Development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assays for rhinovirus detection.

Human rhinoviruses (RVs) belong to the genus Enterovirus of the family Picornaviridae, and are classified into RV-A, -B, and -C species. Two assays were developed to detect RVs by a real-time fluorescent reverse transcription loop-mediated isothermal amplification method: one was designed based on the 5'-untranslated regions (UTRs) of RV-A and -B, and the other was designed based on the 5'-UTR of RV-C. The competence of both assays for the diagnosis of RV infection was tested using isolated viruses and compared with real-time reverse transcription polymerase chain reaction assays on clinical specimens. Neither assay demonstrated cross-reactivity with other tested enteroviruses, and they detected 19 out of 21 tested RV-As and seven out of eight tested RV-Cs. The specificity of the assays was 100% for the detection of RVs and their sensitivity for RV-A and RV-C was 86.3% and 77.3%, respectively, on clinical specimens by the combined use of both assays. Considering that both developed assays were highly specific and detected the majority of recently circulating RVs, they are helpful for the diagnosis of RV infection. Consequently, the results generated by these assays will enhance the surveillance of respiratory illness and the study of the roles of RVs associated with clinical features and disease severity.

Development and evaluation of a new real-time RT-PCR assay for detecting the latest H9N2 influenza viruses capable of causing human infection.

The H9N2 subtype of avian influenza A viruses (AIV) has spread among domestic poultry and wild birds worldwide. H9N2 AIV is sporadically transmitted to humans from avian species. A total of 42 laboratory-confirmed cases of non-fatal human infection with the Eurasian Y280 and G1 lineages have been reported in China, Hong Kong, Bangladesh and Egypt since 1997. H9N2 AIV infections in poultry have become endemic in Asia and the Middle East and are a major source of viral internal genes for other AIV subtypes, such that continuous monitoring of H9N2 AIV is recommended. In this study, a new, one-step, real-time RT-PCR assay was developed to detect two major Eurasian H9 lineages of AIV capable of causing human infection. The sensitivity of this assay was determined using in vitro-transcribed RNA, and the detection limit was approximately 3 copies/reaction. In this assay, no cross-reactivity was observed against RNA from H1-15 subtypes of influenza A viruses, influenza B viruses and other viral respiratory pathogens. In addition, this assay could detect the H9 hemagglutinin (HA) gene from artificially reconstituted clinical samples spiked with H9N2 virus without any non-specific reactions. Therefore, this assay is highly sensitive and specific for H9 HA detection. The assay is useful both for diagnostic purposes in cases of suspected human infection with influenza H9N2 viruses and for the surveillance of both avian and human influenza viruses.

Epitope mapping of the hemagglutinin molecule of A/(H1N1)pdm09 influenza virus by using monoclonal antibody escape mutants.

UNLABELLED: We determined the antigenic structure of pandemic influenza A(H1N1)pdm09 virus hemagglutinin (HA) using 599 escape mutants that were selected using 16 anti-HA monoclonal antibodies (MAbs) against A/Narita/1/2009. The sequencing of mutant HA genes revealed 43 amino acid substitutions at 24 positions in three antigenic sites, Sa, Sb, and Ca2, which were previously mapped onto A/Puerto Rico/8/34 (A/PR/8/34) HA (A. J. Caton, G. G. Brownlee, J. W. Yewdell, and W. Gerhard, Cell 31:417-427, 1982), and an undesignated site, i.e., amino acid residues 141, 142, 143, 171, 172, 174, 177, and 180 in the Sa site, residues 170, 173, 202, 206, 210, 211, and 212 in the Sb site, residues 151, 154, 156, 157, 158, 159, 200, and 238 in the Ca2 site, and residue 147 in the undesignated site (numbering begins at the first methionine). Sixteen MAbs were classified into four groups based on their cross-reactivity with the panel of escape mutants in the hemagglutination inhibition test. Among them, six MAbs targeting the Sa and Sb sites recognized both residues at positions 172 and 173. MAb n2 lost reactivity when mutations were introduced at positions 147, 159 (site Ca2), 170 (site Sb), and 172 (site Sa). We designated the site consisting of these residues as site Pa. From 2009 to 2013, no antigenic drift was detected for the A(H1N1)pdm09 viruses. However, if a novel variant carrying a mutation at a position involved in the epitopes of several MAbs, such as 172, appeared, such a virus would have the advantage of becoming a drift strain. IMPORTANCE: The first influenza pandemic of the 21st century occurred in 2009 with the emergence of a novel virus originating with swine influenza, A(H1N1)pdm09. Although HA of A(H1N1)pdm09 has a common origin (1918 H1N1) with seasonal H1N1, the antigenic divergence of HA between the seasonal H1N1 and A(H1N1)pdm09 viruses gave rise to the influenza pandemic in 2009. To take precautions against the antigenic drift of the A(H1N1)pdm09 virus in the near future, it is important to identify its precise antigenic structure. To obtain various mutants that are not neutralized by MAbs, it is important to neutralize several plaque-cloned parent viruses rather than only a single parent virus. We characterized 599 escape mutants that were obtained by neutralizing four parent viruses of A(H1N1)pdm09 in the presence of 16 MAbs. Consequently, we were able to determine the details of the antigenic structure of HA, including a novel epitope.

Establishment of Reference Reagents for Single-Radial-Immunodiffusion Assay on the 2022/23 Seasonal Influenza Vaccine in Japan and Their Quality Validation.

Potency tests for influenza vaccines are currently performed using a single-radial immunodiffusion (SRID) assay, which requires a reference antigen and anti-hemagglutinin (HA) serum as reference reagents. Reagents must be newly prepared each time a strain used for vaccine production is modified. Therefore, establishing reference reagents of consistent quality is crucial for conducting vaccine potency tests accurately and precisely. Here, we established reference reagents for the SRID assay to conduct lot release tests of quadrivalent influenza vaccines in Japan during the 2022/23 influenza season. The potency of reference antigens during storage was confirmed. Furthermore, we evaluated the cross-reactivity of each antiserum raised against the HA protein of the 2 lineages of influenza B virus toward different lineages of influenza B virus antigens to select a suitable procedure for the SRID assay for accurate measurement. Finally, the intralaboratory reproducibility of the SRID assay using the established reference reagents was validated, and the SRID reagents had sufficient consistent quality, comparable to that of the reagents used for testing vaccines during previous influenza seasons. Our study contributes to the quality control of influenza vaccines.

Evaluation of reverse transcription loop-mediated isothermal amplification assays for rapid diagnosis of pandemic influenza A/H1N1 2009 virus.

Two genetic diagnosis systems using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) technology were evaluated: one for detecting the HA gene of the pandemic influenza A/H1N1 2009 virus (H1pdm RT-LAMP) and the other for detecting the matrix gene of the influenza A virus (TypeA RT-LAMP). The competence of these two RT-LAMP assay kits for the diagnosis of the pandemic influenza A/H1N1 2009 virus was compared using real-time RT-PCR assays developed recently on viruses isolated and clinical specimens collected from patients with suspected infection. TypeA RT-LAMP and H1pdm RT-LAMP showed almost the same sensitivity as real-time RT-PCR for viruses isolated. The sensitivity and specificity of TypeA RT-LAMP and H1pdm RT-LAMP were 96.3% and 88.9%, respectively, for clinical specimens. Considering that the ability of the two RT-LAMP assay kits for detection of the pandemic influenza A/H1N1 2009 virus was comparable to that of the real-time RT-PCR assays, and that the assays were completed within 1 hr and did not require any expensive equipment, these two RT-LAMP assays are promising rapid diagnostic tests for the pandemic influenza A/H1N1 2009 virus at the hospital bedside.

Rapid discrimination of oseltamivir-resistant 275Y and -susceptible 275H substitutions in the neuraminidase gene of pandemic influenza A/H1N1 2009 virus by duplex one-step RT-PCR assay.

Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus caused significant outbreaks worldwide last year (2009). A number of oseltamivir-resistant A/H1N1pdm viruses possessing an H275Y substitution in the neuraminidase (NA) protein were reported sporadically in several countries, including Japan, but they were sensitive to zanamivir and did not spread in the community. In this study, to monitor rapidly and simply oseltamivir-resistant A/H1N1pdm viruses possessing H275Y, a duplex one-step RT-PCR assay (H275Y RT-PCR assay) was developed based on an endpoint genotyping analysis method. H275Y RT-PCR assay evaluated using several subtypes/types of influenza A and B viruses and other respiratory pathogenic viruses and shown to have high sensitivity and high specificity. Forty-four clinical specimens were tested after RNA purification using the H275Y RT-PCR assay, resulting in one clinical specimen being found to contain a virus possessing the H275Y mutation. Seventy-three clinical isolates were then tested with the H275Y assay by using clinical isolates in the cultured supernatants of cells directly, without RNA purification, and the results were consistent with the NA sequencing. Since the H275Y RT-PCR assay could detect the H275Y mutation in clinical isolates without RNA purification, as well as a H275Y mutated virus in clinical specimens after RNA purification, the assay was considered a powerful tool for surveillance screening of oseltamivir-resistant A/H1N1pdm virus activity.

Rapid detection of an I38T amino acid substitution in influenza polymerase acidic subunit associated with reduced susceptibility to baloxavir marboxil.

BACKGROUND: The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved in February 2018 for the treatment of influenza virus infection in Japan. In vitro studies have revealed that an I38T substitution in the polymerase acidic subunit (PA) is associated with reduced susceptibility of influenza viruses to baloxavir. OBJECTIVES: Development of a rapid and simple method for monitoring influenza A(H1N1)pdm09, A(H3N2), and B viruses possessing the I38T substitution in PA. METHODS: Three assays were developed based on RNase H2-dependent PCR (rhPCR) and named A/H1pdm PA_I38T rhPCR, A/H3 PA_I38T rhPCR, and B PA_I38T rhPCR. The assays were evaluated using cDNAs synthesized from in vitro-transcribed PA gene RNA controls, RNAs purified from viruses isolated in the 2017/2018 and 2018/2019 influenza seasons, and RNAs purified from clinical specimens collected in the 2018/2019 influenza season. RESULTS: The assays developed in this study accurately discriminated PA I38 and PA T38 with high sensitivity. CONCLUSIONS: Our assays should be considered a powerful tool for monitoring the emergence of baloxavir-resistant influenza viruses.

Publisher Correction: Clinical evaluation of fully automated molecular diagnostic system "Simprova" for influenza virus, respiratory syncytial virus, and human metapneumovirus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Clinical evaluation of fully automated molecular diagnostic system "Simprova" for influenza virus, respiratory syncytial virus, and human metapneumovirus.

Influenza virus, respiratory syncytial virus, and human metapneumovirus commonly cause acute upper and lower respiratory tract infections, especially in children and the elderly. Although rapid antigen detection tests for detecting these infections have been introduced recently, these are less sensitive than nucleic acid amplification tests. More recently, highly sensitive point-of-care testings (POCTs) have been developed based on nucleic acid amplification tests, which are easy to use in clinical settings. In this study, loop-mediated isothermal amplification (LAMP)-based POCT "Simprova" to detect influenza A and B viruses, respiratory syncytial virus, and human metapneumovirus was developed. Simprova system is fully automated and does not require skilled personnel. In addition, positive results can be achieved faster than with PCR. In this study, the accuracy of the POCT was retrospectively analyzed using 241 frozen stocked specimens. Additionally, the usability of the Simprova at clinical sites was assessed in a prospective clinical study using 380 clinical specimens and compared to those of real-time PCR and rapid antigen detection test. The novel LAMP-based POCT demonstrated high sensitivity and specificity in characterizing clinical specimens from patients with influenza-like illnesses. The Simprova is a powerful tool for early diagnosis of respiratory viral infections in point-of-care settings.

Loop-mediated isothermal amplification-based diagnostic assay for monkeypox virus infections.

Monkeypox virus (MPXV) causes a smallpox-like disease in non-human primates and humans. This infection is endemic to central and western Africa. MPXV is divided into two genetically different groups, Congo Basin and West African MPXV, with the former being the more virulent. A real-time quantitative MPXV genome amplification system was developed for the diagnosis of MPXV infections using loop-mediated isothermal amplification (LAMP) technology. Primers used for genome amplification of Congo Basin (C-LAMP), West African (W-LAMP), and both Congo Basin and West African (COM-LAMP) MPXV by LAMP were designed according to the nucleotide sequences of the Congo Basin-specific D14L gene, the West African-specific partial ATI gene, and the partial ATI gene that is shared by both groups, respectively. The sensitivity and specificity of the LAMP were evaluated with nested PCR using peripheral blood and throat swab specimens collected from Congo Basin MPXV or West African MPXV-infected monkeys. The sensitivity and specificity of COM-LAMP, C-LAMP, and W-LAMP were 80% (45/56) and 100% (64/64); 79% (19/24) and 100% (24/24); and 72% (23/32) and 100% (40/40), respectively. The viremia level determined by LAMP assays increased with increases in the severity of the monkeypox-associated symptoms. The newly developed LAMP assay was confirmed to be a rapid, quantifiable, and highly sensitive and specific system effective in the diagnosis of MPXV infections. The LAMP assays made it possible to discriminate between Congo Basin and West African MPXV. The LAMP developed in this study is useful not only for diagnosis of but also for the assessment of MPXV infections.

Novel virus discovery in field-collected mosquito larvae using an improved system for rapid determination of viral RNA sequences (RDV ver4.0).

In this study, we improved a method for rapid determination of viral RNA sequences (RDV) to overcome the limitations of previous versions. The RDV ver4.0 method can detect RNA sequences with at least 1,000 copies as starting material. A novel virus, which was isolated from field-collected Aedes aegypti larvae in the Phasi Charoen district of Thailand using C6/36 cells, was identified using the RDV ver4.0 protocol. The virus was named Phasi Charoen virus (PhaV). We used a high-throughput pyrosequencing approach to obtain more information about the genome sequence of PhaV. Analysis of a phylogenic tree based on amino acid sequences strongly suggested that PhaV belongs to the family Bunyaviridae.

Epidemiological study of hantavirus infection in the Samara Region of European Russia.

European Russia is a highly endemic area of hemorrhagic fever with renal syndrome (HFRS), a rodent-borne zoonotic disease, caused by hantaviruses. In total, 145 small mammals of four species (Myodes glareolus, Apodemus flavicollis, A. agrarius, and A. uralensis) were trapped in the Samara region of European Russia in August 2005 and examined for the presence of hantavirus (HV). Anti-HV antibodies were found in six of 68 (8.8%) M. glareolus and in one of 19 (5.3%) A. flavicollis by indirect immunofluorescent antibody assay (IFA). The Puumala virus (PUUV), which is one of the hantavirus species, was detected in the lungs of seven M. glareolus by RT-PCR. The virus S-segment was extremely similar (96.2% to 99.3%) to the sequence found in a fatal case of HFRS in the Samara region. Phylogenetic analyses of S and M segments showed that the Samara PUUVs form a cluster within the Russian Volga lineage and apparently differ from other European PUUVs. Anti-PUUV antibodies were found in blood sera from seven HFRS patients and from one undiagnosed patient from the Samara region, using IFA and an enzyme-linked immunosorbent assay (ELISA). These data suggest that the bank vole M. glareolus is a primary natural reservoir and vector for PUUV, which is the main causative agent of HFRS in humans in the Samara region.

Development and Evaluation of New Real-Time RT-PCR Assays for Identifying the Influenza A Virus Cluster IV H3N2 Variant.

From 2005 to July 6, 2018, a total of 435 swine-origin influenza A H3N2 variant virus (H3N2v) infections in humans were reported in the USA. The largest H3N2v outbreak in the USA occurred in 2011-2012. This virus obtained the HA gene from the human seasonal H3N2 influenza A viruses (seasonal H3N2) via human-to-swine transmission in the mid-1990s and was classified as Cluster IV H3N2v. For early detection of public health threats associated with Cluster IV H3N2v distinct from seasonal H3N2, we developed highly specific and sensitive one-step real-time RT-PCR assays directly targeting the HA genes of Cluster IV H3N2v and seasonal H3N2. These assays are useful for the systematic surveillance and identification of Cluster IV H3N2v.

Propagation of Rhinovirus C in Differentiated Immortalized Human Airway HBEC3-KT Epithelial Cells.

Rhinoviruses (RVs) are classified into three species: RV-A, B, and C. Unlike RV-A and -B, RV-C cannot be propagated using standard cell culture systems. In order to isolate RV-Cs from clinical specimens and gain a better understanding of their biological properties and pathogenesis, we established air⁻liquid-interface (ALI) culture methods using HBEC3-KT and HSAEC1-KT immortalized human airway epithelial cells. HBEC3- and HSAEC1-ALI cultures morphologically resembled pseudostratified epithelia with cilia and goblet cells. Two fully sequenced clinical RV-C isolates, RV-C9 and -C53, were propagated in HBEC3-ALI cultures, and increases in viral RNA ranging from 1.71 log10 to 7.06 log10 copies were observed. However, this propagation did not occur in HSAEC1-ALI cultures. Using the HBEC3-ALI culture system, 11 clinical strains of RV-C were isolated from 23 clinical specimens, and of them, nine were passaged and re-propagated. The 11 clinical isolates were classified as RV-C2, -C6, -C9, -C12, -C18, -C23, -C40, and -C53 types according to their VP1 sequences. Our stable HBEC3-ALI culture system is the first cultivable cell model that supports the growth of multiple RV-C virus types from clinical specimens. Thus, the HBEC3-ALI culture system provides a cheap and easy-to-use alternative to existing cell models for isolating and investigating RV-Cs.

Development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primer for influenza virus and respiratory syncytial virus.

Influenza virus and respiratory syncytial virus cause acute upper and lower respiratory tract infections, especially in children and the elderly. Early treatment for these infections is thought to be important, so simple and sensitive detection methods are needed for use at clinical sites. Therefore, in this study, real-time reverse transcription loop-mediated isothermal amplification assays with quenching primer for influenza virus and respiratory syncytial virus were developed. Evaluation of a total of 113 clinical specimens compared to real-time RT-PCR assays showed that the novel assays could distinguish between the types and subtypes of influenza virus and respiratory syncytial virus and had 100% diagnostic specificity. The diagnostic sensitivity of each assay exceeded 85.0% and the assays showed sufficient clinical accuracy. Furthermore, positive results could be obtained in around 15 min using the novel assays in cases with high concentrations of virus. The developed assays should be useful for identifying influenza virus and respiratory syncytial virus cases not only in experimental laboratories but also in hospital and quarantine laboratories.

Detection of antibodies against SARS-Coronavirus using recombinant truncated nucleocapsid proteins by ELISA.

Severe acute respiratory syndrome (SARS) is a lifethreatening emerging respiratory disease caused by the coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is highly antigenic and may be a suitable candidate for diagnostic applications. We constructed truncated recombinant N proteins (N1 [1-422 aa], N2 [1- 109 aa], and N3 [110-422 aa]) and determined their antigenicity by Western blotting using convalescent SARS serum. The recombinants containing N1 and N3 reacted with convalescent SARS serum in Western blotting. However, the recombinant with N2 did not. In ELISA using N1 or N3 as the antigens, positive results were observed in 10 of 10 (100%) SARS-CoV-positive human sera. None of 50 healthy sera gave positive results in either assay. These data indicate that the ELISA using N1 or N3 has high sensitivity and specificity. These results suggest that the middle or C-terminal region of the SARS N protein is important for eliciting antibodies against SARS-CoV during the immune response, and ELISA reactions using N1 or N3 may be a valuable tool for SARS diagnosis.

Characterization and epitope mapping of monoclonal antibodies to the nucleocapsid protein of severe acute respiratory syndrome coronavirus.

The sudden emergence of severe acute respiratory syndrome (SARS) at the end of 2002 resulted in 774 reported deaths from more than 8000 cases worldwide. As no effective vaccines or antiviral agents are available, the most effective measure to prevent the expansion of a SARS epidemic is the rapid diagnosis and isolation of SARS patients. To establish specific diagnostic methods, we generated nine clones of monoclonal antibodies to nucleocapsid protein (NP) of SARS-coronavirus (SARS-CoV). On immunofluorescent antibody assay and Western blotting analysis, none of the monoclonal antibodies showed cross-reactivity to authentic and recombinant NPs of human coronavirus (HCoV) 229E strain. To determine the region on the NP molecule where the monoclonal antibodies bind, we generated four truncated recombinant NPs and analyzed the reactivity between monoclonal antibodies and truncated NPs. Two monoclonal antibodies reacted with a truncated NP covering from amino acid residues 111 to 230, and seven reacted with another truncated NP covering from amino acid residues 221 to 340. Epitope mapping analysis indicated that monoclonal antibody SN5-25 recognized the amino acid sequence Q(245)TVTKK(250) On SARS-NP. Within the epitope, Q245, T246, V247, K249, and K250 appeared to form an essential motif for monoclonal antibody SN5-25 to bind. The information about binding sites and epitopes of monoclonal antibodies may be useful for the development of new diagnostic methods for SARS and for analyzing the function of N protein of SARS-CoV.

Genetic and antigenic analyses of a Puumala virus isolate as a potential vaccine strain.

Puumala virus (PUUV), a causative agent of hemorrhagic fever with renal syndrome (HFRS), is prevalent in Europe and European Russia. No vaccine has been developed for PUUV-associated HFRS, primarily because of the low viral yield in cultured cells. A PUUV strain known as DTK/Ufa-97 was isolated in Russia and adapted for growth in Vero E6 cells maintained in serum-free medium. The DTK/Ufa-97 strain produced a higher viral titer in serum-free medium, suggesting that it may prove useful in the development of an HFRS vaccine. When PUUV-infected Vero E6 cells were grown in serum-free medium, the DTK/Ufa-97 strain yielded more copies of intracellular viral RNA and a higher viral titer in the culture fluid than did the Sotkamo strain. Phylogenetic analysis revealed that PUUVs can be classified into multiple lineages according to geographical origin, and that the DTK/Ufa-97 strain is a member of the Bashkiria-Saratov lineage. The deduced amino acid sequences of the small, medium, and large segments of the DTK/Ufa-97 strain were 99.2% to 100%, 99.3% to 99.8%, and 99.8% identical, respectively, to those of the Bashkirian PUUV strains and 96.9%, 92.6%, and 97.4% identical, respectively, to those of the Sotkamo strain, indicating that the PUUVs are genetically diverse. However, DTK/Ufa-97 and other strains of PUUV exhibited similar patterns of binding to a panel of monoclonal antibodies against Hantaan virus. In addition, diluted antisera (i.e., ranging from 1:160 to 1:640) specific to three strains of PUUV neutralized both homologous and heterologous viruses. These results suggest that the DTK/Ufa-97 strain is capable of extensive growth and is antigenically similar to genetically distant strains of PUUV.