Development of a vesicular stomatitis virus pseudotyped with herpes B virus glycoproteins and its application in a neutralizing antibody detection assay.

Herpes B virus (BV) is a zoonotic virus and belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). BV typically establishes asymptomatic infection in its natural hosts, macaque monkeys. However, in humans, BV infection causes serious neurological diseases and death. As such, BV research can only be conducted in a high containment level facility (i.e., biosafety level [BSL] 4), and the mechanisms of BV entry have not been fully elucidated. In this study, we generated a pseudotyped vesicular stomatitis virus (VSV) expressing BV glycoproteins using G-complemented VSV∆G system, which we named VSV/BVpv. We found that four BV glycoproteins (i.e., gB, gD, gH, and gL) were required for the production of a high-titer VSV/BVpv. Moreover, VSV/BVpv cell entry was dependent on the binding of gD to its cellular receptor nectin-1. Pretreatment of Vero cells with endosomal acidification inhibitors did not affect the VSV/BVpv infection. The result indicated that VSV/BVpv entry occurred by direct fusion with the plasma membrane of Vero cells and suggested that the entry pathway was similar to that of native HSV. Furthermore, we developed a VSV/BVpv-based chemiluminescence reduction neutralization test (CRNT), which detected the neutralization antibodies against BV in macaque plasma samples with high sensitivity and specificity. Crucially, the VSV/BVpv generated in this study can be used under BSL-2 condition to study the initial entry process through gD-nectin-1 interaction and the direct fusion of BV with the plasma membrane of Vero cells.IMPORTANCEHerpes B virus (BV) is a highly pathogenic zoonotic virus against humans. BV belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). By contrast to HSV, cell entry mechanisms of BV are not fully understood. The research procedures to manipulate infectious BV should be conducted in biosafety level (BSL)-4 facilities. As pseudotyped viruses provide a safe viral entry model because of their inability to produce infectious progeny virus, we tried to generate a pseudotyped vesicular stomatitis virus bearing BV glycoproteins (VSV/BVpv) by modification of expression constructs of BV glycoproteins, and successfully obtained VSV/BVpv with a high titer. This study has provided novel information for constructing VSV/BVpv and its usefulness to study BV infection.

Macacine alphaherpesvirus 1 (B Virus) Infection in Humans, Japan, 2019.

Two human patients with Macacine alphaherpesvirus 1 infection were identified in Japan in 2019. Both patients had worked at the same company, which had a macaque facility. The rhesus-genotype B virus genome was detected in cerebrospinal fluid samples from both patients.

Analysis of antiviral drug properties of thymidine kinase of herpes B virus using recombinant herpes simplex virus 1.

IMPORTANCE: Zoonotic infection of humans with herpes B virus (BV) causes severe neurological diseases. Acyclovir (ACV) and ganciclovir (GCV), most frequently used as anti-herpes drugs, are recommended for prophylaxis and therapy in human BV infection. In this study, we examined the property of BV thymidine kinase (TK) against anti-herpes drugs using a recombinant herpes simplex virus type 1 (HSV-1) carrying BV TK gene. We found that HSV-1 carrying BV TK was similarly sensitive to GCV as HSV-1 carrying varicella zoster virus TK. In addition, we demonstrated that BV TK was not mutated in the GCV- and ACV-resistant HSV-1 carrying BV TK, suggesting that ACV- or GCV-resistant BV might be rare during treatment with these antiviral drugs. These data can provide a new insight into the properties of BV TK in terms of the development of drug resistance.

Non-Omicron breakthrough infection with higher viral load and longer vaccination-infection interval improves SARS-CoV-2 BA.4/5 neutralization.

The immune responses to SARS-CoV-2 variants in COVID-19 cases are influenced by various factors including pre-existing immunity via vaccination and prior infection. Elucidating the drivers for upgrading neutralizing activity to SARS-CoV-2 in COVID-19 cases with pre-existing immunity will aid in improving COVID-19 booster vaccines with enhanced cross-protection against antigenically distinct variants, including the Omicron sub-lineage BA.4/5. This study revealed that the magnitude and breadth of neutralization activity to SARS-CoV-2 variants after breakthrough infections are determined primarily by upper respiratory viral load and vaccination-infection time interval. Extensive neutralizing breadth, covering even the most antigenically distant BA.4/5, was observed in cases with higher viral load and longer time intervals. Antigenic cartography depicted a critical role of the time interval in expanding the breadth of neutralization to SARS-CoV-2 variants. Our results illustrate the importance of dosing interval optimization as well as antigen design in developing variant-proof booster vaccines.

Different efficacies of neutralizing antibodies and antiviral drugs on SARS-CoV-2 Omicron subvariants, BA.1 and BA.2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2 has spread in many countries, replacing the earlier Omicron subvariant BA.1 and other variants. Here, using a cell culture infection assay, we quantified the intrinsic sensitivity of BA.2 and BA.1 compared with other variants of concern, Alpha, Gamma, and Delta, to five approved-neutralizing antibodies and antiviral drugs. Our assay revealed the diverse sensitivities of these variants to antibodies, including the loss of response of both BA.1 and BA.2 to casirivimab and of BA.1 to imdevimab. In contrast, EIDD-1931 and nirmatrelvir showed a more conserved activities to these variants. The viral response profile combined with mathematical analysis estimated differences in antiviral effects among variants in the clinical concentrations. These analyses provide essential evidence that gives insight into variant emergence's impact on choosing optimal drug treatment.

Vaccination-infection interval determines cross-neutralization potency to SARS-CoV-2 Omicron after breakthrough infection by other variants.

Background: The immune profile against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has dramatically diversified due to a complex combination of exposure to vaccines and infection by various lineages/variants, likely generating a heterogeneity in protective immunity in a given population. To further complicate this, the Omicron variant, with numerous spike mutations, has emerged. These circumstances have created the need to assess the potential of immune evasion by Omicron in individuals with various immune histories. Methods: The neutralization susceptibility of the variants, including Omicron and their ancestors, was comparably assessed using a panel of plasma/serum derived from individuals with divergent immune histories. Blood samples were collected from either mRNA vaccinees or from those who suffered from breakthrough infections of Alpha/Delta with multiple time intervals following vaccination. Findings: Omicron was highly resistant to neutralization in fully vaccinated individuals without a history of breakthrough infections. In contrast, robust cross-neutralization against Omicron was induced in vaccinees that experienced breakthrough infections. The time interval between vaccination and infection, rather than the variant types of infection, was significantly correlated with the magnitude and potency of Omicron-neutralizing antibodies. Conclusions: Immune histories with breakthrough infections can overcome the resistance to infection by Omicron, with the vaccination-infection interval being the key determinant of the magnitude and breadth of neutralization. The diverse exposure history in each individual warrants a tailored and cautious approach to understanding population immunity against Omicron and future variants. Funding: This study was supported by grants from the Japan Agency for Medical Research and Development (AMED).

Temporal maturation of neutralizing antibodies in COVID-19 convalescent individuals improves potency and breadth to circulating SARS-CoV-2 variants.

Antibody titers against SARS-CoV-2 slowly wane over time. Here, we examined how time affects antibody potency. To assess the impact of antibody maturation on durable neutralizing activity against original SARS-CoV-2 and emerging variants of concern (VOCs), we analyzed receptor binding domain (RBD)-specific IgG antibodies in convalescent plasma taken 1-10 months after SARS-CoV-2 infection. Longitudinal evaluation of total RBD IgG and neutralizing antibody revealed declining total antibody titers but improved neutralization potency per antibody to original SARS-CoV-2, indicative of antibody response maturation. Neutralization assays with authentic viruses revealed that early antibodies capable of neutralizing original SARS-CoV-2 had limited reactivity toward B.1.351 (501Y.V2) and P.1 (501Y.V3) variants. Antibodies from late convalescents exhibited increased neutralization potency to VOCs, suggesting persistence of cross-neutralizing antibodies in plasma. Thus, maturation of the antibody response to SARS-CoV-2 potentiates cross-neutralizing ability to circulating variants, suggesting that declining antibody titers may not be indicative of declining protection.

Development of an RT-LAMP Assay for the Rapid Detection of SFTS Virus.

Detection of severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) during the early phase of the disease is important for appropriate treatment, infection control, and prevention of further transmission. The reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification method that amplifies the target sequence under isothermal conditions. Here, we developed an RT-LAMP with a novel primer/probe set targeting a conserved region of the SFTSV L segment after extraction of viral RNA (standard RT-LAMP). Both the Chinese and Japanese SFTSV strains, including various genotypes, were detected by the standard RT-LAMP. We also performed RT-LAMP using the same primer/probe set but without the viral RNA extraction step (called simplified RT-LAMP) and evaluated the diagnostic efficacy. The sensitivity and specificity of the simplified RT-LAMP were 84.9% (45/53) and 89.5% (2/19), respectively. The simplified RT-LAMP can detect SFTSV in human sera containing >103.5 copies/mL viral RNA. The two RT-LAMP positive but quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) negative samples were positive in the conventional RT-PCR, suggesting that there was no false positive reaction in the RT-LAMP. Both the standard and simplified RT-LAMP are useful for detecting the SFTSV genome in patients during the early phase of the disease.

Clinical COVID-19 diagnostic methods: Comparison of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and quantitative RT-PCR (qRT-PCR).

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is a major public health concern. Accurate and rapid diagnosis of COVID-19 is critical for disease control. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a nucleic acid amplification assay similar to reverse transcription-polymerase chain reaction (RT-PCR), the former being a simple, low cost, and rapid method. OBJECTIVES: This study aimed to compare the RT-LAMP assay with RT-PCR using the Loopamp™ SARS-CoV-2 Detection Kit. STUDY DESIGN: One hundred and fifty-one nasopharyngeal swab and 88 sputum samples obtained from individuals with suspected or confirmed COVID-19 were examined. RESULTS: RT-LAMP had high specificity (98.5 % (95 % CI: 96.9-100 %)), sensitivity (87.0 % (95 % CI: 82.8-91.3 %)), positive predictive value (97.9 % (95 % CI: 96.1-99.7 %)), negative predictive value (90.2 % (95 % CI: 86.4-94.0 %)), and concordance rate (93.3 % (95 % CI: 90.1-96.5 %)). Nasopharyngeal and sputum samples positive in RT-LAMP contained as few as 10.2 and 23.4 copies per 10 µL, respectively. RT-LAMP showed similar performance to RT-PCR for samples with cycle threshold value below 36. CONCLUSIONS: These results indicate that RT-LAMP is a highly reliable and at least equivalent to RT-PCR in utility, and potentially applicable in settings that are more diverse as a point-of-care tool.

Assessment of SARS-CoV-2 infectivity of upper respiratory specimens from COVID-19 patients by virus isolation using VeroE6/TMPRSS2 cells.

BACKGROUND: An outbreak of novel coronavirus (SARS-CoV-2)-associated respiratory infectious diseases (COVID-19) emerged in 2019 and has spread rapidly in humans around the world. The demonstration of in vitro infectiousness of respiratory specimens is an informative surrogate for SARS-CoV-2 transmission from patients with COVID-19; accordingly, viral isolation assays in cell culture are an important aspect of laboratory diagnostics for COVID-19. METHODS: We developed a simple and rapid protocol for isolating SARS-CoV-2 from respiratory specimens using VeroE6/TMPRSS2 cells, a cell line that is highly susceptible to the virus. We also investigated a correlation between isolation of SARS-CoV-2 and viral load detected by real-time RT-PCR (rRT-PCR) using N2 primer/probe set that has been developed for testing of COVID-19 in Japan. RESULTS: The SARS-CoV-2 isolation protocol did not require blind passage of inoculated cells and yielded the results of viral isolation within 7 days after inoculation. Specimens with cycle threshold (Ct) values of <20.2, determined by rRT-PCR, were predicted to be isolation-positive. On the other hand, 6.9% of specimens with Ct values >35 were virus isolation-positive, indicating that low viral loads (high Ct values) in upper respiratory specimens do not always indicate no risk of containing transmissible virus. CONCLUSION: In combination with rRT-PCR, the SARS-CoV-2 isolation protocol provides a means for assessing the potential risk of transmissible virus in upper respiratory specimens.

Descriptive epidemiology of rickettsial infections in Japan: Scrub typhus and Japanese spotted fever, 2007-2016.

OBJECTIVE: This study aimed to describe the epidemiological and clinical characteristics of endemics of two rickettsial diseases, scrub typhus (ST) and Japanese spotted fever (JSF), in Japan. METHODS: We conducted a retrospective, descriptive epidemiological assessment of cases notified via national surveillance from 2007-2016. RESULTS: Over the 10-year period, 4185 ST and 1765 JSF cases were notified; of these, 20 (0.48%) cases of ST and 16 (0.91%) cases of JSF were fatal at the time of reporting. The elderly had higher notification rates and fatalities. While the annual number of ST notifications was stable and cases were reported from a broad geographic range, the number of JSF reports increased three-fold, expanding from the southwest to the east. The seasonality of ST varied by region and was more common during spring/summer in the north and autumn/winter in the south; 78% of cases occurred during autumn/winter, mainly in the southern region. Most of the fatal ST cases occurred in the spring/summer and occurred in the northern region. CONCLUSION: Our analysis identified seasonal and regional variations in the distribution of rickettsiosis. These variations were most likely to be related to the ecology of the vectors and etiological agents. Knowing the recent epidemiological and clinical features of ST and JSF can support clinical diagnosis and guide preventative activities against these vector-borne diseases.

Correlates of laboratory-confirmed measles in Japan, 2011-2015.

BACKGROUND: With the progressive decline in the incidence of measles in Japan, its diagnosis has become challenging, with fewer physicians having experience in examining measles patients. We aimed to determine the correlates of laboratory-confirmed measles to help physicians improve their measles diagnosis. METHODS: This study was conducted using the National Epidemiological Surveillance of Infectious Disease (NESID) system data during 2011-2015. Among clinically suspected measles patients reported to NESID, measles virus (MV)-positive patients were compared with MV-negative patients. The odds ratios (OR) and associated 95% confidence intervals (CI) were determined using logistic regression. RESULTS: A total of 4168 laboratory-tested patients were notified to NESID. We analysed 618 MV-positive patients (median age, 17 years; interquartile range [IQR], 4-30 years) and 600 MV-negative (median age, 10 years; IQR, 1-29 years) patients after excluding those that met the exclusion criteria or were reported during the rubella epidemic period (the 18th epidemiological week of 2012 to the 46th week of 2013). Having an epidemiological link with a measles patient within 14 days of onset (OR, 14.9; 95% CI, 10.0-23.3), a history of recent international travel (OR, 11.7; 95% CI, 6.9-19.9), and unvaccinated/unknown vaccination status for measles-containing vaccine (MCV; OR, 3.7; 95% CI, 2.3-5.7) were significantly associated with MV-positive status. International travel (adjusted OR, 10.2; 95% CI, 5.9-17.7) and unvaccinated/unknown MCV vaccination status (adjusted OR, 5.8; 95% CI, 3.5-9.8) remained significantly associated with MV-positive status after adjusting for age, sex, and each other. CONCLUSION: In low-incidence Japan, having an epidemiological link, international travel, and lack of MCV vaccination were correlates of laboratory-confirmed measles. The findings of this study could potentially improve the clinical diagnosis of measles, which can lead to more efficient testing and earlier laboratory confirmation.

Correction to: A loop-mediated isothermal amplification assay for the detection and quantification of JC polyomavirus in cerebrospinal fluid: a diagnostic and clinical management tool and technique for progressive multifocal leukoencephalopathy.

In the original publication of article [1], '20 × 101 copies', which is in the sentence 'As seen in Fig. 4, the sensitivity of the specimens containing equal to or more than 20 × 101 copies in 2 µL of extracted DNA (equivalent to ≥3.0 × 103 copies/mL CSF) was 100% (29/29)' changes to '2.0 × 101 copies' in results section. The publisher apologizes to the readers and authors for the inconvenience.

A loop-mediated isothermal amplification assay for the detection and quantification of JC polyomavirus in cerebrospinal fluid: a diagnostic and clinical management tool and technique for progressive multifocal leukoencephalopathy.

BACKGROUND: JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunosuppressed patients. PML usually has a poor prognosis. Detection and quantification of the JCV genome in cerebrospinal fluid (CSF) is an efficacious tool for the diagnosis and management of PML, for which proper therapeutic interventions are required. METHODS: A loop-mediated isothermal amplification (LAMP) assay was applied for the quantitative detection of JCV. The LAMP assay was evaluated for the efficacy in diagnosis of PML in comparison with the TaqMan-based quantitative real-time PCR (qPCR) assay using 153 CSF specimens collected from patients with suspected PML. RESULTS: The LAMP assay showed no cross-reactivity against other polyomavirus plasmids, viral DNA, and viral RNA, which causes encephalitis, and detected 1 copy of the standard DNA per reaction. Among 50 qPCR-positives, 42 specimens (containing JCV genome ranged from 3.2 × 100 to 3.2 × 106 copies/reaction) showed positive reactions and 8 specimens (containing 0.9 to 19.9 copies/reaction) showed negative in the LAMP assay. Furthermore, 3 of 103 qPCR-negative specimens showed positive reactions in the LAMP assay. The sensitivity, specificity, positive predictive value, and negative predictive values of the LAMP assay were 84% (42/50), 97% (100/103), 93% (42/45), and 93% (100/108), respectively. The kappa statistic was 0.83. The JCV loads determined by the LAMP assay showed a strong positive correlation with those determined by the qPCR assay for 33 specimens with copy numbers of ≥1 copies/reaction (r = 0.89). Additionally, the LAMP assay could monitor the JCV genome copy number in CSF for sequential samples equivalently to qPCR assay. CONCLUSIONS: The newly developed LAMP assay is highly specific against JCV and detect the JCV genome in the sample DNA containing 20 or more copies of JCV genome per reaction with 100% sensitivity (n = 29), which corresponds to ≥3 × 103 copies/mL of CSF. The LAMP assay is useful for the diagnosis and offers valuable information for the evaluation and management of PML in the clinical setting.

Respiratory Syncytial Virus Surveillance System in Japan: Assessment of Recent Trends, 2008-2015.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract illness in infants and young children. In Japan, surveillance for RSV infection started in 2003 based on approximately 3,000 pediatric sentinel sites. In recent years, RSV notifications have increased, but the interpretation of trends has been challenging due to a suspected increase in testing frequency and the expansion of the insurance coverage for RSV testing to include certain outpatients in late 2011. Therefore, we evaluated RSV surveillance data during 2008-2015, considering the number of sites that reported at least one RSV case during a surveillance year and restricting to sites that had continuous reporting status since 2008. While annual RSV notifications had increased, the number of sites reporting also increased. And the same magnitude of increase was not observed when the number of cases reported was restricted to the 1,372 sites that had continuous reporting status since 2008. Additionally, in the year following the insurance expansion, RSV notifications increased more remarkably for clinics than for hospitals. These results suggested that some of the recent increases in notifications might be due to an increase in testing frequency.

Human Parainfluenza Virus Type 3 Infections in Patients with Hematopoietic Stem Cell Transplants: the Mode of Nosocomial Infections and Prognosis.

There have been a few prospective and comprehensive surveillance studies on the respiratory viral infections (RVIs) among patients undergoing hematopoietic stem cell transplantation (HSCT). A 2-year prospective cohort surveillance study of symptomatic and asymptomatic RVIs was performed in hospitalized HSCT patients. Oropharyngeal (OP) swab samples were serially collected each week from 1 week before and up to 100 days after HSCT and were tested for virus isolation with cell culture-based viral isolation (CC-based VI) and a multiplex PCR (MPCR). A total of 2,747 OP swab samples were collected from 250 HSCT patients (268 HSCT procedures). Among these patients, 79 had RVIs (CC-based VI, n = 63; MPCR, n = 17). The parainfluenza virus type 3 (PIV3) accounted for 71% (57/80) of the cases of RVIs. Some PIV3 infections were asymptomatic and involved a longer virus-shedding period. The PIV3 was often cultured from samples taken before the onset of a respiratory disease. The PIV3 infections were attributed to the transmission of nosocomial infections. PIV3 infections before engraftment will more likely result in the development of lower respiratory tract infections and worse outcomes. A real-time monitoring of respiratory viral infections in the HSCT ward among patients with or without respiratory symptoms is required for the prevention of nosocomial RVIs, especially of PIV3 infections.

Epidemiology of Imported Malaria Cases in Japan, 2006-2014: A Sentinel Traveler Surveillance Approach.

Malaria continues to be imported into Japan. To better assess the risk of imported malaria, we describe malaria species, suspected country/area of infection (accounting for the number of travelers), demographic characteristics, clinical manifestation, and healthcare access, based on the national surveillance data from 2006 to 2014. Among 557 cases, the median age was 33 years (range: 1-83 years), and 76% were male; 306 (55%) were classified as Japanese based on the reported name. The majority were Plasmodium falciparum infections (58%), followed by Plasmodium vivax infections (30%). Most P. falciparum cases were acquired in Africa and P. vivax in Asia/Oceania. Notification rates per 10,000 Japanese travelers for P. falciparum were highest for Africa, P. vivax were highest for Asia/Oceania, and high for both species for Papua New Guinea. Ten percent of the cases were clinically severe at the time of notification. Nearly 80% of severe cases were P. falciparum infections, and among P. falciparum cases, Japanese ethnicity was associated with severe case status (P = 0.03). Plasmodium falciparum cases among Japanese cases showed that older age (≥ 50 years) was associated with severe case status (odds ratio = 5.4; 95% confidence interval = 1.9-15.2), adjusted for sex and healthcare access. More informative assessments are possible by accounting for the number of travelers. Older Japanese represent an important demographic to target prevention and early treatment efforts for malaria.

Association of the Emergence of Acyclovir-Resistant Herpes Simplex Virus Type 1 With Prognosis in Hematopoietic Stem Cell Transplantation Patients.

Background: Antiviral-resistant herpes simplex virus type 1 (HSV-1) has been recognized as an emerging clinical problem among patients undergoing hematopoietic stem cell transplantation (HSCT). Methods: A prospective observational study was conducted at a hematological center over a 2-year period. Oropharyngeal swab samples were serially collected each week from 1 week before and up to 100 days after HSCT and were tested for virus isolation. The HSV-1 isolates were tested for sensitivity to acyclovir (ACV). The prognosis of patients with ACV-resistant (ACVr) HSV-1 and the genetic background of the ACVr HSV-1 isolates were assessed. Results: Herpes simplex virus type 1 was isolated in 39 of 268 (15%) HSCT patients within 100 days after transplantation. Acyclovir-resistant HSV-1 emerged in 11 of these 39 patients (28%). The 100-day death rates of HSCT patients without HSV-1 shedding, those with only ACV-sensitive HSV-1 shedding, and those with ACVr HSV-1 shedding were 31%, 39%, and 64%, respectively. Patients with HSV-1, including ACVr HSV-1, shedding showed a significantly higher mortality rate. Relapsed malignancies were a significant risk factor for the emergence of ACVr HSV-1. Acyclovir resistance was attributable to viral thymidine kinase and DNA polymerase mutations in 6 and 5 patients, respectively. Conclusions: Herpes simplex virus type 1, including ACVr HSV-1, shedding was associated with poorer outcome in HSCT patients, even if HSV disease did not always occur. Patients with relapsed malignancies were at especially high risk for the emergence of ACVr HSV-1.