Heterosexual Transmission of a Murine AIDS Virus

Heterosexual transmission of a murine leukemia virus mixture named LP-BM5 MuLV, which is known as the murine AIDS virus, was investigated. Our results indicated that the heterosexual transmission of LP-BM5 MuLV occurs in both directions with high frequency and that the frequencies of virus transmission in the cervix and penis are higher than those in other genital organs. The results suggested that infection by LP-BM5 MuLV via heterosexual transmission may initially take place at particular retrovirus-sensitive sites (cells) in the genital organs.Human immunodeficiency virus (HIV) infection is now pandemic. In many countries, HIV has been spread mainly by heterosexual transmission (3, 5). For the prevention of HIV infection, as well as for the development of vaccines against HIV, it is of a great importance to understand the mechanisms of the heterosexual transmission of retroviruses. Since it is difficult to investigate the mechanisms of heterosexual transmission of HIV in humans experimentally, an animal model with a retrovirus which induces an acquired immunodeficiency syndrome like human AIDS would be useful. A murine leukemia virus mixture called LP-BM5 MuLV induces a severe acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice (10). The mixture includes a replication-competent ecotropic virus, mink cell focus-inducing virus, and a replication-defective virus which has been considered to be involved in the pathogenesis of MAIDS (4). With many similarities to human AIDS patients, mice infected with the LP-BM5 MuLV mixture develop splenomegaly, systemic lymphadenopathy, and severe immunodeficiency (4, 11). We previously reported that maternal transmission of LP-BM5 MuLV occurs via mother’s milk with high frequency (12). In the present study, we demonstrate that the heterosexual transmission of LP-BM5 MuLV also occurs with high frequency via genital organs.C57BL/10 (B10) mice were purchased from Japan SLC Inc., Shizuoka, Japan. All mice were specific-pathogen free and were housed in an air-conditioned room. They were given autoclaved water and sterilized pelleted feed. An SC-1 clone chronically infected with LP-BM5 MuLV, the G6 cell line, was kindly supplied by H. C. Morse III, National Institutes of Health, Bethesda, Md. Virus was prepared from the supernatant of G6 cells as previously described (12). The virus preparation was stored at −70°C until use. B10 mice were inoculated by the intraperitoneal route with 0.3 ml of the LP-BM5 MuLV preparation. To increase the frequency of sexual contacts and to avoid pregnancy in the female mice, all male mice were sterilized by vasectomy under anesthesia with pentobarbital (Nembutal). The vasectomized male mice were mated with female mice at least 4 weeks postoperation, since sperm are usually kept alive for 2 to 3 weeks in spermiducts. Excised genital organs were crushed with plastic sticks in 1 ml of lysis buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% sodium dodecyl sulfate, and proteinase K (0.5 mg/ml). Spleen cells were lysed after hemolysis with 0.83% NH₄Cl. Lysed samples were incubated at 50°C for 3 h. DNA was extracted three times with phenol-chloroform, precipitated with cold ethanol, treated with RNase and proteinase K, and dissolved in 0.1 ml of H₂O. LP-BM5 MuLV defective virus genome was detected by Southern blot hybridization combined with PCR as described previously (12). In brief, template DNAs (1 μg per tube) were added to a cocktail adjusted to final concentrations of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, 200 μM deoxynucleoside triphosphate, 100 pmol of each primer (5′-CCTCTTCCTTTATCGACACT-3′ [sense] and 5′-ATTAGGGGGGGAATAGCTCG-3′ [antisense]), and 2 U of Taq DNA polymerase (Boehringer Mannheim) in a total volume of 100 μl and were subjected to 32 cycles of amplification. In each cycle of PCR, the mixture was denatured at 95°C for 1 min (5 min for the first cycle), annealed at 55°C for 3 min, and extended at 72°C for 1 min. The PCR-amplified products were subjected to gel electrophoresis (1.5% agarose) and transferred to a Hybond N+ membrane (Amersham) by the alkaline blotting method. Hybridization was achieved with a 5′ ³²P-labeled probe (5′-TGTCAAAGGGACCAGTTAAG-3′) at 45°C overnight in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.5% sodium dodecyl sulfate–100 μg of salmon sperm DNA per ml. Hybridized membranes were washed twice in 2× SSC at 37°C for 10 min and then in 0.5× SSC at 45°C for 30 min. DNA derived from uterine cervices of uninfected mice was used as a negative control. The limit of sensitivity was approximately 10 copies per tube, as assessed by Southern blot analysis with plasmid DNAs (1/10 of the PCR product).Concanavalin A (ConA) was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Responder spleen cells (2 × 10⁵) were cultured with ConA (5 μg/ml) in 96-well flat-bottomed microculture plates in 0.2 ml of culture medium at 37°C in 7.5% CO₂. The culture medium consisted of RPMI 1640 supplemented with 10% fetal calf serum, penicillin (5,000 IU/100 ml), streptomycin (5,000 μg/100 ml), nonessential amino acids, sodium pyruvate (11.0 mg/100 ml), 2-mercaptoethanol (5 × 10⁻⁵ M), and l-glutamine (29.2 mg/100 ml). On day 2, cultures were pulsed with 1 μCi of [³H]thymidine and incubated for an additional 12 to 18 h. Incorporation of [³H]thymidine into responder spleen cells was quantitated by liquid scintillation counting. Determinations were performed in triplicate; standard errors of the means were generally <5% and therefore have not been indicated.As illustrated in Fig. ​Fig.1,1, in order to investigate the heterosexual transmission of LP-BM5 MuLV from male to female mice, normal male mice were inoculated with LP-BM5 MuLV and vasectomized 1 week later. At 5 weeks after virus inoculation, they were mated with uninfected female mice. After 8 weeks of breeding, female mice were sacrificed and their vaginae, cervices uteri, corpora uteri, inguinal lymph nodes, and spleens were removed and stored at −70°C until use. In the opposite direction, to investigate virus transmission from female to male, normal female mice were inoculated with LP-BM5 MuLV and then mated with uninfected, vasectomized male mice as described above. After 8 weeks of breeding, male mice were sacrificed and their penes, prepuces, inguinal lymph nodes and spleens were removed and stored at −70°C until use. Figure ​Figure22 shows the detection by PCR of the LP-BM5 defective virus genome in genital organs and spleens that were taken from mice mated with their virus-infected counterparts. It was demonstrated that although the defective virus genome was detected in both spleens and genital organs in some male mice (2 of 17 [see Table ​Table1]),1]), as shown in Fig. ​Fig.2,2, lanes 3 and 4, the defective virus genome was detected only in the genital organs, not the spleens (Fig. ​(Fig.2,2, lanes 5 and 6), from most of the male mice. In contrast, all of the female mice were positive for defective virus genome only in the genital organs (Fig. ​(Fig.2,2, lanes 1 and 2). None of the mice examined were positive for the virus genome only in the spleens (this issue is discussed below). It should be noted here that the efficacy of PCR amplification, which was measured by experiments using the mixture of genomic DNA and plasmid DNA containing the defective virus, did not differ among the genital organs and spleens. By using the above strategy, the heterosexual transmission of LP-BM5 MuLV was investigated according to the protocol shown in Fig. ​Fig.1.1. Open in a separate windowFIG. 1Experimental design for examination of heterosexual transmission of the MAIDS virus in B10 mice. i.p., intraperitoneal.Open in a separate windowFIG. 2Detection of the LP-BM5 MuLV defective virus genome by PCR in genital organs and spleens. The template DNAs (1 μg) derived from female or male mice which were bred with LP-BM5 MuLV-infected mice were amplified by PCR. Samples were prepared from either female (lanes 1 and 2) or male (lanes 3 to 6) mice. Lanes 1, 3, and 5, spleen; lane 2, uterine cervix; lanes 4 and 6, penis (from two representative male mice). The PCR products (5 μl) were applied to a 1.5% agarose gel and analyzed by Southern blotting with a probe for the defective virus (12).

TABLE 1

Heterosexual transmission of LP-BM5 MuLV

Illumination of Murine Gammaherpesvirus-68 Cycle Reveals a Sexual Transmission Route from Females to Males in Laboratory Mice

Transmission is a matter of life or death for pathogen lineages and can therefore be considered as the main motor of their evolution. Gammaherpesviruses are archetypal pathogenic persistent viruses which have evolved to be transmitted in presence of specific immune response. Identifying their mode of transmission and their mechanisms of immune evasion is therefore essential to develop prophylactic and therapeutic strategies against these infections. As the known human gammaherpesviruses, Epstein-Barr virus and Kaposi''s Sarcoma-associated Herpesvirus are host-specific and lack a convenient in vivo infection model; related animal gammaherpesviruses, such as murine gammaherpesvirus-68 (MHV-68), are commonly used as general models of gammaherpesvirus infections in vivo. To date, it has however never been possible to monitor viral excretion or virus transmission of MHV-68 in laboratory mice population. In this study, we have used MHV-68 associated with global luciferase imaging to investigate potential excretion sites of this virus in laboratory mice. This allowed us to identify a genital excretion site of MHV-68 following intranasal infection and latency establishment in female mice. This excretion occurred at the external border of the vagina and was dependent on the presence of estrogens. However, MHV-68 vaginal excretion was not associated with vertical transmission to the litter or with horizontal transmission to female mice. In contrast, we observed efficient virus transmission to naïve males after sexual contact. In vivo imaging allowed us to show that MHV-68 firstly replicated in penis epithelium and corpus cavernosum before spreading to draining lymph nodes and spleen. All together, those results revealed the first experimental transmission model for MHV-68 in laboratory mice. In the future, this model could help us to better understand the biology of gammaherpesviruses and could also allow the development of strategies that could prevent the spread of these viruses in natural populations.     

Rescue and Transmission of a Replication-Defective Variant of Moloney Murine Leukemia Virus

We have described a clone of mouse cells, termed "8A," which appears to be infected with a replication-defective variant of Moloney murine leukemia virus (MuLV) (Rein et al., J. Virol. 25:146-156, 1978). Clone 8A cells release virus particles which do not form plaques in the standard XC test. However, approximately 10(2) particles per ml of clone 8A supernatant do form plaques in a modified XC test (the "complementation plaque assay"), in which the assay cells are coinfected with the XC-negative, nondefective amphotropic MuLV as well as the test virus. Superinfection of clone 8A cells themselves with amphotropic MuLV results in the production of approximately 10(5), rather than approximately 10(2), particles per ml which register in the complementation plaque assay. This increase is due to the rescue of replication-defective ecotropic MuLV from clone 8A cells by amphotropic MuLV since (i) this ecotropic MuLV can only form XC plaques in cells which are coinfected with amphotropic MuLV; and (ii) it is possible to transmit this defective variant, rescued from superinfected clone 8A cells, to a fresh clone of normal mouse cells. The time course of production of the rescued MuLV particles by superinfected clone 8A cells is virtually identical to that of rescue from these cells of murine sarcoma virus. Amphotropic MuLV superinfection of "NP-N" cells, which contain a "non-plaque-forming" variant of N-tropic MuLV (Hopkins and Jolicoeur, J. Virol. 16:991-999, 1975), also increases the titer of particles registering in the complementation plaque assay; thus, NP-N cells, like clone 8A cells, contain a rescuable defective variant of ecotropic MuLV.     

Vaccinia virus Transmission through Experimentally Contaminated Milk Using a Murine Model

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects dairy cattle and humans. Previous studies have detected the presence of viable virus particles in bovine milk samples naturally and experimentally contaminated with VACV. However, it is not known whether milk contaminated with VACV could be a route of viral transmission. However, anti-Orthopoxvirus antibodies were detected in humans from BV endemic areas, whom had no contact with affected cows, which suggest that other VACV transmission routes are possible, such as consumption of contaminated milk and dairy products. Therefore, it is important to study the possibility of VACV transmission by contaminated milk. This study aimed to examine VACV transmission, pathogenesis and shedding in mice orally inoculated with experimentally contaminated milk. Thirty mice were orally inoculated with milk containing 10⁷ PFU/ml of VACV, and ten mice were orally inoculated with uncontaminated milk. Clinical examinations were performed for 30 consecutive days, and fecal samples and oral swabs (OSs) were collected every other day. Mice were euthanized on predetermined days, and tissue and blood samples were collected. Nested-PCR, plaque reduction neutralization test (PRNT), viral isolation, histopathology, and immunohistochemistry (IHC) methods were performed on the collected samples. No clinical changes were observed in the animals. Viral DNA was detected in feces, blood, OSs and tissues, at least in one of the times tested. The lungs displayed moderate to severe interstitial lymphohistiocytic infiltrates, and only the heart, tonsils, tongue, and stomach did not show immunostaining at the IHC analysis. Neutralizing antibodies were detected at the 20^th and 30^th days post infection in 50% of infected mice. The results revealed that VACV contaminated milk could be a route of viral transmission in mice experimentally infected, showing systemic distribution and shedding through feces and oral mucosa, albeit without exhibiting any clinical signs.     

Turnover of T Cells in Murine Gammaherpesvirus 68-Infected Mice

Respiratory challenge of C57BL/6 mice with murine gammaherpesvirus 68 induces proliferation of T lymphocytes early after infection, as evidenced by incorporation of the DNA precursor bromodeoxyuridine. Using pulse-chase analysis, splenic and peripheral blood activated T lymphocytes were found to continue dividing for at least a month after the initial virus challenge. The results are in accord with the idea that T cells are stimulated for a substantial time after the acute, lytic phase of virus infection is resolved.     

广东省实验小鼠自然感染鼠诺如病毒的调查

目的了解广东省实验小鼠自然感染小鼠诺如病毒（murine norovirus,MNV）的情况。方法随机抽取广东省7个繁育设施的小鼠206只,应用逆转录-聚合酶链反应（RT-PCR）方法检测其感染MNV的情况。结果共检测小鼠盲肠内容物206份,阳性样本为77份,阳性率为37.38%。3个设施的小鼠感染MNV,各品系小鼠易感性差异无显著。结论证实广东省小鼠存在MNV感染,部分设施小鼠MNV感染率很高,需加强动物的饲养管理。RT-PCR方法可以应用于MNV感染检测。     

Inflammatory Lesions of Cochlea in Murine Cytomegalovirus-Infected Mice with Hearing Loss

We sought to determine the distribution of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and lymphocytes in the cochlea of mice infected with murine cytomegalovirus (MCMV). For this purpose, 16 newborn mice were divided equally into model and control (uninfected) groups. In model group, 10 μl of MCMV was injected into the brain of each mouse whereas in control group, 10 μl of physiological saline was injected. Fourteen days after the injection of MCMV, the auditory brainstem response audiometry was performed. Later, the mice were killed and the acoustic capsule samples were collected for polymerase chain reaction analysis, histopathological and immunohistochemical studies. Compared with control mice, the incubation period was longer (F = 13.797; P = 0.003) and the amplitude was lower (F = 5.095; P = 0.043) in model group mice. It indicated that the intracerebral injection of MCMV caused hearing loss in mice. Histopathological examination of cochleas revealed increased levels of lymphocytic infiltration in the membrana vestibularis. Higher levels of TNF-α and IL-6 in scala tympani were detected by immunohistochemical staining. Taken together, the hearing loss in mice could be related with the inflammatory changes occurring in cochlea after inception of MCMV infection.     

Endurance Training Inhibits Insulin Clearance and IDE Expression in Swiss Mice

Introduction

Endurance training improves peripheral insulin sensitivity in the liver and the skeletal muscle, but the mechanism for this effect is poorly understood. Recently, it was proposed that insulin clearance plays a major role in both glucose homeostasis and insulin sensitivity. Therefore, our goal was to determine the mechanism by which endurance training improves insulin sensitivity and how it regulates insulin clearance in mice.

Methods

Mice were treadmill-trained for 4 weeks at 70–80% of maximal oxygen consumption (VO₂ max) for 60 min, 5 days a week. The glucose tolerance and the insulin resistance were determined using an IPGTT and an IPITT, respectively, and the insulin decay rate was calculated from the insulin clearance. Protein expression and phosphorylation in the liver and the skeletal muscle were ascertained by Western blot.

Results

Trained mice exhibited an increased VO₂ max, time to exhaustion, glucose tolerance and insulin sensitivity. They had smaller fat pads and lower plasma concentrations of insulin and glucose. Endurance training inhibited insulin clearance and reduced expression of IDE in the liver, while also inhibiting insulin secretion by pancreatic islets. There was increased phosphorylation of both the canonical (IR-AKT) and the non-canonical (CaMKII-AMPK-ACC) insulin pathways in the liver of trained mice, whereas only the CaMKII-AMPK pathway was increased in the skeletal muscle.

Conclusion

Endurance training improved glucose homeostasis not only by increasing peripheral insulin sensitivity but also by decreasing insulin clearance and reducing IDE expression in the liver.     

Local IL-17 Production Exerts a Protective Role in Murine Experimental Glomerulonephritis

IL-17 is a pro-inflammatory cytokine implicated in the pathogenesis of glomerulonephritis and IL-17 deficient mice are protected from nephrotoxic nephritis. However, a regulatory role for IL-17 has recently emerged. We describe a novel protective function for IL-17 in the kidney. Bone marrow chimeras were created using wild-type and IL-17 deficient mice and nephrotoxic nephritis was induced. IL-17 deficient hosts transplanted with wild-type bone marrow had worse disease by all indices compared to wild-type to wild-type bone marrow transplants (serum urea p<0.05; glomerular thrombosis p<0.05; tubular damage p<0.01), suggesting that in wild-type mice, IL-17 production by renal cells resistant to radiation is protective. IL-17 deficient mice transplanted with wild-type bone marrow also had a comparatively altered renal phenotype, with significant differences in renal cytokines (IL-10 p<0.01; IL-1β p<0.001; IL-23 p<0.01), and macrophage phenotype (expression of mannose receptor p<0.05; inducible nitric oxide synthase p<0.001). Finally we show that renal mast cells are resistant to radiation and produce IL-17, suggesting they are potential local mediators of disease protection. This is a novel role for intrinsic cells in the kidney that are radio-resistant and produce IL-17 to mediate protection in nephrotoxic nephritis. This has clinical significance as IL-17 blockade is being trialled as a therapeutic strategy in some autoimmune diseases.     

Development of the Polar Filament-Polaroplast Complex in a Microsporidian Parasite*

SYNOPSIS. The development of the polar filament in a microsporidian parasite was studied in the electron microscope. The polar filament is a peculiar and complex organelle with intricate anatomical relationships to other structures in the mature spore. The characteristic ultrastructure of the formative and mature stages of the polar filament made it possible to trace its development and study the interactions among various organelles during its formation. In sporoblasts the polar filament develops sequentially from 3 different regions. The base of the filament appears first and is derived from a dense body. The anterior part of the filament is formed from electron dense material located in the perinuclear cisterna and in agranular endoplasmic reticulum. The base and the anterior part of the filament move toward each other and fuse. Subsequently, the posterior part of the filament develops from the posterior part of the Golgi complex. The polar sac and the polaroplast surrounding the anterior segment of the filament are formed from the anterior region of the Golgi complex.     

Latest Progress in Microsporidian Genome Research

Microsporidia are obligate intracellular pathogens of medical and ecological importance whose genomes have been studied extensively over the last decade. Such studies have focused on the remarkably reduced gene content that characterizes all known species, and some have unraveled the mechanisms that are involved in their extreme genome compaction. In the last year, a large number of new genome sequences from several divergent members of the group have been finally released and analyzed, and these have revealed the presence of many features that were previously unsuspected to exist within the group. This study aims to shortly review the most recent progress in the field of microsporidian genomics, highlighting the importance of the most recently released genome data for our understanding of the biology and evolution of this important group of parasites.     

Structure and Distribution of Endogenous Nonecotropic Murine Leukemia Viruses in Wild Mice

Virtually all of our present understanding of endogenous murine leukemia viruses (MLVs) is based on studies with inbred mice. To develop a better understanding of the interaction between endogenous retroviruses and their hosts, we have carried out a systematic investigation of endogenous nonecotropic MLVs in wild mice. Species studied included four major subspecies of Mus musculus (M. m. castaneus, M. m. musculus, M. m. molossinus, and M. m. domesticus) as well as four common inbred laboratory strains (AKR/J, HRS/J, C3H/HeJ, and C57BL/6J). We determined the detailed distribution of nonecotropic proviruses in the mice by using both env- and long terminal repeat (LTR)-derived oligonucleotide probes specific for the three different groups of endogenous MLVs. The analysis indicated that proviruses that react with all of the specific probes are present in most wild mouse DNAs tested, in numbers varying from 1 or 2 to more than 50. Although in common inbred laboratory strains the linkage of group-specific sequences in env and the LTR of the proviruses is strict, proviruses which combine env and the LTR sequences from different groups were commonly observed in the wild-mouse subspecies. The “recombinant” nonecotropic proviruses in the mouse genomes were amplified by PCR, and their genetic and recombinant natures were determined. These proviruses showed extended genetic variation and provide a valuable probe for study of the evolutionary relationship between MLVs and the murine hosts.     

Identification of a Microsporidian Polar Tube Protein Reactive Monoclonal Antibody

ABSTRACT. The microsporidia are characterized by spores containing a single polar tube that coils around the sporoplasm. When triggered by appropriate stimuli, the polar tube rapidly discharges out of the spore forming a hollow tube. The sporoplasm passes out of the spore through this tube serving as a unique vehicle of infection. Due to the unusual functional and solubility properties of the polar tube, the proteins comprising it are likely to be members of a protein family with a highly conserved amino acid composition among the various microsporidia. Polar tube proteins were separated from the majority of other proteins in glass bead disrupted spores of Glugea americanus using sequential 1% sodium dodecyl sulfate (SDS) and 9M urea extractions. The resultant spore pellet demonstrated broken, empty spore coats and numerous polar tubes in straight and twisted formations by negative stain transmission electron microscopy. After subsequent incubation of the pellet with 2% dithiothreitol (DTT), empty spore coats were still observed but the polar tubes were no longer present in the pellet. The DTT supernatant demonstrated four major protein bands by SDS-PAGE: 23, 27, 34 and 43 kDa. Monoclonal antibodies were produced to these proteins using Hunter's Titermax adjuvant. Mab 3C8.23.1 which cross-reacted with a 43-kDa antigen by immunoblot analyis, demonstrated strong reactivity with the polar tube of G. americanus spores by immunogold electron microscopy. This antibody will be useful in further characterization of polar tube proteins and may lead to novel diagnostic and therapeutic reagents.     

Expanded Regulatory T Cells in Chronically Friend Retrovirus-Infected Mice Suppress Immunity to a Murine Cytomegalovirus Superinfection

It is still unclear whether expanded and activated regulatory T cells (Tregs) in chronic viral infections can influence primary immune responses against superinfections with unrelated viruses. Expanded Tregs found in the spleens of chronically Friend virus (FV)-infected mice decreased murine cytomegalovirus (mCMV)-specific CD8⁺ T cell responses during acute mCMV superinfection. This suppression of mCMV-specific T cell immunity was found only in organs with FV-induced Treg expansion. Surprisingly, acute mCMV infection itself did not expand or activate Tregs.