Viral sequences required for neurovirulence of poliovirus

Polioviruses of reduced neurovirulence contain point mutations in the viral RNA that are responsible for the attenuated phenotype. Two such point mutations have been identified in the genomes of the Sabin live oral vaccine strains, one in the 5′-noncoding region of the viral RNA, and one in capsid polypeptide VP3.     

Transgenic animal models of tauopathies

Tauopathies are a group of neurodegenerative disorders that include Alzheimer's disease, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) and other related diseases with prominent tau pathology. Research advances in the last several decades have characterized and defined tau neuropathologies of both neuron and glia in these diverse disorders and this has stimulated development of animal models of tauopathies. Indeed, animal models ranging from invertebrate species such as C. elegan to Drosophila melanogaster and mammalian transgenic mouse models of tauopathies have been generated and reported. This review summarizes the salient features of many of the known models of tauopathies.     

Experience in Canada with the new revised monkey neurovirulence test for oral poliovirus vaccine

Nine years of experience in our laboratory, using more than 1500 cynomolgus monkeys in 138 tests, has shown that the new neurovirulence test (NVT) adopted by the World Health Organization (WHO) for live, oral monovalent vaccine of each poliovirus type, was a reproducible and sensitive assay likely to ensure the safety of this vaccine in humans. Our findings were the following: (1) when the test vaccine and the appropriate homotypic reference vaccine were tested in a single group of monkeys, the concurrent use of the reference vaccine considerably increased the reproducibility of the NVT; (2) in the assessment of the degree of attenuation of each lot of vaccine, the use of 12 monkeys for types 1 and 2 vaccines and 20 monkeys for type 3 vaccine (inoculated intraspinally each for reference and test vaccine) was satisfactory; (3) the virus dose used per monkey (10(5.6) to 10(6.6) pfu per monkey) was found not to be critical, i.e. the lower virus dose yielded mean lesion scores in the central nervous system of monkeys at least as high or higher than the tenfold higher virus dose; (4) the statistical analysis of our data showed that the old intrathalamic (IT) assay was considerably less sensitive than the new intraspinal (IS) assay, i.e., a test vaccine with a twofold increase in monkey neurovirulence would have a 41% chance of failing in the IT test (using 30 monkeys per vaccine), while this chance increased to 99% in the WHO IS assay (using 12 or 20 monkeys per vaccine). Since the introduction of the WHO NVT in Canada, the laboratory findings in monkeys were confirmed by vaccine experience in humans; the number of vaccine-associated paralytic poliomyelitis cases in the population showed a further decline.     

Transgenic animal models of cardiovascular disease

Transgenic experimentation has become a crucial part of hypertension and atherosclerosis research, and is growing more important in several other areas of cardiovascular disease. It has recently made a particular contribution to understanding the role of the renin-angiotensin system in controlling hypertension. The study of blood pressure regulation, cardiac hypertrophy, atherogenesis and thrombosis are also benefiting from the transgenic approach.     

Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing.

Intracerebral inoculation of mice with poliovirus type 2 Lansing induces a fatal paralysis, while most other poliovirus strains are unable to cause disease in the mouse. To determine the molecular basis for Lansing virus neurovirulence, we determined the complete nucleotide sequence of the Lansing viral genome from cloned cDNA. The deduced amino acid sequence was compared with that of two mouse-avirulent strains. There are 83 amino acid differences between the Lansing and Sabin type 2 strain and 179 differences between the Lansing and Mahoney type 1 strain scattered throughout the genome. To further localize Lansing sequences important for mouse neurovirulence, four intertypic recombinants were isolated by exchanging DNA restriction fragments between the Lansing 2 and Mahoney 1 infectious poliovirus cDNA clones. Plasmids were transfected into HeLa cells, and infectious recombinant viruses were recovered. All four recombinant viruses, which contained the Lansing capsid region and different amounts of the Mahoney genome, were neurovirulent for 18- to 21-day-old Swiss-Webster mice by the intracerebral route. The genome of neurovirulent recombinant PRV5.1 contained only nucleotides 631 to 3413 from Lansing, encoding primarily the viral capsid proteins. Therefore, the ability of Lansing virus to cause paralysis in mice is due to the viral capsid. The Lansing capsid sequence differs from that of the mouse avirulent Sabin 2 strain at 32 of 879 amino acid positions: 1 in VP4, 5 in VP2, 4 in VP3, and 22 in VP1.     

Human poliovirus receptor gene expression and poliovirus tissue tropism in transgenic mice.

Expression of the human poliovirus receptor (PVR) in transgenic mice results in susceptibility to poliovirus infection. In the primate host, poliovirus infection is characterized by restricted tissue tropism. To determine the pattern of poliovirus tissue tropism in PVR transgenic mice, PVR gene expression and susceptibility to poliovirus infection were examined by in situ hybridization. PVR RNA is expressed in transgenic mice at high levels in neurons of the central and peripheral nervous system, developing T lymphocytes in the thymus, epithelial cells of Bowman's capsule and tubules in the kidney, alveolar cells in the lung, and endocrine cells in the adrenal cortex, and it is expressed at low levels in intestine, spleen, and skeletal muscle. After infection, poliovirus replication was detected only in neurons of the brain and spinal cord and in skeletal muscle. These results demonstrated that poliovirus tissue tropism is not governed solely by expression of the PVR gene nor by accessibility of cells to virus. Although transgenic mouse kidney tissue expressed poliovirus binding sites and was not a site of poliovirus replication, when cultivated in vitro, kidney cells developed susceptibility to infection. Identification of the changes in cultured kidney cells that permit poliovirus infection may provide information on the mechanism of poliovirus tissue tropism.     

A mouse model for poliovirus neurovirulence identifies mutations that attenuate the virus for humans.

A mutation in the genome of poliovirus type 3 that is known to reduce neurovirulence in humans similarly reduces neurovirulence in mice when incorporated into a mouse-adapted-human poliovirus recombinant. Viral recombinants with a uracil at nucleotide position 472 in the 5'-noncoding regions of their genomes are unable to replicate in the mouse brain. Viral recombinants with a cytosine at this position are neurovirulent in mice. Neurovirulence of poliovirus in mice may therefore prove to be a useful indicator of the genetic stability of new attenuating mutations created by site-directed mutagenesis.     

Alcohol and genetics: new animal models.

In recent years, it has become increasingly evident that there is a genetic component to alcoholism. Attempts to isolate alcoholism genes have met with modest success, in part because alcoholism is a multigenic trait. Recently, experimental animal models and novel genetic manipulations have provided several clues as to the specific genes involved in alcoholism, and extensive research has identified many genes that might influence responses to alcohol. Although not all of these might be proven to influence drug sensitivity, research has provided evidence for the involvement of a few genes. Ultimately, findings from animal models that investigate the function of specific genes could aid the development of pharmacotherapies to treat alcohol dependence.     

The neurovirulence of human and animal rotaviruses in cercopithecus monkeys

Cercopithecus monkeys were inoculated according to the specifications of neurovirulence safety test for live rubella virus vaccine with RIT 4237, a rotavirus vaccine candidate. RIT 4237 is a high passage level of the Nebraska Calf Diarrhoea Virus (NCDV). The histological findings in the first test indicated some involvement of the central nervous system. The same test was therefore repeated with RIT 4237, with a lower passage level of the NCDV strain, and with the 'Wa' strain, a human virus grown in tissue culture. Clinical signs and histological findings were concordant and demonstrated that all the viruses were moderately neurovirulent. As in the poliovirus neurovirulence test, the histological lesions depended mainly upon a correct inoculation in the lumbar cord. RIT 4237 was found to have the same degree of neurovirulence as the low-passage NCDV or as the 'Wa' strain.     

Transgenic mice carrying a tetracycline-inducible, truncated transforming growth factor beta receptor (TbetaRII)

The transforming growth factor-betas (TGFbetas) have multiple roles, making genetic analysis of their functions difficult. We therefore developed transgenic mouse lines to disrupt TGFbeta signaling using a mechanism that is inducible, reversible, and cell-type specific. The transgenic mouse lines carry an EGFP-pBi-DeltaTbetaRII construct (PTR). The DeltaTbetaRII element codes for a dominant-negative receptor that is known to disrupt TGFbeta signaling. The DeltaTbetaRII has a c-myc tag. The transgene was silent in the PTR mice, with expression of both EGFP and DeltaTbetaRII occurring when the PTR mice were crossed with mice that express the tetracycline transactivator (CMV-tTA). The expression of EGFP was repressed by the addition of doxycycline to the drinking water of the PTRxCMV-tTA mice. The PTR mice were then crossed with neuron-specific-tTA mice. Expression of the DeltaTbetaRII transgene in these mice led to an upregulation of native TGFbeta receptor expression, suggesting that neurons can modulate their responsiveness to TGFbetas.     

Osteopenic mice: animal models of the aging skeleton

While our understanding of the developmental biology of the skeleton, like that of virtually every other subject in biology, has been transformed by recent advances in human and mouse genetics, we still know very little, in molecular and genetic terms, about skeletal physiology. Thus, among the many questions that are largely unexplained are the following: why is osteoporosis mainly a women's disease? How is bone mass maintained nearly constant between the end of puberty and the arrest of gonadal functions? Molecular genetics has emerged as a powerful tool to study previously unexplored aspects of the physiology of the skeleton. Among mammals, mice are the most promising animals for this experimental work. This has been previously demonstrated e.g. through the tremendous impact of the different osteopetrotic models on our molecular understanding of osteoclastic bone resorption. Until recently the only way of studying bone loss situations and osteoporosis in mice was by using ovariectomy with all its limitations. Today, however, we have access to more sophisticated osteoporotic mouse-models from four different origins: Transgenic mice (HSV-TK), knock-out mice (OPG), inbred-strains (SAMP6), and through physiological modulation (icv application). These new models have already taught us several important lessons. The first is, that bone remodeling is more than just an autocrine/paracrine process. Multiple experimental evidence has demonstrated that the latter regulation exists, but genetics prove that there is no functional cross-control between resorption and formation. The second lesson is, that remodeling is, at least in part, subject to central regulation. Thus, osteoporosis is partly a central or hypothalamic disease. However, the most dramatic change and the most important advantage we feel is, that today we have models to test a new hypothesis regarding the etiology of osteoporosis before it turns to dogma. Taken together, mouse-studies may lead to a shift in our physiological understanding of skeleton biology and to the emergence of novel paradigms. These, in turn, should help us to devise new treatments for degenerative diseases of the skeleton such as osteoporosis and its associated clinical problems.     

Transgenic models to study disorders of respiratory control in newborn mice

Recent studies described the in vivo respiratory phenotype of mutant newborn mice with targeted deletions of genes involved in respiratory control development. Whole-body flow barometric plethysmography is the noninvasive method of choice for studying unrestrained newborn mice. The main characteristics of the early postnatal development of respiratory control in mice are reviewed, including available data on breathing patterns and on hypoxic and hypercapnic ventilatory responses. Mice are very immature at birth, and their instable breathing is similar to that of preterm infants. Breathing pattern abnormalities with prolonged apneas occur in newborn mice that lack genes involved in the development of rhythmogenesis. Some mutant newborn mice have blunted hypoxic and hypercapnic ventilatory responses whereas others exhibit impairments in responses to hypoxia or hypercapnia. Furthermore, combined studies in mutant newborn mice and in humans have helped to provide pathogenic information on genetically determined developmental disorders of respiratory control in humans.     

Transgenic animals as models for human disease

Transgenic animals, especially mice, have been used quite extensively as models for various human diseases. At first, the level of scientific inquiry was driven by the need to establish the model. In many cases, these models may be considered quite crude because of their limitations. More recently, transgenic models of disease have become more refined and are currently being used to study the pathological mechanisms behind the disease rather than to just provide a model of the disease. Using some examples from the recent literature, we will document the current level and complexity of inquiry using transgenic animals. New techniques and techniques that may prove promising will be discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Keratin genes, epidermal differentiation and animal models for the study of human skin diseases.

The examples shown here illustrate the power of gene targeting to the epidermis as a means of developing animal models for the study of human skin diseases. In this short review, I have focused on contributions which stem predominantly from my own laboratory [31, 32, 34, 37, 47]. However, other laboratories have also contributed heavily to the development of this technology [28-30, 35, 36]. The opportunities for these models are vast: there are a myriad of human skin diseases which have been characterized extensively at a biological level, but whose aetiology is presently unknown. A combined knowledge of (a) the biochemistry of epidermal differentiation; (b) epidermal-specific gene expression; and (c) transgenic mouse technology has provided the foundation for these and future studies in this area.     

Mutational analysis of the cellular receptor for poliovirus.

To identify sequences of the cellular poliovirus receptor (PVR) involved in viral infection, mutant PVR cDNAs were constructed and assayed for biological activity in mouse L cells. To confirm that mutant PVRs reached the cell surface, an immunological tag, consisting of part of CH3 from human immunoglobulin G1, was engineered into the PVR. Deletion of PVR amino acids 256 to 320 or 385 to the carboxy terminus yielded receptors that were able to support poliovirus infection. PVRs lacking amino acids 40 to 136 or 137 to 256 were expressed at the cell surface but were not active as receptors for poliovirus. The results show that immunoglobulin-type domain 3 and the extreme carboxy terminus of the PVR are not required for viral receptor function, but sequences within the two amino-terminal domains contribute to the initiation of poliovirus infection.     

Biological basis of rabies virus neurovirulence in mice: comparative pathogenesis study using the immunoperoxidase technique.

The CVS strain of fixed rabies virus causes acute, fatal encephalomyelitis in young adult ICR mice. Variant RV194-2, which was selected from CVS virus in cell culture with a neutralizing antiglycoprotein monoclonal antibody, has a single amino acid change in the glycoprotein. The infections caused by CVS virus and RV194-2 virus were compared in mice for 14 days postinoculation of 5 x 10(7) PFU into the right masseter muscle. All CVS virus-infected mice died (mean time to death, 7.9 days), compared with a mortality rate of 8.5% for RV194-2 virus-infected mice. RV194-2 virus spread to the ipsilateral trigeminal ganglion during the first 2 days postinoculation, and both viruses spread to the ipsilateral motor nucleus of the trigeminal nerve in the pons. Both viruses spread centrifugally and caused infection of bilateral trigeminal ganglia on day 3. The viruses spread throughout the central nervous system (CNS) at similar rates, but CVS virus infected many more neurons than did RV194-2 virus. Rabies virus antigen was observed in only occasional CNS neurons after day 6 of RV194-2 virus infection. By this time, CVS virus had caused severe widespread infection. In this model, virulence depends on improved efficiency of viral spread between CNS neurons rather than the rate of spread or topographical distribution of the infection.