Setsuko Kitaoka

Comparative efficacy of broad-spectrum antiviral agents as inhibitors of rotavirus replication in vitro.

Several nucleoside analogues which have previously been established as broad-spectrum antiviral agents, i.e. ribavirin, vidarabine, pyrazofurin, tubercidin, carbodine, (S)-9-(2,3-dihydroxypropyl)adenine [(S)-DHPA], carbocyclic 3-deazaadenosine (C-c3 Ado), (RS)-3-adenine-9-yl-2-hydroxypropanoic acid [(RS)-AHPA] isobutyl ester and neplanocin A were compared for their potency and selectivity as inhibitors of human rotavirus (strains Wa, KUN and MO) replication in vitro. As the most efficacious inhibitors emerged (S)-DHPA, C-c3 Ado, (RS)-AHPA isobutyl ester and neplanocin A, with a minimum inhibitory concentration of 60, 1.4, 1.2 and 0.2 micrograms/ml, and a selectivity index of greater than 3, 70, 80 and greater than 20, respectively. As has been postulated for their antiviral action in general, these adenosine analogues probably owe their anti-rotavirus activity to inhibition of S-adenosylhomocysteine hydrolase, a key enzyme in regulating methylations including those that are required for the maturation of viral mRNA.

Effect of tunicamycin on human rotavirus morphogenesis and infectivity

SummaryIn the presence of tunicamycin, decreases in the number of double-shelled human rotavirus particles coincided with a reduction in cell lysates infectivity. The majority of particles within the endoplasmic reticulum were membrane bound, “enveloped” particles.

Further observations on the morphogenesis of human rotavirus in MA 104 cells.

SummaryHuman rotavirus “KUN” strain was cultivated in a fetal rhesus monkey kidney cell line, MA 104 cells. Four types of virus particles in cells infected with KUN strain were clearly identified: nucleoid cores, single-shelled particles, double-shelled particles, and membrane band, “enveloped” particles. “Enveloped” particles were found only in the thin sections of infected cells. When first visible, the virus precursors appeared at the ribosome free membrane of rough endoplasmic reticulum (RER), increasing in size while simultaneously being coated with nucleocapsid, inner shell. Single-shelled particles were also synthesized within bundles of filaments of viroplasm in the cytoplasma. During subsequent virus maturation two types of “budding” processes were observed. Double-shelled particles arising at the RER membrane entered the cisternae of the RER through an exocytosis-like process. In contrast, the “enveloped” particles developed in the cisternae by being completely enclosed with RER membrane, and later during cytolysis released the single-shelled particles. These “enveloped” virus particles appeared to be the result of inefficient virus maturation at the last stage of outer capsid formation.

Elevated serum levels of neutrophil elastase in patients with influenza virus-associated encephalopathy

We examined serum levels of various cytokines, chemokines, growth factors, and adhesion molecules in patients with uncomplicated influenza (n=20) and influenza virus-associated encephalopathy (IE) (n=18) to understand the underlying mechanism of IE. We found that IL-1β, IL-2, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, G-CSF, GM-CSF, TNF-α, TIMP-1, MMP-9, sE-selectin, and neutrophil elastase were elevated significantly in sera from patients with uncomplicated influenza and those with IE, compared with normal controls (n=20). Of note, neutrophil elastase, sE-selectin, IL-8, and IL-13 were elevated significantly in IE as compared with uncomplicated influenza. In the present study, for the first time, we found that serum levels of neutrophil elastase were increased in patients with IE compared with uncomplicated influenza, which suggested that cerebral endothelial damage in the development of IE was mediated by neutrophil elastase. The present study implied that anti-elastase agents are possibly an effective therapeutic protocol for IE, but this needs further elucidation.

Onset of Hemophagocytic Lymphohistiocytosis during Piperacillin-Tazobactam Therapy in Three Children with Acute Focal Bacterial Nephritis

Hemophagoytic lymphohistiocytosis (HLH) is a rare life-threatening disorder caused by overactivation of the immune system, associated with infections, autoimmune disorders, and malignancies. The pathological hallmark of HLH is phagocytosis of blood cells and platelets by activated macrophages and histiocytes. In this report, we describe the onset of HLH in three children, aged 2, 5 and 7 years old, during the treatment of acute focal bacterial nephritis (AFBN) with an antibiotic, piperacillin-tazobactam (PIPC-TAZ). AFBN is acute localized bacterial infection of the kidney without abscess formation. PIPC-TAZ was chosen for the treatment of AFBN, because it not only has indications for complicated urinary tract infections, but also covers most of the causative bacteria of urinary tract infections, including β-lactamase-producing Escherichia coli. The clinical courses of the three patients were similar, and they were treated with PIPC-TAZ and amikacin (AMK) for AFBN. Fever went down 2 to 5 days later, and AMK was discontinued by day 6. However, fever recurred on 13 to 15 days after introduction of PIPC-TAZ therapy, even though all of the patients had no signs of recurrence of AFBN. The clinical features and laboratory tests of two patients fulfilled the criteria of HLH, whereas the other patient had initiated therapy before fulfilling the criteria. Cessation of PIPC-TAZ combined with corticosteroid therapy improved clinical symptoms. HLH of our patients was probably induced by PIPC-TAZ, as judged by the timing of the onset of HLH and the positivity of the drug-lymphocyte stimulation test. In conclusion, prolonged antibiotic therapy with PIPC-TAZ could be a cause of HLH.

Efficient isolation of human metapneumovirus using MNT-1, a human malignant melanoma cell line exhibiting early and distinct cytopathic effect

Isolation of human metapneumovirus (HMPV) from clinical specimens is currently inefficient because of the lack of a cell culture system in which a distinct cytopathic effect (CPE) occurs. The cell lines LLC‐MK2, Vero and Vero E6 are used for isolation of HMPV; however, the CPE in these cell lines is subtle and usually requires a long observation period and sometimes blind passages. Thus, a cell line in which an early and distinct CPE occurs following HMPV inoculation is highly desired by clinical virology laboratories. In this study, it was demonstrated that, in the human malignant melanoma cell line MNT‐1, obvious syncytium formation occurs shortly after inoculation with HMPV‐positive clinical specimens. In addition, the growth and efficiency of isolation of HMPV were greater using MNT‐1 than using any other conventional cell line. Addition of this cell line to our routine viral isolation system for clinical specimens markedly enhanced isolation frequency, allowing isolation‐based surveillance. MNT‐1 has the potential to facilitate clinical and epidemiological studies of HMPV.

In response to letter to Editor by Nosaka et al. on our paper: Elevated serum levels of neutrophil elastase in patients with influenza virus-associated encephalopathy. J Neurol Sci 2015;349:190-195.

We appreciate your attention [1] to ourmanuscript [2]. Since our results are based on statistical analysis, we think it is difficult to compare the result of their three patients with our data. Moreover, their patients are diagnosed as HHV-6 associated encephalopathy. It needs further clarification if Influenza and HHV-6 viruses could induce the same reaction in the endothelium and immune system. However, I agree to their suggestion that an analysis of temporal changes in serum levels of neutrophil elastase (NE) is important for evaluating if NE levels are useful in predicting the severity of influenza virus-associated encephalopathy (IE). Unfortunately we did not checked temporal changes asmentioned in the limitation inDiscussion,which remains to be performed. The clinical types of IE have been discussed recently, although the basic mechanisms differentiating those types are still obscure. We tried to see if the types of IE or severity of IE is correlated with the levels of NE. As described in Results, there were no significant differences in NE levels between in patients with and without sequelae. Themean value and SE of NE in patients with acute necrotizing encephalopathy (ANE) (n = 4), acute encephalopathy with febrile convulsive status epilepticus (AEFCSE) (n = 7), acute hemorrhagic shock encephalopathy (n = 2) and others (n = 5) were 391.0 ± 94.9, 496.3 ± 64.4, 1217.0 ± 417.0, and 377.2 ± 151.3, respectively. However, there was no statistical significance between those types of encephalopathies (p = 0.1, KruskalWallis), which may reflect limited sample size. Only thing we could clearly mention is that NE levels of acute hemorrhagic shock encephalopathy were significantly higher than those of AEFCSE (p = 0.04, Mann-Whitney U test). In order to confirm if anti-elastase agents have a therapeutic potential, we needs further study using a larger patient cohort.

Perinatal Coxsackievirus B3 Infection with Transient Thrombocytopenia

Coxsackievirus (Cox) B is the second common picornaviruses, after echovirus, detected from children younger than 2 months of age. Neonates who present with Cox B3 infection in the first week are known to have severe illness such as myocarditis or menigoencephalitis. Severity is commonly associated with perinatal vertical transmission. Here, we report a neonatal case of Cox B3 infection with severe thrombocytopenia through horizontal transmission. The patient was a preterm infant born without asphyxia by selective cesarean section. From his 6(th) day of life, the patient had recurrent episodes of apnea. At that time, the laboratory investigations revealed a profound thrombocytopenia without any evidence of inflammation. Thus, neonatal alloimmune thrombocytopenia (NAIT) was suspected, and the patient received transfusion of immunoglobulin and platelets. Thereafter, the patient had no further episodes of apnea, and platelet counts of the patient increased gradually. Later, the possibility of NAIT was ruled out by the result of the platelet antigen genotyping of the patient and his parents. Culture obtained from his nasopharynx was positive for Cox B3. We thus speculate that the patient was exposed to the virus from his mother because she had a febrile episode at her 5(th) day after delivery, and her Cox B3 infection was confirmed by serology. Assuming that the thrombocytopenia was a complication of Cox B3 infection, the immunoglobulin transfusion might have provided a neutralizing antibody against Cox B3. It is important to consider the possibility of enterovirus infection as a differential diagnosis whenever unexplained thrombocytopenia was observed in neonates.

Transient Creatine Kinase Elevation Followed by Hypocomplementemia in a Case of Rotavirus Myositis

We report an infant case of rotavirus myositis, a rare complication of rotavirus infection. Complement levels of the patient were normal when serum creatine kinase (CK) level was at its peak and then decreased when the CK level became normalized. In a previous case report of rotavirus myositis, transient decrease of serum albumin, immunoglobulin, and complement levels was reported. The authors speculated that intravascular complement activation was caused by rotavirus and resulted in the pathogenesis of myositis, although complement levels at onset were not measured by the authors. In this report, however, we demonstrate that the complement activation of our patient is a result of, rather than the cause of, skeletal muscle damage.