 30 passages. Leukemia virus or defective sarcoma virus segregated from the competent MuSV with low and equal frequencies only when new mouse cells were infected.Examination of the individual cells within foci formed by spread of viral infection showed that some cells produced only competent virus; other cells from the foci produced MuSV and MuLV; others were transformed nonproducers containing defective MuSV that could be rescued by superinfection with MuLV; and still others were transformed but MuSV could not be rescued from them.No evidence was found for the presence of a helper virus in excess of the concentration of sarcoma virus and competence appears to be a property of the virion itself. The data suggest that the competent MuSV is similar to the helper-independent strain of the Rous avian sarcoma virus. It is not known why this virus is negative in the XC assay, although the MuLV that segregates from it is positive in this assay.In the search for a helper virus, a form of MuSV was found that did not morphologically transform the cells it infected, nor was it produced by them, but both transformation and release of MuSV appeared on superinfection with MuLV.     

Neutralization kinetics of sensitive and resistant subtype B primary human immunodeficiency virus type 1 isolates

The aim of the study was to determine if sensitive and resistant human immunodeficiency virus type 1 (HIV-1) subtype B primary isolates have different neutralization kinetics. Neutralization assays were undertaken where either the time allowed for virus to react with antibodies or the subsequent period of this mixture's exposure to target cells were varied. The relative neutralization sensitivity/resistance is a reproducible property of the isolates. In a minority of combinations, the titre falls exponentially for as long as the free virions are exposed to antibody. In the remainder, neutralization kinetics shows deviations which may be attributed to events occurring after the virus-antibody mixture is added to the target cells: significant neutralization with minimal exposure of the free virions to antibody; a plot where reduction in virus titre is parallel to the duration of the incubation phase of the assay. Neutralization rate constants are similar for primary HIV-1 SF33, HIV-1 SF162, and HIV-1 89.6, reaching 5 x 10(5)-1 x 10(6)/M sec for the monoclonal antibody IgG1 b12. However, although increased antibody levels produced greater reductions in virus titre the rate of neutralization was not proportional to the antibody concentration. Neutralization of either the free virion or cell-associated virus does not correlate with the resistance/sensitivity of primary subtype B isolates. The target cells play an active role, so that in designing neutralization assays with primary isolates of HIV-1, events following the virus-antibody complex binding to the cell surface have to be taken into consideration.     

Some properties of an avirulent Newcastle disease virus

Summary Newcastle disease virus was isolated from healthy birds using chick kidney cultures. The virus strain involved was exceptionally attenuated in character and appeared to be transmitted by the enteric route. It caused no clinical illness even in day old chicks and did not regularly kill embryonated eggs. The virus is relatively thermostable and its haemagglutinin is exceptionally stable at 56 C. This easily observed property may be useful in genetic studies of the virus.     

Behavior of poliovirus strains of different virulence under solid overlays

Summary Strains of type 2 poliovirus, Neva (highly virulent) and Khab and Ovch (moderately virulent) differed by their characteristics of accumulation when grown in monolayer under agar. Production of the former under agar and in liquid medium did not depend on the dose of inoculum, production of the latter with small infecting doses was less under agar than in liquid medium. When gelatine was added to the agar, no reduction in the final yield of the Khab and Ovch strains was observed. A delay in accumulation of the Khab virus under agar began on the second day, reached maximum early on the third day after which it disappeared. It was more marked in acidified solid medium. In the alkaline medium there was no delay. The same relationship was observed under methylcellulose overlay.A reduction in the final yield of the Khab and Ovch strains under agar was not due to (1) direct interaction of the virus with the agar; (2) different adsorption of virus on cells from the liquid and agar media; (3) different sensitivity to virus of cells in contact for 3 days with the liquid or agar media. Ratio of infectivity of RNA to intact virus was higher in the Khab strain preparations under agar than in the Khab preparations from the liquid medium and the Neva preparations from the liquid or agar media.After treatment with concentrated urea, acid buffer and sonication preparations of the Khab virus under agar gave a marked increase in the virus titer. The Khab virus grown under agar was retained in small-pore filters to a greater extent than the Neva virus grown under similar conditions. The former was also sedimented more completely at 16,000g for 60 minutes.It is suggested that the Khab strain is present as agglomerates of virus particles in solid overlays, particularly in acid medium.Some tests of intratypic differentiation are likely to be associated with this property of virus. Thus, differentiation between the Khab and Neva strains by sensitivity to the neutralizing effect of immune serum included into the agar overlay was not reproducible if gelatine was added to the overlay.     

Heterocellular induction of interferon by negative-sense RNA viruses

The infection of cells by RNA viruses is associated with the recognition of virus PAMPs (pathogen-associated molecular patterns) and the production of type I interferon (IFN). To counter this, most, if not all, RNA viruses encode antagonists of the IFN system. Here we present data on the dynamics of IFN production and response during developing infections by paramyxoviruses, influenza A virus and bunyamwera virus. We show that only a limited number of infected cells are responsible for the production of IFN, and that this heterocellular production is a feature of the infecting virus as opposed to an intrinsic property of the cells.     

Functional Analysis of vif Genes Derived from Various Primate Immunodeficiency Viruses

Replication property in cells of human and simian immunodeficiency viruses (HIVs and SIVs) lacking intact vif gene was evaluated. Of 10 vif mutants constructed in vitro of the major four HIV/SIV groups, only those derived from HIV-1 and HIV-2/SIVmac displayed replication defect. The cell lines non-permissive for the vif mutants of HIV-1 and SIVmac were found to be different. To determine whether Vif is exchangeable between HIV-1 and SIVmac, chimeric virus clones with respect to the vif gene were constructed and virus replication in the cells non-permissive for the vif mutant viruses was monitored. Productive infection in these cells of chimeric viruses clearly indicated that Vif is functionally exchangeable, and that Vifs of different virus origin act through a similar mechanism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ultrastructure of Herpesvirus simiae (Herpes B ivurs).

Herpesvirus simiae, isolated in BSC-1 cultures from the lung of a bonnet monkey (Macaca radiata) which died in an outbreak of respiratory disease, was examined by electron microscopy at 24 and 48 hr after inoculation of monkey kidney cells with 100 TCID₅₀ of virus. Characteristic intranuclear, intracytoplasmic and extracellular virus particles were seen, as well as envelopment of the virus at the nuclear membrane, a property which has been reported for other members of the herpesvirus group. However, there were some special features, such as the development of dense material which surrounded the particles and their release from the infected cells which appeared to be effected by reverse pinocytosis.