Search

Consumption of raw or unpasteurized milk is a risk for the consumers because indirect contaminations such as fecal-cross-contamination could occur and determine the presence of enteric viruses. In this study, milk was treated with several temperature and time combinations chosen by performing a preliminary experiment to evaluate the intervals needed to inactivate Hepatitis A virus (HAV) HM175 strain, noroviruses genogroups I and II (GI and GII), and simian rotavirus SA11 at different temperatures. Results were obtained by measuring the genome copies and infectious units by real-time PCR and plaque assays respectively. At 85 °C, one minute and two minutes were needed to achieve 6.6 log10 ± 0.2 and 8 log10 ± 0 reductions of genome copies of HAV respectively. Similar genome copies reduction was also observed for noroviruses (GI and GII) and simian rotavirus. At higher temperatures, 90 s (s) at 90 °C and 60 s at 95 °C were needed to achieve 8 log10 ± 0 reductions of the genome copies of all studied viruses. Significant higher sensitivity of the infectious units of both HAV and simian rotavirus to heat treatment of milk than their genome copies was observed. At boiling point of milk (100.5 °C), 40 s were needed to achieve 8 log10 ± 0 reductions of genome copies of all the studied viruses, while 10 s were needed to achieve 6 log10 ± 0 reductions of the infectious units of HAV and simian rotavirus. Significant higher reduction of infectious units than genome copies was observed confirming that genome copies reduction does not correspond to infectious virus.

SARS-Cov-2 (severe acute respiratory disease coronavirus 2), which causes Coronavirus Disease 2019 (COVID19) was first detected in China in late 2019 and has since then caused a global pandemic. While molecular assays to directly detect the viral genetic material are available for the diagnosis of acute infection, we currently lack serological assays suitable to specifically detect SARS-CoV-2 antibodies. Here we describe serological enzyme-linked immunosorbent assays (ELISA) that we developed using recombinant antigens derived from the spike protein of SARS-CoV-2. Using negative control samples representing pre-COVID 19 background immunity in the general adult population as well as samples from COVID19 patients, we demonstrate that these assays are sensitive and specific, allowing for screening and identification of COVID19 seroconverters using human plasma/serum as early as two days post COVID19 symptoms onset. Importantly, these assays do not require handling of infectious virus, can be adjusted to detect different antibody types and are amendable to scaling. Such serological assays are of critical importance to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. Sensitive and specific identification of coronavirus SARS-Cov-2 antibody titers may, in the future, also support screening of health care workers to identify those who are already immune and can be deployed to care for infected patients minimizing the risk of viral spread to colleagues and other patients.

ImportanceParticulate respirators such as N95 masks are an essential component of personal protective equipment (PPE) for front-line workers. This study describes a rapid and effective UVC irradiation system that would facilitate the safe re-use of N95 respirators and provides supporting information for deploying UVC for decontamination of SARS-CoV-2 during the COVID19 pandemic.ObjectiveTo assess the inactivation potential of the proposed UVC germicidal device as a function of time by using 3M® 8211 - N95 particulate respirators inoculated with SARS-CoV-2.DesignA germicidal UVC device to deliver tailored UVC dose was developed and snippets (2.5cm2) of the 3M-N95 respirator were inoculated with 106 plaque-forming units (PFU) of SARS-CoV-2 and were UV irradiated. Different exposure times were tested (0-164 seconds) by fixing the distance between the lamp (10 cm) and the mask while providing an exposure of at least 5.43 mWcm-2.SettingThe current work is broadly applicable for healthcare-settings, particularly during a pandemic such as COVID-19.ParticipantsNot applicable.Main Outcome(s) and Measure(s)Primary measure of outcome was titration of infectious virus recovered from virus-inoculated respirator pieces after UVC exposure. Other measures included the method validation of the irradiation protocol, using lentiviruses (biosafety level-2 agent) and establishment of the germicidal UVC exposure protocol.ResultsAn average of 4.38×103 PFUml-1(SD 772.68) was recovered from untreated masks while 4.44×102 PFUml-1(SD 203.67), 4.00×102 PFUml-1(SD 115.47), 1.56×102 PFUml-1(SD 76.98) and 4.44×101 PFUml-1(SD 76.98) was recovered in exposures 2s,6s,18s and 54 seconds per side respectively. The germicidal device output and positioning was monitored and a minimum output of 5.43 mWcm-2 was maintained. Infectious SARS-CoV-2 was not detected by plaque assays (minimal level of detection is 67 PFUml-1) on N95 respirator snippets when irradiated for 120s per side or longer suggesting 3.5 log reduction in 240 seconds of irradiation.Conclusions and RelevanceA scalable germicidal UVC device to deliver tailored UVC dose for rapid decontamination of SARS-CoV-2 was developed. UVC germicidal irradiation of N95 snippets inoculated with SARS-CoV-2 for 120s per side resulted in 100% (3.5 log in total) reduction of virus. These data support the reuse of N95 particle-filtrate apparatus upon irradiation with UVC and supports use of UVC-based decontamination of SARS-CoV-2 virus during the COVID19 pandemic.

The 2015 rupture of the Fundao dam near the district of Bento Rodrigues in Mariana, Minas Gerais, Brazil, released around 50 million m3 of iron ore tailings. The first tributary of the Rio Doceto receive this waste was the Gualaxo do Norte River. Many groups in Brazil and from around the world have studied the environmental and social impacts of this disaster. However, relationships between the introduction of mining waste, the geological complexity of the area, and the presence of pathogenic organisms have not yet been investigated. The present study aimed to measure the concentrations of enteric pathogens along the Gualaxo do Norte River after the environmental disaster and to correlate their abundance with the presence of metals and semimetals coming from both mining tailings and geological sources. For this purpose, we collected water samples from 27 stations along the entire basin during a hydrological year. The concentrations of metals and semimetals measured in this study were generally within limits established by national and international legislation, except for those of iron and manganese. Positive correlations between the human adenovirus (HAdV) and arsenic, barium, iron, lead, manganese, and nickel were confirmed, allowing us to observe that there is an abundance of the potentially infectious virus present in the studied sites containing metal/semimetal concentrations. These studies indicate the importance of investigations that consider viral enteric pathogens complexed with metals and may favor the stability and prolongation of the infectivity of such pathogens in water destined for human and animal usage.

Among the most leading causes of mortality across the globe are infectious diseases which have cost tremendous lives with the latest being coronavirus (COVID-19) that has become the most recent challenging issue. The extreme nature of this infectious virus and its ability to spread without control has made it mandatory to find an efficient auto-diagnosis system to assist the people who work in touch with the patients. As fuzzy logic is considered a powerful technique for modeling vagueness in medical practice, an Adaptive Neuro-Fuzzy Inference System (ANFIS) was proposed in this paper as a key rule for automatic COVID-19 detection from chest X-ray images based on the characteristics derived by texture analysis using gray level co-occurrence matrix (GLCM) technique. Unlike the proposed method, especially deep learning-based approaches, the proposed ANFIS-based method can work on small datasets. The results were promising performance accuracy, and compared with the other state-of-the-art techniques, the proposed method gives the same performance as the deep learning with complex architectures using many backbone.

Various treatments and agents had been reported to inactivate RNA viruses. Of these, thermal inactivation is generally considered an effective and cheap method of sample preparation for downstream assays. The purpose of this study is to establish a safe inactivation method for SARS-CoV-2 without compromising the amount of amplifiable viral genome necessary for clinical diagnoses. In this study, we demonstrate the infectivity and genomic stability of SARSCoV- 2 by thermal inactivation at both 56°C and 65°C. The results substantiate that viable SARS-CoV-2 is readily inactivated when incubated at 56°C for 30 min or at 65°C for 10 min. qRT-PCR of specimens heat-inactivated at 56°C for 30 min or 65°C for 15 min revealed similar genomic RNA stability compared with non-heat inactivated specimens. Further, we demonstrate that 30 min of thermal inactivation at 56°C could inactivate viable viruses from clinical COVID-19 specimens without attenuating the qRT-PCR diagnostic sensitivity. Heat treatment of clinical specimens from COVID-19 patients at 56°C for 30 min or 65°C for 15 min could be a useful method for the inactivation of a highly contagious agent, SARS-CoV-2. Use of this method would reduce the potential for secondary infections in BSL2 conditions during diagnostic procedures. Importantly, infectious virus can be inactivated in clinical specimens without compromising the sensitivity of the diagnostic RT-PCR assay.

The presence of pathogenic viruses in drinking water is a major public health concern. Although viability RT-qPCR methods were developed to quantify infectious viruses, they may not always reflect viral infectivity, therefore leading to false-positive results. In this study, sodium deoxycholate (SD) pre-treatment was used to improve the efficiency of viability RT-qPCR methods with respect to exclusive quantification of infectious viruses. The ability of SD pre-treatment to enhance the penetration of three viability markers, namely, ethidium monoazide (EMA, 100 µM), propidium monoazide (PMA, 100 µM), and cis-dichlorodiammineplatinum (CDDP, 1000 µM), into heat-treated (90 °C for 1 min) Aichi virus at various concentrations (0.01–0.5%) was evaluated. The optimal SD concentration was found to be 0.1% for all markers. EMA/PMA/CDDP-RT-qPCR with 0.1% SD pre-treatment was significantly more effective than without SD pre-treatment in determining AiV inactivation after heat (50, 60, 70, 80, or 90 °C for 1 min) or chlorine treatment (1 mgCl2/L for 1, 2, 5, or 10 min). Among the viability RT-qPCR methods tested, CDDP-RT-qPCR with SD pre-treatment (SD-CDDP-RT-qPCR) was the most effective in reflecting viral infectivity. Performance testing of SD-CDDP-RT-qPCR in concentrated drinking water samples did not reveal any significant effects of SD-CDDP treatment. Thus, SD-CDDP-RT-qPCR could be a useful tool for monitoring infectious virus presence in drinking water.

Despite the success of antiretroviral therapy in suppressing HIV, life-long therapy is required to avoid HIV reactivation from long-lived viral reservoirs. Currently, there is intense interest in searching for therapeutic interventions that can purge the viral reservoir to achieve complete remission in HIV patients off antiretroviral therapy. The evaluation of such interventions relies on our ability to accurately and precisely measure the true size of the viral reservoir. In this review, we assess the most commonly used HIV reservoir assays, as a clear understanding of the strengths and weaknesses of each is vital for the accurate interpretation of results and for the development of improved assays. The quantification of intracellular or plasma HIV RNA or DNA levels remains the most commonly used tests for the characterization of the viral reservoir. While cost-effective and high-throughput, these assays are not able to differentiate between replication-competent or defective fractions or quantify the number of infected cells. Viral outgrowth assays provide a lower bound for the fraction of cells that can produce infectious virus, but these assays are laborious, expensive and substantially underestimate the potential reservoir of replication-competent provirus. Newer assays are now available that seek to overcome some of these problems, including full-length proviral sequencing, inducible HIV RNA assays, ultrasensitive p24 assays and murine adoptive transfer techniques. The development and evaluation of strategies for HIV remission rely upon our ability to accurately and precisely quantify the size of the remaining viral reservoir. At this time, all current HIV reservoir assays have drawbacks such that combinations of assays are generally needed to gain a more comprehensive view of the viral reservoir. The development of novel, rapid, high-throughput assays that can sensitively quantify the levels of the replication-competent HIV reservoir is still needed.

Background Machupo virus (MACV) is a member of the Mammarenavirus genus, Arenaviridae family and is the etiologic agent of Bolivian hemorrhagic fever, which causes small outbreaks or sporadic cases. Several other arenaviruses in South America Junín virus (JUNV) in Argentina, Guanarito in Venezuela, Sabiá in Brazil and Chapare in Bolivia, also are responsible for human hemorrhagic fevers. Among these arenaviruses, JUNV caused thousands of human cases until 1991, when the live attenuated Candid #1 vaccine, was used. Other than Candid #1 vaccine, few other therapeutic or prophylactic treatments exist. Therefore, new strategies for production of safe countermeasures with broad spectrum activity are needed. Findings We tested a tri-segmented MACV, a potential vaccine candidate with several mutations, (r3MACV). In cell culture, r3MACV showed a 2-log reduction in infectious virus particle production and the MACV inhibition of INF-1β was removed from the construct and produced by infected cells. Furthermore, in an animal experiment, r3MACV was able to protect 50% of guinea pigs from a simultaneous lethal JUNV challenge. Protected animals didn’t display clinical symptoms nor were virus particles found in peripheral blood (day 14) or in organs (day 28 post-inoculation). The r3MACV provided a higher protection than the Candid #1 vaccine. Conclusions The r3MACV provides a potential countermeasure against two South America arenaviruses responsible of human hemorrhagic fever. Electronic supplementary material The online version of this article (10.1186/s12985-018-1009-4) contains supplementary material, which is available to authorized users.

A robust measure of the size of the latent HIV reservoir is essential to quantifying the effect of interventions designed to deplete the pool of reactivatable, replication competent proviruses. In addition to the ability to measure a biologically relevant parameter, any assay designed to be used in a clinical trial needs to be reproducible and scalable. The need to quantify the number of resting CD4+ T cells capable of releasing infectious virus has led to the development of the quantitative viral outgrowth assay (VOA). The assay as originally described has a number of features that limit its scalability for use in clinical trials; however recent developments reducing the time and manpower requirements of the assay, while importantly improving reproducibility mean that it is becoming much more practical for it to enter into more widespread use. This review describes the background to VOA development and the practical issues that they present in utilising them in clinical trials. It describes the innovations that have made their usage more practical and the limitations that still exist.

In July 1962, the author joined the Food Research Institute (FRI), then at the University of Chicago, to become its food virologist. There was a limited record of waterborne viral disease outbreaks at the time; recorded data on foodborne outbreaks were fewer still. Laboratory environmental (water and wastewater) virology was in its infancy, and food virology was in gestation. Detection of viruses was most often attempted by inoculation of primary primate cell cultures, with observation for plaque formation or cytopathic effects. Focus was initially on enteroviruses and reoviruses. Environmental and food samples had to be liquefied if not already in liquid form; clarified to remove solids, bacteria, and fungi; and concentrated to a volume that could be tested in cell culture. Cytotoxicity was also a concern. Studies at the FRI and some other laboratories addressed all of these challenges. The FRI group was the World Health Organization’s Collaborating Center for Food Virology for many years. Other topics studied were virus inactivation as functions of temperature, time, matrix, disinfectants, and microbial action; peroral and ex-vivo infectivity; and the suitability of various virus surrogates for environmental monitoring and inactivation experiments. Detection of noroviruses and hepatitis A virus required molecular methods, most often RT-PCR. When it was found that inactivated virus often gave the same RT-PCR signal as that of infectious virus, sample treatments were sought, which would prevent false-positive test results. Many laboratories around the world have taken up food and environmental virology since 1962, with the result that a dedicated journal has been launched.

Human Adenovirus 41 (Ad41) is an important human enteric pathogen and widely prevalent in the environment. The aim of this study was to assess the survival of Ad41 based on genome stability and infectivity in different types of manure and three types of biosolids. For viral survival studies, Ad41 was added to pelletized poultry litter (PL), alum-treated poultry litter (AL), raw poultry litter (RPL), liquid dairy manure (DM), swine manure (SM), and three types of biosolids 1, 2, 3. All samples were stored at 20 or 4°C and analyzed every 10 days for up to 60 days. Quantification PCR (qPCR) standard curves were generated for PL, AL, biosolids 1, and DM to measure the number of viral genomic copies remaining in the samples. To study the infectivity, all contaminated manure/biosolids samples were added to mammalian cell culture and viral mRNA was detected using one-step RT–PCR. Overall, Ad41 viral genomes were stable at both 20 and 4°C and there was no significant loss of viral DNA after 60 days in PL, AL, biosolids type 1, and DM. However, infectivity was lost almost immediately in high pH biosolids type 2 and 3, and infectivity decreased quickly in DM, with estimated T90 of 4.3 and 8.7 days at 20 and 4°C, respectively. Ad41 had ~1.9 log loss of infectivity after added in SM and biosolids type 1 at day 0, and estimated T90 was 12.5 and 28.6 days for biosolids type 1, and 19.1 and 51.0 days for SM at 20 and 4°C, respectively. Ad41 maintained infectivity in all three poultry litter, and after 60 days incubation, there were significantly more infectious virus in PL, AL, and RPL than biosolids 1, SM, and DM at 20°C.

Antibody engineering techniques permit the exploitation of the highly specific and diverse nature of the humoral immune system. Intracellular antibodies, consisting minimally of the antigen-binding domains of the parent immunoglobulin protein, may be used to manipulate microbial and cellular target molecules. Applications of this technology described to date include inhibition of replication of an infectious virus, downregulation of growth factor receptors, and inactivation of oncoproteins in cell culture systems. The ability to functionally manipulate virtually any target molecule using intracellular antibodies is clearly beneficial to basic science and molecular medicine.

Mice persistently infected with lymphocytic choriomeningitis virus (LCMV) exhibit impaired learning ability. In this report, we determined whether clearance of the virus was associated with restoration of behavioral function. Neonatal Balb/cByJ mice were persistently infected with LCMV and tested as adults in a nonconditional spatial discrimination task. The presence of viral proteins in neurons was confirmed immunohistochemically and infectious virus was quantified in the blood by plaque assay. LCMV-infected adult mice made more errors in a Y-maze avoidance task compared to sham-inoculated controls. After the initial behavioral analysis, infected and control mice received a dose of cytotoxic T-lymphocytes sufficient to clear virus from these mice. Following complete clearance of the virus, mice were re-tested in the behavioral task, 5 months after the original test. No reversal of the learning deficit was seen following viral clearance; mice that had been cleared of the virus and those that remained persistently infected behaved similarly. These data indicate that persistent LCMV infection of the CNS lasting up to 7 months results in discriminated learning impairments that are not reversed by subsequent anti-viral immunocytotherapy.

Herpes group viruses encode a serine proteinase that is essential for the production of infectious virus. This chapter summarizes our current understanding of its biochemical and enzymatic properties, and considers its potential as a target for antivirals.

Human T cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus-1 (HIV-1) are representative human retroviruses. To develop prophylactic vaccines and more effective medicines, small animal models for the virus infection are useful if they can be infected with the viruses. We constructed a transgenic (Tg) rat expressing human CRM1 (hCRM1), a cellular cofactor of Rex, and showed that T cells derived from Tg rats allowed production of HTLV-1 as efficiently as human T cells. However, viral load in Tg rats was not elevated so much. Our trials of various infection methods including oral administration to neonates failed to improve viral load, suggesting another barrier. During exploration we found lower rate of infection to rat dendritic cells (DC) than human DC, and administration of cyclosporine A (CSA) and As2O3 restored the infection rates. These results suggest the presence of inhibitor(s) that act during the entry process in rat DC. We also constructed Tg rats expressing human CD4, CCR5, CRM1, and CyclinT1, a cofactor of Tat to develop a model for HIV-1 infection. T cells and macrophages of the Tg rats supported production of infectious virus at levels approximately 10- 40% of that detected in human cells. HIV-1 was efficiently infected to macrophages but not to CD4+ T cells. CSA and As2O3 restored the infection rates, suggesting the presence of inhibitor(s) that act during the entry process in rat CD4+ T cells.

Three teams of researchers have developed cell culture systems for the modeling of hepatitis C viral infection and the subsequent generation of infectious virus particles—an important breakthrough in the study of this disease.

After infection with human cytomegalovirus, rabbit lung fibroblasts showed, during the four week period of the experiment, cytopathic changes and virus-specific antigens demonstrable by fluorescent antibody. Infectious virus could be recovered from the infected cells by co-cultivation with human lung fibroblasts.

Adeno 7 virus replicated well in human diploid (LEP) cells but only to a low degree in green monkey kidney (GMK) cells at 37° C; it did not replicate in either system at 29° C. At 37° C SV40 virus replicated well in GMK cells but only moderately in LEP cells; at 29° C it did not replicate in either system. SV40-adeno 7 hybrid grew in both GMK cells and LEP cells at 37° C. At 29° C this virus replicated in GMK cells but not in LEP cells. While the formation of V-antigen generally corresponded to the infectious virus production in the respective system, considerable differences were encountered in the T-antigens production. Adeno 7 T-antigen was detected earlier and in a higher percentage of GMK cells than in the fully permissive LEP cells and its formation was only slightly influenced by the incubation temperature. SV40 T-antigen was more efficiently formed in GMK cells than in LEP cells. At 29° C SV40 T-antigen was only found in GMK cells and was detected later than at 37° C. The difference in the formation of SV40 T-antigen in GMK cells infected with SV40 and SV40-adeno 7 hybrid virus was further analyzed. The results obtained suggest that an early step of the virus-cell interaction, but neither virus attachment nor penetration, was involved.

Replication kinetics of HIV was studied under single growth cycle conditions by backtitrating infectious virus released or remained cell-associated at each time point. Under these conditions HIV seems to replicate faster than previously estimated. The amount of cell-associated virus always exceeds the one detectable in the medium.

Summary A line of mouse neuroblastoma cells which was chronically infected with the neurotropic strain (JHM) of MHV, a member of the coronavirus group, was established. These cells, designated NJ, exhibited typical MHV cytopathic effects (CPE) at all passage levels along with the continual production of infectious virus. Most cells were positive for viral antigen by immunofluorescence. Viral particles consistent with the morphology of MHV were found by electron microscopy. The uninfected neuroblastoma cell line did not contain a detectable population of cells resistant to JHM, and persistence did not elicit the production of interferon. No plaque morphology or temperature sensitive mutants were selected for in the NJ culture, and we were unable to detect the presence of either a defective or defective interfering virus population. The addition of low concentration antiviral antibody modulated the infection to a carrier culture with viral antigen in the cytoplasm of the cells, but no infectious virus was produced, and the cells lacked both surface viral antigen and CPE. Possible mechanisms of viral persistencein vitro are discussed.

Infectious SV40 virions could be rescued from permissive TC7 cells within one to three subcultures following cotransfection with two cellular DNAs, each containing a complementary portion of the SV40 genome. SV40 virions could also be rescued by transfection of TC7 cells with cellular DNAs from a variety of SV40 transformed cells containing complete genome equivalents but not from cells containing subgenomes alone or defective genomes. Infectious virus was not rescued if the transfecting DNA species was treated with DNAase or if the DEAE-dextran pretreatment of the recipient cells was omitted.

The immunogenicity of influenza A strain A/Northern Territory/60/68 for CSL mice when delivered by the ocular, nasal and subcutaneous routes was determined according to the median protective dose, PD50, i.e. the dose of infectious virus required to induce inhibition of multiplication of a standard intranasal challenge dose of 10(4.5) median tissue-culture-infectious doses (TCID50) of homologous virus three weeks after vaccination (PD50). For mice inoculated by the ocular route, an immunizing dose of 10(2.89) TCID50 per animal was required. For anaesthetized mice vaccinated intranasally and unanaesthetized mice vaccinated subcutaneously these figures are less than 10(2.00) and greater than 10(6.00) TCID50 per animal, respectively. The lower immunogenicity of virus delivered by the ocular route compared with the intranasal route can be correlated with a lowered capacity of ocularly administered virus to replicate in the murine respiratory tract. The immunogenicity of A/Ann Arbor/6/60-ca administered in two identical doses, was also determined for (a) the intraocular route, (b) the intranasal route with anaesthetized animals and (c) the intranasal route with unanaesthetized animals, using the parental A/Ann Arbor/6/60 as the challenge virus. Two doses were required because ca viruses have been shown to be poor immunogens in the same animal model. The PD50 for the ocular route was 10(2.83) TCID50 per animal compared with 10(2.71) for the intranasal route using unanaesthetized animals and 10(1.36) for the intranasal route using anaesthetized animals. Administration of living attenuated vaccine viruses by the ocular route is thus an effective means of inducing immunity to influenza viruses in the respiratory tract of mice.

Transformation of a clonal line of BHK21 cells by type 12 adenovirus (Ad 12) is described. A transformation rate of approximately 2 × 10−5 per initially infected cell was observed after high multiplicity infection. A study of the fate of the abortively infected cells revealed, however, that only a small fraction survived the infection and initiated growth of a colony. Of these rare surviving colonies, nearly half contained transformed cells. Of three BHK21 sublines tested, one yielded transformed cells only from colonies growing in soft agar suspension, a second yielded transformants only as foci in monolayers, while the third did not yield detectable transformants by either method. The adenovirus-transformed cells were distinguished from the parental BHK21 cells by the following characteristics: The latter property is most likely due to blockage of a step late in the adenovirus replicative cycle, since 88% of the cells, which synthesized Ad2-specific structural antigens did not yield infectious virus. A similar shift from a productive to a largely abortive response to Ad 2 was seen in BHK21 cells simultaneously infected with Ad 12 and Ad2. This finding supports the view that the restriction of Ad2 replication in Ad 12-transformed cells, like the other characteristics mentioned, is associated with continued activity of at least part of the Ad12 genome.

Quality measurements (number of virions per plaque forming units) were made on vesicular stomatitis virus indicating a virion: p.f.u. ratio of approximately 1. This highly infectious virus was produced by careful control of the inoculum size to avoid the production of the small inhibitory particles associated with VSV. The effect of virus aggregation on titer was investigated, as well as the effect of the dispersal techniques on viral infectivity. Thermal inactivation studies at various temperatures ranging from −57°C to 56°C showed the virus to be relatively stable when stored as a cell lysate or diluted 1∶:10 in nutrient media.

Viruses were isolated from sheep faeces using lamb kidney cultures and examined by negative contrast electron microscopy. Nine agents were morphologically indistinguishable from adenoviruses and their physical and chemical characteristics were also constistent with those of agents of the adenovirus group. Antiserum to 3 of the 9 isolates was prepared and cross neutralisation tests indicated that the 3 were serologically distinct. However 5 of the 6 remaining agents were related to the prototype strains. The sixth strain produced little infectious virus and was not typed.

Cis-dichlorodiammineplatinum (II) partially inhibits the replication of SV 40 virus in green monkey kidney cells. The results of both immunofluorescence and complement-fixation tests indicated that the production of the T antigen of SV 40 was unaltered, but V antigen production was suppressed. The partial block of V antigen synthesis was most marked in the early period after infection, subsequently it was less pronounced. Both infectious virus production and V antigen formation were most markedly inhibited in cells treated with DDP both before and after infection.

The buoyant density of the Bucyrus strain of equine arteritis virus was studied by isodensity centrifugation on three types of gradients. The distribution patterns of infectivity were dependent on the type of gradient used. In sucrose gradients a single peak of infectivity at densities approximating 1.17 g/ml was obtained. From this gradient the total recovery of infectious virus varied between 80 and 90%. In cesium chloride gradients a rather broad band appeared in the form of a “saddled” peak with a range of densities from 1.180 to 1.215 g/ml, the total recovery being 90%. In potassium tartrate (KT) gradients two peaks were formed, one at 1.17 and another at 1.24 g/ml; the total recovery was 50%. Recentrifugation of fractions from KT gradient on sucrose gradients resulted in a changed distribution of infectivity for particles of higher density (1.24 g/ml).

Hamster embryo fibroblasts were transformed by the SV40-Adeno 7 hybrid virus. The transfomed cells, denoted KE SP2, contained the SV40 tumor (T) antigen demonstrable by both complement-fixation reaction and immunofluorescence tests, but no infectious virus. Attempts to rescue the SV40-derived marker from the transformed cells failed. The KE SP2 cells induced tumors in adult hamsters; sera from these animals contained antibodies reacting with the SV40 T antigen, but not with the SV40 or Adeno 7 viral (V) antigens. Immunofluorescence tests with monkey kidney cells infected with the prototype Adeno 7 (Gomen) virus revealed also the presence of antibodies reactive with the Adeno 7 T antigen. Cells containing the determinant for its synthesis were not lost after one passage in hamster or after a long history of passagesin vitro.

The authors have studied the transmissibility of the virus MHV-3 by the transplacental route from experimentally infected pregnant mice to their fetuses at term, delivered by caesarean section and likewise by the transplacental or vaginal route from the infected mother to mice spontaneously delivered and entrusted to healthy wet-nurses. In both cases, in spite of the severe maternal infection, no virus was detected in the liver of the young. Other experiments have demonstrated the full susceptibility of mice, in the first hours after birth, to the infection experimentally induced by the intraperitoneal incoulation of virus. The obtained results allow the conclusion that infectious virus does not pass to the young by the transplacental or vaginal route.

It has been found that certain mutants of TMV with known amino acid exchanges produce abnormally low quantities of infectious virus particlesin vivo at 32°C, although the particles themselves are stable at even higher temperatures. A correlation between temperature sensitivity of the mutantsin vivo and the inability of their A-Proteins to reaggregate to rodsin vitro at 30°C is demonstrated. It is concluded that the amino acid exchanges in coat protein lead to temperature sensitivity of the mutants described.

MANY captive gibbons are naturally exposed to infectious type-C virus based on the prevalence of animals with humoral antibodies reactive to the virus1,2 and isolation of virus from tissue of leukaemic3,4 and normal gibbons5. A closely related infectious type-C virus has also been isolated from a woolly monkey with a spontaneous fibrosarcoma6,7 which implies that a common virus could infect unrelated non-human primates. Several reports recently have identified infectious primate type-C virus8,9, viral components10 and humoral antibody reactive to this virus in leukaemic11 and normal humans12 suggesting that man also may be susceptible to infection by a related virus. In an attempt to identify a possible source for infectious primate type-C virus that might infect primates as well as other animals, we decided to investigate whether healthy captive gibbons were shedding viruss ince this non-human primate is frequently displayed at zoos or kept as pets. This report presents evidence that gibbons can have long term viraemia with type-C virus without detectable immune response. These viraemic animals actively shed infectious virus in urine and faeces.

Infectious virus was isolated fromHippeastrum plants by density gradient centrifugation and examined with the electron microscope. The average lengths of the flexuous particles were 643±24 nm.

Defective particles can serve as templates for their replication by helper infectious virus. While both the “long T” and “short T” defective particles in VSV preparations are capable of homotypic interference, only the larger “long T” particle is capable of heterotypic interference.

SIMIAN virus 40 (SV40) is oncogenic for hamsters1 and Mastomys2. In vitro studies have shown that SV40 is capable of transforming cells derived from hamster3, mouse4, rabbit4, pig4, cow5, monkey6, human7, guinea-pig8 and rat9. We have established and studied continuous lines of uninfected and Rauscher leukaemia virus (RLV) infected rat embryo (RE) cells10. Rat embryo cells exposed to RLV have produced infectious virus and complement-fixing (CF) antigen characteristic of the murine leukaemia-sarcoma virus complex for more than 18 months. This communication describes increased transformation efficiency of SV40 in RLV infected RE cells (RLV-RE) compared with uninfected RE cells.

INFLUENZA has long been considered to be of relatively short duration in the infected individual1. The optimal period for isolating the virus from the upper respiratory tract is reported to be during the first 48 h of the disease2, and viral antigen is not detectable in these cells beyond 8 days after infection3. These reports, coupled with the general failure to isolate influenza virus from normal individuals between epidemics, suggest that the period of association between influenza virus and its host cell is relatively short, and that the virus is eliminated rapidly. Studies involving the infection of avian lung cells have, however, shown that not all infected cells are killed, and that some cells continue to release virus for prolonged periods4. We now have evidence that influenza virus can persistently infect human lung cells, infectious virus being released from the cells for up to 7 weeks after initial infection.

Parallel induction of sister chromatid exchanges and infectious virus from SV40-transformed cells by alkylating agents

Immunization against Marek's disease using virus specific antigens free from infectious virus

INFECTIOUS pancreatic necrosis virus (IPNV) is a highly infectious virus of salmonid fishes, which has been isolated from virtually every trout-producing area of the world. Infections have been transmitted through water1, by injection2 and by protozoan parasites3. Wolf et al.4 established the important epidemiological concept of vertical transmission when they demonstrated that eggs from carrier brood stock developed epizootics of IPN. We have now investigated whether free living warm blooded animals which have eaten IPNV-infected animals can possibly transfer infectious virus or become actively infected with the agent.

AN oncorna-type virus (M-PMV) was detected in a spontaneous breast tumour of a female rhesus monkey1,2. This virus is morphologically and biochemically similar to oncogenic RNA viruses of other species3–5. Using in vitro tissue culture assays and electron microscopy we have observed proliferation of infectious virus in hyperplastic lymph nodes of M-PMV inoculated infant rhesus monkeys.

SIMIAN virus SV40, a DNA tumour virus, causes a productive infection in both monkey and human cells, leading to lysis of the cells and release of infectious virus. In murine systems, however, the virus transforms the cells, leading to new virus-specific antigens on the transformed cells1. These transformed cells induce tumours in mice which then respond to the tumour specific antigen2,3. I report here protection of mice by SV40 when given with syngeneic myeloma tumour cells.

LATENTLY infected sensory ganglia have been thought to be the source of virus for various clinical manifestations of recurrent herpetic disease in man1,2. In direct support of this concept, we recently showed that herpes simplex virus can induce a latent infection in the spinal ganglia of mice3. This murine infection has not, however, been shown to be accompanied by recurrent disease. Recurrent herpetic eye infection can be produced in the rabbit4. If sensory ganglia are involved in recurrent disease, then trigeminal ganglia from rabbits undergoing such recurrent infection would be expected to harbour latent virus. We now report that herpes simplex virus does indeed induce latent infection in trigeminal ganglia of rabbits presenting recurrent eye infection. As in the experiments with mice, infectious virus could not be recovered directly; it was only found when ganglia were established as organ cultures in vitro.

SEVERAL antigens of adenovirus type 5 have been separated from infectious virus particles by chromatography on DEAE columns1,2. They differ in their serological specificity, and some of them have been correlated with haemagglutinins3. Differences in the elution properties between adenovirus serotypes have also been demonstrated in several instances2,4,5.

To investigate further the interaction of cytomegalovirus (CMV) with blood leukocytes, we studied the kinetics of leukocyte infection during sublethal murine CMV (MCMV) infection. Three-week old Balb/c mice were infected ip with 105 plaque-forming-units of MCMV, and mononuclear and granulocyte-enriched leukocyte fractions were studied 0, 1, 3, 5, 7, 14, and 20 days post-infection using in situ nucleic acid hybridization, detection of antigen by monoclonal antibodies, and culture for infectious virus. By day 3, immediate-early MCMV antigens were detected in the leukocytes of all infected animals (mean of 200 antigen-positive cells/106 leukocytes). By day 5, antigens, nucleic acid, and infectious virus were detected in the leukocytes of all animals and were present until day 14. By each method, peak infection was observed on day 5. At peak infection, more cells contained antigens (mean of 248 antigen-positive cells/106 leukocytes) than contained nucleic acid (mean of 64 nucleic acid positive cells/106 leukocytes) (p

The interactions between respiratory viruses and mononuclear leukocytes in the natural course of infections with these agents is not completely understood. We have attempted to look at these cells directly for evidence of production of new antigen and/or infectious progeny. Peripheral blood mononuclear leukocytes (PBML) separated into adherent (M) and non-adherent (Ly) populations were infected with purified respiratory syncytial virus (RSV) or parainfluenza type 3 (PI3) at a MOI of 0.5-0.6. Since M's have been reported to become more macrophage-like over time in tissue culture, experiments were done infecting the M's 1, 2, 4 and 7 d after harvest. Cells were washed after 2 hrs of viral adsorption and every 24 hrs for the duration of these experiments. Antigen production was measured in supernatants by ELISA and in cells by indirect fluorescence (IF); infectious progeny were assayed on Hep-2 monolayers. With both viruses new antigen and infectious virus production were detected in M by 48 hrs and in Ly fractions by 96 hrs after infection. The youngest M's (day 1) were poor producers of new virus although the most striking syncytia formation with PI3 occurred in these cells. RSV caused no definite syncytia but did lead to cell rounding by 96 hrs in the M's; this was most marked in the cells infected 7 d after harvest. For both viruses 10-20% of M's were positive by IF in contrast to only 2-3% of Ly's. Further investigation of this model may be helpful in elucidating aspects of the pathophysiology of infection with these agents.

The virus of ectromelia induces an enormous multiplication ofMusculomyces in the peritoneum of the white mouse. In mice inoculated withMusculomyces alone the resulting scanty infection remains confined to the peritoneal cavity. In the presence of the virus of ectromeliaMusculomyces causes a systemic infection. It does not invade the cells, growth is confined strictly to their surface.