Molecular Epidemiology of Human Metapneumovirus in East Japan before and after COVID-19, 2017-2022.

Human metapneumovirus (hMPV) is genetically classified into two major subgroups, A and B, based on attachment glycoprotein (G protein) gene sequences. The A2 subgroup is further separated into three subdivisions, A2a, A2b (A2b1), and A2c (A2b2). Subgroup A2c viruses carrying 180- or 111-nucleotide duplications in the G gene (A2c 180nt-dup or A2c 111nt-dup ) have been reported in Japan and Spain. The coronavirus disease 2019 (COVID-19) pandemic disrupted the epidemiological kinetics of other respiratory viruses, including hMPV. In this study, we analyzed the sequences of hMPV isolates in Tokyo and Fukushima obtained from 2017 to 2022, i.e., before and after the COVID-19 pandemic. Subgroup A hMPV strains were detected from 2017 to 2019, and most cases were A2c 111nt-dup, suggesting ongoing transmission of this clade, consistent with global transmission dynamics. Subgroup B viruses, but not subgroup A viruses, were detected in 2022 after the COVID-19 peak. Phylogenetic analysis showed that the subgroup B viruses were closely related to strains detected in Yokohama from 2013 to 2016, and strains detected in Fukushima in 2019, suggesting the reappearance of local endemic viruses in East Japan.

Evidence of the simultaneous replications of active viruses in specimens positive for multiple respiratory viruses.

IMPORTANCE: Since the pandemic of coronavirus diseases 2019, the use of real-time PCR assay has become widespread among people who were not familiar with it in virus detection. As a result, whether a high real-time PCR value in one time test indicates virus transmissibly became a complicated social problem, regardless of the difference in assays and/or amplification conditions, the time and number of diagnostic test during the time course of infection. In addition, the multiple positives in the test of respiratory viruses further add to the confusion in the interpretation of the infection. To address this issue, we performed virus isolation using pediatric SARI (severe acute respiratory infections) specimens on air-liquid interface culture of human bronchial/tracheal epithelial cell culture. The result of this study can be a strong evidence that the specimens showing positivity for multiple agents in real-time PCR tests possibly contain infectious viruses.

Construction of a recombinant vaccine expressing Nipah virus glycoprotein using the replicative and highly attenuated vaccinia virus strain LC16m8.

Nipah virus (NiV) is a highly pathogenic zoonotic virus that causes severe encephalitis and respiratory diseases and has a high mortality rate in humans (>40%). Epidemiological studies on various fruit bat species, which are natural reservoirs of the virus, have shown that NiV is widely distributed throughout Southeast Asia. Therefore, there is an urgent need to develop effective NiV vaccines. In this study, we generated recombinant vaccinia viruses expressing the NiV glycoprotein (G) or fusion (F) protein using the LC16m8 strain, and examined their antigenicity and ability to induce immunity. Neutralizing antibodies against NiV were successfully induced in hamsters inoculated with LC16m8 expressing NiV G or F, and the antibody titers were higher than those induced by other vaccinia virus vectors previously reported to prevent lethal NiV infection. These findings indicate that the LC16m8-based vaccine format has superior features as a proliferative vaccine compared with other poxvirus-based vaccines. Moreover, the data collected over the course of antibody elevation during three rounds of vaccination in hamsters provide an important basis for the clinical use of vaccinia virus-based vaccines against NiV disease. Trial Registration: NCT05398796.

Development of a duplex real-time RT-PCR assay for the detection and identification of two subgroups of human metapneumovirus in a single tube.

Human metapneumovirus (hMPV) is a common cause of respiratory infections in children. Many genetic diagnostic assays have been developed, but most detect hMPV regardless of the subgroup. In this study, we developed a real-time RT-PCR assay that can detect and identify the two major subgroups of hMPV (A and B) in one tube. Primers and probes were designed based on the sequences of recent clinical isolates in Japan. The assay showed comparable analytical sensitivity to a previously reported real-time RT-PCR assay and specific reactions to hMPV subgroups. The assay also showed no cross-reactivity to clinical isolates of 19 species of other respiratory viruses. In a validation assay using post-diagnosed clinical specimens, 98% (167/170) positivity was confirmed for the duplex assay, and the three specimens not detected were of low copy number. The duplex assay also successfully distinguished the two major subgroups for all 12 clinical specimens, for which the subgroup had already been determined by genomic sequencing analysis. The duplex assay described here will contribute to the rapid and accurate identification and surveillance of hMPV infections.

Markerless bacterial artificial chromosome manipulation method by red proteins of phage λ mediated homologous recombination utilizing fluorescent proteins for both positive and counter selection.

Manipulating viral genomes is an essential technique in reverse genetics and recombinant vaccine development. A strategy for manipulating large viral genomes involves introducing their entire genome into bacterial artificial chromosomes and employing Escherichia coli genetic tools. For sequence manipulation on bacterial artificial chromosomes (bacterial artificial chromosomes recombineering), a well-established method that relies on the Escherichia coli strain GS1783, and the template plasmid, pEPKan-S, is often used. This method, known as markerless DNA manipulation, allows for the generation of a recombinant bacterial artificial chromosome that does not retain the selection markers used during recombination. Although this method is highly innovative, there remains room for improvement as the plasmid is currently only available for positive selection. Additionally, differentiating true recombinants from false negatives often proves time-consuming. Consequently, an improved method for bacterial artificial chromosomes recombineering, which utilizes fluorescent proteins, has been developed. This method's core comprises three plasmids containing the I-SceI recognition site, antibiotic resistance genes (ampicillin, kanamycin, and zeocin), and fluorescent genes (YPet, mOrange, and mScarlet). The success or failure of Red recombination can be confirmed via fluorescent signals. To validate this method, the Lassa virus genes were introduced into the bacterial artificial chromosomes, containing the entire genome of the vaccinia virus strain LC16m8. Consequently, the expression of fluorescent protein genes contributed to positive selection, such as blue-white screening and counter-selection during the first and second Red recombination.

Rapid whole genome sequencing methods for RNA viruses.

RNA viruses are the etiological agents of many infectious diseases. Since RNA viruses are error-prone during genome replication, rapid, accurate and economical whole RNA viral genome sequence determination is highly demanded. Next-generation sequencing (NGS) techniques perform whole viral genome sequencing due to their high-throughput sequencing capacity. However, the NGS techniques involve a significant burden for sample preparation. Since to generate complete viral genome coverage, genomic nucleic acid enrichment is required by reverse transcription PCR using virus-specific primers or by viral particle concentration. Furthermore, conventional NGS techniques cannot determine the 5' and 3' terminal sequences of the RNA viral genome. Therefore, the terminal sequences are determined one by one using rapid amplification of cDNA ends (RACE). However, since some RNA viruses have segmented genomes, the burden of the determination using RACE is proportional to the number of segments. To date, there is only one study attempting whole genome sequencing of multiple RNA viruses without using above mentioned methods, but the generated sequences' accuracy compared to the reference sequences was up to 97% and did not reach 100% due to the low read depth. Hence, we established novel methods, named PCR-NGS and RCA-NGS, that were optimized for an NGS machine, MinION. These methods do not require nucleic acid amplification with virus-specific PCR primers, physical viral particle enrichment, and RACE. These methods enable whole RNA viral genome sequencing by combining the following techniques: (1) removal of unwanted DNA and RNA other than the RNA viral genome by nuclease treatment; (2) the terminal of viral genome sequence determination by barcoded linkers ligation; (3) amplification of the viral genomic cDNA using ligated linker sequences-specific PCR or an isothermal DNA amplification technique, such as rolling circle amplification (RCA). The established method was evaluated using isolated RNA viruses with single-stranded, double-stranded, positive-stranded, negative-stranded, non-segmented or multi-segmented genomes. As a result, all the viral genome sequences could be determined with 100% accuracy, and these mean read depths were greater than 2,500×, at least using either of the methods. This method should allow for easy and economical determination of accurate RNA viral genomes.

Single Amino Acid Substitution in the Receptor Binding Domain of Spike Protein Is Sufficient To Convert the Neutralization Profile between Ethiopian and Middle Eastern Isolates of Middle East Respiratory Coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes MERS, which is endemic in the Middle East. The absence of human cases in Africa despite the presence of MERS-CoV suggests virological differences between MERS-CoVs in Africa and the Middle East. In fact, in the laboratory, recombinant MERS-CoV carrying the spike (S) protein of Ethiopian isolates exhibits attenuated properties, being more easily neutralized and replicating slower than viruses carrying the S protein of Middle Eastern isolate, EMC. In this study, to identify the amino acids that define the different virological features between Ethiopian and Middle Eastern MERS-CoVs, neutralization titers and viral replication were evaluated using recombinant MERS-CoVs carrying amino acid substitution(s) in the S protein. A single amino acid difference introduced into the receptor binding domain was sufficient to reverse the difference in the neutralizing properties of the S protein between Ethiopian and Middle Eastern MERS-CoVs. Furthermore, amino acid mutations in the S1 and S2 regions of S protein were collectively involved in slow viral replication. Since even a single amino acid difference in S protein can reverse the viral properties of MERS-CoV, it should be noted that multiple mutations may induce a significant change. Careful monitoring of genetic alterations in MERS-CoVs in Africa is therefore required to detect the emergence of virulent strains generated by a few genetic differences. IMPORTANCE There have been no reported cases of human Middle East respiratory syndrome (MERS) in Africa, despite the presence of MERS coronavirus (MERS-CoV). Previous studies have shown that recombinant MERS-CoV carrying the S protein of an Ethiopian isolate replicated slower and was more easily neutralized relative to MERS-CoV carrying the S protein of a Middle Eastern isolate. In this study, we investigated the amino acid(s) in S protein associated with the different viral characteristics between Ethiopian and Middle Eastern MERS-CoVs. The results revealed that a single amino acid difference in the receptor binding domain was sufficient to reverse the neutralization profile. This implies that slight genetic changes can alter the predominant population of MERS-CoV, similar to the transition of variants of severe acute respiratory syndrome coronavirus-2. Careful genetic monitoring of isolates is important to detect the spread of possible virulent MERS-CoVs generated by mutation(s).

Inhibition of cellular RNA methyltransferase abrogates influenza virus capping and replication.

Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces, called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S-adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.

Hydrophobic Alpha-Helical Short Peptides in Overlapping Reading Frames of the Coronavirus Genome.

In this study, we show that the coronavirus (CoV) genome may encode many functional hydrophobic alpha-helical peptides (HAHPs) in overlapping reading frames of major coronaviral proteins throughout the entire viral genome. These HAHPs can theoretically be expressed from non-canonical sub-genomic (sg)RNAs that are synthesized in substantial amounts in infected cells. We selected and analyzed five and six HAHPs encoded in the S gene regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Two and three HAHPs derived from SARS-CoV-2 and MERS-CoV, respectively, specifically interacted with both the SARS-CoV-2 and MERS-CoV S proteins and inhibited their membrane fusion activity. Furthermore, one of the SARS-CoV-2 HAHPs specifically inhibited viral RNA synthesis by accumulating at the site of viral RNA synthesis. Our data show that a group of HAHPs in the coronaviral genome potentially has a regulatory role in viral propagation.

Neutralizing mAbs against SFTS Virus Gn Protein Show Strong Therapeutic Effects in an SFTS Animal Model.

Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease with a high case fatality rate caused by the SFTS virus, and currently there are no approved specific treatments. Neutralizing monoclonal antibodies (mAbs) against the virus could be a therapeutic agent in SFTS treatment, but their development has not sufficiently been carried out. In the present study, mouse and human mAbs exposed to the viral envelope proteins Gn and Gc (16 clones each) were characterized in vitro and in vivo by using recombinant proteins, cell culture with viruses, and an SFTS animal model with IFNAR-/- mice. Neutralization activities against the recombinant vesicular stomatitis virus bearing SFTS virus Gn/Gc as envelope proteins were observed with three anti-Gn and six anti-Gc mAbs. Therapeutic activities were observed among anti-Gn, but not anti-Gc mAbs with neutralizing activities. These results propose an effective strategy to obtain promising therapeutic mAb candidates for SFTS treatment, and a necessity to reveal precise roles of the SFTS virus Gn/Gc proteins.

Ten Nearly Complete Genome Sequences of Human Orthorubulavirus 4 Isolated from Pediatric Inpatients in Fukushima, Japan.

We report 10 nearly complete genomic sequences of human orthorubulavirus 4, also called human parainfluenza virus 4 (HPIV4), isolated from pediatric inpatients with respiratory infections in Fukushima, Japan, by using an air-liquid interface culture of human bronchial and tracheal epithelial cells.

Characterization of Keterah orthonairovirus and evaluation of therapeutic candidates against Keterah orthonairovirus infectious disease.

The species Keterah orthonairovirus is a member of the genus Orthonairovirus. Few studies have focused on this species, and there remains no treatment for Issyk-Kul fever, an infectious disease caused by a Keterah orthonairovirus. This study was performed to characterize this species using two viruses, Issyk-Kul virus (ISKV) and Soft tick bunyavirus (STBV), in cell culture and type I interferon receptor knockout (IFNAR-/-) mice and to evaluate the efficacy of serum transfusion using a mouse model of ISKV infection. The two viruses replicated in many kinds of mammal- and tick-derived cell lines but showed few different characteristics in tropism and antigenicity against anti-viral sera in cell culture. Neither virus caused clinical signs in wild-type mice, but both caused lethal infection in IFNAR-/- mice. ISKV caused more acute death than STBV in IFNAR-/- mice. In both viral infections in IFNAR-/- mice, macroscopic abnormalities were prominent in the liver. Similar levels of viral genome between ISKV- and STBV-infected IFNAR-/- mice were observed in blood, liver, lymphoid tissues and adrenal gland at moribund stages. Hematologic abnormalities in IFNAR-/- mice infected with these viruses, including leukopenia and thrombocytopenia, and biochemical abnormalities indicating liver damage were prominent. In addition, blood levels of many kinds of cytokines and chemokines such as granulocyte colony-stimulating factor, interleukin-6, tumor necrosis factor-α, interferon gamma-induced protein 10 and monocyte chemoattractant protein-1 were elevated. ISKV-immunized serum transfusion after infection delayed the time to death of IFNAR-/- mice. Thus, the present study showed that the species Keterah orthonairovirus could proliferate in most mammal-derived cell lines and cause severe liver lesions and death in IFNAR-/- mice and that serum transfusion might be effective in treatment against Issyk-Kul fever.

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.

A highly attenuated vaccinia virus strain LC16m8-based vaccine for severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) caused by a species Dabie bandavirus (formerly SFTS virus [SFTSV]) is an emerging hemorrhagic infectious disease with a high case-fatality rate. One of the best strategies for preventing SFTS is to develop a vaccine, which is expected to induce both humoral and cellular immunity. We applied a highly attenuated but still immunogenic vaccinia virus strain LC16m8 (m8) as a recombinant vaccine for SFTS. Recombinant m8s expressing SFTSV nucleoprotein (m8-N), envelope glycoprotein precursor (m8-GPC), and both N and GPC (m8-N+GPC) in the infected cells were generated. Both m8-GPC- and m8-N+GPC-infected cells were confirmed to produce SFTSV-like-particles (VLP) in vitro, and the N was incorporated in the VLP produced by the infection of cells with m8-N+GPC. Specific antibodies to SFTSV were induced in mice inoculated with each of the recombinant m8s, and the mice were fully protected from lethal challenge with SFTSV at both 103 TCID50 and 105 TCID50. In mice that had been immunized with vaccinia virus strain Lister in advance of m8-based SFTSV vaccine inoculation, protective immunity against the SFTSV challenge was also conferred. The pathological analysis revealed that mice immunized with m8-GPC or m8-N+GPC did not show any histopathological changes without any viral antigen-positive cells, whereas the control mice showed focal necrosis with inflammatory infiltration with SFTSV antigen-positive cells in tissues after SFTSV challenge. The passive serum transfer experiments revealed that sera collected from mice inoculated with m8-GPC or m8-N+GPC but not with m8-N conferred protective immunity against lethal SFTSV challenge in naïve mice. On the other hand, the depletion of CD8-positive cells in vivo did not abrogate the protective immunity conferred by m8-based SFTSV vaccines. Based on these results, the recombinant m8-GPC and m8-N+GPC were considered promising vaccine candidates for SFTS.

Terminal Genome Sequences of the Soft Tick Bunyavirus.

The complete genome sequence of the soft tick bunyavirus (STBV) was obtained using the Sanger sequencing technique. Comparison with other viral sequences revealed that STBV has unique sequences in the terminal regions that are highly conserved among the genus Orthonairovirus.

Efficient functional screening of a cellular cDNA library to identify severe fever with thrombocytopenia syndrome virus entry factors.

The identification of host cell factors for virus entry is useful for the molecular explanation of viral tropisms and often leads to a more profound understanding of virus-induced diseases. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS virus. No countermeasures against the disease exist. In this report, we show an efficient method using virus-like particles for the functional screening of a cellular cDNA library to identify SFTS virus entry factors. Two variants encoding dendritic cell-specific ICAM-3 grabbing non-integrin related (DC-SIGNR), a calcium-dependent lectin known to enhance SFTS virus infection, were successfully identified from a human liver cDNA library. We will discuss applications for yet unidentified factor(s) for SFTS virus entry and for entry factor(s) for other viruses related to SFTS virus.

Multiple acquired portosystemic shunts secondary to primary hypoplasia of the portal vein in a cat.

A 6-year 5-month-old spayed female Scottish Fold cat presented with a one-month history of gait abnormalities, increased salivation, and decreased activity. A blood test showed hyperammonemia and increased serum bile acids. Imaging tests revealed multiple shunt vessels indicating acquired portosystemic shunt. Histopathologic analysis of liver biopsy showed features consistent with liver hypoperfusion, such as a barely recognizable portal vein, increased numbers of small arterioles, and diffuse vacuolar degeneration of hepatocytes. These findings supported the diagnosis of primary hypoplasia of the portal vein/microvascular dysplasia, (PHPV/MVD). To our knowledge, this is the first case of feline PHPV/MVD that developed multiple acquired portosystemic shunts and presented with hepatic encephalopathy.

Influence of atopic dermatitis on reproduction and uterine natural killer cells.

The causal relationship between severe allergic conditions and successful pregnancy remains unclear. We aimed to evaluate reproductive performance in an experimental mouse model of atopic disease (AD), and the appearance of uterine natural killer (uNK) cells that have crucial roles in placental formation was examined. In the NC/Nga pregnant mice with moderate skin allergic lesions and an 8.6-fold elevation of plasma IgE, significant differences were not detected in the reproductive indices of the number of normal fetuses, abortion rate and placental size. There were few uNK cells in the placenta of AD mice, and they showed a significant decrease regarding the immature subtype as compared with controls. These findings revealed that AD disturbs uNK cell differentiation and provides disadvantageous effects on placental formation, although it does not arrest the pregnancy process. It may be possible that specific immunological conditions behind AD operate favorably to recover the reproductive performance.