Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage.

The Nairoviridae family of the Bunyavirales order comprises tick-borne, trisegmented, negative-strand RNA viruses, with several members being associated with serious or fatal diseases in humans and animals. A notable member is Crimean-Congo hemorrhagic fever virus (CCHFV), which is the most widely distributed tick-borne pathogen and is associated with devastating human disease, with case fatality rates averaging 30%. Hazara virus (HAZV) is closely related to CCHFV, sharing the same serogroup and many structural, biochemical, and cellular properties. To improve understanding of HAZV and nairovirus multiplication cycles, we developed, for the first time, a rescue system permitting efficient recovery of infectious HAZV from cDNA. This system now allows reverse genetic analysis of nairoviruses without the need for high-level biosafety containment, as is required for CCHFV. We used this system to test the importance of a DQVD caspase cleavage site exposed on the apex of the HAZV nucleocapsid protein arm domain that is cleaved during HAZV infection, for which the equivalent DEVD sequence was recently shown to be important for CCHFV growth in tick but not mammalian cells. Infectious HAZV bearing an uncleavable DQVE sequence was rescued and exhibited growth parameters equivalent to those of wild-type virus in both mammalian and tick cells, showing this site was dispensable for virus multiplication. In contrast, substitution of the DQVD motif with the similarly uncleavable AQVA sequence could not be rescued despite repeated efforts. Together, these results highlight the importance of this caspase cleavage site in the HAZV life cycle but reveal the DQVD sequence performs a critical role aside from caspase cleavage.IMPORTANCE HAZV is classified within the Nairoviridae family with CCHFV, which is one of the most lethal human pathogens in existence, requiring the highest biosafety level (BSL) containment (BSL4). In contrast, HAZV is not associated with human disease and thus can be studied using less-restrictive BSL2 protocols. Here, we report a system that is able to rescue HAZV from cDNAs, thus permitting reverse genetic interrogation of the HAZV replication cycle. We used this system to examine the role of a caspase cleavage site, DQVD, within the HAZV nucleocapsid protein that is also conserved in CCHFV. By engineering mutant viruses, we showed caspase cleavage at this site was not required for productive infection and this sequence performs a critical role in the virus life cycle aside from caspase cleavage. This system will accelerate nairovirus research due to its efficiency and utility under amenable BSL2 protocols.

Hazara nairovirus elicits differential induction of apoptosis and nucleocapsid protein cleavage in mammalian and tick cells.

The Nairoviridae family within the Bunyavirales order comprise tick-borne segmented negative-sense RNA viruses that cause serious disease in a broad range of mammals, yet cause a latent and lifelong infection in tick hosts. An important member of this family is Crimean-Congo haemorrhagic fever virus (CCHFV), which is responsible for serious human disease that results in case fatality rates of up to 30 %, and which exhibits the most geographically broad distribution of any tick-borne virus. Here, we explored differences in the cellular response of both mammalian and tick cells to nairovirus infection using Hazara virus (HAZV), which is a close relative of CCHFV within the CCHFV serogroup. We show that HAZV infection of human-derived SW13 cells led to induction of apoptosis, evidenced by activation of cellular caspases 3, 7 and 9. This was followed by cleavage of the classical apoptosis marker poly ADP-ribose polymerase, as well as cellular genome fragmentation. In addition, we show that the HAZV nucleocapsid (N) protein was abundantly cleaved by caspase 3 in these mammalian cells at a conserved DQVD motif exposed at the tip of its arm domain, and that cleaved HAZV-N was subsequently packaged into nascent virions. However, in stark contrast, we show for the first time that nairovirus infection of cells of the tick vector failed to induce apoptosis, as evidenced by undetectable levels of cleaved caspases and lack of cleaved HAZV-N. Our findings reveal that nairoviruses elicit diametrically opposed cellular responses in mammalian and tick cells, which may influence the infection outcome in the respective hosts.

Cerebroretinal microangiopathy with calcifications and cysts (CRMCC).

Extensive intracranial calcifications and leukoencephalopathy are seen in both Coats plus and leukoencephalopathy with calcifications and cysts (LCC; Labrune syndrome). Coats plus syndrome is additionally characterized by the presence of bilateral retinal telangiectasia and exudates while LCC shows the progressive formation of parenchymal brain cysts. Despite these apparently distinguishing features, recent evidence suggests that Coats plus and LCC represent the same clinical entity with a common primary pathogenesis involving a small vessel obliterative microangiopathy. Here, we describe eight previously unreported cases, and present an update on one of the original Coats plus patients to highlight the emerging core clinical features of the "cerebroretinal microangiopathy with calcification and cysts" (CRMCC) phenotype.

A severity scoring tool to assess the neurological features of neuronopathic Gaucher disease.

Type III Gaucher disease is one of the three recognized subtypes of Gaucher disease, an inherited deficiency of lysosomal glucocerebrosidase. Phenotypically there is a wide spectrum of visceral and neurological manifestations. Enzyme replacement is effective in managing the visceral disease; however, the neurological manifestations remain a more challenging obstacle. There is an unfulfilled need to reliably monitor neurological disease and its response to treatment. A severity scoring tool was developed through neurological domain identification, item generation and tool formation. Domain identification was established based on a retrospective single centre study (n = 15) and a systematic review of publications. Forty-seven patients with neuronopathic Gaucher disease were then assessed using the tool to establish the clinical and statistical reliability of each domain. Judgement quantification of the tool was established through a process of content validity involving five European experts. Content validity is considered to be most effective when undertaken systematically. Concurrent validity and feasibility of the tool was also highlighted. This process allowed a revised and validated version of the tool to be developed.

Guideline for the diagnosis and management of glutaryl-CoA dehydrogenase deficiency (glutaric aciduria type I).

Glutaryl-CoA dehydrogenase (GCDH) deficiency is an autosomal recessive disease with an estimated overall prevalence of 1 in 100 000 newborns. Biochemically, the disease is characterized by accumulation of glutaric acid, 3-hydroxyglutaric acid, glutaconic acid, and glutarylcarnitine, which can be detected by gas chromatography-mass spectrometry of organic acids or tandem mass spectrometry of acylcarnitines. Clinically, the disease course is usually determined by acute encephalopathic crises precipitated by infectious diseases, immunizations, and surgery during infancy or childhood. The characteristic neurological sequel is acute striatal injury and, subsequently, dystonia. During the last three decades attempts have been made to establish and optimize therapy for GCDH deficiency. Maintenance treatment consisting of a diet combined with oral supplementation of L: -carnitine, and an intensified emergency treatment during acute episodes of intercurrent illness have been applied to the majority of patients. This treatment strategy has significantly reduced the frequency of acute encephalopathic crises in early-diagnosed patients. Therefore, GCDH deficiency is now considered to be a treatable condition. However, significant differences exist in the diagnostic procedure and management of affected patients so that there is a wide variation of the outcome, in particular of pre-symptomatically diagnosed patients. At this time of rapid expansion of neonatal screening for GCDH deficiency, the major aim of this guideline is to re-assess the common practice and to formulate recommendations for diagnosis and management of GCDH deficiency based on the best available evidence.

The aetiology of neurological complications of organic acidaemias--a role for the blood-brain barrier.

The blood-brain barrier (BBB) metabolically isolates the central nervous system (CNS) from the circulation and protects it against fluctuations of hydrophilic nutrients in plasma and from intoxication. Recent studies have shown that dicarboxylic acids (DCAs) are transported across the blood-brain barrier at very low rates. In organic acidaemias, neurological complications are common. We hypothesize that, as a result of the very limited efflux, in certain organic acidaemias there is pathological accumulation of DCAs (e.g. glutarate, 3-hydroxyglutarate, D-2- and L-2-hydroxyglutarate, methylmalonate) in the brain secondary to the metabolic block. At high concentrations some of these compounds may become neurotoxic. Treatment should be aimed at preventing the accumulation of these compounds using our understanding of the properties of the BBB.

GAMT deficiency: features, treatment, and outcome in an inborn error of creatine synthesis.

BACKGROUND: Guanidinoactetate methyltransferase (GAMT) deficiency is an autosomal recessive disorder of creatine synthesis. The authors analyzed clinical, biochemical, and molecular findings in 27 patients. METHODS: The authors collected data from questionnaires and literature reports. A score including degree of intellectual disability, epileptic seizures, and movement disorder was developed and used to classify clinical phenotype as severe, moderate, or mild. Score and biochemical data were assessed before and during treatment with oral creatine substitution alone or with additional dietary arginine restriction and ornithine supplementation. RESULTS: Intellectual disability, epileptic seizures, guanidinoacetate accumulation in body fluids, and deficiency of brain creatine were common in all 27 patients. Twelve patients had severe, 12 patients had moderate, and three patients had mild clinical phenotype. Twenty-one of 27 (78%) patients had severe intellectual disability (estimated IQ 20 to 34). There was no obvious correlation between severity of the clinical phenotype, guanidinoacetate accumulation in body fluids, and GAMT mutations. Treatment resulted in almost normalized cerebral creatine levels, reduced guanidinoacetate accumulation, and in improvement of epilepsy and movement disorder, whereas the degree of intellectual disability remained unchanged. CONCLUSION: Guanidinoactetate methyltransferase deficiency should be considered in patients with unexplained intellectual disability, and urinary guanidinoacetate should be determined as an initial diagnostic approach.

Disorders of intermediary metabolism: toxic leukoencephalopathies.

Myelination starts in the latter half of gestation. It is initiated by oligodendrocyte progenitor cells. Three sequential steps can be distinguished: (1) initial ensheathment of axons by premyelin sheaths generated by oligodendrocyte progenitor cells; (2) initial insertion of myelin basic protein (MBP) into transitional sheaths; and (3) generation of mature MBP-rich myelin. Different inborn errors of metabolism can interfere with different stages of these physiological processes, causing white-matter diseases, i.e. toxic leukoencephalopathies. Some inborn errors of metabolism disturb the formation of myelin by being toxic to oligodendrocytes or by interference with the biosynthesis of cholesterol and lipids, e.g. globoid cell leukodystrophy and phenylketonuria. Remethylation defects, e.g. methylenetetrahydrofolate reductase deficiency, cobalamin C, D, E, F and G defects, interfere with the expression, processing and insertion of MBP. The concept of excitotoxicity, which has been developed in neurons, has recently been modified and has been extended to the oligodendroglial lineage. Mitochondriopathies and cerebral organic acid disorders may cause secondary excitotoxicity resulting in toxic encephalopathies, which may affect both neurons and oligodendrocytes. This review aims to present relevant diseases, summarizing recent knowledge on mechanisms and formulating testable hypotheses of pathophysiology leading to new and improved treatment strategies.

Glutaric aciduria type I and kynurenine pathway metabolites: a modified hypothesis.

Glutaric aciduria type I is an inborn error of organic acid metabolism that demonstrates a particular temporal vulnerability (acute encephalopathic episodes in infancy) and a spatial vulnerability (acute striatal necrosis, focused on the putamen). Excitotoxic mechanisms involving 3-hydroxyglutaric acid as the major neurotoxin have been suggested. This paper proposes a role for metabolites of the kynurenine pathway in the pathogenic process and modifies the hypothesis of Heyes. Deficiency of glutaryl-CoA dehydrogenase blocking the glutarate pathway and activation of indoleamine 2,3-dioxygenase in macrophages/monocytes by intercurrent inflammation may increase flux down the kynurenine pathway towards the production of quinolinic acid. Quinolinic acid is neurotoxic and is an endogenous agonist at N-methyl-D-aspartate receptors. Synergistic excitation of these receptors by quinolinic acid and 3-hydroxyglutaric acid, which alone does not have sufficient potency, may be involved in the pathogenesis of striatal necrosis.

Dyskinesias and associated psychiatric disorders following streptococcal infections.

BACKGROUND: The classical extrapyramidal movement disorder following beta haemolytic streptococcus (BHS) infection is Sydenham's chorea (SC). Recently, other post-streptococcal movement disorders have been described, including motor tics and dystonia. Associated emotional and behavioural alteration is characteristic. AIMS: To describe experience of post-streptococcal dyskinesias and associated co-morbid psychiatric features presenting to a tertiary referral centre 1999-2002. METHODS: In all patients, dyskinetic movement disorders followed BHS pharyngeal infection. BHS infection was defined by pharyngeal culture of the organism, or paired streptococcal serology. Movement disorders were classified according to international criteria, and validated by experienced child neurologists. Psychiatric complications were defined using ICD-10 criteria using a validated psychiatric interview. RESULTS: In the 40 patients, the following dyskinetic movement disorders were present: chorea (n = 20), motor tics (n = 16), dystonia (n = 5), tremor (n = 3), stereotypies (n = 2), opsoclonus (n = 2), and myoclonus (n = 1). Sixty five per cent of the chorea patients were female, whereas 69% of the tic patients were male. ICD-10 psychiatric diagnoses were made in 62.5%. Using the same psychiatric instrument, only 8.9% of UK children would be expected to have an ICD-10 psychiatric diagnosis. Emotional disorders occurred in 47.5%, including obsessive-compulsive disorder (27.5%), generalised anxiety (25%), and depressive episode (17.5%). Additional psychiatric morbidity included conduct disorders (27.5%) and hyperkinetic disorders (15%). Psychiatric, movement, and post-streptococcal autoimmune disorders were commonly observed in family members. At a mean follow up of 2.7 years, 72.5% had continuing movement and psychiatric disorders. CONCLUSION: Post-streptococcal dyskinesias occur with significant and disabling psychiatric co-morbidity and are potential autoimmune models of common "idiopathic" movement and psychiatric disorders in children. Multiple factors may be involved in disease expression including genetic predisposition, developmental status, and the patient's sex.

The effect of genotype on the natural history of eIF2B-related leukodystrophies.

BACKGROUND: Recessive mutations in the five eucaryotic initiation factor 2B (eIF2B) subunits have been found in leukodystrophies of variable age at onset and severity. OBJECTIVES: To evaluate the clinical spectrum of eIF2B-related disorders and search for a phenotype-genotype correlation. METHODS: Ninety-three individuals (78 families) with an undetermined leukodystrophy were selected on MRI-based criteria of childhood ataxia with central hypomyelination/vanishing white matter (CACH/VWM) for EIF2B genes analysis. RESULTS: Eighty-nine percent of individuals with MRI criteria of CACH/VWM have a mutation in one of the eIF2B beta to epsilon subunits. For 83 individuals (68 families), 46 distinct mutations (90% missense) in four of the five eIF2B subunits (beta, gamma, delta, epsilon) were identified. Sixty-four percent were in the epsilon subunit, a R113H substitution was found in 71% of eIF2B epsilon-mutated families. A large clinical spectrum was observed from rapidly fatal infantile to asymptomatic adult forms. Disease severity was correlated with age at onset (p < 0.0001) but not with the type of the mutated subunit nor with the position of the mutation within the protein. Mutations R113H in the epsilon subunit and E213G in the beta subunit were significantly associated with milder forms. CONCLUSIONS: The degree of eIF2B dysfunction, which is involved in the regulation of protein synthesis during cellular stress, may play a role in the clinical expression of eIF2B-related disorders.

Infantile Parkinsonism-dystonia and elevated dopamine metabolites in CSF.

Two girls and one boy are described, with severe infantile parkinsonism-dystonia. This syndrome is usually caused by endogenous dopamine deficiency but in these patients was associated with elevated dopamine metabolites in CSF and an unusual eye movement disorder: ocular flutter together with saccade initiation failure. Pyramidal tract signs also emerged in the course of the disease in two patients. This combination of symptoms and biochemical findings suggests a unique pathogenic mechanism.

A comparison between parents' and therapists' views of their child's individual seating systems.

Children with cerebral palsy are often prescribed adaptive seating systems for use in their wheelchairs for the purposes of improving posture and to help prevent the development of long-term deformity. However, clinical experience indicates that parents and the therapists who advocate the use of these systems do not always agree about the wheelchairs. This study discusses the development of questionnaires for both parents and therapists to measure differences in their opinions about the wheelchairs. The questions were developed through clinical experience, validation was through interviews to discuss topics important to the participants, and repeated application of the questionnaire ensured consistency. The reliability of the questions appears satisfactory and the interview responses demonstrate that the questions selected are important to both groups of stakeholders. However, it was found that parental concerns over their children's seating systems concentrated on functional and day-to-day management issues, whereas therapist concerns focused on technical issues and postural management. Both groups of stakeholders agreed that the questionnaires would be a useful precursor to attending a seating clinic appointment, as it could aid communication between the parent and provider and improve the efficiency and satisfaction of such an appointment. It appears the questionnaire has potential as an outcome measurement tool.

Neurological outcome of patients with ornithine carbamoyltransferase deficiency.

BACKGROUND: Ornithine carbamoyltransferase (OCT) deficiency is the commonest of the inherited urea cycle disorders. AIMS: To determine the long term neurological and cognitive outcome of continuously treated surviving patients. METHODS: Twenty eight surviving children (five boys) with OCT deficiency who had been treated continuously with a low protein diet and alternative pathway therapy were identified. Those aged 5-16 years had a detailed neurological examination and psychometric testing. RESULTS: Four presented in the neonatal period and four were treated prospectively following antenatal diagnosis. Median (range) age at diagnosis for the later onset group was 19 (2-144) months; median time between onset of symptoms and diagnosis was 10 (2-48) months. Nine children had had less than three episodes of hyperammonaemic encephalopathy, the others more. Seven had focal abnormalities on neurological examination; 14 had global cognitive impairment; four had a normal IQ but specific learning difficulties. Sixteen underwent neuroimaging which was normal in three, showed focal abnormalities of the cerebral hemispheres in six, and global cerebral atrophy in seven. CONCLUSION: Eighteen of 28 surviving children with OCT deficiency had disabling neurological complications. Plasma ammonia at diagnosis was the only factor that predicted this outcome. While most neurological complications could be attributed to hyperammonaemic encephalopathy, other mechanisms may also contribute to the neurological abnormalities.

Infantile Alexander disease: spectrum of GFAP mutations and genotype-phenotype correlation.

Heterozygous, de novo mutations in the glial fibrillary acidic protein (GFAP) gene have recently been reported in 12 patients affected by neuropathologically proved Alexander disease. We searched for GFAP mutations in a series of patients who had heterogeneous clinical symptoms but were candidates for Alexander disease on the basis of suggestive neuroimaging abnormalities. Missense, heterozygous, de novo GFAP mutations were found in exons 1 or 4 for 14 of the 15 patients analyzed, including patients without macrocephaly. Nine patients carried arginine mutations (four had R79H; four had R239C; and one had R239H) that have been described elsewhere, whereas the other five had one of four novel mutations, of which two affect arginine (2R88C and 1R88S) and two affect nonarginine residues (1L76F and 1N77Y). All mutations were located in the rod domain of GFAP, and there is a correlation between clinical severity and the affected amino acid. These results confirm that GFAP mutations are a reliable molecular marker for the diagnosis of infantile Alexander disease, and they also form a basis for the recommendation of GFAP analysis for prenatal diagnosis to detect potential cases of germinal mosaicism.

Diagnostic dilemmas in four infants with nephrotic syndrome, microcephaly and severe developmental delay.

We present four cases with nephrotic syndrome, microcephaly and severe developmental delay. In the differential diagnosis the Galloway-Mowat syndrome, PEHO syndrome, ARC syndrome and the carbohydrate-deficient glycoprotein (CDG) syndrome are considered and discussed. One case may fall into the Galloway-Mowat spectrum and another case was diagnosed with the CDG syndrome. This case is the third report of a nephrotic syndrome as a part of the CDG syndrome. Two remaining cases with cerebellar and brain stem atrophy, and without major histopathological changes in the kidney were left without a definite unifying diagnosis and may well represent a different unknown condition. Although microcephaly and nephrotic syndrome with or without hiatus hernia has been equated with Galloway-Mowat syndrome in the literature, the brain and renal pathology in these reported cases has been very variable. It is likely that this group as a whole is aetiologically heterogeneous.

Homozygous thermolabile variant of the methylenetetrahydrofolate reductase gene: a potential risk factor for hyperhomocysteinaemia, CVD, and stroke in childhood.

In this study of 118 children (median age 5.1 years; range 6 months to 17 years) with ischaemic stroke or transient ischaemic attack (TIA), 22 children (19%) were homozygous for the thermolabile variant of the methylenetetrahydrofolate reductase allele (t-MTHFR), compared with nine of 78 (12%) of a reference population (p=0.18, OR 1.76, 95% CI 0.76 to 4.04). Of those with cerebrovascular disease (CVD), 17 of 84 were homozygous for the t-MTHFR allele (p=0.13 compared with the reference population (OR 1.95, 95% CI 0.81 to 4.65). There was a significant (p<0.025) increment of plasma total homocysteine concentration in homozygotes for the t-MTHFR allele compared with heterozygotes, negatives for the t-MTHFR allele, and control children with no history of stroke. In four of 12 homozygotes for the t-MTHFR allele, plasma homocysteine levels were raised, compared with three of 38 of those who were negative or heterozygous (p=0.047; OR 5.8, 95% CI 1.1 to 31.2). Homozygotes for the t-MTHFR allele were significantly more likely to have a recurrent event than those who were negative or heterozygous (Cox regression p=0.031, hazard ratio 2.18, 95% CI 1.08 to 4.42). These data suggest that homozygosity for the t-MTHFR allele is associated with raised homocysteine levels in children and is a risk factor for primary and secondary stroke and TIA.

How broad is the phenotype of Hallervorden-Spatz disease?

Magnetic resonance imaging (MRI) has enabled ante mortem diagnosis of Hallervorden Spatz disease (HSD). Childhood-onset cases are the most common type and usually present with progressive dystonia and dementia. The duration of illness is 15 to 20 years, leading to death. Presentation in adulthood and infancy have also been reported, however again the progression is usually inexorable. We present a 30-year-old woman who developed cognitive and motor developmental delay from the age of 8 months. There was further cognitive decline in her late teenage years with seizures and then more recent motor decline with dystonia. The imaging appearance was of iron deposition in the globus pallidus and substantia nigra leading to a diagnosis of HSD. The increased availability of MRI has allowed more cases of HSD to be diagnosed in life but as our case illustrates classification of the disease may need to be further examined.