TCID50 Of Virus Research Articles

Genetic and pathogenic characterization of a Russian subtype 2 PRRSV-1 isolate

Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure and respiratory problems. Data about the virulence and pathogenicity of subtype 2 PRRSV-1 strains are limited. The main purposes of this investigation were to characterize the full genome sequence of the subtype 2 PRRSV-1 WestSib13 strain and to compare the pathogenicity with that of the subtype 1 PRRSV-1 Lelystad strain. Comparison of the whole genome sequence of the WestSib13 strain with that of PRRSV-1 prototype strains revealed a 76.2% (subtype 1 Lelystad virus) and 79.0% (subtype 3 Lena virus) identity, respectivelyThe virulence and pathogenicity of the European subtype 2 PRRSV strain WestSib13 and the European subtype 1 PRRSV strain Lelystad were compared in 3-week-old piglets upon inoculation of 105.4 TCID50 of virus. Non-infected animals (control group) as well as animals infected with the Lelystad strain were clinically healthy until 14days post challenge. In contrast, animals infected with the WestSib13 strain demonstrated dyspnea starting at 3days post-inoculation (dpi). All piglets in this group died between 5 and 8 dpi. During that period, fever was not observed in WestSib13-infected animals. Viremia was detected in animals from both infected groups starting from 2 dpi. Viral loads in serum and lungs upon euthanasia were significantly higher (3 log10) in the WestSib13-infected than in the LV-infected animals.Taken together, this study provides the full genome sequence and the unusual virological and clinical outcome (high level viremia without fever) of the novel WestSib13 strain.

Attenuation of tick-borne encephalitis virus using large-scale random codon re-encoding.

Large-scale codon re-encoding (i.e. introduction of a large number of synonymous mutations) is a novel method of generating attenuated viruses. Here, it was applied to the pathogenic flavivirus, tick-borne encephalitis virus (TBEV) which causes febrile illness and encephalitis in humans in forested regions of Europe and Asia. Using an infectious clone of the Oshima 5–10 strain ("wild-type virus"), a cassette of 1.4kb located in the NS5 coding region, was modified by randomly introducing 273 synonymous mutations ("re-encoded virus"). Whilst the in cellulo replicative fitness of the re-encoded virus was only slightly reduced, the re-encoded virus displayed an attenuated phenotype in a laboratory mouse model of non-lethal encephalitis. Following intra-peritoneal inoculation of either 2.105 or 2.106 TCID50 of virus, the frequency of viraemia, neurovirulence (measured using weight loss and appearance of symptoms) and neuroinvasiveness (detection of virus in the brain) were significantly decreased when compared with the wild-type virus. Mice infected by wild-type or re-encoded viruses produced comparable amounts of neutralising antibodies and results of challenge experiments demonstrated that mice previously infected with the re-encoded virus were protected against subsequent infection by the wild-type virus. This constitutes evidence that a mammalian species can be protected against infection by a virulent wild-type positive-stranded RNA virus following immunisation with a derived randomly re-encoded strain. Our results demonstrate that random codon re-encoding is potentially a simple and effective method of generating live-attenuated vaccine candidates against pathogenic flaviviruses.

Virus quantitation by transmission electron microscopy, TCID50, and the role of timing virus harvesting: A case study of three animal viruses

Quantitation of viruses is practised widely in both basic and applied virology. Infectious titration in cell cultures, the most common approach to it, is quite labour-intensive and alternative protocols are therefore sought. One of the alternatives is transmission electron microscope (TEM) quantitation using latex particles at a known concentration as a reference for counting virus particles. If virus TCID50 is determined in parallel, the ratio of infectious to non-infectious virus particles may be established. This study employs such an approach to compute the number of virus particles and TCID50, and establish their correlation for three viruses: Canine adenovirus 1 (CAdV-1), Feline calicivirus (FCV) and Bovine herpesvirus 1 (BoHV-1). Each of the viruses was grown in five replicates until complete cytopathology was recorded (time 0), then frozen. They were thawed, filter-sterilised and left for additional periods of 16, 32 and 48h at 37°C. At each time point, the infectious ability of the virus was characterised by TCID50 and the number of virions quantified by TEM, in order to evaluate the influence of timing on virus harvest. The virus particle count determined by TEM did not change for any of the viruses throughout the experiment. The relationship between virus particle counts with TCID50 at time 0 showed good linearity response; their ratio was almost constant. The virus particle-to-TCID50 ratio varied between 146 and 426 (mean±SD: 282±103) for CAdV-1, between 36 and 79 (57±18) for FCV and between 110 and 249 (167±53) for BoHV-1. The proportion of non-infectious particles did not change throughout the experiment for either CAdV-1 or BoHV-1. However, a decrease in virus infectious ability disclosed by TCID50 indicated that the fraction of non-infectious particles in FCV increased 300,000 times when time 0 and 48h were compared. The quantitation of viruses with TEM is a simple and rapid protocol for virus quantitation but account must be taken of the type of virus and harvesting time as virus counts need not necessarily correlate with virus infectious ability.

Epizootic hemorrhagic disease virus infection of type I interferon receptor deficient mice

Type I interferon receptor deficient (IFNAR−/−) mice were infected with an Israeli isolate of serotype 7 of epizootic hemorrhagic disease virus (EHDV; Orbivirus, Reoviridae). Two out of two mice that received 5×105 50% tissue culture infectious doses (TCID50) by intraperitoneal injection died or were euthanized in a moribund state on day 5 after infection. One mouse out of three that had been inoculated with 5×102 TCID50 died on day 7 while the remaining mice did not show any clinical signs and survived until the end of the experiment. Spleens of all dead mice were highly positive in an EHDV real-time RT-PCR (quantification cycle values ≤15) and contained ≥105.8 TCID50 of virus per ml of homogenate. The viral RNA content and virus titer in the spleens of the two surviving mice, on the other hand, were over 100-fold lower. Different from data reported for BTV, the outcome of EHDV infection of IFNAR−/− mice is dose-dependent and subclinical infections can occur.

Classical swine fever virus in plasma and peripheral blood mononuclear cells of acutely infected swine.

The distribution of classical swine fever virus (CSFV) in plasma, monocytes, T and B lymphocytes in peripheral blood was monitored during experimentally induced acute classical swine fever infection in piglets. Six piglets were infected with 10(3.8) TCID50 of virus and blood samples taken up to 18 days post-inoculation (p.i.). Infectious virus was detected in monocytes, T and B lymphocytes to similar titres in five of the six infected piglets. Infectious virus was detected earlier in plasma than in any of the mononuclear cell subpopulations. No significant difference was observed in the period of time in which virus could be isolated from the three cell subpopulations. While a progressive lymphopenia developed, a marked B cell depletion was observed. However, B cells were apparently replaced by non-IgM-bearing mononuclear cells, as the proportion 'total lymphocyte/total leucocytes' remained unaltered throughout the experiment. Virus titres in plasma and peripheral blood mononuclear cells showed a tendency to increase as the disease progressed to its outcome.

Isolation from an Ant Myrmecia gulosa of Two Inducible O-Glycosylated Proline-rich Antibacterial Peptides

Reported here is the isolation and characterization of two antibacterial peptides synthesized in an ant Myrmecia gulosa in response to bacterial challenge. The peptides were purified by reversed-phase high performance liquid chromatography and characterized by peptide sequencing and mass spectrometry. Both peptides were formed from 16 amino acids, were rich in proline ( approximately 30%), and had N-acetylgalactosamine O-linked to a conserved threonine. The activity of a synthetic non-glycosylated isoform was markedly reduced demonstrating that glycosylation was necessary for maximum activity. The peptides were active only against growing Escherichia coli. They were inactive against stationary cells, Gram-positive bacteria, the yeast Candida albicans, two species of mammalian cells, and bovine pestivirus.

An Enzyme Immunoassay Based Micro-neutralization Test for Titration of Antibodies to Human Cytomegalovirus (CMV) and its Correlation with Direct ELISA Measuring CMV IgG Antibodies

Abstract.An ELISA—based micro-neutralization (Nt) test in MRC-5 cells for titration of neutralizing antibodies against human cytomegalovirus (CMV) in human plasma and preparations of immune globulins was developed to eliminate microscopic reading of cytopathic effect (CPE), a process that is subjective and time consuming. Un-neutralized CMV from the Nt reaction and grown in MRC—5 cells as per the standard micro—Nt test was coated in the same plates by various methods and CMV antigen was quantified by polyclonal or monoclonal CMV antibodies. Optimal coating of plates with CMV antigen (100 TCID 50of virus grown on MRC—5 cells for 7 days) was obtained by freezing/thawing of virus infected MRC—5 cells in phosphate buffered saline, pH 7.2. The CMV antigen treated sequentially with CMV monoclonal antibody to late nuclear protein antigen, goat anti-mouse IgG3 alkaline phosphatase conjugate and phosphatase substrate gave an absorbance of 1 at 410 nm wavelength whereas uninfected MRC—5 cells treated under similar conditions did not show any absorbance. The optimal Nt reaction occurred at 37°C for 1–2 h and was unaffected by complement. At 4°C, CMV was inactivated in 1–2 h. The antibody titres were affected by the virus dose used in the Nt test over a range of 20 to 798 TCID 50. When the titre was determined against a reference serum, the effect of virus dose on the Nt titre was reduced. Complete neutralization of virus read microscopically correlated with ELISA absorbance of < 0.1. CPE produced by approximately 1 TCID 50of CMV showed an absorbance of 0.1 or more. The correlation coefficient (r) between Nt titres and CMV IgG antibodies determined by ELISA was 0.69 ( P< 0.001) for 257 human plasma samples and 0.85 ( P< 0.001) for 50 immune globulin preparations.

Replication and expression of a recombinant Sindbis virus in mosquitoes.

A recombinant Sindbis virus, TE/3'2J/ANTI-S, containing LaCrosse virus small segment cDNA in antisense orientation, was inoculated into Aedes triseriatus mosquitoes. Virus replication and LAC-ANTI-S RNA expression were analysed temporally and spatially. TE/3'2J/ANTI-S virus titre peaked at 5.0 log10 TCID50 in heads 6-9 days post infection (p.i.) and decreased to 3.4 log10 TCID50 by 37 days p.i. Salivary glands contained 4.4 log10 TCID50 of virus 6 days p.i.; titres were lower in other organs. LAC-ANTI-S RNA levels paralleled virus titre. SIN E1 antigen was detected in many mosquito organ systems, but in specific cells and tissues of some organs. TE/3'2J/ANTI-S virus exhibited different cellular tropisms in salivary glands of Aedes and Culex mosquitoes.

Transmission of a Newly Recognized Virus (Bunyaviridae, Bunyavirus) Isolated from Aedes albopictus (Diptera: Culicidae) in Potosi, Missouri

Aedes albopictus (Skuse) mosquitoes collected in Potosi, Mo., were tested for their ability to transmit a newly recognized Bunyamwera sero group virus isolated from the same mosquito population. Mosquitoes were fed artificial blood meals containing 4.5-6.2 log10 TCID50 of virus per ml. After 7-29 d at 25 degrees C, 79-99% of the mosquitoes had disseminated infections and 0-26% transmitted virus to fluid-filled capillary tubes. Transmission was first observed after 7 d of extrinsic incubation. Tests failed to detect transovarial transmission in 5,145 progeny from ovarian cycles 2-4. Following parenteral inoculation with 5.3-6.0 log10 TCID50 of virus, four of nine adult hamsters developed viremia. Ten of 16 suckling mice died following intracerebral inoculation of 5.0 log10 TCID50 of virus (fifth Vero cell passage); the average survival time was 8.8 d (SD, 3.5). No mortality occurred in 10 suckling mice inoculated with 3.6 log10 TCID50 of virus (second Vero cell passage).

Transmission of Experimental Rhinovirus Colds in Volunteer Married Couples

Communicability of rhinovirus type 16 or type 55 was studied in 24 childless couples; on partner (the donor) was infected with laboratory-grown virus. Initially, both partners lacked antibody to the challenge agent. Rates of transmission between partners were 41% and 33% for type 16 and type 55, respectively. These rates are similar to those determined in epidemiologic studies of natural rhinovirus infection. Although the mucosa of the anterior nares was shown to be highly susceptible to infection (less than one 50% tissue culture infective dose [TCID50]), transmission rarely occurred unless (1) at least 1,000 TCID50 of virus was recovered from the donor's nasal washing, (2) the donor had virus on his hands and anterior nares, (3) he was at least moderately symptomatic, and (4) he spent many hours with his spouse. Since person-to-person transfer of rhinovirus was so dependent upon time spent together and shedding of large amounts of virus by the donor, it seems possible that the chain of infection could be interrupted by environmental manipulation.