Pseudomonas aeruginosa proteases: purification procedures for an enzymatic standard.

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.

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.

Endocytosis constitute the infectious route of HIV-1 entry in human and rabbit monocytes lacking the CD4 receptor.

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.

Kinetics of p24 antigenemia, IgM, IgG antibodies to p24 and p41 and Ig virus isolation in rabbits experimentally infected with HIV-1.

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.

Infection of rabbits with human immunodeficiency virus.

An important requirement for the development of a vaccine against the human immunodeficiency virus (HIV-1), the causative agent of AIDS, is a readily available animal model that would allow possible immunogens to be evaluated. The only species to have been infected with HIV-1 so far is the chimpanzee. However, the scarcity of this animal and its designation as an endangered species place severe restrictions on its use as an animal model. Attempts to infect mice, rats, hamsters, guinea-pigs, musk shrews, and rabbits with HIV-1 or infected cells have all been unsuccessful. We now report that the intraperitoneal inoculation of rabbits with HIV-1 or chronically infected H9 cells consistently induces a persistent infection.

The morphogenesis of BLV (bovine leukemia virus) cultivated on FLK (fetal lamb kidney): an ultrastructural study.

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.

Human immunodeficiency virus (HIV): an ultrastructural study.

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.

Prevalence of antibody to human coronaviruses 229E, OC43 and neonatal calf diarrhea coronavirus (NCDCV) in patients of Northern Italy.

A seroepidemiological study for detection of antibody to human coronaviruses OC43, 229E, and neonatal calf diarrhea coronavirus (NCDCV), has been carried out using sera collected from hospitalized patients or healthy persons through routine laboratory tests in Northern Italy. Patients tested were children and adults with different pathological diseases. Antibody detection was performed by using an indirect immunoperoxidase staining technique (for all viruses) and, in the case of OC43 and NCDCV, antibody detection was obtained even with a hemagglutination inhibition test and a plaque reduction neutralization assay. Results obtained show a significant difference in the prevalence of antibody to 229E between children and adult group. Furthermore, a different titer was observed, within the two groups, between patients affected by hematological diseases (leukemia) and patients with other diseases. Finally, our data seem to confirm previous studies reporting a very high prevalence of antibody to coronavirus OC43 but a less detectable seropositivity to coronavirus 229E.

Bovine leukemia virus inhibiting activity in fetal calf serum.

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.

Antibody to bovine leukemia virus in sera from leukemic patients.

A new cell line, persistently infected with Bovine Leukemia Virus (BLV), was obtained by co-cultivation of lymphocytes from adult leukemic bovine with normal calf kidney cells. The presence of BLV was demonstrated directly in the lymphocytes and in the new cell line by electron microscopy, and by ELISA of cell extracts. An immunoperoxidase staining technique was used for detection of BLV-infected cells at each new passage of the line, in order to confirm the persistence of viral infection. Fixed preparations of infected cells were used to study human sera for the presence of specific antibody to BLV. Human sera were collected from different groups of patients, and a specific antibody response to BLV was detected in only 3 out of 33 sera from leukemic patients. No positivity was detected in control sera.

Diagnosis of acute non-bacterial gastroenteritis by rotavirus detection and serology.

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.

Reactivity of human coronavirus OC43 and neonatal calf diarrhoea coronavirus membrane-associated antigens.

Human embryonic lung fibroblast cultures and Vero cell cultures infected with cell culture-adapted strains of human coronavirus (HCV) OC43 or neonatal calf diarrhoea coronavirus (NCDCV) were shown to possess highly cross-reactive membrane-associated antigens (MAA) by the indirect fluorescent antibody technique (IFAMA). MAA appeared 3 h post-infection, concurrently with the appearance of cytoplasmic antigens. Electron microscopic observations of cell cultures infected with either coronavirus strain and labelled with the immunoperoxidase antibody (IPA) technique for MAA detection showed that MAA consisted mainly of a strongly labelled, discontinuous, brush-like layer of amorphous material, strictly associated with the infected cell membrane. By light microscopy, reactivity of MAA with homologous and heterologous immune serum was similar to that of antigens detected by IPA in ethanol-fixed infected cells. IPA and IFAMA, but not haemagglutination-inhibiting (HI) and neutralizing (Nt) antibody, were strongly decreased by absorption of immune sera with trypsin-treated glutaraldehyde-fixed cell cultures infected with homologous virus. MAA IgG antibodies were detected by IFAMA in both human and animal sera. Sera from infants showing an HI and Nt, but not an IPA, antibody response to HCV OC43 were also free of detectable IFAMA antibody to HCV OC43.

Serodiagnosis of respiratory synctial virus infections in infants and young children by the immunoperoxidse technique.

The immunoperoxidase antibody (IPA) technique was used to develop two new tests for serodiagnosis of respiratory syncytial virus infections: a microneutralization test based on the reduction of the number of infected cells and an IPA test for determination of virus-specific immunoglobulin G (IgG). Neutralizing antibody was determined both in the presence and absence of complement. In a group of 24 infants and young childres, ages less than 1 to 36 months, with acute respiratory syncytial virus infection, serodiagnosis was made by the IPA-IgG test in 20 cases, by neutralization test with addition of complement in 19 cases, and by neutralization test without addition of complement in 17 cases. Complement fixation detected only 12 cases of infection. All four cases not serologically diagnosed were infants less than 1 month old. Neutralization test antibody titers in the presence of complement were usually 4- to 16-fold higher than titers obtained without addition of complement. Both IPA-IgG and neutralization test (in the presence of complement) appear very efficient in serologically detecting respiratory syncytial virus infections in infants older than 1 month and give rapid results (IPA-IgG takes 2 h to complete, and the neutralization test takes 24 h). However, IPA-IgG is simpler to perform.

Immunoglobulin G to virus-specific early antigens in congenital, primary, and reactivated human cytomegalovirus infections.

Immunoglobulin G antibody to human cytomegalovirus (CMV)-specific early antigens (EA-Ab) was determined by the immunoperoxidase antibody technique in several cases of congenital, primary, and reactivated CMV infections. Mothers of congenitally infected infants and a group of leukemic children and pregnant women were also studied. In 11 cases of congenital infection, CMV EA-Ab was always associated with CMV excretion whether immunoglobulin M antibody was present or not. Nine mothers of congenitally infected infants had CMV EA-Ab for several months after delivery, but association with CMV elimination was not established when urine and/or saliva were tested for virus isolation. In all nine cases of primary CMV infection, CMV EA-Ab was present, and in five its detection was associated with CMV isolation. In one case, disappearance of EA-Ab occurred when virus excretion ceased. In five cases of reactivated CMV infections, a consistent association between CMV EA-Ab and virus isolation was found. Six of 31 leukemia children had CMV EA-Ab, and virus was isolated from 3 of these. Four of 28 pregnant women showed EA-Ab in their serum, but tests for isolation were not done. These data suggest that CMV EA-Ab is not a marker of a current primary CMV infection, as previously reported, but a marker of an active CMV replication which can take place in primary as well as in congenital and reactivated CMV infections.