The production of antibody-producing hybridomas to the Lassa virus

The work deals with obtaining hybrid cell lines producing monoclonal antibodies to Lassa arenavirus. To obtain preparations for the screening of hybridomas by indirect immunofluorescence techniques, the dynamics of the accumulation of Lassa virus antigen in cell cultures Vero and 4647 was studied. The maximum accumulation of the virus antigen in Vero cells was shown to occur on day 3 after inoculation with a dose of 1.0 PFU/ml. The influence of different doses of gamma radiation on the infectious and antigenic activity of the virus was studied. The expediency of using a dose of 20.0 kGy for the irradiation of the virus was shown. The optimum schedule for the immunization of BALB/c mice was worked out, which made it possible to obtain activated mouse spleen cells used for the construction of hybridomas. The capacity of hybrid cells, injected into syngeneic mice, for the generation of tumors was shown.     

Molecular Diagnostics for Lassa Fever at Irrua Specialist Teaching Hospital,Nigeria: Lessons Learnt from Two Years of Laboratory Operation

Background

Lassa fever is a viral hemorrhagic fever endemic in West Africa. However, none of the hospitals in the endemic areas of Nigeria has the capacity to perform Lassa virus diagnostics. Case identification and management solely relies on non-specific clinical criteria. The Irrua Specialist Teaching Hospital (ISTH) in the central senatorial district of Edo State struggled with this challenge for many years.

Methodology/Principal Findings

A laboratory for molecular diagnosis of Lassa fever, complying with basic standards of diagnostic PCR facilities, was established at ISTH in 2008. During 2009 through 2010, samples of 1,650 suspected cases were processed, of which 198 (12%) tested positive by Lassa virus RT-PCR. No remarkable demographic differences were observed between PCR-positive and negative patients. The case fatality rate for Lassa fever was 31%. Nearly two thirds of confirmed cases attended the emergency departments of ISTH. The time window for therapeutic intervention was extremely short, as 50% of the fatal cases died within 2 days of hospitalization—often before ribavirin treatment could be commenced. Fatal Lassa fever cases were older (p = 0.005), had lower body temperature (p<0.0001), and had higher creatinine (p<0.0001) and blood urea levels (p<0.0001) than survivors. Lassa fever incidence in the hospital followed a seasonal pattern with a peak between November and March. Lassa virus sequences obtained from the patients originating from Edo State formed—within lineage II—a separate clade that could be further subdivided into three clusters.

Conclusions/Significance

Lassa fever case management was improved at a tertiary health institution in Nigeria through establishment of a laboratory for routine diagnostics of Lassa virus. Data collected in two years of operation demonstrate that Lassa fever is a serious public health problem in Edo State and reveal new insights into the disease in hospitalized patients.     

Dose-Response Model for Lassa Virus

This article develops dose-response models for Lassa fever virus using data sets found in the open literature. Dose-response data were drawn from two studies in which guinea pigs were given subcutaneous and aerosol exposure to Lassa virus. In one study, six groups of inbred guinea pigs were inoculated subcutaneously with doses of Lassa virus and five groups of out-bred guinea pigs were similarly treated. We found that the out-bred subcutaneously exposed guinea pig did not exhibit a dose-dependent trend in response. The inbred guinea pigs data were best fit by an exponential dose-response model. In a second study, four groups of out-bred guinea pigs were exposed to doses of Lassa virus via the aerosol route. In that study, aerosol diameter was less than 4.5 μ m and both mortality and morbidity were used as endpoints. The log-probit dose-response model provided a somewhat better fit than the Beta-Poisson model for data with mortality as the endpoint, but the Beta-Poisson is considered the best fit model because it can be derived using biological considerations. Morbidity data were best fit with an exponential dose-response model.     

Interleukin-12 exhibits potent antiviral activity in experimental herpesvirus infections.

The prophylactic and therapeutic efficacy of interleukin-12 was studied by using murine models of herpes simplex virus infection. Prophylactic administration consisted of two intraperitoneal doses of interleukin-12 given 48 and 24 h prior to infection. Therapeutic intraperitoneal administration of interleukin-12 commenced 6 h after the mice were infected with herpes simplex virus and was continued daily for a total of 5 days. Interleukin-12 therapy improved the survival rates of mice with systemic herpes simplex virus infection compared with those of placebo-treated infected mice. Subcutaneous administration of interleukin-12 also improved the rate of survival of mice after systemic herpes simplex virus infection, although higher doses were required to give comparable effects. Combined prophylactic and therapeutic administration of interleukin-12 produced the greatest effect on survival after an otherwise lethal systemic infection. Intraperitoneal administration of interleukin-12 for 2 days before and 3 days after systemic infection with herpes simplex virus resulted in survival of 80% of the mice. These surviving mice were resistant to subsequent reinfection with herpes simplex virus. Such resistance was apparently specific for herpes simplex virus infection, since a second group of survivors succumbed to a lethal infection with murine cytomegalovirus. Infectious virus was recovered from lumbar ganglia explants dissected from survivors of prophylactic interleukin-12 therapy and cultured for 5 days in vitro, suggesting that interleukin-12 treatment did not prevent the establishment of latent herpes simplex virus infection. One action of interleukin-12 may be to enhance natural killer cell-mediated clearance of the virus. However, interleukin-12 therapy was also effective in mice carrying the beige mutation, which reduces natural killer cell lytic activity, suggesting that interleukin-12 has additional activities in vivo.     

Therapy and long-term prophylaxis of vaccinia virus respiratory infections in mice with an adenovirus-vectored interferon alpha (mDEF201)

An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201) was evaluated for efficacy against lethal vaccinia virus (WR strain) respiratory infections in mice. mDEF201 was administered as a single intranasal treatment either prophylactically or therapeutically at doses of 10(6) to 10(8) plaque forming units/mouse. When the prophylactic treatment was given at 56 days prior to infection, it protected 90% of animals from death (100% protection for treatments given between 1-49 days pre-infection), with minimal weight loss occurring during infection. Surviving animals re-challenged with virus 22 days after the primary infection were protected from death, indicating that mDEF201 did not compromise the immune response against the initial infection. Post-exposure therapy was given between 6-24 h after vaccinia virus exposure and protection was afforded by a 10(8) dose of mDEF201 given at 24 h, whereas a 10(7) dose was effective up to 12 h. Comparisons were made of the ability of mDEF201, given either 28 or 1 day prior to infection, to inhibit tissue virus titers and lung infection parameters. Lung, liver, and spleen virus titers were inhibited to nearly the same extent by either treatment, as were lung weights and lung hemorrhage scores (indicators of pneumonitis). Lung virus titers were significantly (>100-fold) lower than in the placebo group, and the other infection parameters in mDEF201 treated mice were nearly at baseline. In contrast, viral titers and lung infection parameters were high in the placebo group on day 5 of the infection. These results demonstrate the long-acting prophylactic and treatment capacity of mDEF201 to combat vaccinia virus infections.     

Effective vaccine for lassa fever

Lassa fever has been estimated to cause 5,000 deaths annually in West Africa. Recently, war in the zone where Lassa fever is hyperendemic has severely impeded control and treatment. Vaccination is the most viable control measure. There is no correlation between antibody levels and outcome in human patients, and inactivated vaccines produce high titers of antibodies to all viral proteins but do not prevent virus replication and death in nonhuman primates. Accordingly, we vaccinated 44 macaques with vaccinia virus-expressed Lassa virus structural proteins separately and in combination, with the object of inducing a predominantly TH1-type immune response. Following Lassa virus challenge, all unvaccinated animals died (0% survival). Nine of 10 animals vaccinated with all proteins survived (90% survival). Although no animals that received full-length glycoprotein alone had a high titer of antibody, 17 of 19 survived challenge (88%). In contrast, all animals vaccinated with nucleoprotein developed high titers of antibody but 12 of 15 died (20% survival). All animals vaccinated with single glycoproteins, G1 or G2, died, but all those that received both single glycoproteins (G1 plus G2) at separate sites survived, showing that both glycoproteins are independently important in protection. Neither group had demonstrable antibody levels prior to challenge. We demonstrate that in primates, immune responses to epitopes on both glycoproteins are required to protect against lethal challenge with Lassa virus without having untoward side effects and that this protection is likely to be primarily cell mediated. We show that an effective, safe vaccine against Lassa virus can and should be made and that its evaluation for human populations is a matter of humanitarian priority.     

Pathophysiology of thrombocytopenia associated with HIV infection in homosexual men. A preliminary report

The pathophysiology of HIV associated immune thrombocytopenia (HIV-ITP) and its response to AZT was investigated. Using autologous 111Indium-labelled platelets, platelet kinetic were analysed in two patient groups. Group 1 (untreated) was comprised of 13 patients with HIV-ITP. Group 2 (AZT) was comprised of 6 patients with a history of HIV-ITP prior to starting AZT. These patients were studied following a rise in their platelet count on AZT. Platelet survivals in both groups were shortened compared to controls, however there was no significant difference between the 2 groups. However platelet turnover rates were significantly depressed in Group 1 compared to Group 2 (P less than 0.05) and control values (P less than 0.05). The platelet count correlated with platelet turnover in Group 1 but not in AZT treated patients. Thus patients with HIV-ITP appear to have both shortened survivals and suppressed platelet production. The mechanism of the latter is unknown, but the increased turnover rate seen with AZT suggests it is a direct or indirect effect of the HIV virus.     

Lassa viral dynamics in non-human primates treated with favipiravir or ribavirin

Lassa fever is an haemorrhagic fever caused by Lassa virus (LASV). There is no vaccine approved against LASV and the only recommended antiviral treatment relies on ribavirin, despite limited evidence of efficacy. Recently, the nucleotide analogue favipiravir showed a high antiviral efficacy, with 100% survival obtained in an otherwise fully lethal non-human primate (NHP) model of Lassa fever. However the mechanism of action of the drug is not known and the absence of pharmacokinetic data limits the translation of these results to the human setting. Here we aimed to better understand the antiviral effect of favipiravir by developping the first mathematical model recapitulating Lassa viral dynamics and treatment. We analyzed the viral dynamics in 24 NHPs left untreated or treated with ribavirin or favipiravir, and we put the results in perspective with those obtained with the same drugs in the context of Ebola infection. Our model estimates favipiravir EC₅₀ in vivo to 2.89 μg.mL^-1, which is much lower than what was found against Ebola virus. The main mechanism of action of favipiravir was to decrease virus infectivity, with an efficacy of 91% at the highest dose. Based on our knowledge acquired on the drug pharmacokinetics in humans, our model predicts that favipiravir doses larger than 1200 mg twice a day should have the capability to strongly reduce the production infectious virus and provide a milestone towards a future use in humans.     

Genotoxic effect of Anovlar 21, an oral contraceptive, on mouse bone marrow

Anovlar 21, a combination drug containing the oestrogen ethinyloestradiol and the progestin norethisterone acetate, was studied for its in vivo genotoxic effect on the bone marrow cells of Swiss albino mice. The chromosomal aberration assay and the micronucleus test were employed for the study. 0.08, 0.4, 0.8, 1.6, 3.2, 4.8, 6.4 and 8.0 mg/kg/day of the drug was orally administered for 15 consecutive days to mice. Bone marrow preparations were made 24 h after the final feeding. The lowest dose, 0.08 mg/kg, represents the human therapeutic range. Marrow preparations of mice fed 0.8 mg/kg/day for 15 days were made at 6, 12, 24, 48 and 96 h, and 1, 2 and 3 weeks and a time-yield analysis was carried out. Statistically significant increases in chromosomal aberrations were observed in animal groups fed doses of greater than or equal to 0.4 mg/kg/day. In the time-response study, the maximum frequency of aberrations was noted at 24 h, thereafter decreasing gradually with increasing time. But the drug did not induce a significant increase in the number of micronuclei in bone marrow erythrocytes at any of the doses or time intervals studied.     

Induction of micronuclei in bone marrow of mice exposed to 1, 2 or 3 daily doses of urethane

Urethane was studied for its potential to induce micronuclei in bone marrow of CD-1 mice following various dosing and sampling schedules. It was found that: (1) mice dosed for 3 days by gavage with urethane at daily doses of 300, 600 and 1200 mg.kg-1 showed no increases in micronuclei compared to normal control values; (2) mice injected with 3 daily intraperitoneal (i.p.) doses of urethane at 400 and 1200 mg.kg-1 demonstrated substantial and dose-related increases in micronuclei; (3) mice given urethane at i.p. doses of 400 mg.kg-1 for 1, 2 and 3 days showed protocol related micronucleus yields with triple greater than single greater than double dosing, while mice given urethane at i.p. doses of 1200 mg.kg-1 showed a different trend with double greater than single dosing, the triple-dosing regimen proving cytotoxic to the bone marrow; (4) mice exposed i.p. to 400 mg.kg-1 of urethane had more micronuclei when sampled at 24 h than at 48 h after 1 or 2 daily treatments, while mice dosed i.p. with 1200 mg.kg-1 of urethane showed more micronuclei at 48 h than at 24 h after a single treatment, the double-dosing protocol being toxic to the marrow; (5) female mice were more susceptible to bone-marrow micronucleus induction than males with both quantitative and qualitative sex differences noted, depending on dose, regimen and sample.     

Effect of relative humidity, atmospheric temperature, and suspending medium on the airborne survival of human rotavirus

The Wa strain of human rotavirus, grown in MA-104 cells, was suspended either in tryptose phosphate broth or feces from a case of rotaviral diarrhea. It was then aerosolized into a rotating drum using a Collison nebulizer. The drum air was sampled using an all-glass impinger containing tryptose phosphate broth as collecting fluid. At 20 +/- 1 degree C, the virus aerosolized from tryptose phosphate broth was found to survive best at 50 +/- 5% relative humidity, where its half-life was 44.2 +/- 6.3 h. At 30 +/- 5% and 80 +/- 5% relative humidity, the half-life of the virus was 24.5 +/- 3.5 and 3.8 +/- 1.0 h, respectively. At 6 +/- 1 degree C, the airborne survival of the virus at the mid and low relative humidity levels was further enhanced, but at the high relative humidity it remained very similar to that seen at 20 +/- 1 degree C. When aerosols of fecally suspended human rotavirus were held at 20 +/- 1 degree C with 50 +/- 5% relative humidity, nearly 80% of the airborne virus particles remained infectious even at the aerosol age of 24 h. These findings may help in our understanding of the epidemiology of rotaviral infections.     

Genetic diversity among Lassa virus strains

The arenavirus Lassa virus causes Lassa fever, a viral hemorrhagic fever that is endemic in the countries of Nigeria, Sierra Leone, Liberia, and Guinea and perhaps elsewhere in West Africa. To determine the degree of genetic diversity among Lassa virus strains, partial nucleoprotein (NP) gene sequences were obtained from 54 strains and analyzed. Phylogenetic analyses showed that Lassa viruses comprise four lineages, three of which are found in Nigeria and the fourth in Guinea, Liberia, and Sierra Leone. Overall strain variation in the partial NP gene sequence was found to be as high as 27% at the nucleotide level and 15% at the amino acid level. Genetic distance among Lassa strains was found to correlate with geographic distance rather than time, and no evidence of a "molecular clock" was found. A method for amplifying and cloning full-length arenavirus S RNAs was developed and used to obtain the complete NP and glycoprotein gene (GP1 and GP2) sequences for two representative Nigerian strains of Lassa virus. Comparison of full-length gene sequences for four Lassa virus strains representing the four lineages showed that the NP gene (up to 23.8% nucleotide difference and 12.0% amino acid difference) is more variable than the glycoprotein genes. Although the evolutionary order of descent within Lassa virus strains was not completely resolved, the phylogenetic analyses of full-length NP, GP1, and GP2 gene sequences suggested that Nigerian strains of Lassa virus were ancestral to strains from Guinea, Liberia, and Sierra Leone. Compared to the New World arenaviruses, Lassa and the other Old World arenaviruses have either undergone a shorter period of diverisification or are evolving at a slower rate. This study represents the first large-scale examination of Lassa virus genetic variation.     

Mice Lacking Functional STAT1 Are Highly Susceptible to Lethal Infection with Lassa Virus

Lassa fever (LF) is a potentially lethal human disease that is caused by the arenavirus Lassa virus (LASV). Annually, around 300,000 infections with up to 10,000 deaths occur in regions of Lassa fever endemicity in West Africa. Here we demonstrate that mice lacking a functional STAT1 pathway are highly susceptible to infection with LASV and develop lethal disease with pathology similar to that reported in humans.     

Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α1-Antitrypsin Variants

Background

Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P) is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication.

Methodology/Principal Finding

We demonstrate that stable cell lines inducibly expressing S1P-adapted α₁-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of α₁-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific α₁-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different α₁-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor.

Conclusions/Significance

Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.     

Prevalence and Risk Factors of Lassa Seropositivity in Inhabitants of the Forest Region of Guinea: A Cross-Sectional Study

Background

Lassa fever is a viral hemorrhagic fever endemic in West Africa. The reservoir host of the virus is a multimammate rat, Mastomys natalensis. Prevalence estimates of Lassa virus antibodies in humans vary greatly between studies, and the main modes of transmission of the virus from rodents to humans remain unclear. We aimed to (i) estimate the prevalence of Lassa virus–specific IgG antibodies (LV IgG) in the human population of a rural area of Guinea, and (ii) identify risk factors for positive LV IgG.

Methods and Findings

A population-based cross-sectional study design was used. In April 2000, all individuals one year of age and older living in three prefectures located in the tropical secondary forest area of Guinea (Gueckedou, Lola and Yomou) were sampled using two-stage cluster sampling. For each individual identified by the sampling procedure and who agreed to participate, a standardized questionnaire was completed to collect data on personal exposure to potential risk factors for Lassa fever (mainly contact with rodents), and a blood sample was tested for LV IgG. A multiple logistic regression model was used to determine risk factors for positive LV IgG. A total of 1424 subjects were interviewed and 977 sera were tested. Prevalence of positive LV Ig was of 12.9% [10.8%–15.0%] and 10.0% [8.1%–11.9%] in rural and urban areas, respectively. Two risk factors of positive LV IgG were identified: to have, in the past twelve months, undergone an injection (odds ratio [OR] = 1.8 [1.1–3.1]), or lived with someone displaying a haemorrhage (OR = 1.7 [1.1–2.9]). No factors related to contacts with rats and/or mice remained statistically significant in the multivariate analysis.

Conclusions

Our study underlines the potential importance of person-to-person transmission of Lassa fever, via close contact in the same household or nosocomial exposure.     

Modulation of ex vivo cytokine production by splenocytes using in vitro combined therapeutics in murine retroviral model.

Altered levels of Type 1 and Type 2 cytokines are important in retrovirus-induced immunosuppression. The combination of immunostimulatory agents with antiviral drugs alters the course of murine retroviral infections. Previously, it was demonstrated that in vitro treatment of noninfected splenocytes and in vivo treatment of Friend leukemia virus (FLV)-infected mice with the combination of azidothymidine (AZT) and methionine enkephalin (MENK) significantly increases Type 1 cytokine levels and decreases Type 2 cytokines compared with treatment with only AZT. In order to study the effect of the time of initiation of immunomodulation on the course of retroviral infections, we examined the kinetics of cytokine production by isolated splenocytes from infected mice. BALB/c mice were infected with FLV, and spleen cells were removed at specified times postinfection (days 1, 3, 7, 10, and 14). Interleukin (IL)-2, interferon (IFN-gamma, IL-4, and IL-10 production by unstimulated or ConA-stimulated splenocytes treated in vitro with AZT, MENK, or AZT + MENK was determined after 48 h. The capacity of the isolated splenocytes to produce the Type 1 cytokines IL-2 and IFN-gamma in response to stimulation with ConA and combination therapy decreased over the course of infection. These results suggest that MENK treatment initiated later in the course of infection is unable to modulate the cytokine profile and would likely be ineffective in altering the course of FLV induced-disease. The results indicate the necessity to initiate antiretroviral therapy early in infection. Such information may be applicable in designing future regimens for HIV-1 infections in humans.     

Micronucleated erythrocyte frequency in control and azidothymidine-treated Tk+/+, Tk+/- and Tk-/- mice

The first step in the activation of the anti-retroviral nucleoside analogue azidothymidine (AZT) involves its conversion to a 5′-monophosphate. In this study, we have evaluated the role of cytosolic thymidine kinase (Tk), the major enzyme involved in phosphorylating thymidine and its analogues, in the nuclear DNA damage produced by AZT in neonatal mice. Tk^+/+, Tk^+/− and Tk^−/− mice were treated intraperitoneally with 200 mg/kg/day of AZT on postnatal days 1 through 8, and micronuclei were measured in peripheral blood 24 h after the last dose. AZT treatment increased the micronucleus (MN) frequencies to similar extents in both the reticulocytes (RETs) and normochromatic erythrocytes (NCEs) of Tk^+/+ and Tk^+/− mice; AZT did not increase the frequency of micronucleated RETs (MN-RETs) or micronucleated NCEs (MN-NCEs) in Tk^−/− mice. Unexpectedly, neonatal Tk^−/− mice treated with the vehicle had significantly elevated MN frequencies for both RETs and NCEs relative to Tk^+/+ and Tk^+/− mice (e.g., 3.4% MN-RETs and 4.8% MN-NCEs in Tk^−/− mice versus 0.7 and 0.6% MN-RETs and MN-NCEs in neonatal Tk^+/+ mice). Additional assays performed on untreated Tk^−/− mice showed that elevated spontaneous MN frequencies persisted until at least 20 weeks of age, which approaches the average lifespan of Tk^−/− mice. These results indicate that metabolism by Tk is necessary for the genotoxicity of AZT in neonatal mice; however, the genotoxicity of AZT is not altered by reducing the Tk gene dose by half. The elevated spontaneous MN frequencies in Tk^−/− mice suggest the presence of an endogenous genotoxic activity in these mice.     

pH-dependent fusion of eukaryotic cells, caused by arenaviruses, pathogenic and non-pathogenic for humans]

The conditions necessary for fusion from inside (FFWI) of the BHK-21 cell culture affected by the Lassa and Mopeya arenaviruses were studied. The fusion was shown to occur only in the slightly acid medium and at lower pH meanings for the Mopeya virus, than for the Lassa virus.