Some lyssaviruses, including the rabies virus (RABV), cause lethal neurological symptoms in humans. However, the efficacy of commercial vaccines has only been evaluated against RABV. To assess ...cross-reactivity among lyssaviruses, including RABV, sera from rabbits inoculated with human and animal RABV vaccines and polyclonal antibodies from rabbits immunized with expression plasmids of the glycoproteins of all 18 lyssaviruses were prepared, and cross-reactivity was evaluated via virus-neutralization tests using Duvenhage lyssavirus (DUVV), European bat lyssavirus-1 (EBLV-1), Mokola lyssavirus (MOKV), Lagos bat lyssavirus (LBV), and RABV. The sera from rabbits inoculated with RABV vaccines showed cross-reactivity with EBLV-1 and DUVV, both belonging to phylogroup I. However, reactivity with MOKV and LBV in phylogroup II was notably limited or below the detection level. Next, we compared the cross-reactivity of the polyclonal antibodies against all lyssavirus glycoproteins. Polyclonal antibodies had high virus-neutralization titers against the same phylogroup but not different phylogroups. Our findings indicate that a new vaccine should be developed for pre- and post-exposure prophylaxis against lyssaviral infections.
During 2014, henipavirus infection caused severe illness among humans and horses in southern Philippines; fatality rates among humans were high. Horse-to-human and human-to-human transmission ...occurred. The most likely source of horse infection was fruit bats. Ongoing surveillance is needed for rapid diagnosis, risk factor investigation, control measure implementation, and further virus characterization.
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DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The complete genome sequences of 44 Bacillus cereus group isolates collected from diverse sources in Japan were analyzed to determine their genetic backgrounds and diversity levels in Japan. ...Multilocus sequence typing (MLST) and core-genome single-nucleotide polymorphism (SNP) typing data from whole-genome sequences were analyzed to determine genetic diversity levels. Virulence-associated gene profiles were also used to evaluate the genetic backgrounds and relationships among the isolates.
The 44 B. cereus group isolates, including soil- and animal-derived isolates and isolates recovered from hospitalized patients and food poisoning cases, were genotyped by MLST and core-genome SNP typing. Genetic variation among the isolates was identified by the MLST and core-genome SNP phylogeny comparison against reference strains from countries outside of Japan. Exploratory principal component analysis and nonmetric multidimensional scaling (NMDS) analyses were used to assess the genetic similarities among the isolates using gene presence and absence information and isolate origins as the metadata. A significant correlation was seen between the principal components and the presence of genes encoding hemolysin BL and emetic genetic determinants in B. cereus, and the capsule proteins in B. anthracis. NMDS showed that the cluster of soil isolates overlapped with the cluster comprising animal-derived and clinical isolates.
Molecular and epidemiological analyses of B. cereus group isolates in Japan suggest that the soil- and animal-derived bacteria from our study are not a significant risk to human health. However, because several of the clinical isolates share close genetic relationships with the environmental isolates, both molecular and epidemiological surveillance studies could be used effectively to estimate virulence in these important pathogens.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rabies virus (RABV) causes fatal neurological disease. Pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) using inactivated-virus vaccines are the most effective measures to prevent ...rabies. In Japan, HEP-Flury, the viral strain, used as a human rabies vaccine, has historically been propagated in primary fibroblast cells derived from chicken embryos. In the present study, to reduce the cost and labor of vaccine production, we sought to adapt the original HEP-Flury (HEP) to Vero cells. HEP was repeatedly passaged in Vero cells to generate ten- (HEP-10V) and thirty-passaged (HEP-30V) strains. Both HEP-10V and HEP-30V grew significantly better than HEP in Vero cells, with virulence and antigenicity similar to HEP. Comparison of the complete genomes with HEP revealed three non-synonymous mutations in HEP-10V and four additional non-synonymous mutations in HEP-30V. Comparison among 18 recombinant HEP strains constructed by reverse genetics and vesicular stomatitis viruses pseudotyped with RABV glycoproteins indicated that the substitution P(L115H) in the phosphoprotein and G(S15R) in the glycoprotein improved viral propagation in HEP-10V, while in HEP-30V, G(V164E), G(L183P), and G(A286V) in the glycoprotein enhanced entry into Vero cells. The obtained recombinant RABV strain, rHEP-PG4 strain, with these five substitutions, is a strong candidate for production of human rabies vaccine.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Rabies is a fatal encephalitic infectious disease caused by the rabies virus (RABV). RABV is highly neurotropic and replicates in neuronal cell lines
. The RABV fixed strain, HEP-Flury, was produced ...via passaging in primary chicken embryonic fibroblast cells. HEP-Flury showed rapid adaptation when propagated in mouse neuroblastoma (MNA) cells. In this study, we compared the growth of our previously constructed recombinant HEP (rHEP) strain-based on the sequence of the HEP (HEP-Flury) strain-with that of the original HEP strain. The original HEP strain exhibited higher titer than rHEP and a single substitution at position 80 in the matrix (M) protein M(D80N) after incubation in MNA cells, which was absent in rHEP.
, intracerebral inoculation of the rHEP-M(D80N) strain with this substitution resulted in enhanced viral growth in the mouse brain and a significant loss of body weight in the adult mice. The number of viral antigen-positive cells in the brains of adult mice inoculated with the rHEP-M(D80N) strain was significantly higher than that with the rHEP strain at 5 days post-inoculation. Our findings demonstrate that a single amino acid substitution in the M protein M(D80N) is associated with neurovirulence in mice owing to adaptation to mouse neuronal cells.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Nipah virus (NiV) is an emerging zoonotic paramyxovirus that causes severe encephalitis and respiratory disease with a high mortality rate in humans. During large outbreaks of the viral disease, ...serological testing of serum samples could be a useful diagnostic tool, which could provide information on not only the diagnosis of NiV disease but also the history of an individual with previous exposure to the virus, thereby supporting disease control. Therefore, an efficient method for the inactivation of NiV in serum samples is required for serological diagnosis.
We determined the optimal conditions for the inactivation of NiV infectivity in human serum using heating and UV treatment. The inactivation method comprised UV irradiation with a cover of aluminum foil for 30 min and heating at 56 °C for 30 min.
With an optimized protocol for virus inactivation, NiV infectivity in serum samples (containing 6.0 × 10
TCID
) was completely inactivated.
We developed a recommended protocol for the effective inactivation of NiV. This protocol would enable a regional or local laboratory to safely transport or process samples, including NiV, for serological testing in its biosafety level-2 facility.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rabies is a fatal zoonotic, neurological disease caused by rabies lyssavirus (RABV) and other lyssaviruses. In this study, we established novel serological neutralizing tests (NT) based on vesicular ...stomatitis virus pseudotypes possessing all 18 known lyssavirus glycoproteins. Applying this system to comparative NT against rabbit sera immunized with current RABV vaccines, we showed that the current RABV vaccines fail to elicit sufficient neutralizing antibodies against lyssaviruses other than to those in phylogroup I. Furthermore, comparative NT against rabbit antisera for 18 lyssavirus glycoproteins showed glycoproteins of some lyssaviruses elicited neutralizing antibodies against a broad range of lyssaviruses. This novel testing system will be useful to comprehensively detect antibodies against lyssaviruses and evaluate their cross-reactivities for developing a future broad-protective vaccine.
Intracellular antibodies (intrabodies) are expected to function as therapeutics as well as tools for elucidating in vivo function of proteins. In this study, we propose a novel intrabody selection ...method in the cytosol of mammalian cells by utilizing a growth signal, induced by the interaction of the target antigen and an scFv-c-kit growth sensor. Here, we challenge this method to select specific intrabodies against rabies virus nucleoprotein (RV-N) for the first time. As a result, we successfully select antigen-specific intrabodies from a naïve synthetic library using phage panning followed by our growth sensor-based intracellular selection method, demonstrating the feasibility of the method. Additionally, we succeed in improving the response of the growth sensor by re-engineering the linker region of its construction. Collectively, the described selection method utilizing a growth sensor may become a highly efficient platform for selection of functional intrabodies in the future.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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.
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.
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.
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.
This study was supported by grants from the Japan Agency for Medical Research and Development (AMED).
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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