Viral isolation analysis of SARS-CoV-2 from clinical specimens of COVID-19 patients.

Genetic testing using reverse transcriptase real-time polymerase chain reaction (rRT-PCR) is the mainstay of diagnosis of COVID-19. However, it has not been fully investigated whether infectious viruses are contained in SARS-CoV-2 genome-positive specimens examined using the rRT-PCR test. In this study, we examined the correlation between the threshold Cycle (Ct) value obtained from the rRT-PCR test and virus isolation in cultured cells, using 533 consecutive clinical specimens of COVID-19 patients. The virus was isolated from specimens with a Ct value of less than 30 cycles, and the lower the Ct value, the more efficient the isolation rate. A cytopathic effect due to herpes simplex virus type 1 contamination was observed in one sample with a Ct value of 35 cycles. In a comparison of VeroE6/TMPRSS2 cells and VeroE6 cells used for virus isolation, VeroE6/TMPRSS2 cells isolated the virus 1.7 times more efficiently than VeroE6 cells. There was no significant difference between the two cells in the mean Ct value of the detectable sample. In conclusion, Lower Ct values in the PCR test were associated with higher virus isolation rates, and VeroE6/TMPRSS2 cells were able to isolate viruses more efficiently than VeroE6 cells.

Further characterization of the synergistic activation mechanism of cationic channels by M2 and M3 muscarinic receptors in mouse intestinal smooth muscle cells.

In mouse ileal myocytes, muscarinic receptor-mediated cationic current (mIcat) occurs mainly through synergism of M2 and M3 subtypes involving Gi/o-type GTP-binding proteins and phospholipase C (PLC). We have further studied the M2/M3 synergistic pathway. Carbachol-induced mIcat was markedly depressed by YM-254890, a Gq/11 protein inhibitor. However, the mIcat was unaffected by heparin, calphostin C, or chelerythrine, suggesting that mIcat activation does not involve signaling molecules downstream of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown. M2-knockout (KO) mice displayed a reduced mIcat (~10% of wild-type mIcat) because of the lack of M2-Gi/o signaling. The impaired mIcat was insensitive to neuropeptide Y possessing a Gi/o-stimulating activity. M3-KO mice also displayed a reduced mIcat (~6% of wild-type mIcat) because of the lack of M3-Gq/11 signaling, and the mIcat was insensitive to prostaglandin F2α possessing a Gq/11-stimulating activity. These results suggest the importance of Gq/11/PLC-hydrolyzed PIP2 breakdown itself in mIcat activation and also support the idea that the M2/M3 synergistic pathway represents a signaling complex consisting of M2-Gi/o and M3-Gq/11-PLC systems in which both G proteins are special for this pathway but not general in receptor coupling.

Impact of COVID-19 and Closed Transmission of SARS-CoV-2 during the First Wave in Toyama Prefecture, Japan, March 30 to May 18, 2020.

We studied 226 patients in Toyama Prefecture who were notified of COVID-19 during the first wave between March 30 and May 18, 2020. Of the 226 patients, 22 (9.7%) died, most (95%) of whom were aged ≥65 years. A large cluster comprising 59 patients (41 residents and 18 staff members) was identified in a nursing home on April 17. No deaths occurred among staff members; however, 12 of the 41 residents (29%) died. Although the threshold cycle (Ct) values were significantly lower in the 20-64 and ≥65 years age groups than in the <20 years age group, no correlation was found between the Ct values and severity, fatal outcome, or secondary infection. The haplotype network of 145 SARS-CoV-2 isolates (64%) from 226 patients was analyzed. The viral genomes of the case groups differed by less than five nucleotide bases. These data suggest that the SARS-CoV-2 strains, which were initially introduced into Toyama Prefecture in late March and early April 2020, and their closely related strains, identified as lineage B.1.1, circulated during the first wave. The reduced inter-prefectural mobility of local residents may support the lack of strain diversity in SARS-CoV-2 during the first wave of the state of emergency.

Rational in silico design identifies two mutations that restore UT28K SARS-CoV-2 monoclonal antibody activity against Omicron BA.1.

SARS-CoV-2 rapidly mutates and acquires resistance to neutralizing antibodies. We report an in-silico-designed antibody that restores the neutralizing activity of a neutralizing antibody. Our previously generated antibody, UT28K, exhibited broad neutralizing activity against mutant variants; however, its efficacy against Omicron BA.1 was compromised by the mutation. Using previously determined structural information, we designed a modified-UT28K (VH T28R/N57D), UT28K-RD targeting the mutation site. In vitro and in vivo experiments demonstrated the efficacy of UT28K-RD in neutralizing Omicron BA.1. Although the experimentally determined structure partially differed from the predicted model, our study serves as a successful case of antibody design, wherein the predicted amino acid substitution enhanced the recognition of the previously elusive Omicron BA.1. We anticipate that numerous similar cases will be reported, showcasing the potential of this approach for improving protein-protein interactions. Our findings will contribute to the development of novel therapeutic strategies for highly mutable viruses, such as SARS-CoV-2.

Evaluation of SARS-CoV-2 isolation in cell culture from nasal/nasopharyngeal swabs or saliva specimens of patients with COVID-19.

It has been revealed that SARS-CoV-2 can be efficiently isolated from clinical specimens such as nasal/nasopharyngeal swabs or saliva in cultured cells. In this study, we examined the efficiency of viral isolation including SARS-CoV-2 mutant strains between nasal/nasopharyngeal swab or saliva specimens. Furthermore, we also examined the comparison of viral isolation rates by sample species using simulated specimens for COVID-19. As a result, it was found that the isolation efficiency of SARS-CoV-2 in the saliva specimens was significantly lower than that in the nasal/nasopharyngeal swab specimens. In order to determine which component of saliva is responsible for the lower isolation rate of saliva specimens, we tested the abilities of lactoferrin, amylase, cathelicidin, and mucin, which are considered to be abundant in saliva, to inhibit the infection of SARS-CoV-2 pseudotyped viruses (SARS-CoV-2pv). Lactoferrin and amylase were found to inhibit SARS-CoV-2pv infection. In conclusion, even if the same number of viral genome copies was detected by the real-time RT-PCR test, infection of SARS-CoV-2 present in saliva is thought to be inhibited by inhibitory factors such as lactoferrin and amylase, compared to nasal/nasopharyngeal swab specimens.

Activation of SARS-CoV-2 by trypsin-like proteases in the clinical specimens of patients with COVID-19.

SARS-CoV-2 enters host cells through the angiotensin converting enzyme 2 (ACE2) receptor and/or transmembrane protease, serine 2 (TMPRSS2). In this study, we investigated whether proteases increased SARS-CoV-2 infectivity using pseudotyped viruses and clinical specimens from patients with COVID-19. First, we investigated how trypsin increased infectivity using the pseudotyped virus. Our findings revealed that trypsin increased infectivity after the virus was adsorbed on the cells, but no increase in infectivity was observed when the virus was treated with trypsin. We examined the effect of trypsin on SARS-CoV-2 infection in clinical specimens and found that the infectivity of the SARS-CoV-2 delta variant increased 36,000-fold after trypsin treatment. By contrast, the infectivity of SARS-CoV-2 omicron variant increased to less than 20-fold in the clinical specimens. Finally, using five clinical specimens containing delta variants, enhancement of viral infectivity was evaluated in the presence of the culture supernatant of several anaerobic bacteria. As a result, viral infectivities of all the clinical specimens containing culture supernatants of Fusobacterium necrophorum were significantly increased from several- to tenfold. Because SARS-CoV-2 infectivity increases in the oral cavity, which may contain anaerobic bacteria, keeping the oral cavities clean may help prevent SARS-CoV-2 infection.

Long-Term Detection and Isolation of Severe Fever with Thrombocytopenia Syndrome (SFTS) Virus in Dog Urine.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infection caused by the SFTS virus (SFTSV), with a high fatality rate of approximately 30% in humans. In recent years, cases of contact infection with SFTSV via bodily fluids of infected dogs and cats have been reported. In this study, clinical and virological analyses were performed in two dogs in which SFTSV infection was confirmed for the first time in the Toyama prefecture. Both dogs recovered; however, one was severely ill and the other mildly ill. The amount of the SFTSV gene was reduced to almost similar levels in both dogs. In the dogs' sera, the SFTSV gene was detected at a low level but fell below the detection limit approximately 2 weeks after onset. Notably, the SFTSV gene was detected at levels several thousand times higher in urine than in other specimens from both dogs. Furthermore, the gene was detected in the urine for a long period of >2 months. The clinical signs disappeared on days 1 or 6 after onset, but infectious SFTSV was detected in the urine up to 3 weeks later. Therefore, it is necessary to be careful about contact with bodily fluids, especially urine, even after symptoms have disappeared.

Virus evolution and reduced viral viability during treatment of persistent COVID-19 Omicron BA.5 infection in an immunocompromised host.

We present the clinical course of a 72-year-old female with COVID-19 and a history of hematologic stem cell transplantation for acute myeloid leukemia. We performed serial analyses of viral load and whole-genome amplification. The virus growth was evaluated by a real-time polymerase chain reaction assay. Neutralizing activity was measured using a chemiluminescence reduction neutralizing test of SARS-CoV-2 pseudotyped virus. After neutralizing antibody therapy, the cycle threshold value of viral genome was 28. Viruses were no longer isolated in a cell culture. K129R, V722I, and V987F of amino acid mutation in spike protein region were identified, although they soon disappeared. Four months after symptom onset, E340K, K356R, R346T, and E484V mutations appeared and persisted. The viability of the virus decreased over time, with the virus at day 145 having a cycle threshold value of 24 and positive virus isolation, but at a slower growth rate. Neutralizing antibody activity for Omicron BA.5 finally appeared about 4 months after infection. In immunocompromised patients, persistent infection with amino acid mutations can occur without neutralizing antibodies. However, the production of neutralizing antibodies reduces the growth rate of the SARS-CoV-2. Moreover, infection control requires attention to viral dynamics and evolution under different conditions.

Neutralization of Omicron subvariants BA.1 and BA.5 by a booster dose of COVID-19 mRNA vaccine in a Japanese nursing home cohort.

The sustained epidemic of Omicron subvariants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a worldwide concern, and older adults are at high risk. We conducted a prospective cohort study to assess the immunogenicity of COVID-19 mRNA vaccines (BNT162b2 or mRNA-1273) in nursing home residents and staff between May 2021 and December 2022. A total of 335 SARS-CoV-2 naïve individuals, including 141 residents (median age: 88 years) and 194 staff (median age: 44 years) participated. Receptor-binding domain (RBD) and nucleocapsid (N) protein IgG and neutralizing titer (NT) against the Wuhan strain, Alpha and Delta variants, and Omicron BA.1 and BA.5 subvariants were measured in serum samples drawn from participants after the second and third doses of mRNA vaccine using SARS-CoV-2 pseudotyped virus. Breakthrough infection (BTI) was confirmed by a notification of COVID-19 or a positive anti-N IgG result in serum after mRNA vaccination. Fifty-one participants experienced SARS-CoV-2 BTI during the study period. The RBD IgG and NTs against Omicron BA.1 and BA.5 were markedly increased in SARS CoV-2 naïve participants 2 months after the third dose of mRNA vaccine, compared to those 5 months after the second dose, and declined 5 months after the third dose. The decline in RBD IgG and NT against Omicron BA.1 and BA.5 in SARS-CoV-2 naïve participants after the second and the third dose was particularly marked in those aged ≥ 80 years. BTIs during the BA.5 epidemic period, which occurred between 2 and 5 months after the third dose, induced a robust NT against BA.5 even five months after the booster dose vaccination. Further studies are required to assess the sustainability of NTs elicited by Omicron-containing bivalent mRNA booster vaccine in older adults.

Neutralizing Antibody Levels and Epidemiological Characteristics of Patients with Breakthrough COVID-19 Infection in Toyama, Japan.

Breakthrough infection (BI) after coronavirus disease 2019 (COVID-19) vaccination has increased owing to the emergence of novel SARS-CoV-2 variants. In this study, we analyzed the epidemiological information and possession status of neutralizing antibodies in patients with BI using SARS-CoV-2 pseudotyped viruses. Analysis of 44 specimens from patients diagnosed with COVID-19 after two or more vaccinations showed high inhibition of infection by 90% or more against the Wuhan strain and the Alpha and Delta variants of pseudotyped viruses in 40 specimens. In contrast, almost no neutralizing activity was observed against the Omicron BA.1 variant. Many patients without neutralizing activity or BI were immunosuppressed. The results of this study show that contact with an infected person can result in BI, even when there are sufficient neutralizing antibodies in the blood. Thus, sufficient precautions must be taken to prevent infection even after vaccination.

Novel super-neutralizing antibody UT28K is capable of protecting against infection from a wide variety of SARS-CoV-2 variants.

Many potent neutralizing SARS-CoV-2 antibodies have been developed and used for therapies. However, the effectiveness of many antibodies has been reduced against recently emerging SARS-CoV-2 variants, especially the Omicron variant. We identified a highly potent SARS-CoV-2 neutralizing antibody, UT28K, in COVID-19 convalescent individuals who recovered from a severe condition. UT28K showed efficacy in neutralizing SARS-CoV-2 in an in vitro assay and in vivo prophylactic treatment, and the reactivity to the Omicron strain was reduced. The structural analyses revealed that antibody UT28K Fab and SARS-CoV-2 RBD protein interactions were mainly chain-dominated antigen-antibody interactions. In addition, a mutation analysis suggested that the emergence of a UT28K neutralization-resistant SARS-CoV-2 variant was unlikely, as this variant would likely lose its competitive advantage over circulating SARS-CoV-2. Our data suggest that UT28K offers potent protection against SARS-CoV-2, including newly emerging variants.

A False Positive Dengue Fever Rapid Diagnostic Test Result in a Case of Acute Parvovirus B19 Infection.

An outbreak of dengue fever occurred in Japan in August 2014. We herein report the case of a 63-year-old man who presented with a persistent fever in September 2014. Acute parvovirus B19 infection led to a false positive finding of dengue fever on a rapid diagnostic test (Panbio Dengue Duo Cassette(TM)). To the best of our knowledge, there are no previous reports of a false positive result for dengue IgM with the dengue rapid diagnostic test. We believe that epidemiological information on the prevalence of parvovirus B19 is useful for guiding the interpretation of a positive result with the dengue rapid diagnostic test.