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Human coronaviruses, represented by the two prototype strains HCV-OC43 and HCV-229E, are important human respiratory pathogens, also associated with necrotizing enterocolitis. Two previous studies, one describing the electron microscopic observation of doughnut-shaped particles, resembling coronaviruses, in a perivascular inflammatory lesion of brain tissue taken at autopsy from a multiple sclerosis patient, and the other one reporting the isolation of coronaviruses from the brains of two multiple sclerosis patients, suggested the possible association between coronaviruses and human demyelinating diseases. We analysed polyadenylated RNAs extracted from cerebrospinal fluid of twenty randomly selected multiple sclerosis patients and ten patients with other neurological diseases (medullary atrophy, Parkinson's disease, polyneuropathy, senile dementia, headache and toxic polyneuropathy) by reverse transcription and polymerase chain reaction searching for HCV-OC43 and HCV-229E sequences. By hybridization analysis of amplification products, we detected HCV-OC43 polyadenylated RNAs in ten specimens of patients with multiple sclerosis. Furthermore, we found positive hybridization signals for HCV-OC43 in the other neurological diseases, except for the toxic polyneuropathy specimen. Positivity for HCV-229E was observed in seven specimens of multiple sclerosis cerebrospinal fluid; one headache cerebrospinal fluid and the medullary atrophy specimen also resulted positive for HCV-229E. Moreover, using a solid phase technique, we report for the first time the sequence of a cDNA fragment derived from RNA extracted from cerebrospinal fluid of multiple sclerosis patient, belonging to the open reading frame which codes for the HCV-OC43 nucleoprotein N. Furthermore, cDNA sequences revealed the presence of a mixed viral population.

Pseudomonas aeruginosa may cause severe infections in debilitated patients. Strains of this microorganism produce several extracellular space proteins, some of which are believed to be virulence factors. There are experimental correlations between the ability to produce proteases and virulence. Treatment of bacteria with subinhibitory concentrations of antimicrobial agents frequently increases bacterial phagocytosis, intracellular killing, and suppresses the production of bacterial virulence factors, including extracellular enzymes. We suggest a simple method for production, purification and quantitation of Pseudomonas aeruginosa extracellular proteases suitable for use in investigations of their role as virulence factors.

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.

In mice experimentally infected with 1 x 10(5) UI/mouse of HTLV-IIIB IgM antibodies were detected 10-12 days after the infection, reaching peak values two weeks later; the IgM seratiter progressively decreased thereafter and was negative at ten-eleven weeks. HIV p24 antigen was detected ten-fifteen days after infection and reached peak values five-six weeks later. Antigenemia subsequently decreased and showed an oscillating course with a progressive decrease which persisted throughout the observation period. Two weeks after infection we detected IgG antibodies to the major core protein p24; reactivity to gp41 was observed as early as reactivity to p24 and persisted throughout observation period. The IgG antibodies to all HIV epitopes peaked two-three weeks after infection; the time course showed a decrease after ten weeks, progressively decreasing thereafter. After sixty-five weeks of infection the IgG seratiter value was lower but remained positive. Viruses indistinguishable from HIV were isolated from the peripheral blood mononuclear cells of infected mice 30, 60, 180 days after infection. These seroimmunological and virological data confirm that the immunocompetent mouse may serve as a low-cost reproducible model for HIV-1 in vivo research.

To obtain "functionally" CD4 negative human monocytes (0-5 CD4 +/1 x 10(6)/cells), 50 ng/5.10(5) cells of OKT4A were added daily after a pre-incubation with OKT4A (100 ng/5.10(5) cells. In our experimental conditions the blocking the CD4 receptor of human monocytes with OKT4A monoclonal antibody did not prevent HIV-1 infection, although the level of virus replication appeared lower than that in cultures without OKT4A. "Naturally"CD4 negative rabbit monocytes infected with HIV-1 also released a detectable level of virus after 12-15 up 28-30 days. In "naturally" CD4 negative rabbit monocytes and "functionally" CD4 negative human monocytes, the virus particles entering via phagocytosis are not infectious because multiple well defined virions were observed in phagocytic vacuoles and the envelopes of these particles did not appear to interact with the vacuolar membrane. The infectious particles were represented by endocytic vesicles containing only the core of HIV after fusion between the viral envelope and endocytic membrane. Fusion between the viral envelope and plasma membrane on the cellular surface was never observed, in spite of examining greater than 1000 virions bound the surface of human and rabbit macrophage monocytes. The absence of cytopathic effect in the rabbit and human CD4 negative monocytes infected with HIV-1, and conversely the presence of specific sequences of HIV in the genomic DNA may indicate that the macrophages-monocytes serve as an important reservoir for the persistence of HIV in infected hosts, similar to the other related Lentiviruses. Our virological data have also demonstrated that virus infection can be transmitted from rabbit and human infected monocytes to uninfected H9 cells. This preliminary study may offer important evidence for the development and testing of vaccines and compounds that inhibit HIV penetration of susceptible cells.

Two CD4 oligopeptides, corresponding to residues (37–53) and (37–55) of the V1 domain of CD4, which recent structural studies propose as the most likely binding site of HIV-1 gp120, have been chemically synthesized by solid-phase techniques, modified by the addition of two side-chain protected cysteines at both termini and purified by HPLC. Their ability to inhibit the infectivity of human immunodeficiency virus type 1 (HIV-1) (HTLV-IIIB, RF and GB8 strains) in different cell lines was monitored by the production of progeny virus, p24 and reverse transcriptase activity in the culture supernatants and by electron microscopy. The results indicated that the peptides inhibited HIV-1 infectivity in a dose-dependent fashion without any detectable cytotoxicity.

In rabbits experimentally infected with 1.10(5) u.i./ml HIV, IgM antibodies were detected 10-15 days after infection, reaching peak value two weeks later and remaining stable for two weeks long. Then a the IgM serotiters progressively decreased and were negative at ten weeks. HIV p24 antigen was detected ten-fifteen days after infection, reaching peak value five-six weeks later. Antigenemia subsequently decreased and reached a second peak after nine weeks. In our experimental conditions, the antigenemia persisted throughout the observation period. The IgG antibody titer reached a maximum two weeks after infection; the time course showed a decrease after ten weeks, followed by progressively decreasing fluctuating course. After twenty four weeks of infection the serotiter values though lower were always positive. Three-four weeks after infection we detected IgG antibodies to the major core protein p24. Reactivity of IgG antibodies to gp41 was observed earlier than reactivity to p24; these antibodies were detected over six months after infection. Viruses indistinguishable from HIV were isolated from the peripheral blood mononuclear cells of infected rabbits 30, 60 and 180 days after infection. These data further confirm that the rabbit may serve as an economical and reproducible model for HIV infection in which vaccines and antiviral agents could be tested.

Four strains of Pseudomonas aeruginosa clinical isolates were studied for resistance to antipseudomonal beta-lactams and aminoglycosides. Two of these strains were isolated from two different patients before antibiotic treatment, the other two strains, isolated during therapy, developed resistance to many of antipseudomonal beta-lactams and, in addition, to aminoglycoside antibiotics. All the strains produced a constitutive chromosomal beta-lactamase, while the latter two showed a significant reduction in permeability coefficient. Thus, a permeability change may be the major factor involved in the resistance to most antipseudomonal beta-lactams and may be responsible for development of cross-resistance to aminoglycosides.

In the present work we report our preliminary data demonstrating the susceptibility of mouse to HIV infection. IgG antibodies were found in mice intraperitoneally inoculated with H9/HTLV-IIIB infected cells. Infected mice showed a humoral response and the antibodies detection includes specific immunoglobulin directed against the major gene product of HIV. Two weeks after infection with HIV infected cells we detected in mice IgG antibodies to p24, and the reactivity to gp41 was observed as early as reactivity to p24 and persisted throughout observation period. The persistence of specific antibody response to HIV after one year seems to demonstrate the possibility of the use of the mouse as animal model for HIV in vivo experiments. Studies concerning the degree and kinetic of HIV infection in mouse are in progress in our laboratory.

We have obtained a new cell line persistently infected with bovine leukemia virus (BLV). BLV-infected cells can be easily detected by indirect immunofluorescence or immunoperoxidase staining technique. The morphology and number of infected cells seem to be related to serum concentration in culture medium: fetal calf serum (FCS) showed a marked effect on virus growth and virus distribution in cell cultures. All of the commercial lots of FCS tested showed BLV inhibiting activity very similar to that previously described for other viruses.

We studied the distribution, in human sera, of non antibody serum inhibitor active towards human OC43 and bovine NCDCV coronaviruses. Antibodies to coronaviruses, present with high prevalence in human sera, were filtered by Protein A Sepharose CL 4B column. In the present work we tested sera of newborns, infants, young children and adults. Inhibitory activity was revealed in all sera except these from newborns. This non antibody inhibitor was tested by a plaque-reduction assay.

We obtained a clinical isolate of Providencia stuartii showing a high level of resistance to norfloxacin and to other 4-quinolones, whose target is the enzyme DNA-gyrase. This strain showed resistance also to beta-lactam and aminoglycoside antibiotics. In order to detect modification of DNA-gyrase, we performed supercoiling assays in vitro in presence of norfloxacin and ciprofloxacin. Furthermore, outer membrane proteins, which are involved in permeability mechanisms, were analyzed on SDS-polyacrylamide gels. Results showed that both modifications in DNA-gyrase and changes in outer membrane proteins can be held responsible for resistance to 4-quinolones; moreover, these modification are probably supported by a third mechanism of resistance.

Human OC43 and bovine neonatal calf diarrhoea coronavirus (NCDCV) are known to be inhibited by non-antibody factors present in sera of different mammalian species, including man and cattle. Such an inhibitor is also present in fetal calf serum generally used for growing and maintaining cell cultures, thus influencing the viral infectivity titer. Previous reports refer to the effectiveness of phospholipase C treatment in eliminating the inhibiting activity. In order to detect the compound possibly involved in coronavirus growth inhibition (among the phospholipids naturally present in mammalian sera), we tested several phospholipids with hemagglutino-inhibition test and plaque reduction assay. Only phosphatidyl-serine seemed to be effective on Coronavirus OC43 and NCDCV, as detected by plaque-reduction assay, but not by hemagglutino-inhibition test.

A new cell line, obtained by co-cultivation of fetal lamb kidney cells and lymphocytes collected from an adult calf affected by enzootic bovine leukemia, was studied for bovine leukemia virus (BLV) morphogenesis. In this new cell line, called FLK-BLV, persistently infected with BLV, we identified extracellular and intracellular BLV particles. We never observed "budding" particles along the cell surface, and therefore assumed it was a new BLV maturation process in the cell vacuoles. In fact we found mature and non-mature particles connected with the cell-membrane system or cellular debris within vacuoles. We suggest that the viral envelope could be supplied by vacuole membrane. In our samples we also observed a cytopathic effect with syncytia formations similar to those observed on other BLV-producing cell lines.

In the present work we studied non antibody inhibiting activity present in fetal bovine serum and active to Rotavirus infectivity and growth in cell cultures. This inhibitor was revealed by an in vitro neutralization test and characterized by gel filtration and chemical and enzymatic treatments. Furthermore, commercial preparations of bovine serum proteins were tested for inhibitory activity. Our results show that serum inhibition is partially resistant to trypsin and neuraminidase treatments but completely destroyed by KIO4. A similar activity was observed in a commercial serum bovine fraction containing predominantly alpha-globulins. These results seem to indicate that glycoproteins, and their glucidic components are the molecules predominantly involved in serum inhibition towards Rotavirus infectivity.

Two different cases of fatal Herpes simplex type 1 encephalitis are reported. A presumptive diagnosis of herpes encephalitis was made on the basis of clinical symptoms and of neurodiagnostic evidence (EEG - CT) and was confirmed by serological and virological tests. The electron microscopy of the nuclei of the infected cells supported the diagnosis of HSV encephalitis.

Seven clinical isolates of Enterobacteriaceae resistant to third-generation cephalosporins were investigated in order to assess the role played in resistance by permeability barrier and by beta-lactamase production. The addition of subinhibitory concentrations of EDTA increased susceptibility to ceftriaxone in five strains showing that the permeability barrier is involved. All strains produced different amounts of beta-lactamases that were always increased by cefoxitin induction. Hydrolytic activity of investigated enzymes varied in different cephalosporins; ceftriaxone and ceftazidime are the most stable compounds.

Herpes simplex virus (HSV) excretion was studied in patients with dental diseases. Two groups of patients were examined: the first with dental caries and the second without dental decay. Results showed a significantly higher elimination rate of HSV in dental caries patients in comparison with control patients.

In 127 infants and young children suffering from acute non-bacterial gastroenteritis, diagnosis of rotavirus infection was done by virus detection and serology. Human rotavirus (HRV) detection was performed by direct electron microscopy (EM), conventional immune electron microscopy (IEM) and/or solid phase immune electron microscopy ( SPIEM ); rotavirus antigens were detected by indirect double-antibody sandwich (DAS) ELISA and HRV isolation was attempted in MA-104 or LLC-MK2 cell cultures. HRV serology was done on paired sera from all the patients by the indirect immunoperoxidase antibody (IPA) technique for HRV IgG determination, and by an indirect ELISA method using a purified HRV Wa strain as a solid phase. HRV particles were detected by EM and/or IEM in 53 cases (41.7%) and by SPIEM in 5 additional cases; HRV antigens were demonstrated by indirect DAS ELISA in the same 53 cases, whereas 40 cases (31.4%) were positive for HRV isolation in cell cultures. Sixty-four patients (50.3%) seroconverted by IPA and ELISA, including all the cases (58) positive for rotavirus detection in stools and 6 additional cases. Thus, SPIEM appears to be the most sensitive technique for detecting a few virus particles in stool specimens, but HRV serology is the most sensitive method for diagnosing HRV infections retrospectively, when paired sera are drawn at an appropriate time. However, EM possess the great advantage of detecting in fecal specimens viral agents other than rotaviruses, such as adenoviruses, enteric coronaviruses, small round viruses, astroviruses and others.

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.

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.

It is well known that some strains of coronaviruses, such as human OC43 and bovine NCDCV, are highly inhibited by non antibody factors present in sera of different mammalian species and sensitive to phospholipase-C treatment. So far this inhibitor has only been revealed in vitro experimental procedures with coronavirus strains adapted to growth in human lung fibroblast cell cultures. The purpose of this work was to ascertain whether this inhibiting activity was also effective "in vivo". In order to detect the "in vivo" activity, we used suckling mice which have been shown to be the animals most sensitive to infection with these viruses. Our results demonstrated that the same non antibody inhibitor is able to neutralize viral infection both "in vivo" and "in vitro".

A group of 82 patients (77 with urethritis) was studied for the presence of Chlamydia trachomatis infection. The agent was isolated by inoculation on McCoy cell cultures, followed by Giemsa and/or Lugol-iodine staining. The results obtained showed that the frequency of Chlamydia trachomatis infection in the group of patients considered was comparable with that previously reported on other groups. Male patients showed a higher infection rate than females. Different percentages of positivity were obtained in patients with gonococcal infection and in those with post-gonococcal urethritis.

H-9 cells producing HIV were examined by electron microscopy to value the virus-host cell relationships. HIV fine structure was also studied. HIV induces little cellular damages and it can penetrate into the cytoplasm by phagocytosis. Phagocytosis of the virus could play an important role in the mechanism of cellular infection.

A peculiar pattern of antibody response to human coronavirus (HCV) OC43 and neonatal calf diarrhea coronavirus (NCDCV) was observed in a significantly higher proportion of children with gastroenteritis (GE) than controls. It was directed only to OC43 surface antigens and appeared to be quite different form that observed in OC43 respiratory infections, in which an antibody response to both OC43 and NCDCV surface and internal antigens was found. Subsequently, fecal excretion of CV-like particles was detected by electron microscopy in a significantly higher proportion of infants and young children with acute GE as compared to controls. Immune electron microscopy showed a two-way cross-reactivity between HCV OC43 and human enteric coronavirus (HECV), both using immune sera (raised in animals immunized with HECV strains purified from stools of two patients) and patients' convalescent sera. These data show that HECVs are antigenically related to HCV OC43 and could be somewhat involved in the etiology of GE in infants and young children.