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.

Nationwide and long-term molecular epidemiologic studies of mumps viruses that circulated in Japan between 1986 and 2017.

In Japan, major mumps outbreaks still occur every 4-5 years because of low mumps vaccine coverage (30-40%) owing to the voluntary immunization program. Herein, to prepare for a regular immunization program, we aimed to reveal the nationwide and long-term molecular epidemiological trends of the mumps virus (MuV) in Japan. Additionally, we performed whole-genome sequencing (WGS) using next-generation sequencing to assess results from conventional genotyping using MuV sequences of the small-hydrophobic (SH) gene. We analyzed 1,064 SH gene sequences from mumps clinical samples and MuV isolates collected from 25 prefectures from 1986 to 2017. The results showed that six genotypes, namely B (110), F (1), G (900), H (3), J (41), and L (9) were identified, and the dominant genotypes changed every decade in Japan since the 1980s. Genotype G has been exclusively circulating since the early 2000s. Seven clades were identified for genotype G using SH sequence-based classification. To verify the results, we performed WGS on 77 representative isolates of genotype G using NGS and phylogenetically analyzed them. Five clades were identified with high bootstrap values and designated as Japanese clade (JPC)-1, -2, -3, -4, -5. JPC-1 and -3 accounted for over 80% of the total genotype G isolates (68.3 and 13.8%, respectively). Of these, JPC-2 and -5, were newly identified clades in Japan through this study. This is the first report describing the nationwide and long-term molecular epidemiology of MuV in Japan. The results provide information about Japanese domestic genotypes, which is essential for evaluating the mumps elimination progress in Japan after the forthcoming introduction of the mumps vaccine into Japan's regular immunization program. Furthermore, the study shows that WGS analysis using NGS is more accurate than results obtained from conventional SH sequence-based classification and is a powerful tool for accurate molecular epidemiology studies.

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.

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.