The optimum cut-off value to differentiate Echinococcus granulosus sensu stricto from other species of E. granulosus sensu lato using larval rostellar hook morphometry.

Cystic echinococcosis caused by Echinococcus granulosus sensu lato is one of the most important helminth zoonoses in the world; it affects both humans and livestock. The disease is endemic in Argentina and highly endemic in the province of Neuquén. Considerable genetic and phenotypic variation has been demonstrated in E. granulosus, and ten different genotypes (G1-G10) have been identified using molecular tools. Echinococcus granulosus sensu lato may be considered a species complex, comprised of E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-G10). In endemic areas, the characterization of cystic echinococcosis molecular epidemiology is important in order to apply adequate control strategies. A cut-off value for larval large hook total length to distinguish E. granulosus sensu stricto isolates from those produced by other species of the complex was defined for the first time. Overall, 1780 larval hooks of 36 isolates obtained from sheep (n= 11, G1), goats (n= 10, G6), cattle (n= 5, G6) and pigs (n= 10, G7) were analysed. Validation against molecular genotyping as gold standard was carried out using the receiver operating characteristic (ROC) curve analysis. The optimum cut-off value was defined as 26.5 µm. The proposed method showed high sensitivity (97.8%) and specificity (91.1%). Since in most endemic regions the molecular epidemiology of echinococcosis includes the coexistence of the widely distributed E. granulosus sensu stricto G1 strain and other species of the complex, this technique could be useful as a quick and economical tool for epidemiological and surveillance field studies, when fertile cysts are present.

Human immunodeficiency virus type 1-related nucleic acids and papillomavirus DNA in cervicovaginal secretions of immunodeficiency virus-infected women.

OBJECTIVE: To evaluate simultaneous human immunodeficiency virus (HIV)-related nucleic acids and human papillomavirus (HPV)-DNA in cervicovaginal secretions of HIV-seropositive women. METHODS: We collected 47 paired blood and cervicovaginal lavage samples from 124 known HIV-1-seropositive women. Proviral HIV-1 DNA, cell-associated, and cell-free HIV-1 RNA in cervicovaginal secretions were quantitatively evaluated by competitive polymerase chain reaction (PCR) and reverse transcription PCR. Polymerase chain reaction and subsequent restriction fragment length polymorphism analysis of PCR products were used to detect HPV types 6, 11, 16, 18, 31, 33, 35, and 56. RESULTS: Proviral HIV-1 DNA, cell-associated, and cell-free HIV-1 RNA were detected in 52.4% (65 of 124), 38.7% (48 of 124), and 33.9% (42 of 124) of lavage samples, respectively. Human papillomavirus-DNA in cervicovaginal secretions was detected in 64% (79 of 124) of participants. The rate of detection of HPV types of intermediate to high oncogenic risk was higher in HIV-positive women who tested positive for cell-associated (odds ratio [OR] 3.57, 95% confidence interval [CI] 1.17, 11.12) or cell-free (OR 4.63, 95% CI 1.42, 15.51) HIV-1 RNA in cervicovaginal secretions than their counterparts who tested negative. Logistic regression analysis showed that the association between HPV infection and the detection of HIV-1 RNA in cervicovaginal secretions persisted after adjustment for potential confounders such as CD4+ cell counts, HIV-1 RNA in plasma, use of antiretroviral drugs, vaginal infection, and regular condom use. In univariable and multivariable analysis, HPV-DNA detection was associated with amounts of cell-free and cell-associated HIV-1 RNA in cervicovaginal secretions (chi(2) for trend 10.35, and 9.84, P =.001 and.002, respectively). CONCLUSIONS: The rate of HPV detection in the genital tract of HIV-1-seropositive women is associated with the amount of cell-associated and cell-free HIV-1 RNA present in cervicovaginal secretions. The association does not appear to be attributable entirely to the effect of HIV-related immunodepression.

Factors associated with nucleic acids related to human immunodeficiency virus type 1 in cervico-vaginal secretions.

OBJECTIVE: To assess HIV-related nucleic acids in cervico-vaginal secretions and the factors associated with them. DESIGN: Observational study. SETTING: Department of Obstetrics and Gynaecology, University of Pavia, Italy. POPULATION: HIV-positive patients attending a cytology service. METHODS: Paired blood and cervico-vaginal lavage samples were obtained from 122 known HIV-seropositive patients during periodic visits for cytologic screening for lower genital tract neoplasia. Vaginal specimens for the diagnosis of bacterial vaginosis, trichomonas vaginalis and candida infection were also obtained. HIV-1-RNA in plasma, proviral HIV-1-DNA, cell associated and cell-free HIV-1 RNA in cervico-vaginal secretions were quantitatively evaluated by competitive polymerase chain reaction (c-PCR) and reverse transcriptase PCR (cRT-PCR). MAIN OUTCOME MEASURE: Prevalences of HIV related nucleic acids in cervico-vaginal secretions and their univariate and multivariate associations with clinical variables. RESULTS: Proviral HIV-1 DNA, cell-associated and cell-free HIV-1 RNA were detected in 50% (61/122), 37.7% (46/122) and 32.8% (40/122) of the patients, respectively. In logistic regression analysis, the presence of HIV-1 RNA in blood was the factor which correlated best with the detection of HIV-1 DNA (OR = 5.48, 95% CI = 2.28-13.20), cell-associated (OR = 4.85; 95% CI = 1.89-12.45) and cell-free HIV-1 RNA (OR = 4.63, 95% CI = 1.74-12.33) in cervico-vaginal samples. However, between 20% and to 35% of patients who tested negative for blood HIV-1 RNA were positive for either HIV-1 DNA or HIV-1 RNA detection in cervico-vaginal lavages. Bacterial vaginosis was associated with an increased prevalence of cell-associated (OR = 3.58, 95% CI = 1.22-10.54) and cell-free HIV-1 RNA (OR = 2.94, 95% CI = 1.0-8.7) detection in cervico-vaginal secretions. Additional factors associated with increased prevalence of HIV-1 RNA detection were advanced stage of HIV disease and vulvovaginal candidiasis. CONCLUSIONS: Although the presence of HIV-1 RNA in blood is the factor which correlates best with the detection of HIV-related nucleic acids in cervico-vaginal secretions, the shedding of HIV in the genital tract can occur in 20-30% of non-viremic subjects. Bacterial vaginosis and candida infection could have a facilitating role in local HIV viral replication and shedding.

Viral excretion in cervicovaginal secretions of HIV-1-infected women receiving antiretroviral therapy.

A longitudinal study was conducted to evaluate the viral shedding present in cervicovaginal secretions of HIV-1-seropositive women receiving antiretroviral therapy. A total of 128 paired cervicovaginal and blood samples was obtained from 37 women during a median follow-up period of 21 months. A sensitive, competitive, polymerase chain reaction and a reverse transcription polymerase chain reaction were used for the simultaneous quantitation of HIV-1 proviral DNA and RNA in cervicovaginal cells and cell-free RNA in cervicovaginal secretions, as well as HIV-1 RNA in peripheral blood. The cumulative probability of detecting proviral DNA in genital secretions was significantly higher over time in women with detectable viremia than in women in whom HIV-1 RNA was persistently undetectable in plasma (< 50 copies/ml) (P = 0.028 by log-rank test). The presence and amount of proviral DNA, cell-associated RNA and cell-free RNA in the cervicovaginal secretions were positively correlated with the presence of detectable viremia or the number of HIV-1 RNA copies in plasma (Spearman rank correlation, 0.290, 0.279, and 0.305, respectively; all P < 0.01), but no correlation was found with the CD4+ cell count. In addition, vaginal infections were positively correlated with the detection of proviral DNA in cervicovaginal secretions (odds ratio, 2.60; 95% confidence interval, 1.07-5.70). However, the positive correlation between the presence and amount of HIV in cervicovaginal secretions and the viral load in plasma provides no assurance that HIV shedding does not occur in the genital tract of women with undetectable HIV-RNA in plasma.

Prevalence and histologic features of transfusion transmitted virus and hepatitis C virus coinfection in a group of HIV patients.

BACKGROUND: A recently identified DNA transfusion-transmitted virus has been associated with post-transfusion non-A to G hepatitis. AIM: To determine the prevalence of transfusion-transmitted virus in patients with human immunodeficiency virus infection. Its clinical role in the pathogenesis of liver disease was also evaluated in patients with transfusion-transmitted-virus hepatitis C virus coinfection compared with those with hepatitis C Virus infection alone. PATIENTS AND METHODS: We evaluated 312 HIV-hepatitis C virus coinfected patients (225 males, 87 females). All underwent screening for transfusion-transmitted virus DNA using a nested polymerase chain reaction technique. In some transfusion transmitted virus-DNA positive patients, we performed a phylogenetic analysis. In 56 patients (20 transfusion-transmitted-virus-hepatitis C virus and 36 hepatitis C virus alone), liver biopsy was collected. RESULTS: The prevalence of transfusion-transmitted virus was 113/312 (36%). The genotype distribution was similar to that reported in other studies. No difference in liver histology was found between the two groups. CONCLUSION: Transfusion-transmitted virus infection is common in human immunodeficiency virus patients. We found no histologic differences between liver biopsy specimens from patients coinfected with transfusion-transmitted virus plus hepatitis C virus compared with those infected with hepatitis C virus alone. Transfusion-transmitted virus is not clearly associated with a distinct liver injury.

Transfusion-transmitted virus infection in HIV-1-seropositive patients.

OBJECTIVES: To investigate the prevalence, persistence and genome heterogeneity of transfusion-transmitted (TTV) in HIV-1-infected patients, a group at high risk both of contracting blood-borne viruses and having viral persistence relating to immunodepression. METHODS: Plasma samples from 238 HIV-1 seropositive subjects and 226 healthy blood donors were examined for TTV-DNA both by polymerase chain reaction (PCR) using primers from the conserved regions in the N22 clone and PCR using primers deduced from the untranslated region (UTR). Direct DNA sequencing and phylogenetic analysis were used to characterize 27 TTV isolates from HIV-1 patients or healthy controls. RESULTS: Using PCR with the UTR primers, TTV DNA was detected in a very high percentage (> 80%) of samples both from HIV-1 seropositive subjects and from blood donors. Using PCR with N22 primers, shown to detect viral strains associated with hepatitis of unknown etiology, TTV DNA was found in 103 of 238 (43.3%) HIV-1-infected patients and in 22 of 226 (9.7%) blood donors. There was no difference in the prevalence of the TTV DNA in HIV seropositive subjects with regard to clinical features related to immunosuppression, markers of HCV infection or intravenous drug use; presence of TTV DNA was associated significantly only with male gender (P = 0.003). Persistent or intermittent viremia was detected in plasma samples taken up over a period of 19 months in all (15 of 15) HIV-infected patients tested. CONCLUSIONS: The persistence and high frequency of infection detected by PCR with N22 primers in HIV-1 seropositive patients suggest that further clinical investigation of immunocompromised hosts will provide information to clarify the pathogenic role of TTV.

Quantification of HIV-1 proviral DNA in patients with undetectable plasma viremia over long-term highly active antiretroviral therapy.

OBJECTIVES: To assess the prognostic role of proviral DNA in peripheral blood mononuclear cells (PBMC) of patients with undetectable viremia over long-term highly active antiretroviral therapy (HAART). METHODS: Eighty-two human immunodeficiency virus (HIV)-1-infected patients, free of acquired immunodeficiency syndrome (AIDS), received zidovudine plus lamivudine plus indinavir. Levels of plasma HIV-RNA, and PBMC proviral DNA and RNA unspliced (US) transcripts were evaluated by using competitive polymerase chain reaction (cPCR) assays, every 3 months over 1 year. RESULTS: Among patients with undetectable viremia at baseline, 13 of 18 with CD4 cell count 350/mm3 or less and 12 of 16 with CD4 between 351 and 700/mm3, constantly maintained undetectable RNA levels; in these patients, a mean proviral DNA decrease of 0.67 6 0.7 and 1.03 6 0.53 log (P < 0.001), respectively, a significant decrease of RNA-US transcripts (P < 0.001), and significant correlations between decreases of proviral DNA and RNA-US transcripts (P = 0.008 and P < 0.001, respectively) were observed. CONCLUSIONS: Proviral DNA quantitation permits the continued monitoring of HAART in patients with undetectable viremia.

Dominant role of host selective pressure in driving hepatitis C virus evolution in perinatal infection.

The dynamics of the genetic diversification of hepatitis C virus (HCV) populations was addressed in perinatal infection. Clonal sequences of hypervariable region 1 of the putative E2 envelope protein of HCV were obtained from four HCV-infected newborns (sequential samples spanning a period of 6 to 13 months after birth) and from their mothers (all samples collected at delivery). The data show that the variants detected between birth and the third month of life in samples from the four newborns were present in the HCV populations of their mothers at delivery. In the newborns, a unique viral variant (or a small group of closely related variants) remained stable for weeks despite active viral replication. Diversification of the intrahost HCV population was observed 6 to 13 months after birth and was substantially higher in two of the four subjects, as documented by the intersample genetic distance (GD) (P = 0.007). Importantly, a significant correlation between increasing GD and high values for the intersample K(a)/K(s) ratio (the ratio between anoffymous and synonymous substitutions; an index of the action of selective forces) was observed, as documented by the increase of both parameters over time (P = 0.01). These data argue for a dominant role of positive selection for amino acid changes in driving the pattern of genetic diversification of HCV populations, indicate that the intrahost evolution of HCV populations is compatible with a Darwinian model system, and may have implications in the designing of future antiviral strategies.

Quantitative assessment of cell-associated and cell-free virus in cervicovaginal samples of HIV-1-infected women.

OBJECTIVE: To examine the amount of cell-free and cell-associated virus in cervicovaginal secretions (CVS) of HIV-infected women. METHODS: Paired cervicovaginal and blood samples from 61 seropositive women were quantitatively evaluated by competitive polymerase chain reaction (cPCR) and reverse transcription-PCR (cRT-PCR) for: (1) genomic RNA from plasma and cell-free CVS, and (2) unspliced (u/s) RNA transcripts and proviral DNA in cells from secretions. RESULTS: HIV DNA was detected in 42.6%, u/s transcripts in 32.7% and cell-free HIV RNA in 31.1% of 61 cervicovaginal samples. The median copy numbers of HIV DNA, u/s transcripts, and cell-free RNA were 125 copies/10(5) cells, 40 copies/10(5) cells, and 300 copies/mL of secretion, respectively. Nineteen of 26 (73.1%) and 17 of 26 (65.3%) women positive for DNA were also positive for RNA transcripts and cell-free RNA, respectively (P<0.001). A significant correlation between the amounts of cell-free and u/s transcripts was also found (Spearman Rho 0.618, P=0.014). The prevalences of u/s transcripts and cell-free RNA were 42.6% and 53.8% respectively among patients with detectable blood RNA, and 22.9% (P=0.09) and 14.3% (P=0.0017) among patients with undetectable blood RNA. In stepwise logistic regression, cell-free RNA was independently associated with the presence of detectable blood viremia. The amount of HIV DNA was lower among subiects currently under treatment (50 copies/10(5) cells) than in untreated subjects (250 copies/10(5) cells) (P=0.037). CONCLUSIONS: Both cell-free and cell-associated HIV could be detected and quantitated in CVS, providing a means to examine the level of viral activity in the female genital tract.

HIV type 1 intraperitoneal infection of rabbits permits early detection of serum antibodies to Gag, Pol, and Env proteins, neutralizing antibodies, and proviral DNA from peripheral blood mononuclear cells.

The aim of this study is the development of an animal model useful for studying HIV-1 pathogenesis, candidate vaccines, and antiviral drugs. Aseptic thioglycolate peritonitis was induced in six rabbits. After 4 days, four rabbits were infected with 1 ml of HIV-1 stock containing 100 times the MID50. Blood samples were collected every 2 weeks for 8 months. Serum antibodies were tested by ELISA, using as antigen the recombinant protein p24; synthetic peptides of highly conserved regions of p31, gp41, and gp120; and a synthetic peptide of gp120 at the V3 loop region of HIV-1 strains IIIB and MN. Furthermore, neutralizing antibodies were tested by a microscale neutralization assay. Proviral DNA was detected by PCR, and virus isolation was performed by a cocultivation technique using primary rabbit peripheral blood mononuclear cells (PBMCs). All infected rabbits produced antibodies to HIV-1 proteins within 2 weeks and up to 8 months after virus infection. Serum antibodies were directed against the Env (gp120 and gp41), Gag (p24), and Pol (p31) proteins and against two synthetic peptides whose sequence corresponds to gp120 at the V3 loop region of HIV-1 strains IIIB and MN. Neutralizing antibodies were also detected in the sera of infected animals. Proviral DNA was detected in PBMCs by PCR within 4 weeks and up to 8 months after HIV-1 infection. HIV-1 was also isolated from PBMCs of infected animals at 30, 60, and 120 days after infection. Results obtained indicate that HIV-1 intraperitoneal infection of the rabbit permits the early detection of serum antibodies to Gag, Pol, and Env proteins, neutralizing antibodies, and proviral DNA sequences from PBMCs.

Booster PCR: evaluation of an improved method for amplification of a few HIV-1 proviral DNA sequences.

A biphasic polymerase chain reaction designed as booster PCR for the detection of human immunodeficiency virus type-1 (HIV-1) was evaluated in samples containing a very low number of target DNA. We examined DNA samples obtained from chronically infected H9/HTLV-III B cells and purified plasmidic DNA containing the entire HIV-1 genome. By using booster PCR we detected HIV-1 DNA sequences up to 5 infected cells in samples containing about 2 micrograms of genomic DNA, and up to 1 copy of plasmidic DNA in samples containing about 0.5 microgram of genomic DNA. Otherwise by using standard PCR HIV-DNA up 100 infected cells and up to 20 copies from plasmidic DNA could be detected. Our experiments in amplification of HIV-1 proviral DNA have demonstrated that booster PCR enhances sensitivity of detection of standard PCR in small quantities of target sequences at least 20-fold with no loss of specificity.

Specificity and sensitivity of 3rd generation EIA for detection of HCV antibodies among intravenous drug-users.

Serum samples from 487 ambulatory I.V. drug users were screened for HIV and HCV antibodies to determine the prevalence of coinfection in this high risk group for AIDS. For anti-HCV antibody screening we first used a 3rd generation EIA using, as antigen synthetic peptides which were not subjected to false positive results due to antibodies against superoxide dismutase or against yeast proteins (which may copurify with the recombinant proteins employed in the first and second generation test). The specimens that were positive in the screening test were confirmed by a more specific EIA system that detect antibodies to proteins encoded by structural (HCV-st EIA) and non structural (HCV-nst-EIA) regions of the HCV genome. A second confirmation assay was also performed: sera were run in presence or absence of blocking reagents which inhibits antibodies to C200 and C22 HCV epitopes for binding to the solid phase. The sensitivity of the HCV EIA screening for human HCV antibody detection revealed a 100% positivity for HCV infection. The confirmatory strategy presented in this paper revealed an HCV EIA specificity of 98.6%. In this work we demonstrated a significantly higher prevalence (p < 0.001) of HCV exposure in HIV infected individuals compared to the general population. Our experimental data also confirmed that HBV infection in drug-users at high risk for HIV infection was significantly associated with HCV infection (p < 0.001). In contrast, the acquisition of HIV by sexual contact was not a statistically significant risk factor for HCV coinfection.

Immunoblot typing of oxacillin-resistant Staphylococcus aureus clinical isolates.

Recently an immunoblotting system was developed for typing oxacillin-resistant Staphylococcus aureus. Oxacillin-resistant S. aureus (ORSA) isolates recovered during a 4-month period from premature new-borns division of University Hospital of Pavia, with several antimicrobial susceptibility patterns, were evaluated with this typing scheme. Immunoblotting was found to be a useful method with good reproducibility and the ORSA strains isolated at our institution were differentiated in two major groups of organisms clinically and epidemiologically related. Furthermore immunoblot typing showed same specific correlations with susceptibility patterns of ORSA isolates studied.

Effects of propolis flavonoids on virus infectivity and replication.

The effect of five propolis flavonoids on the infectivity and replication of some herpesvirus, adenovirus, coronavirus and rotavirus strains has been studied. Experiments were performed in vitro in cell cultures using the viral plaque reduction technique. The cytotoxicity of flavonoids, including chrysine, kaempferol, acacetin, galangin and quercetin, was evaluated on uninfected monolayers to determine their effect on cell growth and viability. Chrysine and kaempferol caused a concentration-dependent reduction of intracellular replication of herpes-virus strains when monolayers were infected and subsequently cultured in a drug-containing medium. However, virus infectivity was not significantly affected. Acacetin and galangin had no effect on either the infectivity or replication of any of the viruses studied. Quercetin reduced infectivity and intracellular replication, but only at the highest concentrations tested.

Emergence of cross-resistance to imipenem and other beta-lactam antibiotics in Pseudomonas aeruginosa during therapy.

The emergence of resistance to imipenem and other beta-lactams by Pseudomonas aeruginosa was investigated with two pairs of isolates. Two of these isolates were susceptible to imipenem and other beta-lactam antibiotics, such as moxalactam, ceftriaxone and cefotaxime, while the other two had developed resistance to those antibiotics during imipenem therapy. So far imipenem-resistant isolates have not demonstrated cross-resistance to other beta-lactam agents. We examined in these clinical isolates the possible mechanisms of resistance due to permeability modifications, either in outer membrane proteins (porins) or to LPS (lipopolysaccharides) complex. Particularly we analysed possible modification of physico-chemical properties of outer membrane proteins, such as changes in their hydrophobicity and electrical charge. beta-lactamase production was also studied. Results showed that resistance to imipenem may be related to loss or modifications in hydrophobicity of an outer membrane protein of about 46 Kdal; other modifications concerned hydrophobicity of the porin OMP F and, in one strain, the LPS complex appears to be responsible for resistance to other beta-lactam antibiotics together in combination with the production of beta-lactamases.

Modification of norfloxacin inhibition of DNA gyrase induced by a 28 KDal DNA binding protein.

We have previously studied a clinical isolate of Providencia stuartii which showed high levels of resistance to 4-quinolones, aminoglycoside and beta-lactam antibiotics (Landini et al., 1987). DNA gyrase from this isolate was inhibited for 50% of activity at a concentration of 15 microM of norfloxacin, which is about 5-fold higher compared to the 50% inhibitory concentration for a standard DNA gyrase. It has been described that 4-quinolone inhibition of DNA gyrase is caused by their binding to DNA and by the distortion induced in DNA tertiary structure, and that affinity binding of 4-quinolones is different for DNAs in different structures. In order to detect whether the interaction between pAT 153 and a protein able to modify DNA tertiary structure could affect norfloxacin inhibitory concentrations for DNA gyrase we purified from the clinical isolate of Providencia stuartii a DNA binding protein of about 28 KDal which induces changes in supercoiling degree of DNA. Assays of DNA gyrase activity were performed on the complex pAT 153-DNA binding protein-norfloxacin. Results showed an increase from 15 microM to 20 microM of 50% norfloxacin inhibitory concentration for DNA gyrase when pAT 153 was complexed with the 28 KDal protein.

Beta-lactam resistant Pseudomonas aeruginosa strains emerging during therapy: synergistic resistance mechanisms.

The emergence of beta-lactam resistant strains of Pseudomonas aeruginosa during treatment was studied. Three different strains present before treatment persisted with changes in their beta-lactam resistance during treatment. The isolates before and after therapy were studied for beta-lactamase production and permeability barrier. The increased beta-lactam resistance was correlated with an increased permeability barrier. In order to verify if the permeability barrier was correlated with changes in outer membrane proteins, outer membrane preparations were analyzed by SDS-PAGE. Several differences were observed between the OMP profiles of the post therapy and the pre therapy isolates. Furthermore, an analysis of PBPs pattern of strains studied was carried out and alterations in target proteins were observed.

Antiviral activity of Chamaecyparis lawsoniana extract: study with herpes simplex virus type 2.

Ethanolic extract of the green part of Chamaecyparis Lawsoniana was tested for antiviral activity and toxicity in tissue culture. All experiments were carried out in confluent human embryo lung fibroblasts. Treatment of fibroblast cells with extracts after viral inoculation was effective in inhibiting the replication of herpes simplex virus type 2 (HSV-2). The critical time for virus inhibition was 4 to 5 h after virus absorption. The antiviral activity was assayed employing the techniques of viral plaque and yield reduction. Toxicity of Chamaecyparis Lawsoniana in uninfected cells was studied as alteration of cell morphology, cellular viability and inhibition of host cell DNA synthesis. Herpes simplex virus inhibition occurred in presence of extract concentration of 0.5 micrograms/ml, whereas concentrations exceeding 5 micrograms/ml and 140 micrograms/ml were found to be cytotoxic when evaluated with inhibition of host DNA synthesis and cellular viability respectively. Results suggest that further investigations concerning the isolation of substances responsible for antiviral activity and an effort to define the mechanisms of action are warranted.