Yoko Matsuzaki

Background: Kosakonia radicincitans is a plant-associated gram-negative bacterium that rarely causes human infection. Only a few cases of bacteremia have been reported worldwide, and no cases have been documented in Japan. Routine clinical identification methods may misidentify this organism as closely related Enterobacter species. Case Summary: A 77-year-old woman with pancreatic cancer and multiple liver metastases, undergoing chemotherapy, presented with chills and fever 1 week after treatment. Blood cultures were positive in three of four bottles, yielding gram-negative rods. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry suggested K. radicincitans as the top candidate, whereas API 20E identified the isolate as Enterobacter cloacae . Final identification was established by 16S rRNA gene sequencing, which showed 100% identity with the type strain. Whole-genome sequencing of the isolate, designated YUH003903, yielded two contigs of 5.48 Mb and 118 kb. Comparative genome analysis showed 99.2%–99.4% average nucleotide identity with clinical and plant-derived K. radicincitans strains. Single nucleotide polymorphism (SNP)-based phylogenetic analysis did not clearly distinguish clinical isolates from plant-derived strains. The isolate showed low MICs to most antimicrobial agents tested, and the patient recovered after levofloxacin treatment. Conclusion: This is the first reported case of K. radicincitans bacteremia in Japan. Genome analysis indicated that the clinical isolate was highly similar to both clinical and plant-derived strains and revealed no obvious genomic feature specifically associated with human pathogenicity. These findings suggest that K. radicincitans may act as an opportunistic pathogen in susceptible hosts and that accurate identification requires molecular and genomic approaches when routine diagnostic methods yield inconsistent results.

Advances in the epidemiology of seasonal coronavirus (CoV) are important for understanding CoV transmission dynamics and strengthening the strategies against emerging CoV infections. The epidemiology of CoV‐NL63, a seasonal CoV discovered in 2003, is poorly understood, particularly before 2003. To analyze the seroprevalence of CoV‐NL63 in Yamagata, Japan, we performed a microneutralization test using 146 to 379 serum samples collected from four age groups ranging from 0 to 4 years to ≥ 40 years in 1976, 1990, 2010, 2019, and 2022, as well as the Yamagata isolate. We only measured the neutralizing antibodies (NT Abs) against CoV‐NL63, as methods for other seasonal CoVs had not yet been established. The total NT Ab‐positive rate in each year was 68.0%–90.4%. The Ab‐positive rates in 0–4‐year age group were 21.3%–65.2% in each year, whereas the rates for individuals aged above 4 years old were between 73.3% and 100% throughout the study period. We do not understand the influence of cross‐reactive immunity among seasonal CoVs. This study shows that seroprevalence against CoV‐NL63 increased with age in the younger generation and then plateaued at a high level, suggesting that reinfection may occur and that CoV‐NL63 was already transmitted among infants and children before CoV‐NL63 had been identified.

CM2 is a single-pass transmembrane protein located in the viral envelope and is essential for the growth of influenza C virus. We previously reported that CM2 is involved in viral genome packaging and uncoating. We also demonstrated that alanine substitutions at residues 47–48, 67–69, 73–75, 85–87 and 113–115 in the cytoplasmic domain of CM2 significantly reduced its expression levels. Herein, we analysed whether these mutations affected viral replication. The alanine-substituted recombinant viruses rCM2-Ala67–69, 73–75 and 85–87 exhibited significantly reduced replication compared with recombinant wild-type viruses (rWT), and the amount of CM2 protein in virions and infected cells was also reduced compared with that in rWT. In addition, the amount of viral RNA within the particles of these mutant viruses was significantly lower than that in rWT. Furthermore, the amount of viral RNA-NP complexes transported into the nucleus of infected cells was reduced in rCM2-Ala73–75 and 85–87 viruses. In contrast, recombinant viruses with mutations at positions 113–115 could not be rescued, and virus-like particles containing this mutation showed suppressed genome packaging and uncoating and reduced expression of the CM2 protein. These results suggested that the cytoplasmic region of CM2 contributes to efficient packaging and uncoating of the genome through its stable expression.

Background: We have monitored influenza C virus infection using the cell culture method due to its advantage in detecting infectious virus. Application of multiplex polymerase chain reaction (PCR) improves the detection rate of multiple pathogens from the same clinical specimen, and it can be difficult to determine whether the influenza C virus is the cause of the patient’s symptoms. This study aimed to evaluate the impact of co-pathogens in children infected with the influenza C virus based on viral load in clinical specimens. Methods: We enrolled 11 patients in whom influenza C virus and co-pathogens were isolated by cell culture. Pathogens were further detected by multiplex real-time PCR and next-generation sequencing (NGS). Viral load was determined by quantitative real-time PCR, and its association with the patients’ symptoms was investigated. Results: Co-pathogens not isolated in cell culture, including rhinovirus, adenovirus and bocavirus, were detected in 5 of the 11 specimens by real-time PCR. Additionally, adenovirus and human metapneumovirus, which were not detected by real-time PCR, were detected by next-generation sequencing, but both had very low read numbers. Children whose specimens had influenza C virus levels of ≥10⁴ copies/μL showed typical symptoms of influenza C virus infection, such as coughing and nasal discharge. When the viral load of a co-pathogen was greater than that of influenza C virus, atypical symptoms such as persistent fever were likely due to the co-pathogens, suggesting that co-infection contributed to symptom severity. Conclusion: Knowledge of viral load can help in evaluating the etiologic role of the virus in influenza C-infected children with co-pathogens.

To clarify the epidemiology of enterovirus D68 (EV‐D68), an enterovirus rarely identified in the 20th century, we performed seroepidemiological analysis against EV‐D68 using sera collected in 1976, 1985, 1990, 1999, 2009, and 2019, as well as Yamagata isolate (EVD68/Yamagata.JPN/2023‐89), in Yamagata, Japan. The neutralizing antibody (Ab)‐positive rates for those under 20 years old were 61.0%, 82.5%, 84.3%, 46.7%, 50.5%, and 67.9%, in each year, whereas the rates for those above 20 years old were between 93.4% and 99.1%. Generally, geometric mean titers (GMTs）increased with age among children and the total GMT in each year was 25.4, 49.2, 37.2, 30.8, 29.5, and 33.9, from 1976 to 2019, respectively. The findings in this Yamagata‐based study showed that the seroprevalence of EV‐D68 over the last four decades has increased with age among children, as a susceptible group, and then reaches a plateau of over approximately 80% among adults. This study clearly revealed that EV‐D68 was stably transmitted among children in the 20th century, when EV‐D68 detection was quite rare.

We identified a child coinfected with influenza B viruses of B/Yamagata and B/Victoria lineages, in whom we analyzed the occurrence of genetic reassortment. Plaque purification was performed using a throat swab specimen from a 9-year-old child, resulting in 34 well-isolated plaques. The genomic composition of eight gene segments (HA, NA, PB1, PB2, PA, NP, M, and NS genes) for each plaque was determined at the lineage level. Of the 34 plaques, 21 (61.8%) had B/Phuket/3073/2013 (B/Yamagata)-like sequences in all gene segments, while the other 13 (38.2%) were reassortants with B/Texas/02/2013 (B/Victoria)-like sequences in 1–5 of the 8 segments. The PB1 segment had the most B/Victoria lineage genes (23.5%; 8 of 34 plaques), while PB2 and PA had the least (2.9%; 1 of 34 plaques). Reassortants with B/Victoria lineage genes in 2–5 segments showed the same level of growth as viruses with B/Yamagata lineage genes in all segments. However, reassortants with B/Victoria lineage genes only in the NA, PB1, NP, or NS segments exhibited reduced or undetectable growth. We demonstrated that various gene reassortments occurred in a child. These results suggest that simultaneous outbreaks of two influenza B virus lineages increase genetic diversity and could promote the emergence of new epidemic strains.

To investigate the antigenic changes in parechovirus 1 (PeVA1), seroepidemiological analyses were performed against the Harris strain (Harris), isolated in 1956, and PeVA1/Yamagata.JPN/2021‐4785, isolated in 2021, using immune sera and 207 and 237 human serum specimens collected in 2021 and 1976, respectively. Although rabbit immune sera showed the highest neutralization antibody (NT‐Ab) titers against the immunized viruses at 1:12 800–1:102 400, they were cross‐reactive at 1:400–1:800. All 62 Yamagata isolates obtained between 2001 and 2021 (Yamagata strains), belonging to phylogenetic lineage 1B, reacted more strongly (mostly 4–64 times) to antiserum against PeVA1/Yamagata.JPN/2021‐4785 than to antiserum against Harris, belonging to phylogenetic lineage 1 A. Human serum specimens obtained in 2021 showed higher NT‐Ab titers against PeVA1/Yamagata.JPN/2021‐4785, whereas those obtained in 1976 had similar NT‐Ab titers against both strains. These findings suggested that Yamagata strains and Harris were antigenically cross‐reactive, although there were differences. There are still high NT‐Abs titers present against Harris in 2021 in particular, indicating that PeVA1 has been in circulation with high immunity in the population. In conclusion, this study suggested that PeVA1 has been endemically perpetuated with only minor antigenic changes as well as with high immunity over several decades in the community.

The emergence and spread of antiviral-resistant influenza viruses are of great concern. To minimize the public health risk, it is important to monitor antiviral susceptibilities of influenza viruses. Analyses of the antiviral susceptibilities of influenza A and B viruses have been conducted globally; however, those of influenza C and D viruses are limited. Here, we determined the susceptibilities of influenza C viruses representing all six lineages (C/Taylor, C/Yamagata, C/Sao Paulo, C/Aichi, C/Kanagawa, and C/Mississippi) and influenza D viruses representing four lineages (D/OK, D/660, D/Yama2016, and D/Yama2019) to RNA polymerase inhibitors (baloxavir and favipiravir) by using a focus reduction assay. All viruses tested were susceptible to both drugs. We then performed a genetic analysis to check for amino acid substitutions associated with baloxavir and favipiravir resistance and found that none of the viruses tested possessed these substitutions. Use of the focus reduction assay with the genotypic assay has proven valuable for monitoring the antiviral susceptibilities of influenza C and D viruses as well as influenza A and B viruses. Antiviral susceptibility monitoring of all influenza virus types should continue in order to assess the public health risks posed by these viruses.