Virological Assays Research Articles

Empaquetage sélectif du génome des influenzavirus A.

Electron microscopy of influenza A virus (IAV) and three-dimensional reconstruction of their interior by electron tomography, combined with genetic, biochemical and virology assays, has revealed that genome packaging of IAVs is a selective process, the molecular mechanisms of which start to be unveiled. The eight genomic viral RNAs (vRNAs) most likely form a supramolecular complex maintained by base-pairings within the strain-dependent packaging signals of each vRNA. Visualization of viral ribonucleoproteins inside cells also brought new insights about spatio-temporal assembly of the supramolecular complexes, prior to their incorporation into budding virions. Altogether, these data improve our understanding of the rules governing packaging of the IAV genome and offer clues for optimization of vaccine seeds production. Genetic reassortment events between different IAVs, which can lead to severe pandemics, are probably also affected by the rules that govern genome packaging.

Luciferase Time-based, High-throughput Screening Assay for the Discovery of HIV-1 Inhibitors

With the enhancement of high-throughput screening (HTS) for target-based antiretroviral discovery, fluorescence has been considered the best bioassay. Bioluminescence by genetic reporters has maintained a major place among cell-based virological assay formats. Luminescent bioassays have a wide variety of applications due to their high sensitivity and linearity, even for a range of complex biological samples. In this target-based method, we used an HTS bioluminescence assay and highlighted the detection capabilities of this time-of-addition experiment. Hence, the development of a cell-based assay that uses a phenotypic drug discovery approach based on bioluminescence by stable reporter cells is described. Utilizing this screening method, a bioluminescent, target-based, time-of-addition experiment was performed by adding antiretrovirals with known mechanisms of action to analyze their drug targets by measuring the length of time until the antiretrovirals lost their efficacy in an HIV-1 replication assay. Depending on the viral replication target, the antiretrovirals lost activity at different times: fusion inhibitors acted for 0-2 h; retrotranscriptase inhibitors acted efficiently for the first 4 h; integrase inhibitors acted for 18 h; and protease inhibitors were inactivated after 15 h. The target-based, bioluminescence assay identifies the mode of action of antiretroviral drugs and provides valuable information about drug targets that inhibit HIV replication. This assay delimits the time (hours) for which the addition of an antiviral can be delayed before losing its antiviral activity relative to the replication cycle of HIV. The target of an antiviral compound can be identified both by comparing the relative time until it loses efficacy to that of the reference drugs and by the time elapsed when the antiretroviral loses activity compared to a known control.

Anti-HIV-1 activity of the G-quadruplex ligand BRACO-19.

A dynamic G-quadruplex region has been previously shown to form in the long terminal repeat (LTR) promoter of the HIV-1 integrated DNA genome. Inhibition of promoter activity and antiviral effects have been observed when this region was stabilized by BRACO-19, a trisubstituted acridine derivative that binds G-quadruplexes. Here, we aimed at characterizing the antiviral mechanism of action of BRACO-19 by analysing its activity towards a broad range of HIV-1 strains, host cells and infection modes. The antiviral activity of BRACO-19 in cell lines and primary cells infected or persistently infected by HIV-1 strains was evaluated at different times post-infection. Virucidal, viral binding, time-dependent and drug-dependent assays were performed to identify the viral target step. Circular dichroism, UV spectroscopy and a reverse transcriptase (RT) stop assay were used to assess RNA G-quadruplex folding and inhibition of RT processing. Thorough virological assays demonstrated that BRACO-19 acts both at the reverse transcription and the post-integration level during the virus life cycle. This behaviour was rationalized by the observation that a G-quadruplex-forming sequence identical to that of the LTR DNA is present at the 3'-end of the virus RNA genome. Biophysics and biomolecular testing showed that this region has the ability to fold into very stable G-quadruplex structures that are even more stabilized by BRACO-19, therefore inhibiting the reverse transcription process at the template level. Our findings strongly support the activity of BRACO-19 at the viral G-quadruplex level and therefore strengthen the use of viral G-quadruplexes as new anti-HIV-1 targets.

Detection of a phylogenetically divergent eel virus European X (EVEX) isolate in European eels (Anguilla anguilla) farmed in experimental tanks in Italy

There is very little scientific literature about the impact of Eel virus European X (EVEX) on eels held under artificial reproduction condition, and its effective role in eel decline is uncertain and still unclear. Following a disease outbreak of European eels, farmed in an experimental hatchery in Italy, a sample of the affected animals was analyzed to determine the cause of illness. Virological isolation, PCR assay and molecular characterization revealed the presence of EVEX in the diseased animals. The complete N, P, M and G gene regions were amplified by using novel primers specific for EVEX. The EVEX strain identified in this study was phylogenetically close to the known EVEX isolates; however, this Italian isolate diverges and localizes in a separated branch of this cluster. In addition, some unique amino acid changes in three out of four analyzed viral proteins differentiated the Italian virus from reported EVEX sequences which, although few in number, do cover different geographical areas and a 30-year time span. Molecular data are needed to gain further insight into the genetic variability of EVEX, to better define its phylogenetic and evolutionary relationships and to unravel the existence of different circulating strains. Examination of samples caught in the same area failed to detect the presence of EVEX infection. These results strongly indicate that the presence and risk factors associated with EVEX infection are not well established and further study should be performed in this field, in particular on stress and temperature dependence for disease development.

Vertical transmission of arboviruses in mosquitoes: A historical perspective

Arthropod-borne viruses (arboviruses) are mainly transmitted horizontally among vertebrate hosts by blood-feeding invertebrate vectors, but can also be transmitted vertically in the vector from an infected female to its offspring. Vertical transmission (VT) is considered a possible mechanism for the persistence of arboviruses during periods unfavorable for horizontal transmission, but the extent and epidemiological significance of this phenomenon have remained controversial. To help resolve this question, we reviewed over a century of published literature on VT to analyze historical trends of scientific investigations on experimental and natural occurrence of VT in mosquitoes. Our synthesis highlights the influence of major events of public health significance in arbovirology on the number of VT publications. Epidemiological landmarks such as emergence events have significantly stimulated VT research. Our analysis also reveals the association between the evolution of virological assays and the probability of VT detection. Increased sensitivity and higher-throughput of modern laboratory assays resulted in enhanced VT detection. In general, VT contribution to arbovirus persistence is likely modest because vertically infected mosquitoes are rarely observed in nature. Taken together, however, our results call for caution when interpreting VT studies because their conclusions are context- and method-dependent.

Standardization of Quantitative Virological Assays for Antiretroviral Clinical Trials

The AIDS Clinical Trial Group (ACTG) was formed and funded by the National Institute of Allergy and Infectious Diseases (NIAID) in 1987 to enable multi-center clinical trials for the evaluation of treatments for HIV. As there were no licensed anti-retroviral drugs at that time large numbers of subjects and many testing sites were needed to adequately demonstrate clinical efficacy and make the drugs available for general treatment. Initially trial endpoints were based on evaluation of symptoms some of which were based on subjective judgment by clinicians. Measurements of CD4 positive cells were used to demonstrate progression from asymptomatic to symptomatic disease to AIDS. In order to shorten the length of the trials and obtain objective results it was necessary to identify virus specific markers of efficacy. Some of the first viral markers to be evaluated included HIV-1 p24 antigen as measured in serum, and the infectious outgrowth of HIV from peripheral blood mononuclear cells (PBMCs). These tests paved the way for more sensitive tests using HIV nucleic acid amplification to determine the copies of HIV genome in plasma. HIV RNA continues to be the best surrogate marker used today to evaluate the efficacy of new combinations of antiretroviral drugs.

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world’s population and causes serious liver ailments including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world's population and causes serious liver ailments including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Integrative functional genomics of hepatitis C virus infection identifies host dependencies in complete viral replication cycle.

Recent functional genomics studies including genome-wide small interfering RNA (siRNA) screens demonstrated that hepatitis C virus (HCV) exploits an extensive network of host factors for productive infection and propagation. How these co-opted host functions interact with various steps of HCV replication cycle and exert pro- or antiviral effects on HCV infection remains largely undefined. Here we present an unbiased and systematic strategy to functionally interrogate HCV host dependencies uncovered from our previous infectious HCV (HCVcc) siRNA screen. Applying functional genomics approaches and various in vitro HCV model systems, including HCV pseudoparticles (HCVpp), single-cycle infectious particles (HCVsc), subgenomic replicons, and HCV cell culture systems (HCVcc), we identified and characterized novel host factors or pathways required for each individual step of the HCV replication cycle. Particularly, we uncovered multiple HCV entry factors, including E-cadherin, choline kinase α, NADPH oxidase CYBA, Rho GTPase RAC1 and SMAD family member 6. We also demonstrated that guanine nucleotide binding protein GNB2L1, E2 ubiquitin-conjugating enzyme UBE2J1, and 39 other host factors are required for HCV RNA replication, while the deubiquitinating enzyme USP11 and multiple other cellular genes are specifically involved in HCV IRES-mediated translation. Families of antiviral factors that target HCV replication or translation were also identified. In addition, various virologic assays validated that 66 host factors are involved in HCV assembly or secretion. These genes included insulin-degrading enzyme (IDE), a proviral factor, and N-Myc down regulated Gene 1 (NDRG1), an antiviral factor. Bioinformatics meta-analyses of our results integrated with literature mining of previously published HCV host factors allows the construction of an extensive roadmap of cellular networks and pathways involved in the complete HCV replication cycle. This comprehensive study of HCV host dependencies yields novel insights into viral infection, pathogenesis and potential therapeutic targets.

Performance and logistical challenges of alternative HIV-1 virological monitoring options in a clinical setting of Harare, Zimbabwe.

We evaluated a low-cost virological failure assay (VFA) on plasma and dried blood spot (DBS) specimens from HIV-1 infected patients attending an HIV clinic in Harare. The results were compared to the performance of the ultrasensitive heat-denatured p24 assay (p24). The COBAS AmpliPrep/COBAS TaqMan HIV-1 test, version 2.0, served as the gold standard. Using a cutoff of 5,000 copies/mL, the plasma VFA had a sensitivity of 94.5% and specificity of 92.7% and was largely superior to the VFA on DBS (sensitivity = 61.9%; specificity = 99.0%) or to the p24 (sensitivity = 54.3%; specificity = 82.3%) when tested on 302 HIV treated and untreated patients. However, among the 202 long-term ART-exposed patients, the sensitivity of the VFA decreased to 72.7% and to 35.7% using a threshold of 5,000 and 1,000 RNA copies/mL, respectively. We show that the VFA (either on plasma or on DBS) and the p24 are not reliable to monitor long-term treated, HIV-1 infected patients. Moreover, achieving acceptable assay sensitivity using DBS proved technically difficult in a less-experienced laboratory. Importantly, the high level of virological suppression (93%) indicated that quality care focused on treatment adherence limits virological failure even when PCR-based viral load monitoring is not available.

The anti-influenza virus effect of Phellinus igniarius extract

Herbal medicine has been used in the orient for thousands of years to treat large and small ailments, including microbial infections. Although there are treatments for influenza virus infection, there is no treatment for drug-resistant viruses. It is time that we explored and exploited the multi-component nature of herbal extracts as multi-drug combination therapies. Here, we present data on the anti-influenza virus effect of a medicinal mushroom, Phellinus igniarius. The P. igniarius water extract was effective against influenza A and B viruses, including 2009 pandemic H1N1, human H3N2, avian H9N2, and oseltamivir-resistant H1N1 viruses. Virological assays revealed that the extract may interfere with one or more early events in the influenza virus replication cycle, including viral attachment to the target cell. Therefore, our results provide new insights into the use of P. igniarius as an anti-influenza medicine.

Recurrence of another hepatitis B virus escape mutant comes back in a patient infected with HIV and low CD4+ count

The case of an HIV-positive patient who cleared formerly acute HBV infection and experienced HBV exacerbation before starting antiretroviral therapy (ART) followed by a severe elevation of liver enzymes one month after the introduction of ART is reported. Laboratory tests revealed the presence of a subgenotype A2 carrying at least seven different mutations within the S protein and high lopinavir plasma concentrations. Irrespectively of the many possible mechanisms of HBV reactivation in patients infected with HIV (escape mutants selection, host immune deficiency progression, antiretroviral-related immune reconstitution, glucocorticosteroids, chemotherapy), the message is that HIV patients should be monitored strictly not only by serological and virological assays to recognize early HBV reactivation, but also for drug over-exposure.

Evaluation of an affordable HIV-1 virological failure assay and antiretroviral drug resistance genotyping protocol

HIV-1 RNA viral load is the preferred tool to monitor virological failure during antiretroviral therapy (ART) exposure. Timely detection of virological failure can reduce the prevalence and complexity of HIV-1 drug resistance. This field evaluation further characterizes a two-step approach to identify virological failure, as a measure of ART adherence, and detect HIVDR mutations in the reverse transcriptase (RT) gene of HIV-1. Two hundred and forty-eight (248) samples were tested; 225 from South African HIV-1 participants enrolled in the PharmAccess African Studies to Evaluate Resistance (PASER) cohort, forty of which had paired dried blood spot (DBS) samples and 23 HIV-1 negative samples. A newly developed virological failure assay (ARTA-VFA) was used on all samples, and those with a viral load >5000 RNA copies/ml were genotyped with a shortened RT protocol to detect HIVDR (ARTA-HIVDRultralight). The ARTA-VFA showed good precision and linearity as compared to a commercial reference assay (NucliSENS® EasyQ v1.2, Roche) with an R2 of 0.99. Accuracy studies illustrated standard deviations of <1 log RNA copies/ml for plasma and DBS ARTA-VFA results compared to the reference method. The ARTA-VFA's intended use was to deliver qualitative results either < or >5000 RNA copies/ml. No significant differences in the proportion of results < or > either the 5000 RNA copies/ml or 1000 RNA copies/ml cut-off were noted for plasma indicating either cut-off to be useful. Significant differences were noted in these proportions when DBS were used (P=0.0002), where a 5000 RNA copies/ml cut-off was deemed more appropriate. The sensitivity and specificity of the ARTA-VFA with plasma were 95% and 93% and 91% and 95% for DBS using a 5000 RNA copies/ml cut-off. The ARTA HIVDRultralight assay was reliable for plasma and DBS samples with a viral load >5000 RNA copies/ml, with amplification and sequencing success rates of 91% and 92% respectively for plasma, and 95% and 80% respectively for DBS. HIVDR profiles for plasma and DBS were 100% concordant with the reference assay. This study evaluated a previously described combination of two assays potentially useful in assessing HIV-1 virological failure and resistance, showing good concordance with reference assays. These assays are simple to perform and are affordable, viable options to detect virological failures in certain resource limited settings. The assays’ compatibility with DBS sampling extends the access of HIV-1 virological monitoring to more remote settings.

Considerations in the development of nonhuman primate models of combination antiretroviral therapy for studies of AIDS virus suppression, residual virus, and curative strategies.

Animal models will be critical for preclinical evaluations of novel HIV eradication and/or functional cure strategies in the setting of suppressive combination antiretroviral therapy (cART). Here, the strengths, limitations, and challenges of recent efforts to develop nonhuman primate (NHP) models of cART-mediated suppression for use in studies of persistent virus and curative approaches are discussed. Several combinations of NHP species and viruses that recapitulate key aspects of human HIV infection have been adapted for cART-mediated suppression studies. Different cART regimens incorporating drugs targeting multiple different steps of the viral replication cycle have provided varying levels of virologic suppression, dependent in part upon the host species, virus, drug regimen and timing, and virologic monitoring assay sensitivity. New, increasingly sensitive virologic monitoring approaches for measurements of plasma viral RNA, cell-associated and tissue-associated viral RNA and DNA, and the replication-competent residual viral pool in the setting of cART in NHP models are being developed to allow for the assessment of persistent virus on cART and to evaluate the impact of viral induction/eradication strategies in vivo. Given the vagaries of each specific virus and host species, and cART regimen, each model will require further development and analysis to determine their appropriate application for addressing specific experimental questions.

Field expansion of DNA polymerase chain reaction for early infant diagnosis of HIV-1: The Ethiopian experience.

BackgroundEarly diagnosis of infants infected with HIV (EID) and early initiation of treatment significantly reduces the rate of disease progression and mortality. One of the challenges to identification of HIV-1-infected infants is availability and/or access to quality molecular laboratory facilities which perform molecular virologic assays suitable for accurate identification of the HIV status of infants.MethodWe conducted a joint site assessment and designed laboratories for the expansion of DNA polymerase chain reaction (PCR) testing based on dried blood spot (DBS) for EID in six regions of Ethiopia. Training of appropriate laboratory technologists and development of required documentation including standard operating procedures (SOPs) was carried out. The impact of the expansion of EID laboratories was assessed by the number of tests performed as well as the turn-around time.ResultsDNA PCR for EID was introduced in 2008 in six regions. From April 2006 to April 2008, a total of 2848 infants had been tested centrally at the Ethiopian Health and Nutrition Research Institute (EHNRI) in Addis Ababa, and which was then the only laboratory with the capability to perform EID; 546 (19.2%) of the samples were positive. By November 2010, EHNRI and the six laboratories had tested an additional 16 985 HIV-exposed infants, of which 1915 (11.3%) were positive. The median turn-around time for test results was 14 days (range 14–21 days).ConclusionExpansion of HIV DNA PCR testing facilities that can provide quality and reliable results is feasible in resource-limited settings. Regular supervision and monitoring for quality assurance of these laboratories is essential to maintain accuracy of testing.

Diagnosis and personalized management of hepatitis B including significance of genotypes

Chronic hepatitis B virus (HBV) infection is a leading cause of cirrhosis and hepatocellular carcinoma globally. This article reviews recent therapeutic developments and highlights the significance of HBV genotypes. Seven drugs (two interferons and five oral antiviral drugs) are currently available for the treatment of chronic hepatitis B. Peginterferon has the advantage of finite treatment duration and higher off-treatment durability, but causes more side effects. On-treatment monitoring with hepatitis B surface antigen and HBV DNA levels can predict response and serve as a stopping rule. Oral antiviral therapy carries fewer side effects, but long-term treatment is often required. Regular HBV DNA monitoring is essential for detecting drug resistance and suboptimal responders. HBV is divided into eight genotypes based on a difference in genetic sequence of 8% or more. Genotype C is associated with prolonged immune clearance and a higher risk of progression to cirrhosis and hepatocellular carcinoma. Patients with genotype D HBV infection have low response rate of 7-25% to peginterferon. The availability of effective antiviral therapy and virological assays has transformed HBV treatment in the past two decades. The differences in response rate and side-effect profile call for individualized treatment selection.

Point of care testing of HIV in children younger than 18 months with three different HIV virological assays. Experience from a district hospital in a resource-limited setting

Diagnosis of HIV in children younger than 18 months can be challenging in developing countries because requires the use of HIV virological tests. In this study we describe the experience with three commercial assays, HIV- 1 DNA polymerase chain reaction (PCR) (Roche Amplicor 1.5) with dried blood spot, HIV-1 RNA PCR (Roche COBAS TaqMan) with plasma and reverse transcriptase activity assay (Cavidi Exavir Load 3) with plasma in a rural setting of India. Sensitivity and specificity were 98.1% (95% confidence interval [CI] 90- 100) and 99.3% (95% CI 97.9-99.9) for the HIV- 1 RNA PCR assay, 66.7% (95% CI 29.9-92.5) and 100% (95% CI 96.8-100) for the HIV-1 DNA PCR assay, and 100% (95% CI 48-100) and 98.7% (95% CI 92.8-100) for the reverse transcriptase activity assay respectively. The low sensitivity of the HIV-1 DNA PCR assay in this setting is worrisome and warrants further investigations.

Evaluating Surveillance Strategies for the Early Detection of Low Pathogenicity Avian Influenza Infections

In recent years, the early detection of low pathogenicity avian influenza (LPAI) viruses in poultry has become increasingly important, given their potential to mutate into highly pathogenic viruses. However, evaluations of LPAI surveillance have mainly focused on prevalence and not on the ability to act as an early warning system. We used a simulation model based on data from Italian LPAI epidemics in turkeys to evaluate different surveillance strategies in terms of their performance as early warning systems. The strategies differed in terms of sample size, sampling frequency, diagnostic tests, and whether or not active surveillance (i.e., routine laboratory testing of farms) was performed, and were also tested under different epidemiological scenarios. We compared surveillance strategies by simulating within-farm outbreaks. The output measures were the proportion of infected farms that are detected and the farm reproduction number (Rh). The first one provides an indication of the sensitivity of the surveillance system to detect within-farm infections, whereas Rh reflects the effectiveness of outbreak detection (i.e., if detection occurs soon enough to bring an epidemic under control). Increasing the sampling frequency was the most effective means of improving the timeliness of detection (i.e., it occurs earlier), whereas increasing the sample size increased the likelihood of detection. Surveillance was only effective in preventing an epidemic if actions were taken within two days of sampling. The strategies were not affected by the quality of the diagnostic test, although performing both serological and virological assays increased the sensitivity of active surveillance. Early detection of LPAI outbreaks in turkeys can be achieved by increasing the sampling frequency for active surveillance, though very frequent sampling may not be sustainable in the long term. We suggest that, when no LPAI virus is circulating yet and there is a low risk of virus introduction, a less frequent sampling approach might be admitted, provided that the surveillance is intensified as soon as the first outbreak is detected.

Influenza Viruses Expressing Chimeric Hemagglutinins: Globular Head and Stalk Domains Derived from Different Subtypes

The influenza virus hemagglutinin molecule possesses a globular head domain that mediates receptor binding and a stalk domain at the membrane-proximal region. We generated functional influenza viruses expressing chimeric hemagglutinins encompassing a variety of globular head and stalk combinations, not only from different hemagglutinin subtypes but also from different hemagglutinin phylogenetic groups. These chimeric recombinant viruses possess growth properties similar to those of wild-type influenza viruses and can be used as reagents to measure domain-specific antibodies in virological and immunological assays.

Antiviral Potential of a Proteolytic Inhibitor from Streptomyces Chromofuscus 34–1

ABSTRACTThe novel proteinaceous protease inhibitor, isolated from the culture supernatants of Streptomyces chromopfuscus 34–1 (SS 34–1) demonstrated a specific and selective anti-influenza virus effect. By the use of complementary virological assays it was demonstrated that the expression of the viral haemagglutinin (HA) on the surface of infected cells, the virus-induced cytopathic effect (CPE) and the infectious virus yields (IVY), used as measures of A/Germany/34, strain Rostock (H7N1) (A/Rostock) virus growth, were all reduced at non-toxic concentrations of SS 34–1 in a dose-dependent way. In a single cycle of viral growth, the penetration of viral particles and the late stages of viral replication were the most susceptible to inhibition.The preparation protected mice from mortality in the experimental A/Aichi/2/68 (H3N2) influenza virus infection. The SS 34–1-treatment (0.16 mg/mouse/day) of virus-infected mice led to a considerable reduction of mortality rate (index ofprotection = 71.0 %) and marked prolongation of mean survival time (+ 5.5 days). The lesions of the lungs due to infection as well as lung infectious virus titres were significantly reduced—δ log10 TCID50/ml up to 4.5. The application of SS 34–1 induced a continuous rise of the anti-protease activity but did not cause substantial changes in the lung protease activity of healthy mice. The infection triggered a slight reduction of protease activity in the lungs at 5 and 48 h p.i. and a marked increase at 24 h and 6 day p.i. Protease inhibition in the lungs was reduced at 24 and 48 h p.i. and an increase was observed at 5 h, 6 and 9 day p.i. SS 34–1-treatment brought both activities to normal levels. It was assumed that SS 34–1 exerted its inhibitory effect indirectly, through an increase of the protease-inhibitory activity. Histopathological examination of the lungs showed that under SS 34–1-treatment pulmonary lesions were less severe than those of untreated controls.The isolated novel protease inhibitor was purified by anion-exchange chromatography and reversed phase-HPLC. Analysis by differential scanning calorimetry (DSC) revealed that at pH 7.5 the denaturation temperature was 75 °C and the denaturation itself was irreversible. Analysis by circular dichroism (CD) in the UV region 190–250 nm showed that at pH 7.5 the spectrum presented 2 minima at 208 and 222 nm, typical for an α-helical structure. At pH 2.5 and after heating the spectrum corresponded to an unordered structure.