A Genome Epidemiological Study of SARS-CoV-2 Introduction into Japan.

After the first case of coronavirus disease 2019 (COVID-19) in Japan on 15 January 2020, multiple nationwide COVID-19 clusters were identified by the end of February. The Japanese government focused on mitigating the emerging COVID-19 clusters by conducting active nationwide epidemiological surveillance. However, an increasing number of cases continued to appear until early April 2020, many with unclear infection routes and no recent history of travel outside Japan. We aimed to evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from the COVID-19 cases that appeared until early April 2020 and to characterize their genealogical networks in order to demonstrate possible routes of spread in Japan. Nasopharyngeal specimens were collected from patients, and reverse transcription-quantitative PCR tests for SARS-CoV-2 were performed. Positive RNA samples were subjected to whole-genome sequencing, and a haplotype network analysis was performed. Some of the primary clusters identified during January and February 2020 in Japan descended directly from the Wuhan-Hu-1-related isolates from China and other distinct clusters. Clusters were almost contained until mid-March; the haplotype network analysis demonstrated that the COVID-19 cases from late March through early April may have created an additional large cluster related to the outbreak in Europe, leading to additional spread within Japan. In conclusion, genome surveillance has suggested that there were at least two distinct SARS-CoV-2 introductions into Japan from China and other countries.IMPORTANCE This study aimed to evaluate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome sequences from COVID-19 cases and to characterize their genealogical networks to demonstrate possible routes of spread in Japan. We found that there were at least two distinct SARS-CoV-2 introductions into Japan, initially from China and subsequently from other countries, including Europe. Our findings can help understand how SARS-CoV-2 entered Japan and contribute to increased knowledge of SARS-CoV-2 in Asia and its association with implemented stay-at-home/shelter-in-place/self-restraint/lockdown measures. This study suggested that it is necessary to formulate a more efficient containment strategy using real-time genome surveillance to support epidemiological field investigations in order to highlight potential infection linkages and mitigate the next wave of COVID-19 in Japan.

Development of a multiplex real-time PCR assay for detection of human enteric viruses other than norovirus using samples collected from gastroenteritis patients in Fukui Prefecture, Japan.

There are many varieties of gastroenteritis viruses, of which norovirus (NoV) accounts for over 90% of the viral food poisoning incidents in Japan. However, protocols for rapidly identifying other gastroenteritis viruses need to be established to investigate NoV-negative cases intensively. In this study, a multiplex real-time PCR assay targeting rotavirus A, rotavirus C, sapovirus, astrovirus, adenovirus, and enterovirus was developed using stool samples collected from gastroenteritis patients between 2010 and 2013 in Fukui Prefecture, Japan. Of the 126 samples collected sporadically from pediatric patients with suspected infectious gastroenteritis, 51 were positive for non-NoV target viruses, whereas 27 were positive for NoV, showing a high prevalence of non-NoV viruses in pediatric patients. In contrast, testing in 382 samples of 58 gastroenteritis outbreaks showed that non-NoV viruses were detected in 13 samples, with NoV in 267. Of the 267 NoV-positive patients, only two were co-infected with non-NoV target viruses, suggesting that testing for non-NoV gastroenteritis viruses in NoV-positive samples was mostly unnecessary in outbreak investigations. Given these results, multiplex real-time PCR testing for non-NoV gastroenteritis viruses, conducted separately from NoV testing, may be helpful to deal with two types of epidemiological investigations, regular surveillance of infectious gastroenteritis and urgent testing when gastroenteritis outbreaks occur.

Development of a Practical Method to Detect Noroviruses Contamination in Composite Meals.

Various methods to detect foodborne viruses including norovirus (NoV) in contaminated food have been developed. However, a practical method suitable for routine examination that can be applied for the detection of NoVs in oily, fatty, or emulsive food has not been established. In this study, we developed a new extraction and concentration method for detecting NoVs in contaminated composite meals. We spiked NoV-GI.4 or -GII.4 stool suspension into potato salad and stir-fried noodles. The food samples were suspended in homogenizing buffer and centrifuged to obtain a food emulsion. Then, anti-NoV-GI.4 or anti-NoV-GII.4 rabbit serum raised against recombinant virus-like particles or commercially available human gamma globulin and Staphylococcus aureus fixed with formalin as a source of protein A were added to the food emulsion. NoV-IgG-protein A-containing bacterial complexes were collected by centrifugation, and viral RNA was extracted. The detection limits of NoV RNA were 10-35 copies/g food for spiked NoVs in potato salad and stir-fried noodles. Human gamma globulin could also concentrate other NoV genotypes as well as other foodborne viruses, including sapovirus, hepatitis A virus, and adenovirus. This newly developed method can be used as to identify NoV contamination in composite foods and is also possibly applicable to other foodborne viruses.

[Mutations of gyrA gene and parC gene in fluoroquinolone-resistant Escherichia coli isolates from sporadic diarrheal cases].

We studied 107 isolates of Escherichia coli O153 from sporadic diarrhea cases in Fukui, Toyama, Aichi, and Saga prefectures from 1991 to 2005 for antimicrobial susceptibility and mechanisms of fluoroquinolone resistance, based on standard disk diffusion. Of 12 drugs tested, ampicillin displayed resistance to 72.9% of isolates, streptomycin to 48.6%, tetracycline to 46.7%, sulfisoxazole to 46.7%, trimethoprim/sulfamethoxazole to 29.9%, nalidixic acid (NA) to 29.9%, and ciprofloxacin (CPFX) to 24.3%. Ten of 32 isolates resistant to 3-6 drugs and 16 of 18 isolates resistant to 7-10 drugs were resistant both to NA and CPFX. Mutations of amino acid in quinolone resistance-determining regions of gyrA and parC genes were detected in 24 isolates resistant both to NA and CPFX, and in 1 isolate resistant to NA. The former possessed a combination of double substitution (S83L and D87L) in GyrA and a single substitution (S80I) in ParC. Some 12 of 24 isolates possessed another single substitution (E84V or E84G or A108T) in ParC. The 25 isolates were classified into 4 types as follows. 1 isolate as type 1: GyrA (S83L) and ParC (S80I); 12 isolates as type 2: GyrA (S83L and D87N) and ParC (S80I); 8 isolates as type 3: GyrA (S83L and D87N) and ParC (S80I and E84G/S80R and E84V); and 4 isolate as type 4: GyrA (S83L and D87N) and ParC (S80I and A108T). In the relationship between amino acid mutations and minimal inhibitory concentrations (MIC) of fluoroquinolone, MICs of CPFX, ofloxacin, and norfloxacin showed 1microg/mL, 2microg/mL and 8microg/mL in type 1; 8 approximately 32microg/mL, 8 approximately 32microg/mL and 16 approximately 256microg/mL in type 2; and 32 approximately 256microg/mL' 32 approximately 128microg/mL and 128-->512microg/ mL in types 3 and 4. These results suggest that most of multiple-antimicrobial-resitant E. coli O153 isolates from sporadic diarrhea cases were resistant to fluoroquinolones and possessed mutations at gyrA and parC genes associated with fluoroquinolone resistance.

[Adult cases of viral meningitis caused by echovirus type 13].

Adult cases of viral meningitis caused by echovirus type 13 (E13) were studied. E13 was isolated from 8 of 11 adult patients (73%) with viral meningitis between April and September 2002 in Fukui Prefecture. The mean age was 27.4 +/- 6.4 years (4 males and 4 females). The disease was prevalent among adults, especially younger adults as well as children. The symptoms and signs were as follows; headache (100%), fever (100%), nausea and/or vomiting (88%), Kernig's sign (88%), and increased deep tendon reflexes (50%). The average cell counts in cerebrospinal fluid (CSF) were 118 +/- 111/mm3. Of the 2 patients, polynuclear cells were dominant during the early phase of the disease. The prognosis was good. Since May 2002, the number of patients with viral meningitis caused by E13 has rapidly increased. Most of the reported patients were children. We should consider the possibility of E13 infection as a cause of adult viral meningitis.