Potential Contribution of Cytomegalovirus Infection to Prenatal and Early Neonatal Mortality of Monkeys in the Adler Breeding Center.

Scrapings from the cervical canals and uterine cavities of females with a history of miscarriages, pathological deliveries, and stillbirths were tested for the cytomegalovirus DNA. The incidence of the agent in the females with a history of gestosis and abnormal deliveries was significantly higher than in females without anamnesis of this kind. Parenchymatous organs of stillborn neonates and animals dead during the first month of life were studied. This analysis and studies of the umbilical cords and placentas showed generalized cytomegalovirus infection in 22% dead animals, which objectively proved intrauterine infection.

[Phylogenetic analysis of cytomegaloviruses isolated from man and different primate species].

Institute of Medical Primatology, Russian Academy of Medical Sciences, Sochi The conserved regions of nucleotide sequences were found in primate cytomegaloviruses (CMV). Universal primers were designed for the consensus sequence of a conservative region of the UL56 gene of the betaherpesvirinae subfamily. Amplification, sequencing, and phylogenetic analysis of the fragments of CMV strains isolated from man and different primate species were made. Analysis of sequenced gene fragments showed that the UL56 gene area is most suitable for the phylogenetic analysis of primate CMV and could identify several groups of clusters by the degree of relationship among the viruses of this family.

Laboratory diagnosis of cytomegalovirus in monkeys in the Adler Breeding Center and in humans.

Monitoring of cytomegalovirus markers was carried out in the blood of humans and in the blood, lavage fluid specimens from the throat, and salivary gland tissues of monkeys of different species. Correlation between the percentage of cytomegalovirus infection and age was detected in humans. The virus was most often detected in salivary gland tissues and least so in the blood.

[Development of the PCR-test system for detection primate betaherpesvirinae].

A PCR-test system for detection of primate Betaherpesvirinae viruses was developed. Using alignment of complete genome of human, chimpanzee, and macaque rhesus cytomegalovirus conserve regions of viral genes were found. The oligonucleotide primers for consensus conserve regions of CMV UL56 gene were developed. The conditions of PCR were optimized and primer specificity for cytomegaloviruses of different primate species was confirmed.

[Detection of SV40 in blood samples from healthy subjects in the Russian Federation by RT-PCR].

Simian virus 40 (SV40), a monkey polyoma virus, is believed to have been introduced into the human population with a contaminated vaccine used in 1955-1963. Recent studies have demonstrated the presence of SV40 DNA in certain human tumours and normal tissues. We studied the occurrence of SV49 infections in human populations of Russia by RT-PCR analysis of blood samples taken from healthy subjects in different regions of the country. The results confirm the presence of SV40 in subjects of different age including those born after 1970. The significance of human SV40 infection is unknown.

Epidemiology of SV-40 simian virus in different regions of the Russian federation.

Multiplication of poliomyelitis virus for vaccine production in 1955-1961 was realized in kidney cell culture from M. rhesus naturally infected with SV-40 simian virus. Hence, some lots of the vaccine were contaminated with this virus. It was found that SV-40 is oncogenic for laboratory rodents. Since 1963, in accordance with WHO recommendation, green monkey kidneys containing no SV-40 were used instead of M. rhesus kidneys. Overall vaccination of the population with poliomyelitis vaccine in 1955-1961 led to infection of many humans in Russia and many foreign countries with SV-40. The possibility of horizontal transmission of the virus was demonstrated. As a result, virus (its DNA sequences) was detected in individuals who were never vaccinated. Hundreds of reports, often contradictory, discuss this problem. Our study is based on the analyses of 460 blood specimens from subjects living in different regions of Russia (Krasnodar region, Moscow, Novosibirsk region, Krasnoyarsk territory). The percent of individuals infected with SV-40 varies from 16 to 49%.

[Analysis of DNA of higher primates using inter-SINE PCR].

Two types (MIR and Alu) of short interspersed repeated DNA sequences (SINEs) were used for analysis of genetic relationships among higher primates, and for detection of polymorphism in human genomic DNA. The DNA regions located between the neighboring copies of these SINEs were amplified in polymerase chain reaction with primers complementary to the MIR and Alu consensus sequences (inter-SINE PCR). Comparison of the sets of amplified DNA fragments for different species or individuals provides evaluation of the relationships among them. Using inter-MIR PCR technique, the relationships among the higher primates of the infraorder Catarrhini reported elsewhere were confirmed, pointing to the efficiency of the method for phylogenetic studies. No human DNA polymorphism was revealed with the help of inter-MIR PCR. This polymorphism was detected by means of inter-Alu PCR, which is probably associated with the continuing amplification of Alu elements in human genome.

[Phylogenetic analysis of the mycoplasma isolated from the Javanese macaque (M. fascicularis)].

The nucleotide sequence of the fragment of 16S-aRNA of mycoplasma was determined. The fragment was identified using the PCR method in the urogenital scrape of the Javanese macaque (M. fascicularis). The sequenced fragment of mycoplasma of M. fascicularis was compared to well-known sequences of mycoplasma of mammals. The results of our comparative and phylogenetic analyses of the sequenced DNA fragment revealed that the mycoplasma belonged to the M. primatum species and fell within the same cluster as M. hominis. The mycoplasma M. primatum was for the first time observed in the monkeys M. fascicularis. The pathogenicity of the mycoplasma species with respect to monkeys is being studied.

[Spontaneous urogenital infection in macaques].

Rates of natural infection of macaques with microorganisms that cause urogenital tract infections (UGT)--chlamydias, mycoplasms, ureaplasms and trichomonades--have been assessed. 198 macaques (rhesus, javan and lapunder) aged from newborn to 25 years (born healthy or from females with complicated pregnancy or labour) were examined. High rate of infection (40%) was observed in healthy macaques by PCR assay. In animals born from females with complicated pregnancy or labour (abortion, complicated labour, stillbirth or inflammatory postdelivery complications) rate of pathogens detection was significantly higher (up to 54,5 - 64,5%). In such animals mixed infection with 2 - 4 microorganisms in association was commonly observed. Births of weak, low weight and vitality monkey's calves was observed in infected macaques aged 17 - 25 years. Pathogens' nucleotide sequences that were analogous to ones detected in mothers were detected in calves that died during 1st month of life. This finding can be the evidence of the intrauterine infection of calves.

[The virogenic status of cultured continuous simian lymphoid cells and their immunophenotypical characteristics].

Immunophenotyping of cultured continuous simian lymphoid cultures using a panel containing monoclonal antibodies to human B- and T-cell antigens has been carried out, by employing enzyme immunoassay and flow cytofluorometry. The test cultures showed a wide variety of cells containing B- and T-cell antigens with their varying expression. The cultures were also found to comprise lymphoblasts simultaneously containing B- and T-cell markers. Simian lymphotropic viruses, such as EBV-like and STLV-1 retrovirus, detected apart or simultaneously, have been verified by polymerase chain reaction. Whether there is a possible relationship between the type of cells in the culture and the type of their replicating virus(es) is discussed in the paper.

Study of the prevalence of agents of sexually transmitted diseases in monkeys by the gene diagnosis method.

Carriership of agents of sexually transmitted diseases (Trichomonas, Chlamydia, Mycoplasma, Ureaplasma) is highly prevalent in healthy monkeys living in the Adler Breeding Center. The incidence of these microorganisms is appreciably higher in animals with gestoses and labor abnormalities in comparison with animals with normal genital function. Mixed infection caused by 2-4 agents is much more incident than monoinfection.

No evidence for productive PERV infection of baboon cells in in vivo infection model.

OBJECTIVES: The discovery that pig endogenous retroviruses are infectious for human cells in vitro lead to vehement discussions about the possible risk of infection after clinical xenotransplantation. Since PERV transmission to non-human primate cells in vitro has been observed, similar to human cells, infection studies in non-human primates should represent the best model to analyze a potential PERV transmission after xenotransplantation. However, it is still open to discussion, whether non-human primate cells can be infected productively-similar to human cells- and whether those species are suitable to analyze PERV infection risks in vivo. METHODS: In vitro, only few cell types can be tested for susceptibility. We developed a pig to baboon cell transplantation model with special emphasis on B-cell effective immunosuppression, removal of anti Gal-alpha 1,3-Gal-antibodies, inhibition of the complement cascade and long term survival of transplanted cellular grafts. This model allows us to investigate in vivo, whether any baboon cell types may be permissive for productive PERV infection. The xenograft recipients were investigated for up to 535 days post transplantation. Gal-alpha 1,3-Gal-antibody and complement levels were monitored. Potential PERV transmission was analyzed, not only in PBMC, but in a variety of tissue samples as well as in serum and plasma samples by PCR, RT-PCR and by detection of RT-activity. Moreover, potential PERV specific immune responses were studied by a highly sensitive Western-Blot-assay. RESULTS: Despite several days of extremely low levels of Gal-alpha 1,3-Gal-antibody and complement, and despite of long term xenochimerism, no evidence for PERV infection was obtained in any of the tested tissues or in the tested serum samples. CONCLUSION: This study supplies further evidence for a low susceptibility of baboons towards productive PERV infection after xenotransplantation.

Induction of long-term peripheral microchimerism in non-human primates in a model of xenogeneic peripheral tolerance induction.

Xenotransplantation represents a promising solution to the ever increasing shortage of donor organs in allotransplantation. However, due to different and stronger modes of rejection, successful xenotransplantation will require different organ-protective regimes from those used in allogeneic transplantation today. Since one can not simply increase the dosage of the drugs used, immunomodulation or tolerance induction of the recipient would be the most desirable approach. Transfusion of donor leukocytes has been shown to downregulate recipient responses or even induce peripheral tolerance in small animal models. Since the infusion of donor cells represents a relatively simple approach, as one can purify and compose the inoculum exactly before infusion, we studied whether this approach can be successfully employed in a preclinical swine to non-human primate model of peripheral tolerance induction/immunomodulation. In our model, baboons underwent sequential column adsorption and complement blockade. The animals received only initial immunosuppression with cyclophosphamide. No further immunosuppression was given. Subsequently all animals received 1-3 x 10(10) porcine splenocytes i.v. Development and maintenance of chimerism was analyzed by sequential flow cytometric and PCR analyses. Other parameters studied included effects of the preparatory induction protocol. We could show that a low level of chimerism is maintained in these animals for up to 1.5 years, despite the fact that they received no additional immunosuppression after the initial one. At no time of the experiment did any animal display symptoms of poor health. Thus we demonstrate that the concept of donor leukocyte transfusion is transferable into preclinical xenotransplantation. We are currently conducting organ transplantation experiments into animals thus treated to directly analyze the immunomodulatory effect of the donor cells.

Spontaneous infection of lower primates with hepatitis C virus.

We present serological and virological evidence of spontaneous infection in Old World lower monkeys with human hepatitis C virus or, maybe, antigenically related but genetically different simian virus strain. These data is of both theoretical and practical importance and can serve as the basis for further development of experimental model of hepatitis C on lower monkeys.

Detection of Campylobacter jejuni in healthy monkeys and monkeys with enteric infections by PCR.

Campylobacter were detected by PCR in feces of monkeys of different species (clinically healthy, with diarrhea, and dead from acute enteric infections). High prevalence of these bacteria in monkeys was revealed. The incidence of C. jejuni DNA in monkeys with acute enteric infections was higher than in healthy animals (69.6 and 51.3%, respectively). The highest percentage (92.3) of positive results was observed in Macaca mulatta with enteric diseases and in macaque dead of these diseases. The presence of C. jejuni in monkeys with diarrhea and the absence of pathogenic enterobacteria (Shigella, Salmonella, Yersinia) in feces probably attest to etiological relationship of acute enteric infections with Campylobacter.