Indiscriminate slaughter of pregnant goats for meat in Enugu, Nigeria: Causes, prevalence, implications and ways-out.

BACKGROUND: The indiscriminate slaughter of pregnant goats (SPGs) undermines meat production and food security especially in developing countries. It also connotes animal cruelty, depletion of goat population and may enhance the spread of zoonotic pathogens inhabiting the female reproductive tract during carcass processing. Consequently, this study determined the causes and prevalence of slaughtering pregnant goats for meat in Enugu, Nigeria. The study also estimated the economic losses associated with SPGs, discussed the negative public health consequences and suggested the ways-out. METHODS: Structured, validated and pilot-tested questionnaire was used to ascertain the reasons for SPGs for meat among 78 willing and randomly selected respondents. The questionnaire survey was conducted in the form of interview. Pregnancy statuses of the goats slaughtered were ascertained by visual inspection and palpation of the eviscerated and longitudinally incised uteri and the horns for macroscopic evidence of pregnancy. Ages of the dams were estimated by dentition method. Estimation of the gestational age was performed by crown-rump length method. The study lasted for six months, comprised of three months (December to March) during the dry/hot season and another three months (May to August) during the wet/rainy season. Economic loss estimation was based on the current monetary values of a matured (30 kilogram) goat and one kilogram of chevon in Enugu, Nigeria; which was determined through market survey. Pearson's Chi-square test was used to determine whether there were significant (P<0.05) statistical associations between SPGs and age and season. RESULTS: Major reasons adduced for SPGs were: economic hardship (41%), ignorance of the goat's pregnancy status (21%), increased demand for chevon (13%) and feed scarcity during drought (11%). Of the 1,658 does examined during the six months study, 589 (35.5%) were pregnant. The majority (876/1658, 52.8%) of the female goats slaughtered were in their active reproductive age of ≤ 4 years, while 782 (47.2%) were aged > 4 years. Similarly, majority (1007/1658, 60.7%) of the does/nannies were slaughtered during the dry/hot season. A total of 907 foetuses at first (n = 332, 36.6%), second (n = 486, 53.6%) and third (n = 89, 9.8%) trimesters of gestation were recovered from the 589 PGs. Singleton, twin and triplet pregnancies were observed in 312 (53%), 236 (40%) and 41 (7%) PGs, respectively. About ₦34.44 million ($83,390) would have been earned if the foetuses were born alive and raised to maturity. Additionally, 19,136 kg of chevon, valued at ₦47,841, 000 ($115,838), which would have accrued from the wasted foetuses was also lost. CONCLUSION: Considering the economic, zoonotic and livestock production implications of this work, frantic efforts to reduce SPGs in Enugu, Nigeria is imperative. This could be achieved through advocacy, goat farmers' enlightenment, ante-mortem pregnancy diagnosis, provision of subsidized feed materials during the dry season and strict enforcement of the Nigerian Meat Edict law, which proscribes unapproved slaughter of gravid animals. These measures may improve food safety and security, improve goat reproduction and production, reduce protein malnutrition, limit dissemination of zoonotic pathogens during carcass processing and hence protect public health in Nigeria.

Experiences of cervical cancer screening in HIV-positive women in Zimbabwe.

BACKGROUND: The primary purpose of screening is to detect individuals in danger of cervical cancer so as to prevent further progression of the disease. Cervical cancer remains a global concern, as it ranks as the fourth most commonly diagnosed female malignancy worldwide. It is the commonest female cancer in Zimbabwe. Women living with human immunodeficiency virus (HIV) have a disproportionate risk of invasive cervical cancer, as they are 2-12 times more likely to develop pre-cancerous lesions. As a result of the increased risk, routine screenings are suggested. Few women are screened for cervical cancer in Zimbabwe. OBJECTIVES: This study aimed at describing the experiences of screening for cervical cancer and motivation behind screening. METHOD: The study employed a qualitative research approach. In-depth one to one interviews and focus group discussions were conducted using interview and focus group guides. The study was conducted at an opportunistic infections clinic in Mpilo Central Hospital. Data analysis was performed by using Giorgi's descriptive method of data analysis. RESULTS: The themes that emerged from data analysis were facilitators to screening for cervical cancer, community awareness of cervical cancer screening, free cervical cancer treatment and more screening centres and integrating cervical cancer screening with HIV care. CONCLUSION: In-depth understanding of the factors that enable women to take part in cervical cancer screening is essential so that these factors can be strengthened to improve uptake of cervical cancer screening services.

Molecular detection and characterisation of protozoan and rickettsial pathogens in ticks from cattle in the pastoral area of Karamoja, Uganda.

Ticks and tick-borne diseases (TBDs) significantly affect cattle production and the livelihoods of communities in pastoralist areas. Data on protozoan and rickettsial pathogens in ticks infesting cattle in Uganda is scanty; while it is an indicator of the likelihood of disease transmission and occurrence. A cross-sectional study was conducted amongst cattle in the Karamoja Region, northeastern Uganda, from July through September 2017, to determine the tick species diversity, identify protozoan and rickettsial pathogens in the ticks, and characterise pathogenic species by sequence and phylogenetic analyses. About 50 % of the ticks detected from each predilection site on each animal were collected from 100 purposively-selected cattle from 20 randomly-selected herds. Twelve tick species belonging to the genera Amblyomma, Rhipicephalus and Hyalomma were identified, the most abundant being Amblyomma lepidum (93.9 %), followed by Amblyomma variegatum (2.0 %) and Rhipicephalus evertsi evertsi (1.0 %). Tick species that have not been reported in recent studies amongst cattle in Uganda were found, namely Rhipicephalus pravus, Rhipicephalus praetextatus and Rhipicephalus turanicus. The ticks were grouped into 40 pools, by species and location, and the reverse line blot (RLB) hybridisation assay was used to detect pathogens from the ticks. The most frequently detected tick-borne parasites were Theileria mutans, Theileria velifera and Theileria parva, each observed in 25 % (10/40) of the tick pools. Tick-borne pathogens, namely Babesia rossi, Babesia microti and Theileria sp. (sable) that are not common to, or not known to infect, cattle were identified from ticks. The gene encoding Ehrlichia ruminantium pCS20 region, the Ehrlichia and Anaplasma 16S rRNA gene, and T. parva p67 sporozoite antigen gene were amplified, cloned and sequenced. Seven novel E. ruminantium pCS20 variants were identified, and these grouped into two separate clusters with sequences from other parts of Africa and Asia. The T. parva p67 sequences were of the allele type 1, and parasites possessing this allele type are commonly associated with East Coast fever in eastern Africa. Analysis of the Ehrlichia and Anaplasma 16S rRNA gene sequences showed that they were closely related to Rickettsia africae and to a new Ehrlichia species variant recently found in China. Our R. africae 16S rRNA sequences grouped with R. africae isolates from Nigeria, Egypt and Benin. The information on tick species diversity and pathogens in the various tick species provides an indicator of potential transmission amongst cattle populations, and to humans, and can be useful to estimate disease risk and in control strategies.

Redescription of Cichlidogyrus philander (Monogenea, Ancyrocephalidae) using scanning electron microscopy (SEM) and molecular analysis.

The sclerotized structures of monogeneans have traditionally been studied by light microscopy and different staining techniques. Recently, enzymatic digestion followed by scanning electron microscopy (SEM) has enabled the examination of structural details not visible with light microscopy. In order to obtain better, and more accurate, morphological information on sclerotized structures not affected by mounting medium or cover slip pressure, the sclerites of Cichlidogyrus philander Douëllou, 1993 (Monogenea, Ancyrocephalidae), collected from Pseudocrenilabrus philander (Weber, 1897), were redescribed using SEM. Parasites were collected from Padda Dam, Gauteng, South Africa and soft tissue was digested to release the sclerotized structures. The digested tissue also provided sufficient genetic material for molecular characterization of this species. Cichlidogyrus philander is characterised by a penis with a sharp, curved, lateral termination, an accessory piece with a hook-like extremity that may appear forked terminally, and lack of a visible vagina. The transverse bars have concave and convex surfaces with ribs on the concave surface. The dorsal bar bears fenestrations at the base of the auricles and the ventral and dorsal gripi are dissimilar. Furthermore, the large first pair of uncinuli shows lateral wings on the left side of the base. On top of this wing, a ball-like structure with a small fenestration is visible. Genetic characters derived from the 28S rDNA, the COI mitochondrial DNA and ITS1 rDNA regions distinguish C. philander from all other Cichlidogyrus sequenced species.

Evaluation of the microbial community, acidity and proximate composition of akamu, a fermented maize food.

BACKGROUND: Akamu is a lactic acid-fermented cereal-based food that constitutes a major infant complementary food in most West African countries. The identities of LAB populations from DGGE analysis and conventionally isolated LAB and yeasts from traditionally fermented akamu were confirmed by PCR sequencing analysis. The relationships between pH, acidity and lactic acid levels and proximate composition of the akamu samples were investigated. RESULTS: The LAB communities in the akamu samples comprised mainly Lactobacillus species, including Lb. fermentum, Lb. plantarum, Lb. delbrueckii ssp. bulgaricus and Lb. helveticus, as well as Lactococcus lactis ssp. cremoris. Identified yeasts were Candida tropicalis, Candida albicans, Clavispora lusitaniae and Saccharomyces paradoxus. Low pH (3.22-3.95) was accompanied by high lactic acid concentrations (43.10-84.29 mmol kg⁻¹). Protein (31.88-74.32 g kg⁻¹) and lipid (17.74-36.83 g kg⁻¹ contents were negatively correlated with carbohydrate content (897.48-926.20 g kg⁻¹, of which ≤1 g kg⁻¹ was sugars). Ash was either not detected or present only in trace amounts (≤4 g kg⁻¹). Energy levels ranged from 17.29 to 18.37 kJ g⁻¹. CONCLUSION: The akamu samples were predominantly starchy foods and had pH < 4.0 owing to the activities of fermentative LAB.

Mice transgenic for a human porcine endogenous retrovirus receptor are susceptible to productive viral infection.

Porcine endogenous retrovirus (PERV) is considered one of the major risks in xenotransplantation. No valid animal model has been established to evaluate the risks associated with PERV transmission to human patients by pig tissue xenotransplantation or to study the potential pathogenesis associated with PERV infection. In previous work we isolated two genes encoding functional human PERV receptors and proved that introduction of these into mouse fibroblasts allowed the normally nonpermissive mouse cells to become productively infected (T. A. Ericsson, Y. Takeuchi, C. Templin, G. Quinn, S. F. Farhadian, J. C. Wood, B. A. Oldmixon, K. M. Suling, J. K. Ishii, Y. Kitagawa, T. Miyazawa, D. R. Salomon, R. A. Weiss, and C. Patience, Proc. Natl. Acad. Sci. USA 100:6759-6764, 2003). In the present study we created mice transgenic for human PERV-A receptor 2 (HuPAR-2). After inoculation of transgenic animals with infectious PERV supernatants, viral DNA and RNA were detected at multiple time points, indicating productive replication. This establishes the role of HuPAR-2 in PERV infection in vivo; in addition, these transgenic mice represent a new model for determining the risk of PERV transmission and potential pathogenesis. These mice also create a unique opportunity to study the immune response to PERV infection and test potential therapeutic or preventative modalities.

The potential hazards of xenotransplantation: an overview.

Xenotransplantation, in particular the transplantation of pig cells, tissues and organs into human recipients, may alleviate the current shortage of suitable allografts available for human transplantation. This overview addresses the physiological, immunological and microbial factors involved in xenotransplantation. The issues reviewed include the merits of using pigs as xenograft source species, the compatibility of pig and human organ physiology, and the rejection mechanism and attempts to overcome this immunological challenge. The authors discuss advances in the prevention of pig organ rejection through the creation of genetically modified pigs, more suited to the human micro-environment. Finally, in regard to microbial hazards, the authors review possible viral infections originating from pigs.

Potential of aspirin to inhibit thrombotic microangiopathy in alpha1,3-galactosyltransferase gene-knockout pig hearts after transplantation in baboons.

Hearts from alpha1,3-Galactosyltransferase gene-knockout (GaIT-KO) pigs were transplanted heterotopically into 8 baboons that received an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen and heparin. Three baboons died or were euthanized with beating grafts on 16, 23, and 56 days, respectively, and the remaining 5 grafts functioned for 59-179 days. Hyperacute rejection did not occur, and classical features of acute humoral xenograft or acute cellular rejection were rare. However, thrombotic microangiopathy (TM) developed in all cases; its onset was delayed in 2 baboons that received aspirin. Function of a pig organ in a baboon for a period approaching 6 months has not been reported previously and lends encouragement that the barriers to xenotransplantation will be overcome, but TM requires investigation.

Bone marrow transplantation from alpha1,3-galactosyltransferase gene-knockout pigs in baboons.

BACKGROUND: Successful hematopoietic cell allotransplantation results in donor-specific tolerance, but this approach has been unsuccessful in the wild-type pig-to-baboon xenotransplantation model, as pig cells were lost from the circulation within 5 days. However, after cessation of immunosuppressive therapy on day 28, all baboons demonstrated non-specific unresponsiveness on mixed leukocyte reaction (MLR) for at least 30 days. We have now investigated the transplantation of bone marrow (BM) cells from miniature swine homozygous for alpha1,3-galactosyltransferase gene-knockout (GalT-KO). METHODS: Baboons (n = 3) were pre-treated with whole body and thymic irradiation, anti-thymocyte globulin, and splenectomy, and received immunosuppressive and supportive therapy for 28 days. BM was harvested from GalT-KO swine (n = 3). The baboons were monitored for the presence of pig cells by flow cytometry and colony-forming units (CFUs), and for cellular reactivity by MLR. RESULTS: A mean of 11 x 10(8) BM cells/kg was infused into each baboon. The mean absolute numbers and percentages of pig cells detected in the blood at 2 h and on days 1, 2 and 4, respectively, were 641/microl (9.5%), 132/microl (3.4%), 242/microl (3.9%), and 156/microl (2.9%). One baboon died (from accidental hemorrhage) on day 6, at which time chimerism was present in the blood (2.0%) and BM (6.4%); pig cell engraftment in the BM was confirmed by polymerase chain reaction (PCR) of CFUs. In the two other baboons, blood chimerism was lost after day 5 but returned at low levels (<1%) between days 9 to 16 and 7 to 17, respectively, indicating transient BM engraftment. Both surviving baboons showed non-specific unresponsiveness on MLR until they were euthanized on days 85 and 110, respectively. CONCLUSIONS: By using BM cells from GalT-KO pigs, chimerism was detected at levels comparable with previous studies when 30-fold more growth factor-mobilized peripheral blood progenitor cells had been transplanted. In addition, cellular hyporesponsiveness was prolonged. However, long-term engraftment and chimerism were not achieved.

Structural and functional characteristics of a dominant-negative isoform of porcine MHC class II transactivator.

The MHC class II transactivator, CIITA, is critical for MHC class II gene expression in all species studied to date. We isolated an interferon (IFN)-gamma-inducible isoform of porcine CIITA (pCIITA') encoding a protein of 566 amino acids (aa) with significant homology to human CIITA (hCIITA). Analysis indicated that pCIITA' lacks the entire GTP-binding domain that is important for nuclear translocation and activation of target genes by hCIITA. In pCIITA' this region is replaced by a 14-aa motif with homology to several signalling peptide sequences. Expression of pCIITA' in porcine (ST-IOWA) and human (HeLa) cell lines resulted in suppression of IFN-gamma-stimulated MHC class II gene expression, at the protein and mRNA levels. We also identified two IFN-gamma-inducible variants of hCIITA, hCIITAlo and hCIITA' from Hela cells, both exhibiting dominant-negative suppression of MHC class II gene expression. Interestingly, hCIITA' encodes a predicted protein of 546 aa with a strikingly similar organization to pCIITA' including the 14-aa GTP-binding domain-replacement motif in which 10 out of 14 amino acids are identical to the pig sequence. Expression of hCIITA' and hCIITAlo sequences in Hela cells suppressed IFN-gamma-induced MHC class II gene expression. hCIITAlo, a predicted 303-aa protein with deleted GTP-binding and carboxy-terminal domain, displayed a more subtle suppression of IFN-gamma-induced MHC class II expression. These in vitro data indicate that there may be a role in vivo for isoforms of CIITA that can suppress full-length CIITA-mediated MHC class II gene expression. Both humans and now, potentially, pigs are candidate donors for organ and tissue allografts and xenografts, respectively. Regulation of MHC class II gene expression by manipulation of CIITA isoform expression in humans and pigs may provide a useful strategy for attenuation of T-cell-mediated cellular rejection.

Methods for the exclusion of circoviruses and gammaherpesviruses from pigs.

The use of porcine tissues is being developed as a means to alleviate the shortage of allogeneic tissues and organs available for transplantation. To reduce the possibility of a microorganism of pigs being inadvertently transferred to the recipient of the xenograft, recommendations have been published on the microbiological specifications for organ source pigs. The porcine circoviruses (PCV1 and PCV2) and porcine lymphotropic herpesviruses (PLHV1 and PLHV2) are two infectious agents of pigs which are considered to be of significance for the microbiological safety of xenotransplantation. To ensure the exclusion of these microorganisms from animals destined for use under clinical conditions, reliable breeding methodologies are required. We investigated the efficiency of established derivation procedures for the removal of PCV and PLHV. In comparison with conventionally reared pigs, caesarian and barrier derived animals showed a markedly reduced prevalence of PCVs and PLHVs. Our results indicate that the derivation of animals free of both of these microorganisms is achievable and will enhance the microbiological safety of xenotransplantation.

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.

Posttransplantation lymphoproliferative disease in miniature swine after allogeneic hematopoietic cell transplantation: similarity to human PTLD and association with a porcine gammaherpesvirus.

Posttransplantation lymphoproliferative disease (PTLD) is a major complication of current clinical transplantation regimens. The lack of a reproducible large-animal model of PTLD has limited progress in understanding the pathogenesis of and in developing therapy for this clinically important disease. This study found a high incidence of PTLD in miniature swine undergoing allogeneic hematopoietic stem cell transplantation and characterized this disease in swine. Two days before allogeneic peripheral blood stem cell transplantation, miniature swine were conditioned with thymic irradiation and in vivo T-cell depletion. Animals received cyclosporine daily beginning 1 day before transplantation and continuing for 30 to 60 days. Flow cytometry and histologic examination were performed to determine the cell type involved in lymphoproliferation. Polymerase chain reaction was developed to detect and determine the level of porcine gammaherpesvirus in involved lymph node tissue. PTLD in swine is morphologically and histologically similar to that observed in human allograft recipients. Nine of 21 animals developed a B-cell lymphoproliferation involving peripheral blood (9 of 9), tonsils, and lymph nodes (7 of 9) from 21 to 48 days after transplantation. Six of 9 animals died of PTLD and 3 of 9 recovered after reduction of immunosuppression. A novel porcine gammaherpesvirus was identified in involved tissues. Miniature swine provide a genetically defined large-animal model of PTLD with many characteristics similar to human PTLD. The availability of this reproducible large-animal model of PTLD may facilitate the development and testing of diagnostic and therapeutic approaches for prevention or treatment of PTLD in the clinical setting.

Multiple groups of novel retroviral genomes in pigs and related species.

In view of the concern over potential infection hazards in the use of porcine tissues and organs for xenotransplantation to humans, we investigated the diversity of porcine endogenous retrovirus (PERV) genomes in the DNA of domestic pigs and related species. In addition to the three known envelope subgroups of infectious gamma retroviruses (PERV-A, -B, and -C), classed together here as PERV group gamma 1, four novel groups of gamma retrovirus (gamma 2 to gamma 5) and four novel groups of beta retrovirus (beta 1 to beta 4) genomes were detected in pig DNA using generic and specific PCR primers. PCR quantification indicated that the retroviral genome copy number in the Landrace x Duroc F(1) hybrid pig ranged from 2 (beta 2 and gamma 5) to approximately 50 (gamma 1). The gamma 1, gamma 2, and beta 4 genomes were transcribed into RNA in adult kidney tissue. Apart from gamma 1, the retroviral genomes are not known to be infectious, and sequencing of a small number of amplified genome fragments revealed stop codons in putative open reading frames in several cases. Analysis of DNA from wild boar and other species of Old World pigs (Suidae) and New World peccaries (Tayassuidae) showed that one retrovirus group, beta 2, was common to all species tested, while the others were present among all Old World species but absent from New World species. The PERV-C subgroup of gamma1 genomes segregated among domestic pigs and were absent from two African species (red river hog and warthog). Thus domestic swine and their phylogenetic relatives harbor multiple groups of hitherto undescribed PERV genomes.

Identification of novel porcine endogenous betaretrovirus sequences in miniature swine.

PCR amplification of genomic DNA from miniature swine peripheral blood lymphocytes, using primers corresponding to highly conserved regions of the polymerase (pol) gene, allowed the identification of two novel porcine endogenous retrovirus (PERV) sequences, PMSN-1 and PMSN-4. Phylogenetic analyses of the nucleotide sequences of PMSN-1 and PMSN-4 revealed them to be most closely related to betaretroviruses. The identification of PERVs belonging to the Betaretrovirus genus shows that endogenous retroviruses of this family are more broadly represented in mammalian species than previously appreciated. Both sequences contained inactivating mutations, implying that these particular loci are defective. However, Southern blot analysis showed additional copies of closely related proviruses in the miniature swine genome. Analyses of fetal and adult miniature swine tissues revealed a broad mRNA expression pattern of both PMSN-1 and PMSN-4. The most abundant expression was detected in whole bone marrow c-kit(+) (CD117(+)) progenitor bone marrow cells, fetal liver, salivary gland, and thymus. It appears unlikely that functional loci encoding these novel PERV sequences exist, but this remains to be established. The betaretrovirus sequences described in this report will allow such investigations to be actively pursued.

Susceptibility of the porcine endogenous retrovirus to reverse transcriptase and protease inhibitors.

Porcine xenografts may offer a solution to the shortage of human donor allografts. However, all pigs contain the porcine endogenous retrovirus (PERV), raising concerns regarding the transmission of PERV and the possible development of disease in xenotransplant recipients. We evaluated 11 antiretroviral drugs licensed for human immunodeficiency virus type 1 (HIV-1) therapy for their activities against PERV to assess their potential for clinical use. Fifty and 90% inhibitory concentrations (IC(50)s and IC(90)s, respectively) of five nucleoside reverse transcriptase inhibitors (RTIs) were determined enzymatically for PERV and for wild-type (WT) and RTI-resistant HIV-1 reference isolates. In a comparison of IC(50)s, the susceptibilities of PERV RT to lamivudine, stavudine, didanosine, zalcitabine, and zidovudine were reduced >20-fold, 26-fold, 6-fold, 4-fold, and 3-fold, respectively, compared to those of WT HIV-1. PERV was also resistant to nevirapine. Tissue culture-based, single-round infection assays using replication-competent virus confirmed the relative sensitivity of PERV to zidovudine and its resistance to all other RTIs. A Gag polyprotein-processing inhibition assay was developed and used to assess the activities of protease inhibitors against PERV. No inhibition of PERV protease was seen with saquinavir, ritonavir, indinavir, nelfinavir, or amprenavir at concentrations >200-fold the IC(50)s for WT HIV-1. Thus, following screening of many antiretroviral agents, our findings support only the potential clinical use of zidovudine.

MuLV packaging systems as models for estimating/measuring retrovirus recombination frequency.

Interaction of retrovirus vectors and endogenous retroviruses present in packaging cell lines and target cells may result in the formation of recombinant viruses. Using sensitive RT-PCR assays, we have investigated human and murine gene therapy packaging cell lines for the incorporation of endogenous retrovirus transcripts into murine leukaemia virus (MLV) vector particles and whether vector genomes are incorporated into human endogenous retrovirus (HERV) particles. VL30 endogenous retrovirus sequences were packaged in particles produced by the murine AM12 packaging system. For every seven MLV-derived -galactosidase beta-Gal vector genomes present in the particles, one copy of VL30 was also packaged. Although human FLY packaging cells expressed HERV transcripts (HERV-K, HuRT, type C, and RTVL-H), none was detectable in the MLV vector particles released from the cells. Non-specific packaging of the MLV gag-pol expression vector transcripts was detected in the FLY virions at a low level (one in 17,000 sequences). In other experiments, gag proteins produced by HERV-K particles present in human teratocarcinoma cells did not appear to package MLV-based vectors that expressed Gal transcripts. These findings indicate that retrovirus vectors interact with human packaging cells to produce retrovirus particles that are far less contaminated by endogenous viral sequences or other types of extraneous particles than murine packaging cells.

Productive infection of primary human endothelial cells by pig endogenous retrovirus (PERV).

The potential risk of viral transmission in the setting of xenotransplantation has gained major attention. Different porcine cell types have been shown to release retroviral particles, which are infectious for human cell lines in vitro. However, there are only a few data on whether PERV (pig endogenous retrovirus) is able to infect primary human cells. In this study we have analyzed endothelial cells, vascular fibroblasts, mesangial cells, mononuclear cells, hematopoetic stem cells and bone marrow stromal cells for PERV transmission. We now provide evidence for primary human endothelial cells, vascular fibroblasts, and mesangial cells to be susceptible to PERV transmission. PERV infection was productive in endothelial cells and mesangial cells. Our data confirm and extend former reports concerning the PERV infection of human cells. The PERV infection of different primary human cells represents further significant evidence for a viral risk during xenotransplantation. In this context, special attention should be directed towards productive infection of human endothelial cells: in the setting of xenotransplantation this cell type will have close contact with porcine cells and PERV particles.