Free-Chlorine Disinfection as a Selection Pressure on Norovirus.

Human noroviruses are excreted in feces from infected individuals and included in wastewater. It is critical to remove/inactivate them in wastewater treatment processes, particularly in the disinfection step, before release to aquatic environments. However, the high mutation rates of human noroviruses raise concerns about the emergence of strains that are less susceptible to disinfectants and can survive even after wastewater treatment. This study aimed to demonstrate the strain-dependent susceptibility of norovirus to free chlorine. A population originated from the murine norovirus strain S7-PP3, a surrogate for human noroviruses in environmental testing, was exposed to free chlorine and then propagated in a host cell. This cycle of free chlorine exposure followed by propagation in cells was repeated 10 times, and populations with lower susceptibility to free chlorine were obtained from two independent trials of chlorine exposure cycles. Open reading frame 2 (ORF2) and ORF3 of the murine norovirus genome were analyzed by next-generation sequencing, and a unique nonsynonymous mutation (corresponding to a change from phenylalanine to serine) at nucleotide (nt) 7280 in ORF3, which encodes the minor capsid protein VP2, was found in chlorine-exposed populations from both trials. It was confirmed that all of the clones from the chlorine-treated population had lower susceptibility to free chlorine than those from the control population. These results indicate that exposure to free chlorine and dilution exert different driving forces to form murine norovirus (MNV) quasispecies, and that there is a selective force to form MNV quasispecies under free chlorine exposure.IMPORTANCE This study showed that free chlorine disinfection exerted a selection pressure for murine norovirus (MNV). The strain-dependent viral susceptibility to the disinfectant elucidated in this study highlights the importance of employing less susceptible strains as representative viruses in disinfection tests, because the disinfection rate values obtained from more susceptible strains would be less useful in predicting the virus inactivation efficiency of circulating strains under practical disinfection conditions.

Culture-independent evaluation of nonenveloped-virus infectivity reduced by free-chlorine disinfection.

The inability of molecular detection methods to distinguish disinfected virions from infectious ones has hampered the assessment of infectivity for enteric viruses caused by disinfection practices. In the present study, the reduction of infectivity of murine norovirus S7-PP3 and mengovirus vMC0, surrogates of human noroviruses and enteroviruses, respectively, caused by free-chlorine treatment was characterized culture independently by detecting carbonyl groups on viral capsid protein. The amount of carbonyls on viral capsid protein was evaluated by the proportion of biotinylated virions trapped by avidin-immobilized gel (percent adsorbed). This culture-independent approach demonstrated that the percent adsorbed was significantly correlated with the logarithm of the infectious titer of tested viruses. Taken together with the results of previous reports, the result obtained in this study indicates that the amount of carbonyls on viral capsid protein of four important families of waterborne pathogenic viruses, Astroviridae, Reoviridae, Caliciviridae, and Picornaviridae, is increased in proportion to the received oxidative stress of free chlorine. There was also a significant correlation between the percent adsorbed and the logarithm of the ratio of genome copy number to PFU, which enables estimation of the infectious titer of a subject virus by measuring values of the total genome copy number and the percent adsorbed. The proposed method is applicable when the validation of a 4-log reduction of viruses, a requirement in U.S. EPA guidelines for virus removal from water, is needed along with clear evidence of the oxidation of virus particles with chlorine-based disinfectants.

Regeneration of 5-HT fibers in hippocampal heterotopia of methylazoxymethanol-induced micrencephalic rats after neonatal 5,7-DHT injection.

In order to elucidate the regeneration properties of serotonergic fibers in the hippocampus of methylazoxymethanol acetate (MAM)-induced micrencephalic rats (MAM rats), we examined serotonergic regeneration in the hippocampus following neonatal intracisternal 5,7-dihydroxytryptamine (5,7-DHT) injection. Prenatal exposure to MAM resulted in the formation of hippocampal heterotopia in the dorsal hippocampus. Immunohistochemical and neurochemical analyses revealed hyperinnervation of serotonergic fibers in the hippocampus of MAM rats. After neonatal 5,7-DHT injection, most serotonergic fibers in the hippocampus of 2-week-old MAM rats had degenerated, while a small number of serotonergic fibers in the stratum lacunosum-moleculare (SLM) of the hippocampus and in the hilus adjacent to the granular cell layer of the dentate gyrus (DG) had not. Regenerating serotonergic fibers from the SLM first extended terminals into the hippocampal heterotopia, then fibers from the hilus reinnervated the DG and some fibers extended to the heterotopia. These findings suggest that the hippocampal heterotopia exerts trophic target effects for regenerating serotonergic fibers in the developmental period in micrencephalic rats.

[Case of diabetes mellitus associated with cervical pyogenic spondylitis and meningoencephalitis secondary to retropharyngeal abscess caused by Streptococcus pneumoniae].

A 63-year-old man with diabetes mellitus had undergone insulin therapy for 10 years. He developed symptoms of upper respiratory tract infection and neck pain. After 5 days, he suddenly experienced high fever and consciousness disturbance. Neurological examination detected drowsiness and neck stiffness. Cerebrospinal fluid (CSF) examination revealed pleocytosis with low glucose level. Gram staining and a latex agglutination test of his CSF revealed Streptococcus pneumoniae to be the causative organism of meningoencephalitis in the patient. Gadolinium-enhanced T1-weighted images obtained from a cervical spine MRI showed ring enhancement in the anterior clivus and thickening in the anterior dura matter with specific thickening at the dens of the axis. Based on the diagnosis of cervical pyogenic spondylitis and meningoencephalitis secondary to retropharyngeal abscess caused by Streptococcus pneumoniae, the patient was administered panipenem/betamipron and dexamethasone, following which his neurological symptoms and signs gradually improved. Diabetes mellitus is a factor that predisposes patients to invasive pneumococcal infection. Thus, we conclude that physicians need to be aware of the possible development of cervical pyogenic spondylitis and meningoencephalitis subsequent to Streptococcus pneumoniae infection, and symptoms such as fever and neck pain should be carefully examined.

[Distal hereditary motor neuropathy type II with mutation in heat shock protein 27 gene. A case report].

A 48-year-old man was admitted to our hospital with a tendency to stumble during walking. The family history indicated that the father was diagnosed with Charcot-Marie-Tooth disease (CMT) at the age of 55 and his younger sister (aunt) had similar symptoms that were considered to reflect autosomal dominant inheritance. Examination showed no pes cavus or inverted champagne-bottle thighs. In addition, the patient walked with foot drop due to weakness and atrophy of the distal parts of the lower extremities. Sensory examination revealed no deficits or abnormalities. Nerve conduction study and needle electromyography indicated pure motor axonal neuropathy. The diagnosis of distal hereditary motor neuropathy (distal HMN) type II was made. Genetic analysis detected mutation in the heat shock protein 27 (HSP27) gene. A recent report indicated that mutations in the HSP27 gene cause both distal hereditary motor neuropathy and CMT2F. In Japan, there are only a few reports of distal hereditary motor neuropathy with mutation in the HSP27 gene. Distal HMN should be considered in the differential diagnosis of patients with CMT like distal amyotrophy.