Genetic variations from successive whole genome sequencing during COVID-19 treatment in five individuals.

We report multiple single nucleotide polymorphism taken at different time interval during treatment of COVID-19. Gene sequencing showed mutation within ORF1b at position P314L. Mutation at this point has been shown to impose structural remodelling that increases the affinity for remdesivir binding and may also affect binding affinity for favipiravir.

Evaluating human rabies control in Asia: using 'One Health' principles to assess control programmes for rabies.

Rabies is a global issue, and kills tens of thousands of people every year, despite being a preventable disease. The goal of the World Health Organization (WHO) and the World Organisation for Animal Health (OIE) to eliminate dog-mediated rabies by 2030 requires serious collaborative efforts across Ministries and countries. This cross-sector cooperation includes, but is not limited to, physicians, veterinarians, local authorities, local communities and the media; in other words, a 'One Health' approach. Countries where human rabies has been successfully eliminated still require constant vigilance, in terms of regulations on companion animal ownership and vaccination, the movement of animals from countries where the disease still exists, and the continued raising of public awareness. Rabies-endemic countries need to refocus their efforts towards eliminating the disease once and for all, by working together and sharing their knowledge, experiences and efforts. Rabies control programmes require the efficient management of resources, and surveillance programmes do not need to be specific to one disease. Governments must play a more proactive role by enforcing legislation and allocating funds and resources to help to eliminate human rabies by 2030 throughout the world.

La rage est un problème mondial qui fait des dizaines de milliers de victimes humaines chaque année alors même qu'elle est une maladie évitable. L'objectif que se sont fixé l'Organisation mondiale de la santé (OMS) et l'Organisation mondiale de la santé animale (OIE) d'éliminer la rage transmise par les chiens d'ici 2030 nécessite qu'une véritable collaboration soit en place entre ministères et entre pays différents. Cette coopération intersectorielle comprend la participation de médecins et de vétérinaires ainsi que des administrations et communautés locales et des médias, entre autres intervenants ; autrement dit, il s'agit d'une approche « Une seule santé ¼. Les pays ayant réussi à éliminer la rage humaine doivent continuer à exercer une vigilance permanente, en réglementant la possession et la vaccination d'animaux de compagnie, en surveillant les mouvements d'animaux provenant de pays où la maladie est toujours présente et en sensibilisant sans relâche les populations. Les pays où la rage est endémique doivent recentrer leurs efforts sur l'élimination totale et définitive de la maladie en collaborant entre eux et en partageant leurs connaissances, leurs expériences et leurs efforts. Les programmes de lutte contre la rage nécessitent une gestion efficiente des ressources tandis que les programmes de surveillance ne sont pas nécessairement spécifiques à une seule maladie. Les gouvernements doivent jouer un rôle plus proactif aussi bien en matière de législation que d'allocation des fonds et des ressources afin de contribuer à l'élimination de la rage humaine dans le monde d'ici 2030.

La rabia es un problema planetario, una enfermedad que, pese a ser prevenible, mata a decenas de miles de personas al año. El objetivo que persiguen la Organización Mundial de la Salud (OMS) y la Organización Mundial de Sanidad Animal (OIE), esto es, haber eliminado la rabia transmitida por perros para 2030, exige una seria labor de colaboración entre ministerios y países. En esta cooperación intersectorial deben participar, entre otras instancias, médicos, veterinarios, autoridades locales, comunidades locales y medios de comunicación, lo que supone, dicho de otro modo, aplicar los planteamientos de «Una sola salud¼. Los países donde ya se ha logrado eliminar la rabia humana siguen requiriendo una vigilancia constante, que se plasma en la aplicación de reglamentos sobre la tenencia y vacunación de animales de compañía y el movimiento de animales desde países donde aún existe la enfermedad, junto con una continua labor de sensibilización pública. Los países con rabia endémica deben reorientar sus esfuerzos para eliminar la enfermedad de una vez por todas, trabajando conjuntamente y compartiendo conocimientos, experiencias y actividades. Los programas de lucha antirrábica requieren una gestión eficiente de los recursos, y en este sentido no es preciso que los programas de vigilancia estén centrados específicamente en una sola enfermedad. Los gobiernos deben cumplir una función más activa y resolutiva, haciendo cumplir las leyes y destinando fondos y recursos al objetivo de haber eliminado la rabia humana en todo el mundo para 2030.

Measurement of urinary chemokine and growth factor messenger RNAs: a noninvasive monitoring in lupus nephritis.

Noninvasive molecular tests of urine cells have been developed to monitor the activity of kidney diseases. We evaluate whether measurement of urinary messenger RNA (mRNA) levels of chemokine and growth factor genes could distinguish between diffuse proliferative lupus nephritis (class IV LN) and others and whether it is able to predict the response to therapy. Prebiopsy urine samples were collected from 26 LN patients. Urine specimens were serially collected over a period of 6 months from class IV LN patients who were receiving standard immunosuppressive treatments. Urinary interferon-producing protein 10 and its CXC chemokine receptor (CXCR)3, transforming growth factor-beta (TGF-beta), and vascular endothelial growth factor (VEGF) mRNA levels were analyzed by quantitative real-time polymerase chain reactions. Levels of chemokine or growth factor mRNAs in urine could distinguish class IV LN from others, with a sensitivity of 85% and a specificity of 94%. The receiver-operative characteristic curve demonstrated that urine mRNA levels of these genes could identify active class IV LN with an accuracy greater than the current available clinical markers, namely systemic lupus erythematosus (SLE) disease activity index, proteinuria, renal function, or urinalysis. A significant reduction of interferon-producing protein 10 (IP-10), CXCR3, TGF-beta, and VEGF mRNA levels from baselines was observed in patients who responded to therapy, whereas the levels tended to increase in those who resisted to treatment. Measurement of urinary chemokine and growth factor mRNAs can precisely distinguish class IV LN from others. Temporal association between these markers and therapeutic response is demonstrated. This noninvasive approach serves as a practical tool in diagnosis and management of LN.

Rabies encephalitis following fox bite--histological and immunohistochemical evaluation of lesions caused by virus.

Rabies caused by fox bite is uncommon, most cases being caused by bite of rabid dogs (95%). We report a 45-year-old lady with rabies encephalomyelitis caused by bite of a rabid wild fox (Vulpes vulpes), a species prevalent in the Deccan plateaus of Central India. Though foxes are known to be susceptible to rabies, literature on the pathological changes caused by fox bite rabies in humans is scarce. Unlike the mild histological alterations described in canine rabies, a florid encephalitic process evolved in fox bite rabies, in our case, with intense microglial reaction, neuronophagia and perivascular inflammatory infiltrates despite clinical manifestation as a paralytic rabies. Immunostaining using polyclonal antibodies to the rabies viral nucleocapsid antigen and to the whole virion demonstrated high viral load within neurons with extensive spread along dendritic arborization and axonal tracts. Genomic sequence analysis demonstrated close homology with canine virus strain with only minor variations.

Nucleic-acid sequence based amplification in the rapid diagnosis of rabies.

Current serological tests do not reliably diagnose rabies. We describe a technique based on amplification of nucleic-acid sequences to detect rabies-specific RNA in the saliva and cerebrospinal fluid (CSF) of four living patients with rabies. Rabies RNA could be detected in either saliva or CSF, or both, in all patients and as early as day 2 after onset of symptoms. Both saliva and CSF should be serially tested because not every sample can be expected to be positive. The whole process, including automated extraction, isothermal amplification, and detection can be done within 4 h.

B-cell responses to myelin basic protein and its epitopes in autoimmune encephalomyelitis induced by Semple rabies vaccine.

Semple rabies vaccine is composed of rabies virus-infected sheep or goat brain inactivated with phenol and is administered daily after exposure for 14-21 days. Semple rabies vaccine-induced autoimmune encephalomyelitis (SAE) has clinico-pathological findings of demyelination similar to experimental autoimmune encephalomyelitis (EAE) caused by injection of central nervous system tissue or purified myelin proteins into experimental animals and frequently studied as a model for the human demyelinating disease, multiple sclerosis (MS). T-cell-mediated immune responses play a major role in induction of EAE, and antibody responses enhance disease severity. We studied the antibody responses to myelin basic protein (MBP) in 24 Thai patients with SAE and 77 control individuals to define the linear epitopes in human MBP that are encephalitogenic. Antibody levels were assessed by ELISA using native human MBP or synthetic MBP peptides of 20 amino acids. The major B-cell epitope was MBP61-80 and a minor epitope was MBP106-140 in SAE while in MS the major B-cell epitope is MBP84-96. MBP61-80-specific IgG1 and IgG3 levels were significantly higher in patients than controls while IgG2 and IgG4 were not. The data support the hypothesis that autoreactive Th1 cells induce SAE. The difference in B-cell epitope recognition may be due to differences in the genetic backgrounds of the populations studied or may reflect underlying differences in the pathogenesis of SAE and MS.

Association of HLA and T-cell receptor gene polymorphisms with Semple rabies vaccine-induced autoimmune encephalomyelitis.

Semple rabies vaccine is derived from brain tissue infected with rabies virus that is subsequently inactivated with phenol. Semple rabies vaccine-induced autoimmune encephalomyelitis (SAE) occurs in 1 in 220 immunized individuals. The immune response to myelin basic protein and pathological changes of demyelination in SAE suggest that this disease is the human homologue of experimental autoimmune encephalomyelitis (EAE). SAE and EAE are frequently studied as models for the human demyelinating disease multiple sclerosis. Major histocompatibility complex (MHC) class II and T-cell receptor (TCR) gene polymorphisms play important roles in rodent susceptibility to EAE and were analyzed to determine if the same was true in humans with SAE. HLA-DRB1, HLA-DQB1, and TCRBV gene polymorphisms were studied in Thai individuals with SAE (n = 18), with vaccination without neurological complications (n = 43), and without vaccination (n = 140). The allele frequencies of HLA-DR9 (DRB1*0901) and HLA-DR17 (DRB1*0301) were increased in SAE patients (DR9 = 22%, DR17 = 14%) compared with vaccinated controls (DR9 = 13%, DR17 = 6%) and with unvaccinated controls (DR9 = 9%, DR17 = 4%). The allele frequency of HLA-DQ7 (DQB1*0301) was decreased in SAE patients (8%) compared with vaccinated controls (15%) and with unvaccinated controls (25%). These susceptibilities are distinct from those associated with multiple sclerosis. The frequencies of TCRBV alleles and haplotypes were similar in SAE patients and vaccinated controls. These data suggest that genetic susceptibility associated with MHC class II alleles may have a role in the pathogenesis of SAE and its mechanism may be different from those involved in multiple sclerosis.

Rabies.

Rabies is a complex disease. We still do not understand the mechanisms of clinically diverse furious and dumb types and its fatal course. Moreover clinical symptomatology, once believed to be unique, may be variable, particularly in those patients who develop disease after exposure to virus of the insectivorous or frugivorous bat origin. This review summarizes classic and nonclassic clinical features associated with canine and bat rabies variants and also atypical presentations of rabies survivors. Difference in cellular tropism either at the inoculation site or in the central nervous system or differences in route of spread, or both, may account for these discrepancies. Furthermore, these may affect different sets of neurotransmitters that in turn modulate variable neurobehavioural patterns and neuroendocrine-immune cascades.

Idiopathic hypertrophic cranial pachymeningitis: an unusual cause of subacute and chronic headache.

Three cases of idiopathic hypertrophic cranial pachymeningitis are presented. The diagnosis was based on the CT scan or MRI findings (or both) of thickened enhancing dura. In all cases, meningeal biopsies were performed and microscopic findings were compatible with nonspecific inflammation. All cases presented with subacute and chronic localized headache. Two cases had associated chronic meningitis. One cases presented with a syndrome of multiple cranial nerve involvement (polyneuritis cranialis). Corticosteroids, in the form of prednisolone 60 mg/day, were effective in all cases. Two cases with less severe pachymeningitis received corticosteroids for 2 weeks, then were tapered off in 4 to 6 weeks. A case with extensive lesions needed a long-term low dosage of prednisolone, 5 to 10 mg/day for maintenance therapy. Idiopathic hypertrophic cranial pachymeningitis may be related to the Tolosa-Hunt syndrome, the syndrome of polyneuritis cranialis, and multifocal fibrosclerosis.

Alteration of muscarinic acetylcholine receptors in rabies viral-infected dog brains.

Functions of the muscarinic acetylcholine receptor (mAChR) were studied in rabid dog brains using [3H]quinuclidinyl benzilate (QNB) as a radioligand. Of various brain regions, hippocampus and brainstem were the areas mostly affected in terms of impaired specific binding to [3H]QNB, as compared to other regions, as well as to those of controls. Saturation studies of the hippocampus revealed significantly elevated dissociation equilibrium constant (K(d)) values in both furious (n = 5) (9.80 + or - 2.77 nM) and dumb (n = 6) (6.01 + or - 1.08 nM) types of rabies as compared to 11 controls (2.15 + or - 0.31 nM), whereas the maximum number of receptor sites (B (max)) values were comparable among all subgroups of normal (1.38 + or - 0.10 pmol/mg protein), dumb (1.43 + or - 0.17 pmol/mg protein) and furious (1.28 + or - 0.12 pmol/mg protein) rabies types. Hippocampal K(d) values were comparable between high (fluorescent antibody test-FAT and polymerase chain reaction-PCR positive; n = 4) (7.47 + or - 3.27 nM), and low (FAT-negative and PCR-positive; n = 4) virus amount (8.34 + or - 3.93 nM) but these were significantly higher than controls (n = 4) (1.58 + or - 0.17 nM). Our data suggest a functional derangement of mAChR at specific sites of hippocampus and brainstem which is not dependent on the amount of virus.

Rabies and its prevention.

The increase in world-wide travel means that physicians everywhere require an understanding of rabies and its prevention, to advise intending travellers, or to follow-up on treatment begun overseas. In this article, we discuss measures to prevent rabies.

Immune response to rabies vaccine in Alaskan dogs: failure to achieve a consistently protective antibody response.

Previous studies in Thailand and Tunisia have shown that one injection of dog pre-exposure rabies vaccine does not produce a lasting antibody titre in a significant group of animals. We therefore duplicated the Thai study in a small North American community using healthy, owned dogs. A tissue culture vaccine of known high antigenicity was given intramuscularly as one primary injection and antibody titres were determined by the rapid fluorescent focus inhibition test on days 14, 30, 60, 180 and 360. Titres were less than 0.5 i.u./mL in 27% of dogs bled at 2 months, 24% at 6 months, and in 33% one year after the primary vaccination. In rabies endemic regions, it may be hazardous to rely on the previous vaccine history of a biting dog when making post-exposure management decisions. A retrospective study of antibody levels in previously vaccinated dogs in North America also indicated that a single injection of vaccine often failed to result in adequate titres.

Immune activation in human rabies.

Serum cytokines were compared by enzyme-linked immunosorbent assay among (i) 28 patients with rabies, (ii) 13 patients with non-fatal encephalitis due to other viruses, (iii) 16 patients with immune-mediated neurological diseases, and (iv) 15 patients with non-viral central nervous system infections and non-HTLV-I progressive spastic paraparesis. Levels of soluble interleukin 2 receptor (S-IL2R) were comparable in groups (i)-(iii). Fewer paralytic (1/6) than encephalitic (12/22) rabies patients had elevated S-IL2R. Only one patient with rabies and one with non-fatal viral encephalitis (group ii) had elevated S-CD8. Interleukin 6 (IL-6) was elevated in 5/22 rabies patients with encephalitis and in 0/6 paralytic rabies patients. Four individuals in groups (ii) and (iii) had elevated IL-6. Patterns of cytokine responses in group (iv) were similar to those in groups (i)-(iii). The results suggest defects in immune responsiveness in paralytic rabies.

Diagnosis of rabies by polymerase chain reaction with nested primers.

A simple, sensitive, and specific polymerase chain reaction (PCR) protocol for detection of rabies virus is described. The process consists of sample preparation, reverse transcription, two-step DNA amplification, and detection of the amplified product. RNA was extracted from animal and human brain by phenol-chloroform using guanidinium thiocyanate. Viral RNA was then amplified in a two-step PCR that used two sets of nested primers designed to amplify rabies nucleocapsid (N) sequence. Rabies nucleocapsid sequence was amplified from all brain samples from 95 dogs and 3 humans with rabies confirmed by fluorescent antibody (FAT) and mouse inoculation tests (MIT). Rabies-negative brain samples (110 dogs, 2 humans) were PCR-negative. The process requires < 24 h. Detection of viral RNA was still possible in brain material that was left at room temperature for 72 h. As little as 8 pg of rabies virus RNA could be detected. This technique could have practical applications as a confirmatory test to FAT at busy rabies diagnostic centers.