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Acute intestinal mucositis is a common off-target effect of chemotherapy, leading to co-morbidities such as vomiting, diarrhea, sepsis, and death. We previously demonstrated that the presence of enteric bacteria modulates the extent of jejunal epithelial damage induced by doxorubicin (DXR) in mice. Despite conventional thinking of the crypt as a sterile environment, recent evidence suggests that bacterial signaling influences aISC function. In this study, we labeled aISCs using transgenic Lgr5 -driven fluorescence or with immunostaining for OLFM4. We examined the effect of DXR in both germ free (GF) mice and mice depleted of microbiota using an established antimicrobial treatment protocol (AMBx). We found differences in DXR-induced loss of aISCs between GF mice and mice treated with AMBx. aISCs were decreased after DXR in GF mice, whereas AMBx mice retained aISC expression after DXR. Neither group of mice exhibited an inflammatory response to DXR, suggesting the difference in aISC retention was not due to differences in local tissue inflammation. Therefore, we suspected that there was a protective microbial signal present in the AMBx mice that was not present in the GF mice. 16S rRNA sequencing of jejunal luminal contents demonstrated that AMBx altered the fecal and jejunal microbiota. In the jejunal contents, AMBx mice had increased abundance of Ureaplasma and Burkholderia . These results suggest pro-survival signaling from microbiota in AMBx-treated mice to the aISCs, and that this signaling maintains aISCs in the face of chemotherapeutic injury. Manipulation of the enteric microbiota presents a therapeutic target for reducing the severity of chemotherapy-associated mucositis.

Congenital tumours and tumour-like lesions represent a group of rare disorders in both veterinary and human medicine that arise from tissue remnants and are detected during pregnancy or within the first 2-3 months of life. Different forms of congenital tumours and congenital tumour-like lesions have been reported in calves and their development is poorly understood. They often pose a diagnostic challenge and the referring nomenclature occasionally may be equivocal. Previous reports regarding tumour-like lesions, soft tissue tumours, vascular tumours, round cell tumours and neoplasms of the nervous, peritoneum and urogenital systems are summarized in this review, and the role of genetic factors in the development of these conditions is discussed., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Paneth cells are professional secretory cells that classically play a role in the innate immune system by secreting antimicrobial factors into the lumen to control enteric bacteria. In this role, Paneth cells are able to sense cues from luminal bacteria and respond by changing production of these factors to protect the epithelial barrier. Paneth cells rely on autophagy to regulate their secretory capability and capacity. Disruption of this pathway through mutation of genes, such as Atg16L1, results in decreased Paneth cell function, dysregulated enteric microbiota, decreased barrier integrity, and increased risk of diseases such as Crohn's disease in humans. Upon differentiation Paneth cells migrate downward and intercalate among active intestinal stem cells at the base of small intestinal crypts. This localization puts them in a unique position to interact with active intestinal stem cells, and recent work shows that Paneth cells play a critical role in influencing the intestinal stem cell niche. This review discusses the numerous ways Paneth cells can influence intestinal stem cells and their niche. We also highlight the ways in which Paneth cells can alter cells and other organ systems., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: aISCs (aISCs) are sensitive to acute insults including chemotherapy and irradiation. Regeneration after aISC depletion has primarily been explored in irradiation (IR). However, the cellular origin of epithelial regeneration after doxorubicin (DXR), a common chemotherapeutic, is poorly understood., Methods: We monitored DXR's effect on aISCs by enumerating Lgr5-eGFP + and Olfm4 + crypts, cleaved caspase-3 (CASP3 + ) immunofluorescence, and time-lapse organoid imaging. Lineage tracing from previously identified regenerative cell populations (Bmi1 + , Hopx + , Dll1 + , and Defa6 + ) was performed with DXR damage. Lineage tracing from aISCs was compared with lineage tracing from early progeny cells (transit-amplifying cells arising from aISCs 1 day predamage) in the context of DXR and IR. We compared stem cell and DNA damage response (DDR) transcripts in isolated aISCs and early progeny cells 6 and 24 hours after DXR., Results: Epithelial regeneration after DXR primarily arose from early progeny cells generated by aISCs. Early progeny cells upregulated stem cell gene expression and lacked apoptosis induction (6 hours DXR: 2.5% of CASP3 + cells, p<0.0001). aISCs downregulated stem cell gene expression and underwent rapid apoptosis (6 hours DXR: 63.4% of CASP3 + cells). There was minimal regenerative contribution from Bmi1 + , Hopx + , Dll1 + , and Defa6 + -expressing populations. In homeostasis, 48.4% of early progeny cells were BrdU + , and expressed low levels of DDR transcripts., Conclusions: We show that DXR effectively depleted aISCs in the small intestine and subsequent epithelial regeneration depended on nonquiescent early progeny cells of aISCs. The chemoresistant phenotype of the early progeny cells may rely on a dampened DDR in contrast to aISCs' robust DDR, which facilitates expeditious apoptosis., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Enteric bacteria and/or their products are necessary for doxorubicin (DXR)-induced small intestine mucosal damage. While DXR does not induce gross loss of epithelium, others have shown elevated serum endotoxin after DXR administration. However, the mechanism of movement is unknown. We hypothesized that DXR treatment resulted in increased paracellular translocation of bacteria or bacterial products through the small intestinal epithelium. We measured permeability after DXR administration using transepithelial resistance and macromolecular flux and assessed tight junctional gene expression and protein localization both in vitro using T84 cells and ex vivo using murine jejunum. DXR treatment increased flux of 4 kDa dextrans in mouse jejenum, but increased flux of 4, 10 and 20 kDa dextrans in T84 cells. Following DXR, we observed increased permeability, both in vitro and ex vivo, independent of bacteria. DXR induced increased expression of Cldn2 and Cldn4 in murine small intestine but increased only CLDN2 expression in T84 cells. DXR treatment induced disorganization of tight junctional proteins. We conclude that DXR increases paracellular transit of small macromolecules, including bacterial products, through the epithelium, by altering expression of tight junctional components and dynamic loosening of cellular tight junctions.

Alphaviruses are enveloped viruses with a positive-stranded RNA genome, of the family Togaviridae. In mammals and birds they are mosquito-transmitted and are of veterinary and medical importance. They cause primarily two types of disease: encephalitis and polyarthritis. Here we review attempts to understand the molecular basis of encephalitis and virulence for the central nervous system (CNS) in mouse models. Sindbis virus (SINV) was the first virus to be studied in this way. Other viruses analysed are Semliki Forest virus (SFV), Venezuelan equine encephalitis virus, Eastern equine encephalitis virus and Western equine encephalitis virus. Neurovirulence was found to be associated with damage to neurons in the CNS. It mapped mainly to the E2 region of the genome, and to the nsP3 gene. Also, avirulent natural isolates of both SINV and SFV have been found to have more rapid cleavage of nonstructural proteins due to mutations in the nsP1-nsP2 cleavage site. Immune-mediated demyelination for avirulent SFV has been shown to be associated with infection of oligodendrocytes. For Chikungunya virus, an emerging alphavirus that uncommonly causes encephalitis, analysis of the molecular basis of CNS pathogenicity is beginning. Experiments on SINV and SFV have indicated that virulence may be related to the resistance of virulent virus to interferon action. Although the E2 protein may be involved in tropism for neurons and passage across the blood-brain barrier, the role of the nsP3 protein during infection of neurons is unknown. More information in these areas may help to further explain the neurovirulence of alphaviruses.

Background: Prior studies have shown the prognostic utility of measuring L-lactate in critically ill neonatal foals, both as single (at admission) and serial measurements. Greater prognostic sensitivity and specificity may be achieved by use of the area under the L-lactate versus time curve (LACArea ) over the first 24 hours of hospitalization, which captures both severity and duration of hyperlactatemia. Prior to application of this concept in sick equine neonates, a reference interval for LACArea should be determined., Methods: The concentration of lactate [LAC] was measured in blood obtained via direct jugular venipuncture from clinically normal foals on Days 1 (birth-24 h of age), 3, 7, and 14 following birth at 6-hour intervals for each 24-hour period. LACArea was calculated using the trapezoidal method. Differences in LACArea by Day were determined by MANOVA with a priori Bonferroni correction, P ≤ 0.05., Results: LACArea differed by Day (P = 0.001), being largest on Day 1, followed by Day 3. Days 7 and 14 were smallest and not different from each other., Conclusion: LACArea decreases substantially and predictably over the first week of life in normal neonatal foals. Knowing how LACArea normally changes over the first 2 weeks of life will aid in future study of LACArea as it applies to sick neonatal foals, allowing for consideration of maturational changes potentially unrelated to disease., (© Veterinary Emergency and Critical Care Society 2015.)

Background: A variety of measures of L-lactate concentration ([LAC]) in the blood of critically ill neonatal foals have shown utility as prognostic indicators. These measures, evaluating either the severity of hyperlactatemia or the duration of exposure to hyperlactatemia, perform fairly well and have correctly classified 75-80% of foals examined in several studies. The area under the L-lactate concentration versus time curve (LACArea) encompasses both severity and duration of hyperlactatemia and should improve correct classification of patient survival., Hypothesis/objectives: LACArea is larger in nonsurviving critically ill neonatal foals., Animals: Forty-nine foals admitted for critical illness to 1 of 4 referral hospitals., Methods: Whole blood was obtained at admission and 6, 12, 18, and 24 hours after admission for measurement of L-lactate using a handheld lactate meter. LACArea was calculated for: admission-6, 6-12, 12-18, 18-24 hours, and admission-24 hours using the trapezoidal method and summing the 6-hours interval areas to determine total 24 hours area. Differences between survivors and nonsurvivors were determined using robust regression and Kruskal-Wallis testing, P < .05., Results: LACArea was significantly larger in nonsurviving foals (n = 9) than in surviving foals (n = 40) at all time periods examined., Conclusions and Clinical Importance: Differences in LACArea between surviving and nonsurviving critically ill neonatal foals are large and support further investigation of this method as an improved biomarker for survival in critically ill neonatal foals is indicated., (Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Aims: Glioblastoma multiforme is the most common and most malignant adult brain tumour. Despite numerous advances in cancer therapy there has been little change in the prognosis of glioblastoma multiforme, which remains invariably fatal. We examined the Semliki Forest virus virus-like particle (SFV VLP) expression system encoding interleukin-12 (IL-12) as a therapeutic intervention against the syngeneic RG2 rat glioma model., Methods: Glioma-bearing rats were treated with IL-12-encoding SFV VLPs via an implanted cannula. Animals were treated with 5 × 10⁷ (low-dose) or 5 × 10⁸ (high-dose) VLPs per treatment and the effect on glioma growth and survival was assessed., Results: Low-dose treatment produced a 70% reduction in tumour volume, associated with a significant extension (20.45%) in survival that was dependent upon IL-12 expression. High-dose treatment resulted in an 87% reduction in tumour volume, related to the oncolytic capacity of the SFV VLP system. VLP delivery to the central nervous system (CNS) demonstrated the potential of the vector system to induce lethal pathology that was unrelated to replication-competent virus or high-level IL-12 expression. Treatment-related death was pronounced in high dose-treated animals and appeared to be the result of inflammation, necrosis and oedema at the inoculation site., Conclusion: The efficacy of an IL-12 gene therapy approach for the treatment of the RG2 glioma model has been demonstrated in addition to the oncolytic capacity of the VLP vector system. Despite this, the broad tropism of the SFV-based expression vector may limit use as a CNS gene therapy vector unless this inherent limitation can be overcome., (© 2010 The Authors. Neuropathology and Applied Neurobiology © 2010 British Neuropathological Society.)

Electroporation is the application of high-voltage short-duration pulses to transiently permeabilize cells, permitting the cellular uptake of macromolecules, including nucleic acid. Although much attention has been focused on DNA vaccines, antigen-encoding RNA molecules may also stimulate immunity. Several methods are being examined in an effort to enhance the efficacy of nucleic acid delivery. One such method is the application of electroporation. The present study was designed to develop electroporation for use as a method of RNA delivery in conjunction with the Semliki Forest virus (SFV) RNA vector system for stimulation of immunity. Expression of SFV-based β-galactosidase and luciferase vectors was observed in the muscle after electroporation. Although some tissue damage was induced following intramuscular injection and electroporation with SFV vector RNA encoding LacZ at optimum pulse conditions, immunity to LacZ was efficiently induced. Following two immunizations, there was a higher IgG2a antibody response with the viral vector delivery and a higher IgG1 response in electroporated rSFV-LacZ RNA immunized mice.

Little is known about viral determinants of virulence associated with western equine encephalitis virus (WEEV). Here, we have analysed six North American WEEV isolates in an outbred CD1 mouse model. Full genome sequence analyses showed < or =2.7 % divergence among the six WEEV isolates. However, the percentage mortality and mean time to death (MTD) varied significantly when mice received subcutaneous injections of 10(3) p.f.u. of each virus. Two WEEV strains, McMillan (McM) and Imperial 181 (IMP), were the most divergent of the six in genome sequence; McM caused 100 % mortality by 5 days post-infection, whereas IMP caused no mortality. McM had significantly higher titres in the brain than IMP. Similar differences in virulence were observed when McM and IMP were administered by aerosol, intranasal or intravenous routes. McM was 100 % lethal with an MTD of 1.9 days when 10(3) p.f.u. of each virus was administered by intracerebral inoculation; in contrast, IMP caused no mortality. The presence of IMP in the brains after infection by different routes and the lack of observed mortality confirmed that IMP is neuroinvasive but not neurovirulent. Based on morbidity, mortality, MTD, severity of brain lesions, virus distribution patterns, routes of infection and differences in infection of cultured cells, McM and IMP were identified as high- and low-virulence isolates, respectively.

Alphavirus vectors are high-level, transient expression vectors for therapeutic and prophylactic use. These positive-stranded RNA vectors, derived from Semliki Forest virus, Sindbis virus and Venezuelan equine encephalitis virus, multiply and are expressed in the cytoplasm of most vertebrate cells, including human cells. Part of the genome encoding the structural protein genes, which is amplified during a normal infection, is replaced by a transgene. Three types of vector have been developed: virus-like particles, layered DNA-RNA vectors and replication-competent vectors. Virus-like particles contain replicon RNA that is defective since it contains a cloned gene in place of the structural protein genes, and thus are able to undergo only one cycle of expression. They are produced by transfection of vector RNA, and helper RNAs encoding the structural proteins. Layered DNA-RNA vectors express the Semliki Forest virus replicon from a cDNA copy via a cytomegalovirus promoter. Replication-competent vectors contain a transgene in addition to the structural protein genes. Alphavirus vectors are used for three main applications: vaccine construction, therapy of central nervous system disease, and cancer therapy.

The effect of intranasal (IN) administration of Semliki Forest virus (SFV) recombinant particles expressing interferon-β [IFN-β, a partially effective treatment for multiple sclerosis (MS)] on the progression of experimental autoimmune encephalomyelitis (EAE, a murine model for MS) was investigated. The murine IFN-β gene was cloned from SFV-infected mouse brain by RT-PCR into an SFV-enhanced expression vector, pSFV10-E, from which IFN-β-expressing recombinant particles (rSFV10-E-IFN-β) were prepared. Expression studies using immunohistochemistry and viral inhibition assay in BHK and murine L929 cells confirmed increased expression of IFN-β. High level expression in the central nervous system (CNS) following IN inoculation was confirmed by the excision of olfactory bulbs, brain and spinal cord, and the detection of IFN-β levels in homogenised tissue by ELISA. rSFV10-E-IFN-β particles were administered IN to C57/Bl6 mice that had been induced for EAE using the encephalogenic peptide myelin oligodendrocyte glycoprotein (MOG) 35-55. The progression of EAE was measured by clinical score, weight loss and pathology. As previously shown, treatment with empty rSFV10-E particles moderately exacerbated EAE, as did continuous treatment with rSFV10-E-IFN-β particles. Inhibition of disease with rSFV10-E-IFN-β particles was dependent on the number and timing of treatments. Fewer treatments, administered before the effector stage, led to an improvement in clinical and pathology score. In conclusion, the timing and frequency of IN administration of rSFV10-E-IFN-β particles are critical to disease outcome, with treatment prior to the effector stage being most effective.

An investigation of the role of the 5' untranslated region (UTR) of Semliki Forest virus (SFV) in determining pathogenicity in infected mice was carried out by constructing 5' UTR chimeras. Analysis of 5' UTR sequences showed nucleotide differences between virulent and avirulent strains at positions 21, 35 and 42. Reciprocal chimeras incorporating these changes were constructed from avirulent CA7 and rA7[74], and virulent SFV-4 virus, derived from infectious clones, and avirulent A7 and A7[74] plaque-purified stock virus. Survival rates and neuropathology in intranasally (i.n.) infected mice were analysed. While no statistically significant difference between rates of RNA synthesis was detected between strains in cell culture, an increase in survival of infected mice and a reduction in the severity of brain lesions was observed on substitution of the 5' UTR from a stock avirulent virus into an infectious clone where the remainder of the genome was derived from avirulent virus. However, substitution of a 5' UTR from an avirulent stock virus into an infectious clone where the remainder of the genome was from virulent virus did not affect virulence. These results and other studies suggest that control of virulence is polygenic, and that the SFV 5' UTR acts as a pathogenicity determinant in synergy with other determinants in the genome.

A Semliki Forest virus (SFV) recombinant particle vaccine vector was constructed expressing the viral E1 and E2 envelope proteins of the RA27/3 vaccine strain of rubella virus. This vector induced high titres of antibody after intramuscular administration to Balb/C mice, both following initial vaccination and a boost 4 weeks later. This occurred for antibody as measured by ELISA and as measured by a latex agglutination test. However, co-administration of similar particles expressing the measles virus H protein and the mumps virus HN protein with the rubella protein expressing vector resulted in reduction of the anti-rubella immune response.

Inhibition of tumour angiogenesis has been shown to restrict primary tumour growth and metastatic spread. This study examines the active induction of immune responses against tumour endothelial cells following immunization with recombinant Semliki Forest virus (rSFV) particles encoding murine vascular endothelial growth factor receptor-2 (VEGFR-2). This approach was tested in two murine tumour models, CT26 colon carcinoma and 4T1 metastasizing mammary carcinoma. Tumour growth and metastatic spread were shown to be significantly inhibited in mice that were prophylactically vaccinated or therapeutically treated with rSFV particles coding for VEGFR-2. Microvessel density analysis showed that immunization with rSFV led to significant inhibition of tumour angiogenesis. Therapeutic efficacy was found to be associated with the induction of an antibody response against VEGFR-2. Co-immunization of mice with rSFV particles encoding VEGFR-2 and interleukin (IL)-12 completely abrogated both the antibody response and the antitumour effect. However, co-immunization of mice with VEGFR-2 and IL-4 encoding particles was shown both to induce higher titres of anti-VEGFR-2 antibodies and lead to enhanced survival following tumour challenge when compared to mice vaccinated with VEGFR-2 particles alone. These findings indicate that active immunization with rSFV particles coding for VEGFR-2 can break immunological tolerance and could potentially be used as part of a novel treatment for cancer.

The enhanced Semliki Forest virus vector (SFV10-E), an RNA-based suicide expression vector system, expresses foreign genes at levels up to 10x higher than the original SFV10 vector. This vector has been used previously to express interleukin-12 for a tumour treatment study in a BALB/c murine model. Interleukin-18, an IFN-gamma-inducing cytokine, plays a key role in the early induction of T helper1 (Th1) cell-mediated immune responses in addition to anti-angiogenic activity. In this study, the murine IL-18 gene along with an Ig-kappa leader sequence was cloned into the SFV10-E vector. The pSFV10-E-IL-18 construct was characterised in vitro for levels of expression and secretion, and the production of biologically active IL-18 was confirmed. An in vivo tumour treatment study using high titre rSFV10-E-IL-18 virus-like particles to treat subcutaneous K-BALB and CT26 tumours in BALB/c mice demonstrated therapeutic efficacy including the disappearance of tumour cells in a minority of treated animals. Tumour regression was associated with induction of avascular and suppurative necrosis.

Major virulence determinants of Semliki Forest virus (SFV) lie within the non-structural genes that form the replicase complex proteins. Gene exchange between virulent and avirulent viruses has shown that the nsP3 gene, which has essential 5' conserved domains and a non-essential hypervariable 3' domain, is one of the virulence determinants. This protein plays a role in subgenomic 26S and negative-strand RNA synthesis and is thought to function with nsP1 to anchor replication complexes to cell membrane structures. Studies to date have focused on analysing the effect of mutational changes spread over the whole gene on virulence of the virus. The virulent SFV4 virus, derived from an infectious clone, was utilized to analyse the effect on virulence of large deletions in the hypervariable domain of nsP3. Two viruses with different in-frame deletions that spanned this domain showed reduced rates of RNA synthesis and multiplication in cell culture. In adult BALB/c mice, these viruses were avirulent after intramuscular and intraperitoneal inoculation, and brains sampled from infected mice showed minimal or no evidence of pathology. These deleted viruses had greatly reduced virulence when administered by the intranasal route and brains from infected mice showed lesions that were much less severe than those seen in SFV4 infection. Mice surviving infection with the deleted viruses resisted challenge with the virulent L10 strain, indicating induction of protective immunity. This work establishes that deletions in the nsP3 hypervariable domain attenuate virulence after peripheral inoculation and also reduce virulence after intranasal inoculation.

Members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds have been shown to induce apoptosis in a number of human leukemia cell lines of different haematological lineage, suggesting their potential as anti-cancer agents. In this study, we sought to determine if PBOX-6, a well characterised member of the PBOX series of compounds, is also an effective inhibitor of breast cancer growth. Two estrogen receptor (ER)-positive (MCF-7 and T-47-D) and two ER-negative (MDA-MB-231 and SK-BR-3) cell lines were examined. The 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine reduction in cell viability. PBOX-6 reduced the cell viability of all four cell lines tested, regardless of ER status, with IC(50) values ranging from 1.0 to 2.3 microM. PBOX-6 was most effective in the SK-BR-3 cells, which express high endogenous levels of the HER-2 oncogene. Overexpression of the HER-2 oncogene has been associated with aggressive disease and resistance to chemotherapy. The mechanism of PBOX-6-induced cell death was due to apoptosis, as indicated by the increased proportion of cells in the pre-G1 peak and poly(ADP-ribose) polymerase (PARP) cleavage. Moreover, intratumoural administration of PBOX-6 (7.5 mg/kg) significantly inhibited tumour growth in vivo in a mouse mammary carcinoma model (p=0.04, n=5, Student's t-test). Thus, PBOX-6 could be a promising anti-cancer agent for both hormone-dependent and -independent breast cancers.

The Semliki Forest virus (SFV) vector is an RNA-based suicide expression vector that has been used experimentally for tumour therapy. Recently, a new enhanced vector pSFV10-E has been developed that expresses foreign genes at levels up to 10 times higher than the original vector. Interleukin-12 (IL-12), an immunomodulatory cytokine, plays a key role in the induction of T-helper1 responses. The two IL-12 gene subunits were cloned from mouse splenocytes and inserted into the pSFV10-E and pSFV10 (non-enhanced) vectors. Both constructs expressed and secreted biologically active murine IL-12. Administration of high titre rSFV10-E-IL12 particles intratumourally to treat implanted K-BALB tumours in BALB/c mice demonstrated complete tumour regression in comparison to control or rSFV10-IL12 treated groups. High titre rSFV10-E-IL12 particles were also effective in the CT26 tumour model. Histological and immunohistochemical studies revealed tumour necrosis in addition to aggressive influx of CD4+ and CD8+ T cells and other immune cells. Furthermore, inhibition of primary tumour growth and lung metastases of a metastatic (4T1) tumour model indicated the potential of high titres of rSFV10-E-IL12 particles as an efficient antitumour therapeutic agent.

To assess the potential of immune stimulation in combination with apoptosis induction by Semliki Forest virus (SFV) and its derived vector for tumour treatment, we have utilized the poorly immunogenic and rapidly growing K-BALB and CT26 murine tumour models. Both cell lines underwent apoptosis and expressed viral antigen when infected with the SFV4 strain of SFV, or recombinant SFV (rSFV) virus-like particles (VLPs) encoding the p62-6k viral structural proteins. VLPs were used to immunize groups of BALB/c and BALB/c nu/nu mice prior to subcutaneous tumour induction and treatment. Direct intratumoral injection of VLPs or SFV4 resulted in an immediate and intense inflammatory reaction in immunized groups that was not observed in naive groups until day 5 of treatment, and was not observed in nu/nu groups. A significantly higher level of tumour growth inhibition was observed in immunocompetent groups than in athymic mice. For K-BALB tumours, SFV4 treated groups showed greater inhibition than that observed in VLP-treated groups, with immunization prior to treatment enhancing the overall antitumour effect and immune response. No significant difference was observed in CT26 tumours between VLP and SFV4-treated groups, but prior immunization considerably enhanced the antitumoural response. It is concluded that use of the inherent apoptosis-inducing capability of SFV or its vector, by perfusion in combination with immune stimulation, may have potential for the treatment of rapidly growing tumours.

The alphaviruses Semliki Forest virus (SFV) and Sindbis virus have recently been developed as prototype anti-cancer agents. These are RNA-containing enveloped viruses that code for only 9 proteins of unique sequence. The standard recombinant SFV vector system consists of suicide particles containing recombinant RNA. In addition, alphavirus vectors capable of limited multiplication in the host are also being developed. Several strategies are being adopted to construct prototype SFV vectors for cancer treatment. These include: 1) construction of both prophylactic and therapeutic vaccines to stimulate immunity to tumor-associated antigens, 2) use of apoptosis induction to destroy tumor cells, which includes both the use of the inherent apoptosis-inducing ability of the vector and the action of pro-apoptotic genes cloned into the vector, and 3) expression of cytokines and other immunoregulatory proteins by the vector that enhance anti-tumor immune responses and/or inhibit tumor cell growth. This includes the use of cytokines such as IL-12 that target angiogenesis. Sindbis virus appears to have a natural tropism for tumor cells that may allow targeting both of the wild-type virus and the vector. This approach may also be useful for targeting metastases. For SFV, neurovirulence and/or neurotropism, as well as other tissue damage, may preclude the use of unmodified replication competent wild-type virus in tumor treatment. However, it may be possible to use such a virus in animals that have been vaccinated, using a vector-derived vaccine.

A 3-year-old Wirehaired Fox Terrier was presented to the University Veterinary Hospital, University College Dublin, for evaluation of chronic cough of 8-months duration. Bronchoscopy showed a severely dilated collapsed left principal bronchus filled with highly viscous white mucus. Cytologically, globular lipid-like material and round concentrically laminated crystalline structures were evident within the proteinaceous mucus. These findings resembled the calcospherites and granular caseous debris often observed in human tuberculous patients. A Ziehl-Neelsen-stained cytocentrifuged preparation of material obtained by bronchoalveolar lavage revealed a few acid-fast rods within macrophages, suggestive of tuberculosis. At necropsy, granulomas with caseous necrosis were present in the lung parenchyma, bronchial and mediastinal lymph nodes, liver, pancreas, and mesentery. Granulomas were adherent to both kidney capsules and to the diaphragm. Histologically, there was evidence of mild calcification within caseous granulomas, which was confirmed by von Kossa's stain. Using Ziehl-Neelsen stain, acid-fast rods were identified within granulomas; bacterial culture was positive for Mycobacterium bovis. The cytologic findings in this case have not been reported previously in dogs and demonstrate a possible correlation between tuberculosis and calcospherite-like bodies with caseous, globular material in bronchial mucus, similar to that described in human patients.

We have initiated studies to determine the feasibility of employing the Semliki Forest virus (SFV) expression system as a central nervous system (CNS) vector. We investigated the effects of infecting Balb/c mice intranasally (i.n.) with recombinant SFV particles expressing the enhanced green fluorescent protein (EGFP) reporter gene. EGFP expression was detected by fluorescence microscopy in the olfactory bulb as early as 1 day postinfection. No pathological changes were associated with infection. Viral RNA could be detected in the olfactory mucosa only, whereas fluorescence was detected in axons in the olfactory bulb, indicating that only the expressed protein was present. A vector expressing interleukin 10 (IL-10) was constructed and shown to induce good cytokine expression in cultured cells. IL-10 expression in the nasal passage and olfactory bulb of infected mice was enhanced following i.n. administration of such particles. Mice induced for experimental autoimmune encephalomyelitis (EAE) were treated i.n. with vectors expressing EGFP and IL-10 and with empty vector. The EGFP-expressing and empty vectors were found to exacerbate EAE, whereas that expressing IL-10 ameliorated EAE. It is concluded that the mice showed a significant biological response when treated i.n. with recombinant SFV particles and that such particles administered by the i.n. route have potential as a noninvasive vector for protein delivery to the CNS.

Semliki Forest virus (SFV) infection of mice provides a useful model for the analysis of viral neuropathogenesis. In this study, the roles of interferon (IFN)-gamma and nitric oxide (NO) in the pathogenesis of SFV infection were assessed using mice deficient in inducible nitric oxide synthase (iNOS-/-), an enzyme important in the production of NO, and mice deficient in IFN-gamma receptor (IFN-gammaR-/-). Gene-knockout and wildtype mice were infected intranasally with the avirulent A7 strain of SFV and neuropathological lesions were correlated with levels of IFN-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 in the olfactory bulbs and frontal cortex. Lesions in IFN-gammaR-/- mice were characterized by higher levels of neuronal necrosis than in wildtype mice. The higher levels of neuronal necrosis were associated with increased levels of SFV antigen in neurones and increased numbers of macrophages and B cells. Relative differences in the severity of demyelination between IFN-gammaR-/- and wildtype mice were not detected. Similar levels of neuronal necrosis and SFV antigen labelling occurred in iNOS-/- mice and wildtype mice and levels of demyelination and macrophage infiltration in the iNOS-/- mice were lower than those in the wildtype strain. A rapid, but transient increase in the concentration of IFN-gamma was demonstrated in the frontal cortex of all infected mice samples. IL-10 levels in the frontal cortex and olfactory bulbs of SFV-infected iNOS-/- mice exceeded those present in the wildtype mice. This study, taken with our previous reports, provides further evidence that type 1 T cell responses are important in the control of brain viral clearance and the prevention of neuronal necrosis, but not in the development of demyelination.

Semliki Forest virus (SFV) infection of mice is a useful model of viral neuropathogenesis in animals and avirulent strains such as SFV-A7 induce immune-mediated demyelination and death of neurones by necrosis and apoptosis. Matrix metalloproteinases (MMPs) have been implicated in various diseases including arthritis and cancer in many species. In this report, we show that MMP-2 and MMP-9 expression is induced in the brains of mice infected i.n. with SFV-A7. Treatment of mice with the pan MMP inhibitor GM6001 ameliorated the development of SFV-induced neuropathological lesions via an effect on the integrity of the blood-brain barrier. Low levels of neuronal necrosis and demyelination in GM6001-treated mice correlated with localisation of fibrinogen staining to thin-walled blood vessels and less intense staining of the perivascular neuropil.

Experimental infection of mice with avirulent Semliki Forest virus (SFV) has been used as a model of demyelinating disease in humans. A number of studies have shown that T cells may be important for mediating demyelination, but the role of T cells is still, unclear. Here, we show that neuronal necrosis, but not demyelination, was more severe in interleukin (IL)-12-defective mice compared with wild-type mice and this correlated with higher virus titers in the brain. In contrast, the severity of demyelination and neuronal depletion was reduced in IL-4-defective mice and this correlated with reduced brain virus titers and enhanced SFV-specific IFN-gamma production. The findings indicate that type 1 T cells play a role in the control of SFV replication but not directly in SFV-induced pathology in the CNS.

Mice and lambs were infected with the LI/I, LI/31 or MA54 strain of louping ill virus (LIV) to provide information relevant to testing the efficacy and biosafety of a new generation of flavivirus vaccines based on a Semliki Forest virus (SFV) vector. Whereas clinical signs and neuropathological lesions were consistently severe in mice, the majority of lambs showed lesions of moderate severity and only lambs with severe lesions were clinically affected. For both species, dispersal of viral antigen occurred along neuronal cell processes, and neuronal degeneration and death were confirmed as central events after infection with LIV. In contrast to lambs, in which most lesions remained localized, mice showed widely dispersed lesions which were associated with less intense leucocytic infiltrates. Among the infiltrating cells, histiocytes predominated and apoptotic forms were prominent in severely affected animals. The intranasal route of infection provided an efficient avenue for entry of LIV into the brain and resulted in lesions which were more severe than those produced by subcutaneous or intraperitoneal inoculation., (Copyright Harcourt Publishers Ltd.)

The Semliki Forest virus (SFV) vector is a transient RNA expression vector that has an inherent p53-independent apoptosis-inducing property. It is administered as recombinant SFV particles (rSFV) that undergo 1 round of replication only and express a gene cloned into the multicloning site. In our study we have investigated the ability of the SFV vector to induce apoptosis and inhibit tumour growth in rat prostate cancer (AT3-Neo) cells expressing the Bcl-2 oncogene (AT3-Bcl-2 cells), which normally inhibits apoptosis. rSFV expressing the enhanced green fluorescent protein (EGFP) gene (rSFV-EGFP), or recombinant RNA transfected into cells by electroporation, induced delayed apoptosis in AT3-Bcl-2 cells. SFV-mediated expression of a cloned pro-apoptotic Bax gene by the vector, however, enhanced apoptosis induction both in AT3-Bcl-2 cells and standard BHK-21 cells. Such Bax-expressing particles could be produced only at low titers compared to EGFP-expressing particles under standard conditions for particle production, but lowering the incubation temperature for particle production to 33 degrees C partially alleviated this effect. Bax-expressing particles were shown to inhibit the growth of AT3-Neo and AT3-Bcl-2 tumours in nude mice, as did high titre EGFP-expressing particles. It is concluded that SFV recombinant particles have potential as anti-tumour agents to treat apoptosis-resistant tumours., (Copyright 2001 Wiley-Liss, Inc.)

This study has examined the efficacy following intramuscular administration of a recombinant Semliki Forest virus (rSFV) vaccine, encoding the prME and NS1 proteins of louping ill virus (LIV), in sheep. Administration of rSFV-LIV vaccine resulted in transient detection at the injection site and draining lymph node only and no dissemination to distal sites. In addition, the recombinant vaccine offered complete protection against subcutaneous challenge with LIV, and partial protection following intranasal administration of LIV. Protected animals had no pathological changes normally associated with LIV infection, and had developed high antibody titres. In contrast, the two animals not protected exhibited classical clinical signs and neuropathological lesions of LIV infection. These findings indicate that rSFV-based vaccines have the potential to be developed as effective prototype vaccines for LIV.

Progressive ataxia, with head tremor, developed in 10 captive-born cheetah cubs under six months of age. The condition was usually preceded by coryza and an ocular discharge. Initially the ataxia and weakness affected the hindquarters, then the forelegs, and head tremor developed later. Significant pathological changes were confined to the central nervous system. There was widespread Wallerian degeneration in the funiculi of the spinal cord (except those in the dorsal columns), in the medulla and in the cerebellum. In the cerebellum there was degeneration of Purkinje cells and of the molecular and granular cell layers. There was chromatolysis in the Purkinje cells, the ventral horn cells of the spinal cord and in the neurons of the lateral vestibular nucleus. The olivary nucleus was necrotic. There were foci of inflammatory cells in the molecular layer of the cerebellum and in the medulla. The cause of the disease remains unknown.

This study has examined the persistence, distribution and pathological changes following intramuscular administration of Semliki Forest virus (SFV) vaccine vectors in mice and chickens. Administration of recombinant SFV RNA particles showed persistence at the injection site of mice up to 7 days, transient detection in secondary lymphoid organs and no dissemination to distal sites. In contrast, administration of a layered SFV DNA/RNA vector and a conventional standard naked DNA vector resulted in long-term persistence at the injection site, plasmid DNA being detected at 8 months post-inoculation in mice. Plasmid DNA was found distributed throughout the body, and tissues distal from the site of injection were positive up to 3 months. A similar pattern was observed in chickens. Mild pathological changes were observed at the injection site only, and plasmid DNA or recombinant RNA was not detected in mouse foetuses. These findings indicate that SFV-based vectors have the potential to be developed as safe vaccines.

We have utilised cell cultures and growth of tumours in nude mice to assess further the potential of the Semliki Forest virus (SFV) vector as a cancer therapy agent. This vector is a transient RNA-based expression vector, and we have previously shown that SFV and its derived vector can induce p53-independent apoptosis by expression of the nonstructural region of the virus genome. Apoptosis induction is therefore an inherent property of the vector and is not dependent on heterologous gene expression. SFV recombinant suicide particles (rSFV) were shown to induce apoptosis in H358a cells, which are human non-small cell lung carcinoma cells deleted in p53. EGFP-expressing rSFV also inhibited the growth of developing H358a spheroids. Direct injection of rSFV into H358a tumours subcutaneously implanted as xenografts in nu/nu mice inhibited tumour growth, and in some cases caused complete regression. It is concluded that tumour growth suppression induced by rSFV was due to apoptosis induction and that the vector has an inherent cell death-promoting and antitumour activity. These results, as well as previous work by other authors on targeting and immune stimulation using alphavirus vectors, indicate that SFV recombinant particles in particular have considerable potential for further exploitation as a cancer therapy agent.

Multiple sclerosis (MS) is a chronic, demyelinating disease of the CNS in which autoimmunity to myelin plays a role in pathogenesis. The epidemiology of MS indicates that it may be triggered by a virus infection before the age of adolescence, but attempts to associate a specific virus with MS have produced equivocal results. Many studies of the aetiology of MS have postulated that a persistent virus infection is involved, but transient virus infection may provide a plausible alternative mechanism that could explain many of the inconsistencies in MS research. The most studied animal model of MS is chronic relapsing experimental autoimmune encephalomyelitis (CREAE), which is induced in susceptible animals following injection of myelin components. While CREAE cannot provide information on the initiating factor for MS, it may mimic disease processes occurring after an initial trigger that may involve transient virus infection. The disease process may comprise separate triggering and relapse phases. The triggering phase may involve sensitisation to myelin antigens as a result of damage to oligodendrocytes or molecular mimicry. The relapse phase could be similar to CREAE, or alternatively relapses may be induced by further transient virus infections which may not involve infection of the CNS, but which may involve the recrudescence of anti-myelin autoimmunity. Although current vaccines have a high degree of biosafety, it is suggested that the measles-mumps-rubella vaccine in particular could be modified to obviate any possibility of triggering anti-myelin autoimmunity., (Copyright 2000 John Wiley & Sons, Ltd.)

Louping ill virus (LIV) infection of mice was used as a model to evaluate the protective efficacy of Semliki Forest virus (SFV)-based vaccines in comparison to a standard DNA vaccine and a commercial chemically inactivated vaccine. The recombinant SFV-based vaccines consisted of suicidal particles and a naked layered DNA/RNA construct. The nucleic acid vaccines expressed the spike precursor prME and the nonstructural protein 1 (NS1) antigens of LIV. Three LIV strains of graded virulence for mice were used for challenge. One of these was a naturally occurring antibody escape variant. All vaccines tested induced humoral immunity but gave varying levels of protection against lethal challenge. Only recombinant SFV particles administered twice gave full protection against neuronal degeneration and encephalitis induced by two of the three challenge strains, and partial protection against the highly virulent strain, whereas the other vaccines tested gave lower levels of partial protection.

Semliki Forest virus (SFV) infection of mice is used as a model to study pathogenic processes occurring in viral encephalitis. It has previously been shown that avirulent strains of SFV differ from virulent strains in showing restricted multiplication in neurones and in producing localized rather than widespread lesions in the central nervous system (CNS). Restricted neuronal damage is age-dependent and does not occur in neonatal animals. In this study, cell death mechanisms occurring in the CNS of adult rats infected intranasally (i.n.) with a virulent (SFV4) and an avirulent (A7) strain of SFV have been investigated. Although i.n. infection of rats was less efficient than that of mice, SFV4 reached a higher titre in the CNS of infected animals than A7. Neuronal destruction and leucocytic infiltration occurred throughout the forebrain of SFV4-infected rats. A7-infected rats remained clinically normal although degenerate neurons and inflammatory changes were present primarily in the olfactory system. Following infection with either A7-SFV or SFV4, TUNEL-positive nuclei were seen in areas of leucocytic infiltration and among the poorly differentiated cells of the rostral migratory stream. Migrating cells had condensed nuclear chromatin, compacted cytoplasm and intact cellular membranes, characteristic of apoptosis, and were sparsely immunolabelled for viral antigen. In SFV4-infected rats, large numbers of contiguous neurones in forebrain areas exhibited cytoplasmic eosinophilia and karyolysis and were surrounded by phagocytic cells. Such neurones contained dense intracytoplasmic deposits of viral antigen and showed weak cytoplasmic TUNEL staining; electron microscopy showed membrane disruption, organelle disintegration, irregular chromatin condensation and cytoplasmic aggregation of virus particles. Bcl-2 staining was similar in infected and control rats and was most intense in randomly distributed Purkinje cells in the cerebellum; neurons in the olfactory bulbs were unstained. These findings indicate that during SFV encephalitis, infiltrating leucocytes and neural precursor cells undergo apoptosis whilst productively infected neurons undergo necrosis.

Recombinant Semliki Forest virus (rSFV) vaccines encoding louping ill virus (LIV) genes prME and NS1 were examined. Cells transfected with rSFV-prME RNA showed correct processing of the precursor prME and the release into the medium of M and E proteins in particulate form, whilst rSFV-NS1-transfected cells secreted glycosylated, heat-labile NS1 dimers. Mice immunized with rSFV particles produced antibodies against prME and NS1 that were mainly of the IgG2a subtype, indicating that a T-helper 1 immune response was induced. Immunization with prME- or NS1-encoding particles induced T-cell proliferation. Mice vaccinated intraperitoneally (i.p.) with rSFV-prME and/or rSFV-NS1 were significantly protected from lethal i.p. challenge by two strains of LIV, the virulent LI/31 strain, from which the commercial LIV vaccine is derived, and the less-virulent LI/I antibody-escape variant. Intranasal (i.n.) vaccination was protective for rSFV-prME only against LI/31 challenge and not against challenge with LI/I. Immunization with rSFV-NS1 was protective against i.p. and i.n. challenge with both virus strains when given i.p., but was not protective when given i.n. For unvaccinated mice infected with LIV, all animals showing clinical signs had severe degenerative and inflammatory lesions in the central nervous system. None of the rSFV-vaccinated mice that survived challenge showed central nervous system pathology, with the exception of mild leptomeningitis in a minority of LI/31-infected mice. This suggests that protection following immunization with rSFV must occur at early stages of LIV infection.

Three deletion mutants of the structural protein region of the Semliki Forest virus (SFV) genome, including one which encompassed all the viral structural protein genes, induced apoptosis in BHK cells at 48 h after transfection, as shown by DNA laddering and TUNEL staining, as did the wild-type SFV4 RNA. A similar result was obtained for the SFV1 expression vector, which has a multicloning site inserted in place of the structural protein genes. However, in cells transfected with viral RNA containing a deletion of the nsP2 gene, neither viral RNA synthesis nor the induction of apoptosis occurred. Both SFV1 vector and wild-type SFV4 RNA induced apoptosis in human H358a lung carcinoma cells, which have a homozygous deletion of the p53 gene. It is concluded that the SFV vector encodes a function in the nonstructural coding region which induces p53-independent apoptosis and is dependent on viral RNA synthesis.

Using a murine respiratory challenge model we have previously demonstrated a role for Th1 cells in natural immunity against Bordetella pertussis, but could not rule out a role for antibody. Here we have demonstrated that B. pertussis respiratory infection of mice with targeted disruptions of the genes for the IFN-gamma receptor resulted in an atypical disseminated disease which was lethal in a proportion of animals, and was characterized by pyogranulomatous inflammation and postnecrotic scarring in the livers, mesenteric lymph nodes and kidneys. Viable virulent bacteria were detected in the blood and livers of diseased animals. An examination of the course of infection in the lung of IFN-gamma receptor-deficient, IL-4-deficient and wild-type mice demonstrated that lack of functional IFN-gamma or IL-4, cytokines that are considered to play major roles in regulating the development of Th1 and Th2 cells, respectively, did not affect the kinetics of bacterial elimination from the lung. In contrast, B cell-deficient mice developed a persistent infection and failed to clear the bacteria after aerosol inoculation. These findings demonstrate an absolute requirement for B cells or their products in the resolution of a primary infection with B. pertussis, but also define a critical role for IFN-gamma in containing bacteria to the mucosal site of infection.

The nonstructural region of the genome of the avirulent A7 strain of Semliki Forest virus (SFV) has been sequenced, so that the complete nucleotide sequence is available. Compared to the virulent SFV4 strain (produced from the infectious clone pSP6-SFV4), A7 contains 226 nucleotide changes in the translated region, which result in 47 amino acid changes. The 5' nontranslated region has two nucleotide changes, and the 3' nontranslated region is longer in A7 than SFV4, and contains divergent and repeated sequences. Chimeras containing SFV4 and A7 sequences and an infectious clone of A7, pSP6-CA7, were constructed. The virulence of these was tested by intraperitoneal and intranasal infection of adult BALB/c mice. It was shown that determination of the avirulent phenotype of A7 was polygenic, and required the additive effect of sequences from both the structural and non-structural regions of the SFV genome.

We have investigated the induction of cell death in cultured cells by the virulent SFV4 and avirulent A7 strains of Semliki Forest virus (SFV). In BHK cells, death occurred by a typical apoptotic mechanism, as did the death of oligodendrocytes in glial cell cultures. For cerebellar neuron cultures, virus-induced death was due to necrosis. Although the SFV4 and A7 strains did not differ in the mechanism of induction of cell death, the virulent SFV4 strain did multiply to a higher titre in cultured neurons than the avirulent A7 strain. This is consistent with previous animal studies which indicate that the virulence of SFV strains is controlled by rapidity of multiplication in the CNS, leading to a lethal threshold of damage, rather than differential cell tropism or cell death mechanisms. The immune-mediated demyelination induced by avirulent strains may be triggered by apoptosis of oligodendrocytes, the consequences of which are obscured by death for virulent strains.

Semliki Forest virus (SFV) infection of mice is used as a model to study pathogenic processes occurring in viral encephalitis and demyelinating disease. In this study, the long-term effects of infection by the avirulent M9 mutant of SPV on the central nervous system (CNS) of BALB/c and SJL mice were determined. The presence of infectious virus, viral RNA and cytokine mRNA in the brains of individual mice and the presence of lesions in the spinal cords of the same mice up to 360 days post-infection (d.p.i.) were analysed in order to detect any correlation between these parameters of pathogenesis. Infectious virus could not be detected beyond 7 d.p.i. for either mouse strain. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the presence of the E2 and nsP1 regions of the virus genome and mRNA for interferon-gamma and tumour necrosis factor-alpha. Viral RNA could be detected up to 90 d.p.i. for both mouse strains. Cytokine mRNA could be detected up to 28 d.p.i. for BALB/c mice but up to 360 d.p.i. for SJL mice. Inflammatory lesions, which were associated with cytokine mRNA expression, were not detected in BALB/c mice beyond 28 d.p.i. but were detected in two SJL mice at 90 d.p.i. It is concluded that M9-SFV infection induces long-term prolonged expression of pro-inflammatory cytokines in the CNS of the majority of SJL (but not BALB/c) mice which is not associated with persistence of the virus genome. M9-SFV infection of SJL mice may be a relevant model for the pathogenesis of multiple sclerosis in man.

The onset of growth retardation was investigated in fetal lambs following experimental infection of pregnant ewes with Border Disease virus (BDV) on day 53 of pregnancy. Fetuses from control and infected ewes were harvested at weekly intervals between day 60 and day 95 of gestation and morphometric studies were completed on tibial radiographs and tibial growth cartilage metaphyseal junctions. Mean tibial areas were significantly reduced (P < 0.01) in fetuses from infected ewes at 35 and 42 days after infection and growth cartilage metaphyseal junctions were less mature in fetuses from infected ewes at 42 days after infection. Positive immunostaining for BDV antigen was demonstrated in the brains of all fetuses from infected ewes between 14 and 42 days after infection. Attempts to demonstrate BDV antigen in bone proved unsuccessful. It is concluded that intrauterine growth retardation is an early manifestation of BDV infection in lambs and that the process is initiated shortly following infection of the fetus.

We have examined 26 human AIDS brains obtained at post mortem for infection by human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV), and for dual infection of cells by both viruses. The techniques used were enzyme-linked immunocytochemistry for HCMV and in situ hybridisation using a cDNA probe for HIV. Using these techniques, HCMV infection was detected in 14 brains, HIV infection in 14 brains, and coinfection with HIV and HCMV in 7 brains. Four case of dual HIV/HCMV infection were found where no colocalisation could be detected. In randomly chosen dually infected areas 19.2% of infected cells were coinfected with both viruses. Although cells identified morphologically as macrophages were the most common infected cell type, astrocytes and neurons were both singly and doubly infected with HIV and HCMV. Complete clinical data were available for 4 of the 7 cases with coinfection and each had AIDS dementia complex.

The Semliki Forest virus (SFV) expression vector consists of a plasmid based on the SFV infectious clone. Foreign genes may be inserted into the structural coding region, transcribed as RNA, and expressed in cell culture after transfection. RNA containing inserted sequences may be packaged into virions using a helper systems. This allows efficient infection and expression without chemical transfection, but only one round of multiplication is possible. The biosafety of the system has been increased by the introduction of multiple mutations, specifying a maturation defect, into the helper. Potential vaccines can be constructed by insertion of genes coding for antigenic proteins into the vector. Following insertion of the influenza virus nucleoprotein (NP) into the SFV vector, immunity was induced following injection of packaged or naked RNA into mice. The SFV vector is a "suicide" expression vector that has great potential for the construction of vaccines for both human and veterinary use.