Detailed analysis of low temperature inactivation of respiratory syncytial virus.

Our previous findings indicated that many respiratory syncytial virus (RSV) isolates are unstable at 4 °C compared to 20 °C. Some of the strains completely lose infectivity after 24 h at 4 °C. This study analyzed the inactivation process at 4 °C using a representative strain, RSV/Sendai/851/13. After 24 h of storage at 4 °C, the virus was completely inactivated but retained its ability to attach to and to be taken into host cells. It suggested a reduced fusion ability between the viral and cellular membranes. During storage at 4 °C, the RSV fusion (F) protein underwent a conformational change and was no longer recognized by pre-fusion form-specific antibodies. When the RSV/Sendai/851/13 strain was passaged at 4 °C, a variant with an amino acid substitution, I148T, in the F protein fusion peptide was selected. Also, an amino acid change in G protein demonstrating stability at low temperatures was obtained. These results show that the inactivation of RSV at 4 °C is due to the loss of membrane fusion activity in the F protein, which cannot maintain its pre-fusion state at 4 °C.

Epidemiologic, clinical, and genetic characteristics of influenza C virus infections among outpatients and inpatients in Sendai, Japan from 2006 to 2020.

BACKGROUND: Influenza C virus is a pathogen that causes acute respiratory illness in children. The clinical information about this virus is limited because of the small number of isolated viruses compared to influenza A or B viruses. METHODS: A total of 60 influenza C viruses were isolated by clinical tests using cell culture methods conducted in one hospital and one clinic during the 15 years from 2006 to 2020. These 60 cases were retrospectively analyzed by comparing outpatients and inpatients. Moreover, isolated viruses were analyzed for genomic changes during the study period. RESULTS: All were younger than 7 years, and 73% of inpatients (19 out of 26) were under 2 years of age. A significant difference was found in the frequency of pneumonia, accounting for 45% and 4% of inpatients and outpatients, respectively. Most of the viruses isolated from 2006 to 2012 belonged to the S/A sublineage of the C/Sao Paulo lineage, but three sublineage viruses, including the S/A sublineage with K190N mutation, S/V sublineage, and C/Kanagawa lineage, have cocirculated since 2014. Moreover, S/A sublineage viruses were undergoing reassortment since 2014, suggesting significant changes in the virus, both antigenically and genetically. Of the 10 strains from patients with pneumonia, 7 were in the S/A sublineage, which had circulated from 2006 to 2012. CONCLUSION: Infants under 2 years of age were more likely to be hospitalized with pneumonia. The genomic changes that occurred in 2014 were suggested to affect the ability of the virus to spread.

Variation in Thermal Stability among Respiratory Syncytial Virus Clinical Isolates under Non-Freezing Conditions.

Virus isolates are not only useful for diagnosing infections, e.g., respiratory syncytial virus (RSV), but can also facilitate many aspects of practical viral studies such as analyses of antigenicity and the action mechanisms of antivirals, among others. We have been isolating RSV from clinical specimens from patients with respiratory symptoms every year since our first isolation of RSV in 1964, and isolation rates have varied considerably over the years. As collected clinical specimens are conventionally stored in a refrigerator from collection to inoculation into cells, we hypothesized that certain storage conditions or associated factors might account for these differences. Hence, we evaluated the thermal stability of a total of 64 viruses isolated from 1998 to 2018 upon storage at 4 °C and 20 °C for a defined duration. Interestingly, and contrary to our current understanding, 22 strains (34%) showed a greater loss of viability upon short-term storage at 4 °C than at 20 °C. Thirty-seven strains (57%) showed an almost equal loss, and only five strains (8%) were more stable at 4 °C than at 20 °C. This finding warrants reconsideration of the temperature for the temporary storage of clinical samples for RSV isolation.

Low response in eliciting neuraminidase inhibition activity of sera among recipients of a split, monovalent pandemic influenza vaccine during the 2009 pandemic.

Antibodies against influenza virus neuraminidase (NA) protein prevent releasing of the virus from host cells and spreading of infection foci and are considered the 'second line of defence' against influenza. Haemagglutinin inhibition antibody-low responders (HI-LRs) are present among influenza split vaccine recipients. The NA inhibition (NAI) antibody response in vaccinees is worth exploring, especially those in the HI-LRs population. We collected pre- and post-vaccination sera from 61 recipients of an inactivated, monovalent, split vaccine against A/H1N1pdm09 and acute and convalescent sera from 49 unvaccinated patients naturally infected with the A/H1N1pdm09 virus during the 2009 influenza pandemic. All samples were subjected to haemagglutinin inhibition (HI), NAI and neutralisation assays. Most paired sera from naturally infected patients exhibited marked elevation in the NAI activity, and seroconversion rates (SCR) among HI-LRs and HI-responders (HI-Rs) were 60% and 87%, respectively; however, those from vaccinees displayed low increase in the NAI activity, and the SCR among HI-LRs and HI-Rs were 0% and 12%, respectively. In both HI-LRs and HI-Rs, vaccination with the inactivated, monovalent, split vaccine failed to elicit the NAI activity efficiently in the sera of the naive population, compared with the natural infection. Hence, the improvement of influenza vaccines is warranted to elicit not only HI but also NAI antibodies.

Inhibitory effects of glycopyrronium, formoterol, and budesonide on coronavirus HCoV-229E replication and cytokine production by primary cultures of human nasal and tracheal epithelial cells.

BACKGROUND: Coronavirus 229E (HCoV-229E), one of the causes of the common cold, exacerbates chronic obstructive pulmonary disease (COPD) and bronchial asthma. Long-acting muscarinic antagonists and ß2-agonists and inhaled corticosteroids inhibit the exacerbation of COPD and bronchial asthma caused by infection with viruses, including HCoV-229E. However, the effects of these drugs on HCoV-229E replication and infection-induced inflammation in the human airway are unknown. METHODS: Primary human nasal (HNE) and tracheal (HTE) epithelial cell cultures were infected with HCoV-229E. RESULTS: Pretreatment of HNE and HTE cells with glycopyrronium or formoterol decreased viral RNA levels and/or titers, the expression of the HCoV-229E receptor CD13, the number and fluorescence intensity of acidic endosomes where HCoV-229E RNA enters the cytoplasm, and the infection-induced production of cytokines, including IL-6, IL-8, and IFN-ß. Treatment of the cells with the CD13 inhibitor 2'2'-dipyridyl decreased viral titers. Pretreatment of the cells with a combination of three drugs (glycopyrronium, formoterol, and budesonide) exerted additive inhibitory effects on viral titers and cytokine production. Pretreatment of HNE cells with glycopyrronium or formoterol reduced the susceptibility to infection, and pretreatment with the three drugs inhibited activation of nuclear factor-kappa B p50 and p65 proteins. Pretreatment with formoterol increased cAMP levels and treatment with cAMP decreased viral titers, CD13 expression, and the fluorescence intensity of acidic endosomes. CONCLUSIONS: These findings suggest that glycopyrronium, formoterol, and a combination of glycopyrronium, formoterol, and budesonide inhibit HCoV-229E replication partly by inhibiting receptor expression and/or endosomal function and that these drugs modulate infection-induced inflammation in the airway.

Efficient isolation of human metapneumovirus using MNT-1, a human malignant melanoma cell line with early and distinct cytopathic effects.

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.

Efficient isolation of human parainfluenza viruses 1 and 3 using MNT-1, a human malignant melanoma cell line system that exhibits an apparent cytopathic effect.

Isolation of human parainfluenza virus (HPIV) serotypes 1 and 3 from clinical specimens is not very efficient because of the lack of a cell culture system capable of inducing CPE. In this study, the utility of a melanoma cell line, MNT-1, that allows HPIV growth and displays CPE was demonstrated. In particularly, the efficiency of isolating HPIV1 and HPIV3 using MNT-1 was greater than for cell lines conventionally used for HPIV isolation. Our demonstrated efficacy of HPIV1 and HPIV3 isolation with apparent CPE using the MNT-1 cell culture system has the potential to improve virus isolation from clinical specimens.

Differences in neutralizing antigenicity between laboratory and clinical isolates of HCoV-229E isolated in Japan in 2004-2008 depend on the S1 region sequence of the spike protein.

Human coronavirus (HCoV) is a causative agent of the common cold. Although HCoV is highly prevalent in the world, studies of the genomic and antigenic details of circulating HCoV strains have been limited. In this study, we compared four Japanese isolates with the standard HCoV-229E strain obtained from ATCC (ATCC-VR740) by focusing on the spike (S) protein, a major determinant of neutralizing antigen and pathogenicity. The isolates were found to have nucleotide deletions and a number of sequence differences in the S1 region of the S protein. We compared two of the Japanese isolates with the ATCC-VR740 strain by using virus-neutralizing assays consisting of infectious HCoV-229E particles and vesicular stomatitis virus (VSV)-pseudotyped virus carrying the HCoV-229E S protein. The two clinical isolates (Sendai-H/1121/04 and Niigata/01/08) did not react with antiserum to the ATCC-VR740 strain via the neutralizing test. We then constructed a pseudotype VSV-harboured chimeric S protein with the ATCC S1 and Sendai S2 regions or that with Sendai S1 and ATCC S2 regions and compared them by a neutralization test. The results revealed that the difference in the neutralizing antigenicity depends on the S1 region. This different antigenic phenotype was also confirmed by a neutralizing test with clinically isolated human sera. These results suggest that the HCoV-229E viruses prevalent in Japan are quite different from the laboratory strain ATCC-VR740 in terms of the S sequence and neutralization antigenicity, which is attributed to the difference in the S1 region.

Detection of respiratory viruses in nasopharyngeal secretions and middle ear fluid from children with acute otitis media.

CONCLUSIONS: Our results suggest that various respiratory viruses contribute to the pathogenesis of acute otitis media (AOM). OBJECTIVE: AOM is one of the most common complications of viral upper respiratory tract infections in children. Recently, the importance of respiratory viruses has been stressed as causative agents of AOM. SUBJECTS AND METHODS: A total of 1092 children < or =10 years old (average age 1.38 years) diagnosed as having AOM between 2002 and 2004 were studied. Bacterial and viral cultures of both nasopharyngeal secretions (NPS) and middle ear fluid (MEF) were performed for all 1092 children. Body temperature, changes of the tympanic membrane, and the number of days from the onset of illness were analyzed. RESULTS: Respiratory viruses were detected in 360 of 1092 NPS specimens, including 157 isolates of respiratory syncytial virus and 88 of influenza virus. Among 1092 MEF specimens, 102 were virus-positive, including 43 for respiratory syncytial virus and 29 for influenza virus. In 75 children, respiratory viruses were only detected in MEF. The viral detection rate was higher in children with fever at an early stage of their illness. The tympanic membrane changes associated with viral infection tended to be less severe, while changes were more severe in cases with bacterial infection, especially co-infection with bacteria and viruses.

Acute otitis media associated with cytomegalovirus infection in infants and children.

OBJECTIVE: Acute otitis media (AOM) is one of the most common complications of viral respiratory tract infections in children, but the role of each virus is still to be elucidated. We analyzed AOM associated with infection by cytomegalovirus (CMV), which is known as one of the major causes of viral respiratory tract infection. METHODS: Four hundred and ninety-five children (292 boys and 203 girls) diagnosed as having AOM in 2002 were studied. All of the children were under 6 years old, with the average age being 1.31+/-1.36 years. Bacterial and viral culture of both nasopharyngeal secretions (NPS) and middle ear fluid (MEF) was performed in all 495 children. The levels of glutamyl pyruvic transaminase (GPT) and the serum IgM antibody for CMV were measured. CMV infection was defined on the basis of isolation of this virus by culture and/or positive anti-CMV IgM antibody. NPS and MEF specimens of the subjects diagnosed as having CMV infection were tested for the virus by nested PCR. RESULTS: Twelve of the 495 children were found to have CMV infection. They included 6 boys and 6 girls aged from 3 to 25 months, with the average age being 11+/-7 months. Among 10 children in whom CMV infection was diagnosed by viral culture, CMV was isolated from NPS alone in nine cases and from both NPS and MEF in one case. Nested PCR was performed in all 12 subjects diagnosed as having CMV infection, and all NPS samples were positive, as were 8 MEF samples. We obtained serum samples from 205 children under 2 years of age, including 9 with CMV infection. The mean serum GPT level of 124 children in whom no viruses were detected was 20.7+/-14.4 IU/L. While, the serum GPT levels of 9 children with CMV infection ranged from 10 to 280 IU/L with the average titer being 78.4+/-81.9 IU/L, and the GPT levels of the children with CMV infection were significantly higher than those of the children in whom no viruses were detected (p<0.05). CONCLUSION: Our results suggested that CMV is a causative pathogen of AOM, and that CMV infection should be suspected in patients with AOM and liver dysfunction.

Detection of human coronavirus-NL63 in children in Japan.

Human coronavirus NL63 recently found in The Netherlands has been detected in Japan with a reverse transcription-polymerase chain reaction technique in clinical specimens from pediatric patients with respiratory symptoms. Of 419 specimens that were negative for common respiratory viruses, 5 were positive for human coronavirus NL63, and these specimens were all collected in the first 3 months of 2003.

Detection of human metapneumovirus from children with acute otitis media.

Nasal and middle ear specimens collected from children with acute otitis media were subjected to viral isolation and bacteria culture. All virus-negative specimens underwent reverse transcription polymerase chain reaction to detect human metapneumovirus. Three of 126 middle ear specimens were positive by this assay.