Infectious Units Of Virus Research Articles

Infectivity assays of human rhinovirus-A and -B serotypes.

Infectivity is a fundamental property of viral pathogens such as human rhinoviruses (HRVs). This chapter describes two methods for measuring the infectivity of HRV-A and -B serotypes: end point dilution (TCID50) assay and plaque assay. End point dilution assay is a quantal, not quantitative, assay that determines the dilution of the sample at which 50 % of the aliquots have infectious virus. It can be used for all the HRV-A and -B serotypes and related clinical isolates that grow in cell culture and induce cytopathic effect (CPE), degenerative changes in cells that are visible under a microscope. Plaque assay is a quantitative assay that determines the number of infectious units of a virus in a sample. After an infectious unit of virus infects one cell, the infected cell produces progeny viruses that then infect and kill a circle of adjacent cells. This circle of dead cells detaches from the dish and thus leaves a clear hole in a cell monolayer. Plaque assay works only for HeLa-adapted HRV-A and -B serotypes that can make visible plaques on the cell monolayer. Currently the end point dilution assay and plaque assay have not been developed for the newly discovered HRV-C.

Neuropathology associated with feline immunodeficiency virus infection highlights prominent lymphocyte trafficking through both the blood-brain and blood-choroid plexus barriers

Feline immunodeficiency virus (FIV) infection in the cat is a well-evaluated model of human immunodeficiency virus (HIV)-1 infection in man with both viruses associated with significant neuropathology. Although studies in both HIV and FIV infections have shown that virus enters the brain in the acute stages of disease, little is known of the mechanisms of viral entry. The dissection of this stage is fundamental to the development of therapies that may prevent or modulate central nervous system (CNS) infection. The present study was designed to characterize the early sequential neuropathological changes following infection with FIV(GL8), a strain known to enter the CNS in acute infection. Cats were infected either by the intraperitoneal (n = 13) or intravenous (n = 12) route with 2000 cat infectious units of virus. Histopathological assessments following intraperitoneal infections were at 4 (n = 2), 5 (n = 1), 8 (n = 3), 10 (n = 1), 16 (n = 1), 32 (n = 2), 52 (n = 2), and 104 (n = 1) weeks post infection whereas animals infected intravenously were examined (n = 3) at 1, 4, 10, and 23 weeks post infection. The most significant lesions following both routes of infection were lymphocyte-rich perivascular infiltrates within cerebral and cerebellar meninges, in choroid plexus and spinal cord dura mater and within epineurium of the sciatic nerve. In addition, following intravenous infection perivascular infiltrations were noted in parenchymal blood vessels primarily of cerebral white matter. Infiltrates were composed of CD79+ B cells and CD3+ T cells. The latter population contained a mixture of CD4+ and CD8+ cells. The severity of lesions increased in intensity in the 8-to 16-week period following infection and then began to wane. The evaluation of this large group of cats at multiple time points revealed pathology comparable with that of early stage HIV-1-associated encephalitis. Moreover, in contrast to previous FIV neuropathology studies, transient meningeal, choroid plexus, and parenchymal vascular pathology were consistent significant findings suggesting that, as in HIV-1 infection, blood-brain barrier and choroid plexus brain barrier integrity are both compromised in early infection.

A newly developed adenovirus-mediated transfer of a wild-type p53 gene increases sensitivity to cis-diamminedichloroplatinum (II) in p53-deleted ovarian cancer cells.

A new recombinant adenovirus carrying a wild-type p53 gene (AxCAp53) was developed and the combination effect of p53 gene transfer and cis-diamminedichloroplatinum (II) (CDDP) was examined in an ovarian cancer cell line, SK-OV-3, with deletion of the p53 gene. AxCAp53 showed a high efficiency of gene transduction and increased sensitivity to CDDP in the SK-OV-3 cells. It was found that the sensitivity of the cells to CDDP correlated with the amount of infectious units of virus per cell of AxCAp53 which correlated with p53 protein expression. The results suggest that the combination of CDDP and AxCAp53 may be a potential strategy for the therapy of CDDP-resistant ovarian cancer.

Generation of cytotoxic and humoral immune responses by nonreplicative recombinant Semliki Forest virus.

The Semliki Forest virus (SFV) expression system can be used to package recombinant RNA into infectious suicide particles. Such RNA encodes only the SFV replicase and the heterologous protein but no structural proteins of SFV, and it is thus deficient in productive replication. We demonstrate here that infection of C57BL/6 (H-2b) and BALB/c (H-2d) mice with recombinant SFV expressing the nucleoprotein (NP) of influenza virus (SFV-NP) resulted in efficient priming of influenza virus-specific CD8+ cytotoxic T-cell (CTL) responses. The generated CTLs lysed both homologous (A/PR/8/34) and heterologous (A/HK/68) influenza virus-infected, or peptide-coated, target cells to a similar degree as CTLs induced by wild-type influenza virus in a major histocompatibility complex class I-restricted fashion. As few as 100 infectious units of virus induced a strong CTL response. Induction of CTL by SFV-NP could also be achieved in CD4 gene-targeted mice, demonstrating the independence of the primary CTL response of CD4+ helper T cells. One immunization generated a CTL response that peaked after 1 week, and an additional booster injection generated a CTL memory, which was still detectable after 40 days. SFV-NP immunizations also generated high-titered IgG humoral responses that remained significant after several months. These results demonstrate that the recombinant SFV suicide system is highly efficient in antigen presentation and suggest that it may have a potential as a recombinant vaccine.

Rapid detection of mumps virus by the polymerase chain reaction

A procedure for detecting mumps virus in under 48 h was developed using the PCR. The sensitivity of the PCR amplification reaction and of the detection of the PCR product was significantly improved by: (i) enriching for viral template RNAs by overnight culture of the virus in Vero cells and (ii) substitution of polyacrylamide gel analysis for agarose gel electrophoresis. The technique was capable of detecting 1–20 infectious units of virus or an equivalent of 1–10 pg of mumps virus-specific plasmid DNA.

Detection of Human Hepatitis a Virus in Environmental Water by an Antigen-Capture Polymerase Chain Reaction Method

To detect hepatitis A virus (HAV) in environmental water samples, sensitive and specific methods are needed. An hemi-nested enzymatic amplification procedure was developed. When it was associated with an immuno-capture (IC) step, specificity and sensitivity were increased. The presence of a specific DNA fragment of 318 bp was detected by the analysis of the electrophoresis of the PGR products and confirmed on the autoradiogram of the Southern blot of the gel, using a labeled oligoprobe PAl selected in a very highly conserved region of the genome coding for the capsid protein VPl. The IC/PCR detected less than one infectious unit of virus in 75 µ1 sample. Eleven Seine River samples and two drinking water specimens were monitored by this assay. The IC/PCR brings a significant contribution for increasing our knowledge of the incidence of HAV on public health.

Molecular epidemiology of human hepatitis A virus defined by an antigen-capture polymerase chain reaction method.

We describe an immunoaffinity-linked nucleic acid amplification system (antigen-capture/polymerase chain reaction, or AC/PCR) for detection of viruses in clinical specimens and its application to the study of the molecular epidemiology of a picornavirus, hepatitis A virus (HAV). Immunoaffinity capture of virus, synthesis of viral cDNA, and amplification of cDNA by a polymerase chain reaction (PCR) were carried out sequentially in a single reaction vessel. This approach simplified sample preparation and enhanced the specificity of conventional PCR. AC/PCR detected less than one cell culture infectious unit of virus in 80 microliters of sample. Sequencing of AC/PCR reaction products from 34 virus strains demonstrated remarkable conservation at the nucleotide level among most strains but revealed hitherto unsuspected genetic diversity among human isolates. Epidemiologically related strains were identical or closely related in sequence. Virus strains recovered from epidemics of hepatitis A in the United States and Germany were identical in sequence, providing evidence for a previously unrecognized epidemiologic link between these outbreaks.

Application of Gene Probes to Virus Detection in Water

Gene probes offer a rapid and sensitive method for the detection of viruses in water and other environmental samples. Gene probes are small strands of nucleic acid labeled with radioactive or nonradioactive compounds for their detection. The target organism is identified by the hybridization of the probe to the organism's nucleic acid. Nucleic acid probes are at least 1000-fold more sensitive than serological tests such as enzyme-linked-immunoassay and do not first require cultivation of the virus for detection. Gene probes have been developed for organisms that do not grow in cell culture, and probes have been constructed for most of the major groups of enteric viruses. Gene probes have been applied to the detection of enteric viruses in water, marine sediment and shellfish. Radioactively labeled probes can detect as little as 1-10 infectious units of virus within 48 hours. A current disadvantage of probes is that they cannot determine the infectivity of the viruses; however, they can be used to quickly determine the growth of viruses in cell culture. Further development of nonradioactive probes should place virus detection capabilities into the hands of most water quality laboratories.

Variation in tolerance induction and oncogenicity due to strain of avian leukosis virus

Chickens were inoculated as embryos or orally at hatching with various doses of four strains of avian leukosis virus (ALV) subgroup A (RAV-1, RPL40, RPL41 and RPL42). Viraemia, antibody and tumours in chickens of various groups were compared; ALV shedding was determined, but only in chickens inoculated with virus at hatching. Results indicate that 95% to 100% of chickens embryonally inoculated with 105 infectious units of virus were viraemic at hatching, regardless of the strain of virus used. However, the incidence of viraemia in groups of chickens embryonally inoculated with 100 infectious units of virus varied, depending on the strain of virus, from 5% to 72%. All embryonally inoculated chickens, that had detectable virus at hatching and survived to 16 weeks of age were immunologically tolerant to the virus. ALV-induced tumours ranged from 4% to 47% depending on the strain of virus. Chickens inoculated orally at hatching did not develop immuno-logical tolerance to the virus; 13% to 76% of these chickens, depending on the strain of virus, had antibody by 18 weeks of age. ALV shedding in albumen of eggs or cloacal swabs at 42 weeks of age varied from 7% to 9%. Data from this study indicate that the strain of ALV may influence induction of immunological tolerance and tumours in embryonally inoculated chickens and induction of antibody in nontolerantly infected chickens. The data also suggest that chickens that are viraemic at hatching are probably incapable of breaking tolerance and developing antibody.

ENTEROVIRUS PERSISTENCE IN SAUSAGE AND GROUND BEEF

Persistence of coxsackievirus type A9 suspended in ground beef was found not to be sufficiently affected by extensive bacterial growth, during periods of up to 8 days at 23 or 4 C, to afford any notable degree of protection to the consumer. Longer storage times resulted in marked virus loss. After 2 weeks at both temperatures >90% of the input virus was no longer infective. In the preparation of Thuringer sausage, approximately 85% of the input virus was lost during a 24-hr fermentation at 30 C. Subsequent heating of the prepared sausage at 49 C resulted in a progressive loss of virus. However, after 6 hr at 49 C, an average of 1.1 × 103 infectious units of virus per gram of sausage remained of an initial 7.5 × 105 infectious units per gram.

Feline leukemia and sarcoma viruses: susceptibility of human cells to infection.

Human embryonic cells are highly susceptible to infection with feline leukemia and sarcoma viruses. These viruses were propagated in human cultures without antigenic modification or loss of infectivity for cat or human cells. Virus stocks contained at least 10(6) infectious units of virus per milliliter for human cells. Virus present in 10(-6) dilution of virus stock replicated to detectable amounts as early as 7 days after virus infection. The feline sarcoma virus induced morphological transformation of human cells.

Plaque Formation by 55 Rhinovirus Serotypes

Plaque production by 55 rhinoviruses in several lines of HeLa cells is reported. Forty-nine types produced macroscopic plaques under the conditions described previously employing 30 mM MgCl 2 and 30 μg/ml of DEAE-dextran in overlay and M HeLa cells. Basically three kinds of plaques were observed: clear large and intermediate sized plaques and small turbid plaques. Five rhinoviruses produced plaques only in a sensitive clonal line isolated from the M HeLa line. Rhinovirus type 4 produced plaques after the pretreatment of cells with actinomycin D. Enhancement of plaque formation by MgCl 2 has been demonstrated to date with 17 rhinoviruses. Some rhinoviruses were enhanced either by DEAE-dextran or by dextran sulfate in the overlay. No inhibitors were found in any of 10 bovine sera tested. Rhinovirus type 2 was visualized inside HeLa cells by electron microscopy and there appeared to be a very high ratio of physical particles per infectious unit of virus.

Plaque Formation by 55 Rhinovirus Serotypes

Plaque production by 55 rhinoviruses in several lines of HeLa cells is reported. Forty-nine types produced macroscopic plaques under the conditions described previously employing 30 mM MgCl(2) and 30 mug/ml of DEAE-dextran in overlay and M HeLa cells. Basically three kinds of plaques were observed: clear large and intermediate sized plaques and small turbid plaques. Five rhinoviruses produced plaques only in a sensitive clonal line isolated from the M HeLa line. Rhinovirus type 4 produced plaques after the pretreatment of cells with actinomycin D. Enhancement of plaque formation by MgCl(2) has been demonstrated to date with 17 rhinoviruses. Some rhinoviruses were enhanced either by DEAE-dextran or by dextran sulfate in the overlay. No inhibitors were found in any of 10 bovine sera tested. Rhinovirus type 2 was visualized inside HeLa cells by electron microscopy and there appeared to be a very high ratio of physical particles per infectious unit of virus.

Studies on persistent infections of tissue cultures. III. Some quantitative aspects of host cell-virus interactions.

Efforts were made to obtain information on some of the quantitative aspects of host cell-virus interactions in MCN cultures persistently infected with Newcastle disease, mumps, and 6-6 viruses, and to elicit the mechanism which permits simultaneous maintenance of virus and cells for indefinite periods of time. It was shown by 4 different technics that only between 10 and less than 1 per cent of the cells yield infectious virus, depending upon the agent employed and, possibly, variations in the conditions of the cultures. No evidence was found to indicate production of non-infectious virus materials. Only the cells carrying infectious virus are capable upon transfer to uninfected cultures to transmit the infection. Cells from persistently infected cultures, which are free of infectious virus at the time of transfer, failed to liberate virus at a later time during incubation periods of up to 4 weeks. The virus-producing cells contain at any given moment not more than 1 infectious unit of virus, suggesting a linear mode of production; i.e., as soon as a virus particle is completed, it is released. Upon inoculation of MCN test tube cultures with chick embryo-adapted NDV persistent infection and interference with vesicular stomatitis virus (VSV) is established with considerable delay. In contrast, following transfer of MCN-adapted NDV, in form of MCN(NDV) cells or first allantoic passage seeds derived therefrom, the number of virus-producing cells increases logarithmically, doubling every 6 to 8 hours until a total of about 10(4) is reached. Thereafter their numbers rise in proportion to the increase in total cell population; i.e., doubling approximately every 48 hours. At the time when 10(4) virus-producing cells are present in the culture interference with VSV is solidly established. In order to obtain this result about 10(6) cells must have adsorbed virus particles, or, in other words, at least 10(6) virus units must have totally been produced instead of the 10(4) measured by infectivity assay. The implications of these and previously reported data have been discussed in detail and a scheme of the course of events in persistently infected cultures has been presented.

Equivalence between vaccinia particles counted by electron microscopy and infectious units of the virus.

Summary1. Studies of the relation between the number of infectious or pock-forming units of vaccinia virus and the number of virus particles indicate a ratio of 1.5:1 under conditions described. 2. A modified centri-fugation cell is described with which virus suspensions can be concentrated and the particles then counted by electron microscopy. The virus particles are sedimented directly onto coated electron microscope grids which when removed from the centrifugation cell are ready for shadow-casting and examination. The precision of this method is comparable to that obtained by other technics.

Repeated Vaccination of Man Against the Virus of Equine Encephalomyelitis

Summary The content of neutralizing antibodies has been studied in the serum of man following primary vaccination against the Eastern and Western strains of virus of equine encephalomyelitis. The titer of antibodies effective against the Eastern strain in most instances diminishes so rapidly that within 6 to 9 months the serum possesses little or no more immunizing capacity than that taken before vaccination. In no instance was the titer maintained for this period at the level observed 2 weeks following initial vaccination. In contrast, antibodies for the Western strain, though diminishing markedly in a few instances, were maintained in general at a relatively high level for the same period. One instance of four was found in which antibodies induced by subclinical infection before initial vaccination and augmented by the primary vaccination were lost at about the same rate as those produced in normal individuals by vaccination alone. Response to revaccination with a single dose resulted in every case in the induction of antibodies such that serum taken after 7 days neutralized 100,000 to 1,000,000 infectious units of virus of either strain. This reaction with respect to the Eastern strain was much more rapid than that following the first vaccination. The degree of response to both antigens was markedly greater than that seen at the initial vaccination. General reactions to revaccination were more marked than to first vaccination, though still relatively mild. The findings constitute evidence of the transient nature of at least one immunological factor in resistance to the virus. This finding in connection with results of direct investigation on the duration of immunity in vaccinated animals indicates the advisability of a second course of vaccination within one year under conditions of continued exposure to the virus. The height of the response to this second vaccination indicates the possibility that the interval between the second and subsequent vaccinations may safely be much longer than that between the first and second.