Exploring the threshold for the start of respiratory syncytial virus infection epidemic season using sentinel surveillance data in Japan.

Introduction: An unusual seasonality of respiratory syncytial virus (RSV) infection in Japan is observed in recent years after 2017, becoming challenging to prepare for: a seasonal shift from autumn-winter to summer-autumn in 2017-2019, no major epidemic in 2020, and an unusually high number of cases reported in 2021. Methods: To early detect the start-timing of epidemic season, we explored the reference threshold for the start-timing of the epidemic period based on the number of cases per sentinel (CPS, a widely used indicator in Japanese surveillance system), using a relative operating characteristic curve analysis (with the epidemic period defined by effective reproduction number). Results: The reference values of Tokyo, Kanagawa, Osaka, and Aichi Prefectures were 0.41, 0.39, 0.42, and 0.24, respectively. Discussion: The reference CPS value could be a valuable indicator for detecting the RSV epidemic and may contribute to the planned introduction of monoclonal antibody against RSV to prevent severe outcomes.

Title: Factors associated with time lag between symptom onset and reporting in the first epidemic wave of COVID-19 in Osaka, Japan.

Objectives: Longer reporting lags after symptom onset reportedly exert a substantial impact on onward transmission, increasing outbreak probability. Our study investigated the risk factors associated with reporting lag. Methods: Using active epidemiological surveillance data for all symptomatic cases reported in Osaka Prefecture during the first wave of the coronavirus disease 2019 (COVID-19) epidemic (February 1-May 13, 2020), multivariable regression analyses were implemented to estimate the effects of exposure variables on reporting lag, by controlling for potential confounders. Results: Cases in their 30s showed a longer reporting lag than cases ≥ 80 years old. Cases who lived in areas with a high COVID-19 incidence demonstrated a longer reporting lag. Cases with a history of visiting a nightlife district also showed longer reporting lag than cases without such a history. Healthcare workers and cases with immunodeficiency both displayed shorter reporting lags than others. Conclusion: Identifying newly infected cases as soon as possible and increased testing capacity for all age groups, and for individuals with a history of visiting high infection-risk areas, represented important measures in shortening reporting lags in the first wave period. The evidence from this study may provide lessons for controlling future emerging diseases.

Environmental Sampling for Severe Acute Respiratory Syndrome Coronavirus 2 During a COVID-19 Outbreak on the Diamond Princess Cruise Ship.

During a COVID-19 outbreak on the Diamond Princess cruise ship we sampled environmental surfaces after passengers and crew vacated cabins. SARS-CoV-2 RNA was detected in 58 of 601 samples (10%) from case cabins 1-17 days after cabins were vacated but not from noncase cabins. There was no difference in detection proportion between cabins of symptomatic (15%, 28/189; cycle quantification [Cq], 29.79-38.86) and asymptomatic cases (21%, 28/131; Cq, 26.21-38.99). No SARS-CoV-2 virus was isolated from any of the samples. Transmission risk of SARS-CoV-2 from symptomatic and asymptomatic patients may be similar and surfaces could be involved in transmission.

Haplotype networks of SARS-CoV-2 infections in the Diamond Princess cruise ship outbreak.

The Diamond Princess cruise ship was put under quarantine offshore Yokohama, Japan, after a passenger who disembarked in Hong Kong was confirmed as a coronavirus disease 2019 case. We performed whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly from PCR+ clinical specimens and conducted a phylogenetic analysis of the outbreak. All tested isolates exhibited a transversion at G11083T, suggesting that SARS-CoV-2 dissemination on the Diamond Princess originated from a single introduction event before the quarantine started. Although further spreading might have been prevented by quarantine, some progeny clusters could be linked to transmission through mass-gathering events in the recreational areas and direct transmission among passengers who shared cabins during the quarantine. This study demonstrates the usefulness of haplotype network/phylogeny analysis in identifying potential infection routes.

Descriptive study of COVID-19 outbreak among passengers and crew on Diamond Princess cruise ship, Yokohama Port, Japan, 20 January to 9 February 2020.

An outbreak of coronavirus disease (COVID-19) occurred on the Diamond Princess cruise ship making an international journey, which led to quarantine of the ship at Yokohama Port, Japan. A suspected COVID-19 case was defined as a passenger or crew member who developed a fever or respiratory symptoms, and a confirmed COVID-19 case had laboratory-confirmation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Between 3 and 9 February 2020, 490 individuals were tested for SARS-CoV-2 and 172 were positive (152 passengers (median age: 70 years; interquartile range (IQR): 64-75; males: 45%) and 20 crew (median age: 40 years; IQR: 35-48.5; males: 80%). Other than the Hong Kong-related index case, symptom onset for the earliest confirmed case was 22 January, 2 days after the cruise ship left port. Attack rates among passengers were similar across the decks, while beverage (3.3%, 2/61) and food service staff (5.7%, 14/245) were most affected. Attack rates tended to increase with age. A comprehensive outbreak response was implemented, including surveillance, provision of essential medical care, food and medicine delivery, isolation, infection prevention and control, sampling and disembarkation.

Initial Investigation of Transmission of COVID-19 Among Crew Members During Quarantine of a Cruise Ship - Yokohama, Japan, February 2020.

An outbreak of coronavirus disease 2019 (COVID-19) among passengers and crew on a cruise ship led to quarantine of approximately 3,700 passengers and crew that began on February 3, 2020, and lasted for nearly 4 weeks at the Port of Yokohama, Japan (1). By February 9, 20 cases had occurred among the ship's crew members. By the end of quarantine, approximately 700 cases of COVID-19 had been laboratory-confirmed among passengers and crew. This report describes findings from the initial phase of the cruise ship investigation into COVID-19 cases among crew members during February 4-12, 2020.

Oxidation of Flavone, 5-Hydroxyflavone, and 5,7-Dihydroxyflavone to Mono-, Di-, and Tri-Hydroxyflavones by Human Cytochrome P450 Enzymes.

Biologically active plant flavonoids, including 5,7-dihydroxyflavone (57diOHF, chrysin), 4',5,7-trihydroxyflavone (4'57triOHF, apigenin), and 5,6,7-trihydroxyflavone (567triOHF, baicalein), have important pharmacological and toxicological significance, e.g., antiallergic, anti-inflammatory, antioxidative, antimicrobial, and antitumorgenic properties. In order to better understand the metabolism of these flavonoids in humans, we examined the oxidation of flavone, 5-hydroxyflavone (5OHF), and 57diOHF to various products by human cytochrome P450 (P450 or CYP) and liver microsomal enzymes. Individual human P450s and liver microsomes oxidized flavone to 6-hydroxyflavone, small amounts of 5OHF, and 11 other monohydroxylated products at different rates and also produced several dihydroxylated products (including 57diOHF and 7,8-dihydroxyflavone) from flavone. We also found that 5OHF was oxidized by several P450 enzymes and human liver microsomes to 57diOHF and further to 567triOHF, but the turnover rates in these reactions were low. Interestingly, both CYP1B1.1 and 1B1.3 converted 57diOHF to 567triOHF at turnover rates (on the basis of P450 contents) of >3.0 min-1, and CYP1A1 and 1A2 produced 567triOHF at rates of 0.51 and 0.72 min-1, respectively. CYP2A13 and 2A6 catalyzed the oxidation of 57diOHF to 4'57triOHF at rates of 0.7 and 0.1 min-1, respectively. Our present results show that different P450s have individual roles in oxidizing these phytochemical flavonoids and that these reactions may cause changes in their biological and toxicological properties in mammals.

Cytochrome P450 2A6 and other human P450 enzymes in the oxidation of flavone and flavanone.

1. We previously reported that flavone and flavanone interact spectrally with cytochrome P450 (P450 or CYP) 2A6 and 2A13 and other human P450s and inhibit catalytic activities of these P450 enzymes. In this study, we studied abilities of CYP1A1, 1A2, 1B1, 2A6, 2A13, 2C9 and 3A4 to oxidize flavone and flavanone. 2. Human P450s oxidized flavone to 6- and 5-hydroxylated flavones, seven uncharacterized mono-hydroxylated flavones, and five di-hydroxylated flavones. CYP2A6 was most active in forming 6-hydroxy- and 5-hydroxyflavones and several mono- and di-hydroxylated products. 3. CYP2A6 was also very active in catalyzing flavanone to form 2'- and 6-hydroxyflavanones, the major products, at turnover rates of 4.8 min-1 and 1.3 min-1, respectively. Other flavanone metabolites were 4'-, 3'- and 7-hydroxyflavanone, three uncharacterized mono-hydroxylated flavanones and five mono-hydroxylated flavones, including 6-hydroxyflavone. CYP2A6 catalyzed flavanone to produce flavone at a turnover rate of 0.72 min-1 that was ∼3-fold higher than that catalyzed by CYP2A13 (0.29 min-1). 4. These results indicate that CYP2A6 and other human P450s have important roles in metabolizing flavone and flavanone, two unsubstituted flavonoids, present in dietary foods. Chemical mechanisms of P450-catalyzed desaturation of flavanone to form flavone are discussed.

Site-specific oxidation of flavanone and flavone by cytochrome P450 2A6 in human liver microsomes.

The roles of human cytochrome P450 (P450 or CYP) 2A6 in the oxidation of flavanone [(2R)- and (2S)-enantiomers] and flavone were studied in human liver microsomes and recombinant human P450 enzymes. CYP2A6 was highly active in oxidizing flavanone to form flavone, 2'-hydroxy-, 4'-, and 6-hydroxyflavanones and in oxidizing flavone to form mono- and di-hydroxylated products, such as mono-hydroxy flavones M6, M7, and M11 and di-hydroxy flavones M3, M4, and M5. Liver microsomes prepared from human sample HH2, defective in coumarin 7-hydroxylation activity, were very inefficient in forming 2'-hydroxyflavanone from flavanone and a mono-hydroxylated product, M6, from flavone. Coumarin and anti-CYP2A6 antibodies strongly inhibited the formation of these metabolites in microsomes prepared from liver samples HH47 and 54, which were active in coumarin oxidation activities. Molecular docking analysis showed that the C2'-position of (2R)-flavanone (3.8 Å) was closer to the iron center of CYP2A6 than the C6-position (10 Å), while distances from C2' and C6 of (2S)-flavanone to the CYP2A6 were 6.91 Å and 5.42 Å, respectively. These results suggest that CYP2A6 catalyzes site-specific oxidation of (racemic) flavanone and also flavone in human liver microsomes. CYP1A2 and CYP2B6 were also found to play significant roles in some of the oxidations of these flavonoids by human liver microsomes.

Persistent organic pollutants in red-crowned cranes (Grus japonensis) from Hokkaido, Japan.

The red-crowned crane (Grus japonensis) from eastern Hokkaido is classified as a Special Natural Monument in Japan. In this study, we determined the concentrations of persistent organic pollutants (POPs) in red-crowned crane muscle tissues (n = 47). Polychlorinated biphenyls (PCBs) had the highest median concentration (240ng/g lipid weight), followed by dichlorodiphenyltrichloroethane and its metabolites (DDTs) (150ng/g lipid weight), chlordane-related compounds (CHLs) (36ng/g lipid weight), hexachlorobenzene (HCB) (16ng/g lipid weight), hexachlorocyclohexanes (HCHs) (4.4ng/g lipid weight), polybrominated diphenyl ethers (PBDEs) (1.8ng/g lipid weight), and finally, Mirex (1.5ng/g lipid weight). Additionally, a positive correlation was found among POP concentrations. No sex differences beyond body parameters were observed. Additionally, red-crowned cranes exhibited a high enantiomeric excess of (+)-alpha-HCH, with enantiomer fractions varying from 0.51 to 0.87 (average: 0.69).

Determination of the human cytochrome P450 monooxygenase catalyzing the enantioselective oxidation of 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183).

2,2',3,5',6-Pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183) possess axial chirality and form the aS and aR enantiomers. The enantiomers of these congeners have been reported to accumulate in the human body enantioselectively via unknown mechanisms. In this study, we determined the cytochrome P450 (CYP) monooxygenase responsible for the enantioselective oxidization of PCB 95 and PCB 183, using a recombinant human CYP monooxygenase. We evaluated 13 CYP monooxygenases, namely CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP4F2, and aromatase (CYP19), and revealed that CYP2A6 preferably oxidizes aS-PCB 95 enantioselectively; however, it did not oxidize PCB 183. The enantiomer composition was elevated from 0.5 (racemate) to 0.54. In addition, following incubation with CYP2A6, the enantiomer fraction (EF) of PCB 95 demonstrated a time-dependent increase.

Oxidation of 1-chloropyrene by human CYP1 family and CYP2A subfamily cytochrome P450 enzymes: catalytic roles of two CYP1B1 and five CYP2A13 allelic variants.

1. 1-Chloropyrene, one of the major chlorinated polycyclic aromatic hydrocarbon contaminants, was incubated with human cytochrome P450 (P450 or CYP) enzymes including CYP1A1, 1A2, 1B1, 2A6, 2A13, 2B6, 2C9, 2D6, 2E1, 3A4 and 3A5. Catalytic differences in 1-chloropyrene oxidation by polymorphic two CYP1B1 and five CYP2A13 allelic variants were also examined. 2. CYP1A1 oxidized 1-chloropyrene at the 6- and 8-positions more actively than at the 3-position, while both CYP1B1.1 and 1B1.3 preferentially catalyzed 6-hydroxylation. 3. Five CYP2A13 allelic variants oxidized 8-hydroxylation much more than 6- and 3-hydroxylation, and the variant CYP2A13.3 was found to slowly catalyze these reactions with a lower kcat value than other CYP2A13.1 variants. 4. CYP2A6 catalyzed 1-chloropyrene 6-hydroxylation at a higher rate than the CYP2A13 enzymes, but the rate was lower than the CYP1A1 and 1B1 variants. Other human P450 enzymes had low activities towards 1-chloropyrene. 5. Molecular docking analysis suggested differences in the interaction of 1-chloropyrene with active sites of CYP1 and 2 A enzymes. In addition, a naturally occurring Thr134 insertion in CYP2A13.3 was found to affect the orientation of Asn297 in the I-helix in interacting with 1-chloropyrene (and also 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and caused changes in the active site of CYP2A13.3 as compared with CYP2A13.1.

Size Distribution of Chlorinated Polycyclic Aromatic Hydrocarbons in Atmospheric Particles.

The particle size distribution of chlorinated polycyclic aromatic hydrocarbons (ClPAHs) in particulate matter (PM) in Japan is examined for the first time. PM was collected using a PM0.1 air sampler with a six-stage filter. PM was collected in October 2014 and January 2015 to observe potential seasonal variation in the atmospheric behavior and size of PM, including polycyclic aromatic hydrocarbons (PAHs) and ClPAHs. We found that the concentration of PAHs and ClPAHs between 0.5-1.0 µm and 1.0-2.5 µm markedly increase in January (i.e., the winter season). Among the ClPAHs, 1-ClPyrene and 6-ClBenzo[a]Pyrene were the most commonly occurring compounds; further, approximately 15% of ClPAHs were in the nanoparticle phase (<0.1 µm). The relatively high presence of nanoparticles is a potential human health concern because these particles can easily be deposited in the lung periphery. Lastly, we evaluated the aryl hydrocarbon receptor (AhR) ligand activity of PM extracts in each size fraction. The result indicates that PM < 2.5 µm has the strong AhR ligand activity.

Roles of Human CYP2A6 and Monkey CYP2A24 and 2A26 Cytochrome P450 Enzymes in the Oxidation of 2,5,2',5'-Tetrachlorobiphenyl.

2,5,2',5'-Tetrachlorobiphenyl (TCB) induced type I binding spectra with cytochrome P450 (P450) 2A6 and 2A13, with Ks values of 9.4 and 0.51 µM, respectively. However, CYP2A6 oxidized 2,5,2',5'-TCB to form 4-hydroxylated products at a much higher rate (∼1.0 minute-1) than CYP2A13 (∼0.02 minute-1) based on analysis by liquid chromatography-tandem mass spectrometry. Formation of 4-hydroxy-2,5,2',5'-TCB by CYP2A6 was greater than that of 3-hydroxy-2,5,2',5'-TCB and three other hydroxylated products. Several human P450 enzymes, including CYP1A1, 1A2, 1B1, 2B6, 2D6, 2E1, 2C9, and 3A4, did not show any detectable activities in oxidizing 2,5,2',5'-TCB. Cynomolgus monkey CYP2A24, which shows 95% amino acid identity to human CYP2A6, catalyzed 4-hydroxylation of 2,5,2',5'-TCB at a higher rate (∼0.3 minute-1) than CYP2A26 (93% identity to CYP2A6, ∼0.13 minute-1) and CYP2A23 (94% identity to CYP2A13, ∼0.008 minute-1). None of these human and monkey CYP2A enzymes were catalytically active in oxidizing other TCB congeners, such as 2,4,3',4'-, 3,4,3',4'-, and 3,5,3',5'-TCB. Molecular docking analysis suggested that there are different orientations of interaction of 2,5,2',5'-TCB with the active sites (over the heme) of human and monkey CYP2A enzymes, and that ligand interaction energies (U values) of bound protein-ligand complexes show structural relationships of interaction of TCBs and other ligands with active sites of CYP2A enzymes. Catalytic differences in human and monkey CYP2A enzymes in the oxidation of 2,5,2',5'-TCB are suggested to be due to amino acid changes at substrate recognition sites, i.e., V110L, I209S, I300F, V365M, S369G, and R372H, based on the comparison of primary sequences.

Structure-Function Studies of Naphthalene, Phenanthrene, Biphenyl, and Their Derivatives in Interaction with and Oxidation by Cytochromes P450 2A13 and 2A6.

Naphthalene, phenanthrene, biphenyl, and their derivatives having different ethynyl, propynyl, butynyl, and propargyl ether substitutions were examined for their interaction with and oxidation by cytochromes P450 (P450) 2A13 and 2A6. Spectral interaction studies suggested that most of these chemicals interacted with P450 2A13 to induce Type I binding spectra more readily than with P450 2A6. Among the various substituted derivatives examined, 2-ethynylnaphthalene, 2-naphthalene propargyl ether, 3-ethynylphenanthrene, and 4-biphenyl propargyl ether had larger ΔAmax/Ks values in inducing Type I binding spectra with P450 2A13 than their parent compounds. P450 2A13 was found to oxidize naphthalene, phenanthrene, and biphenyl to 1-naphthol, 9-hydroxyphenanthrene, and 2- and/or 4-hydroxybiphenyl, respectively, at much higher rates than P450 2A6. Other human P450 enzymes including P450s 1A1, 1A2, 1B1, 2C9, and 3A4 had lower rates of oxidation of naphthalene, phenanthrene, and biphenyl than P450s 2A13 and 2A6. Those alkynylated derivatives that strongly induced Type I binding spectra with P450s 2A13 and 2A6 were extensively oxidized by these enzymes upon analysis with HPLC. Molecular docking studies supported the hypothesis that ligand-interaction energies (U values) obtained with reported crystal structures of P450 2A13 and 2A6 bound to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, indole, pilocarpine, nicotine, and coumarin are of use in understanding the basis of possible molecular interactions of these xenobiotic chemicals with the active sites of P450 2A13 and 2A6 enzymes. In fact, the ligand-interaction energies with P450 2A13 4EJG bound to these chemicals were found to relate to their induction of Type I binding spectra.

[Validation Study for Analytical Method of Diarrhetic Shellfish Poisons in 9 Kinds of Shellfish].

A method was developed for the simultaneous determination of okadaic acid, dinophysistoxin-1 and dinophysistoxin-2 in shellfish using ultra performance liquid chromatography with tandem mass spectrometry. Shellfish poisons in spiked samples were extracted with methanol and 90% methanol, and were hydrolyzed with 2.5 mol/L sodium hydroxide solution. Purification was done on an HLB solid-phase extraction column. This method was validated in accordance with the notification of Ministry of Health, Labour and Welfare of Japan. As a result of the validation study in nine kinds of shellfish, the trueness, repeatability and within-laboratory reproducibility were 79-101%, less than 12 and 16%, respectively. The trueness and precision met the target values of notification.

Trends in the enantiomeric composition of polychlorinated biphenyl atropisomers in human breast milk.

For the precise estimation of the risk to human health caused by persistent organic pollutants (POPs), it is important to discuss enantiomer fraction value (EF value) because it is reported that behaviors such as stability and toxicity of enantiomers are quite different in human body. Among POPs, polychlorinated biphenyl (PCB) is known as one of the most persistent compounds in human breast milk samples. The main exposure source of PCB for human body is mostly from food especially in seafood. The contamination of fish and shellfish has been a serious problem for the Japanese, who consume a large amount of fish in their diet. PCBs have 19 congeners which are chlorine-substituted in 3- or 4- ortho positions are known to have enantiomers. In this study, we analyzed PCB 183 (2,2',3,4,4',5',6-hepta CB) in human breast milk and fish samples enantioselectively and revealed the time trends of the EF value. Though EF value of PCB 183 in fish samples sustained close to racemate (EF = 0.5) from 1982 to 2012, that in breast milk increased over time. This fact indicates that (+)-PCB-183 has greater bioaccumulation potential than (-)-PCB-183 in human body; therefore, the toxicity of (+)-PCB-183 should be emphasized.