A Complex-Type N-Glycan-Specific Lectin Isolated from Green Alga Halimeda borneensis Exhibits Potent Anti-Influenza Virus Activity.

Marine algal lectins specific for high-mannose N-glycans have attracted attention because they strongly inhibit the entry of enveloped viruses, including influenza viruses and SARS-CoV-2, into host cells by binding to high-mannose-type N-glycans on viral surfaces. Here, we report a novel anti-influenza virus lectin (named HBL40), specific for complex-type N-glycans, which was isolated from a marine green alga, Halimeda borneensis. The hemagglutination activity of HBL40 was inhibited with both complex-type N-glycan and O-glycan-linked glycoproteins but not with high-mannose-type N-glycan-linked glycoproteins or any of the monosaccharides examined. In the oligosaccharide-binding experiment using 26 pyridylaminated oligosaccharides, HBL40 only bound to complex-type N-glycans with bi- and triantennary-branched sugar chains. The sialylation, core fucosylation, and the increased number of branched antennae of the N-glycans lowered the binding activity with HBL40. Interestingly, the lectin potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells at IC50 of 8.02 nM by binding to glycosylated viral hemagglutinin (KD of 1.21 × 10-6 M). HBL40 consisted of two isolectins with slightly different molecular masses to each other that could be separated by reverse-phase HPLC. Both isolectins shared the same 16 N-terminal amino acid sequences. Thus, HBL40 could be useful as an antivirus lectin specific for complex-type N-glycans.

Buffer-free CAS assay using a diluted growth medium efficiently detects siderophore production and microbial growth.

Siderophores are iron chelators and low-molecular-weight compounds secreted by various microorganisms under low-iron conditions. Many microorganisms produce siderophores in the natural environment as iron is an essential element for many of them. CAS assays are widely used to detect siderophores in cultures of various microorganisms; however, it is necessary to improve their sensitivity for the efficient application to fastidious microorganisms. We developed a simple, high-throughput CAS assay employing a buffer-free CAS reagent and diluted growth medium (10% dR2A) in a 96-well microplate. Using a diluted growth medium in agar plates suitable for iron-restricted conditions supported siderophore production by microorganisms from activated sludge. A buffer-free CAS reagent combined with a diluted growth medium revealed that these microorganisms tended to produce more siderophores or iron chelators than microorganisms under iron-rich conditions. Moreover, this buffer-free CAS assay easily and efficiently detected not only siderophore production but also the growth of fastidious microorganisms.

Phospholipid analysis of two influenza A virus-infected cell lines differing in their viral replication kinetics.

Fluctuations in phospholipid composition in infected cells during influenza A virus replication were analyzed using two different susceptible host cell lines: H292 cells, exhibiting a rapid cytopathic effect, and A549 cells, exhibiting a retarded cytopathic effect. Microarray analysis demonstrated that A549 cells recognized influenza A virus invasion, expression of pathogen recognition genes was affected, and antiviral genes were activated. On the other hand, H292 cells did not display such an antiviral state, and in these cells, rapid virus amplification and a rapid cytopathic effect were observed. Levels of ceramide, diacylglycerol, and lysolipids were higher in virus-infected cells than in the corresponding mock-infected cells at the later stages of infection. The accumulation of these lipids in IAV-infected cells occurred together with viral replication. The relationship between the characteristic features of ceramide, diacylglycerol, and lysolipid in the plasma membrane, where enveloped viruses are released, and their role in viral envelope formation are discussed. Our results indicate that viral replication disturbs cellular lipid metabolism, with consequences for viral replication kinetics.

Easy detection of siderophore production in diluted growth media using an improved CAS reagent.

Siderophores are low molecular weight organic compounds produced by various microorganisms, especially pathogenic bacteria including rhizobacteria, and have a high affinity for iron. Although most microorganisms are thought to secrete siderophores under iron-depleted conditions, it is unclear how many microorganisms produce siderophores in the natural environment. Also, the chrome azurol sulfonate (CAS) assay, which is widely used for the detection of siderophores, needs to be improved for wider applicability. We developed a simple, high-throughput CAS assay in a 96-well microplate with a concentrated CAS reagent and commonly used diluted growth media in the absence of artificial iron depletion. The improved microplate CAS shuttle assay revealed that it could easily detect siderophores released from Pseudomonas (P.) fluorescence, P. putida, Burlkholderia stabilis, and Ottowia oryzae, as models of siderophore-producing bacteria. This CAS shuttle assay employed along with diluted growth media is a promising tool to screen new siderophore-producing bacteria.

Lectins engineered to favor a glycan-binding conformation have enhanced antiviral activity.

Homologues of the Oscillatoria agardhii agglutinin (OAA) lectins contain a sequence repeat of ∼66 amino acids, with the number of tandem repeats varying across family members. OAA homologues bind high-mannose glycans on viral surface proteins, thereby interfering with viral entry into host cells. As such, OAA homologues have potential utility as antiviral agents, but a more detailed understanding of their structure-function relationships would enable us to develop improved constructs. Here, we determined the X-ray crystal structure of free and glycan-bound forms of Pseudomonas taiwanensis lectin (PTL), an OAA-family lectin consisting of two tandem repeats. Like other OAA-family lectins, PTL exhibited a ß-barrel-like structure with two symmetrically positioned glycan-binding sites at the opposite ends of the barrel. Upon glycan binding, the conformation of PTL undergoes a more significant change than expected from previous OAA structural analysis. Moreover, the electron density of the bound glycans suggested that the binding affinities are different at the two binding sites. Next, based on analysis of these structures, we used site-specific mutagenesis to create PTL constructs expected to increase the population with a conformation suitable for glycan binding. The engineered PTLs were examined for their antiviral activity against the influenza virus. Interestingly, some exhibited stronger activity compared with that of the parent PTL. We propose that our approach is effective for the generation of potential microbicides with enhanced antiviral activity.

The OAAH Family: Anti-Influenza Virus Lectins.

High mannose (HM)-binding Oscillatoria agardhii agglutinin homologue (OAAH) lectin family is an important class of anti-viral proteins. The OAAH family lectins show potent anti-influenza virus activity with EC50 of nanomolar levels by binding to HM glycans of the envelope glycoprotein hemagglutinin (HA), thereby inhibiting the viral entry into host cells. No broadly effective neutralizing vaccines for influenza virus are available due to the frequent antigenic drift caused by rapid mutations. Alternatives for vaccines need to be developed to prepare for a possible risk of future emergence of a highly virulent virus. Possible use of antiviral lectins is a simple and useful strategy to prevent viral infection by interfering with the interaction between viral HA and the host sialic acid-containing receptor. High-density glycans of surface HA are primary targets for the lectins to inhibit viral entry. In general, the anti-influenza virus potency of lectins is evaluated by a series of inhibitory assays for infection, such as neutral red dye uptake assay to determine the extent of viral cytopathic effect, and immunofluorescence microscopy to detect the expression of viral proteins in infected cells. Direct interaction between lectins and HA could be evaluated by enzyme-linked immunosorbent assay or surface plasmon resonance analysis.

Development of a recombinant replication-deficient rabies virus-based bivalent-vaccine against MERS-CoV and rabies virus and its humoral immunogenicity in mice.

Middle East respiratory syndrome-coronavirus (MERS-CoV) is an emerging virus that causes severe disease with fatal outcomes; however, there are currently no approved vaccines or specific treatments against MERS-CoV. Here, we developed a novel bivalent vaccine against MERS-CoV and rabies virus (RV) using the replication-incompetent P-gene-deficient RV (RVΔP), which has been previously established as a promising and safe viral vector. MERS-CoV spike glycoprotein comprises S1 and S2 subunits, with the S1 subunit being a primary target of neutralizing antibodies. Recombinant RVΔP, which expresses S1 fused with transmembrane and cytoplasmic domains together with 14 amino acids from the ectodomains of the RV-glycoprotein (RV-G), was developed using a reverse genetics method and named RVΔP-MERS/S1. Following generation of RVΔP-MERS/S1 and RVΔP, our analysis revealed that they shared similar growth properties, with the expression of S1 in RVΔP-MERS/S1-infected cells confirmed by immunofluorescence and western blot, and the immunogenicity and pathogenicity evaluated using mouse infection experiments. We observed no rabies-associated signs or symptoms in mice inoculated with RVΔP-MERS/S1. Moreover, virus-specific neutralizing antibodies against both MERS-CoV and RV were induced in mice inoculated intraperitoneally with RVΔP-MERS/S1. These findings indicate that RVΔP-MERS/S1 is a promising and safe bivalent-vaccine candidate against both MERS-CoV and RV.

High Mannose Binding Lectin (PFL) from Pseudomonas fluorescens Down-Regulates Cancer-Associated Integrins and Immune Checkpoint Ligand B7-H4.

Pseudomonas fluorescens lectin (PFL), which belongs to the high mannose (HM)-binding OAAH (Oscillatoria agardhii agglutinin homologue) lectin family, induces cancer cell death. However, the detailed mechanisms underlying this process have not yet been elucidated. We found that PFL decreased various integrins as well as EGFR in cancer cells by promoting internalization and autophagic degradation of these molecules, subsequently inducing caspase-8 dependent cell apoptosis. As revealed by an ex vivo angiogenesis assay using the rat aortic model, PFL inhibited neovascularization in a dose-dependent manner, which was potentially mediated by down-regulation of endothelium integrins. Interestingly, PFL also down-regulated B7-H4 in cancer cells, which has been implicated as a negative regulator of T cell-mediated immunity. We found that B7-H4 co-localized with ß3 integrin in MKN28 gastric cancer cells. siRNA silencing of B7-H4 in MKN28 cells decreased expression of ß3 integrin, suggesting physical and functional association between these molecules. Direct interaction of PFL with integrin αvß3 or B7-H4 was examined by surface plasmon resonance analysis, which detected high affinity glycan-dependent binding to PFL. These investigations suggest that PFL interaction with cell surface integrins is a key process for the anti-cancer activities of PFL.

Replication-incompetent rabies virus vector harboring glycoprotein gene of lymphocytic choriomeningitis virus (LCMV) protects mice from LCMV challenge.

BACKGROUND: Lymphocytic choriomeningitis virus (LCMV) causes a variety of diseases, including asymptomatic infections, meningitis, and congenital infections in the fetus of infected mother. The development of a safe and effective vaccine against LCMV is imperative. This study aims to develop a new candidate vaccine against LCMV using a recombinant replication-incompetent rabies virus (RV) vector. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have generated a recombinant deficient RV expressing the LCMV glycoprotein precursor (GPC) (RVΔP-LCMV/GPC) which is lacking the RV-P gene. RVΔP-LCMV/GPC is able to propagate only in cells expressing the RV-P protein. In contrast, the LCMV-GPC can be expressed in general cells, which do not express RV-P protein. The ability of RVΔP-LCMV/GPC to protect mice from LCMV infection and induce cellular immunity was assessed. Mice inoculated intraperitoneally with RVΔP-LCMV/GPC showed higher survival rates (88.2%) than those inoculated with the parental recombinant RV-P gene-deficient RV (RVΔP) (7.7%) following a LCMV challenge. Neutralizing antibody (NAb) against LCMV was not induced, even in the sera of surviving mice. CD8+ T-cell depletion significantly reduced the survival rates of RVΔP-LCMV/GPC-inoculated mice after the LCMV challenge. These results suggest that CD8+ T cells play a major role in the observed protection against LCMV. In contrast, NAbs against RV were strongly induced in sera of mice inoculated with either RVΔP-LCMV/GPC or RVΔP. In safety tests, suckling mice inoculated intracerebrally with RVΔP-LCMV/GPC showed no symptoms. CONCLUSIONS/SIGNIFICANCE: These results show RVΔP-LCMV/GPC might be a promising candidate vaccine with dual efficacy, protecting against both RV and LCMV.

A Novel High-Mannose Specific Lectin from the Green Alga Halimeda renschii Exhibits a Potent Anti-Influenza Virus Activity through High-Affinity Binding to the Viral Hemagglutinin.

We have isolated a novel lectin, named HRL40 from the green alga Halimeda renschii. In hemagglutination-inhibition test and oligosaccharide-binding experiment with 29 pyridylaminated oligosaccharides, HRL40 exhibited a strict binding specificity for high-mannose N-glycans having an exposed (α1-3) mannose residue in the D2 arm of branched mannosides, and did not have an affinity for monosaccharides and other oligosaccharides examined, including complex N-glycans, an N-glycan core pentasaccharide, and oligosaccharides from glycolipids. The carbohydrate binding profile of HRL40 resembled those of Type I high-mannose specific antiviral algal lectins, or the Oscillatoria agardhii agglutinin (OAA) family, which were previously isolated from red algae and a blue-green alga (cyanobacterium). HRL40 potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells with half-maximal effective dose (ED50) of 2.45 nM through high-affinity binding to a viral envelope hemagglutinin (KD, 3.69 × 10-11 M). HRL40 consisted of two isolectins (HRL40-1 and HRL40-2), which could be separated by reverse-phase HPLC. Both isolectins had the same molecular weight of 46,564 Da and were a disulfide -linked tetrameric protein of a 11,641 Da polypeptide containing at least 13 half-cystines. Thus, HRL40, which is the first Type I high-mannose specific antiviral lectin from the green alga, had the same carbohydrate binding specificity as the OAA family, but a molecular structure distinct from the family.

Anti-influenza virus activity of high-mannose binding lectins derived from genus Pseudomonas.

Lectin PFL binding high-mannose glycan derived from Pseudomonas fluorescens and other homologous lectins: PML derived from Pseudomonas mandelii and PTL derived from Pseudomonas taiwanensis were examined for antiviral activity. The cDNA of these lectin genes were synthesized, cloned, expressed in Escherichia coli. The expressed lectins were purified by gel filtrations, and supplied to cultures infected with several strains of influenza virus. These three lectins have inhibited propagation of influenza viruses with a similar extent, 50% of inhibition-dose was around ten nanomolar concentration. An immunofluorescent microscopy, a microarray analysis, and several infection experiments with different time periods of lectin addition or using the competitor substrates indicated that binding of these lectins with high-mannose glycan on HA protein of influenza virus could block the virus entry into the host cells, thereby resulting in inhibition of the virus propagation. These Pseudomonas-derived lectins would be protential and attractive antiviral agents targeting glycoproteins of enveloped viruses including influenza virus.

Rabies vaccination at a virus-inoculated site as an alternative option to rabies immunoglobulin.

Combined active and passive immunization has been established to be an optimal strategy for rabies post-exposure prophylaxis (PEP). Prompt administration of vaccine and rabies immunoglobulin (RIG) can reliably prevent the disease. However, RIG is unavailable and unaffordable in the majority of cases. On the basis of a model experiment using hamsters, we demonstrated that vaccine injection at the wound site in the same manner as administration of RIG provided protective efficacy that was not inferior to the current optimal PEP, a combination of vaccination and RIG. Further study is needed to determine whether it can replace the use of RIG.

High mannose-binding Pseudomonas fluorescens lectin (PFL) downregulates cell surface integrin/EGFR and induces autophagy in gastric cancer cells.

BACKGROUND: Pseudomonas fluorescens lectin (PFL) belongs to a recently discovered anti-HIV lectin family and induces anoikis-like cell death of MKN28 gastric cancer cells by causing α2 integrin internalization through recognition of high mannose glycans; however, the detailed anti-cancer mechanism is not fully elucidated. METHODS: Cell adherence potency of MKN28 upon PFL treatment was assessed using a colorimetric assay. Cell surface molecules to which PFL bound were identified by peptide mass finger printing with Matrix Assisted Laser Desorption/Ionization-time of flight mass spectrometry and their cellular localization determined by immunofluorescence microscopy. Gene and protein expression in PFL-treated MKN28 cells were evaluated by microarray analysis and western blot, and the function of these genes was evaluated by siRNA knock-down. A proliferation assay measured the sensitivity of PFL-treated cancer cells to anti-cancer drugs. The effect of PFL on subcutaneous MKN28 tumor growth and hepatic tumor formation in BALB/c nude mice was evaluated. RESULTS: The strength of MKN28 cell adherence in vitro to the extracellular matrix was impaired by PFL treatment, consistent with the observation that PFL induces rapid downregulation of surface integrins. PFL also was found to bind to cell surface epidermal growth factor receptor (EGFR). Surface EGFR molecules were endocytosed following PFL binding, and were degraded in a time-dependent fashion. This degradation process was largely the result of autophagy, as revealed by the increased expression of autophagic proteins. PFL-induced EGFR degradation was partly inhibited by RAB7 siRNA as well as LC3 siRNA, and internalized EGFR colocalized with ATG9 at 48 h post-PFL treatment, suggesting that these proteins contribute to dynamic degradation induced by PFL. PFL-induced decrease in surface EGFR rendered MKN28 cells susceptible to gefitinib, a selective inhibitor of EGFR tyrosine kinase. In vivo experiments showed that PFL-treated MKN28-EGFP cells injected in the portal vein of BALB/c nude mice failed to form tumor colonies on the liver, and intratumoral injection of PFL significantly inhibited tumor growth. CONCLUSION: PFL-mediated downregulation of integrin and EGFR contributes to the inhibition of tumor growth in vitro and in vivo. This novel anti-cancer mechanism of PFL suggests that this lectin would be useful as an anti-cancer drug or an adjuvant for other drugs.

Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin.

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC50 of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC50s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells.

Rabies virus glycoprotein variants display different patterns in rabies monosynaptic tracing.

Rabies virus (RV) has been widely used to trace multi-synaptic neuronal circuits. The recent development of glycoprotein-deficient rabies virus (RV-ΔG) expressing various proteins has enabled analyzes of both the structure and function of neuronal circuits. The main advantage of RV-ΔG is its ability to trace monosynaptic circuits by the complementation of rabies virus glycoprotein (RVG), but it has the disadvantage of cytotoxicity. Several strain variants of RV have different biological characteristics, such as synaptic spreading and cytotoxicity, mainly due to amino acid mutations in RVG. We developed an improved protocol for the production of a highly attenuated strain of RV-ΔG and assessed whether RVG variants affect rabies monosynaptic tracing and the health of infected neurons. We demonstrated that (1) rabies monosynaptic tracing with RVG variants traced different subsets of presynaptic partners, (2) RVG of the attenuated strain also labeled astrocytes, and (3) the cytotoxicity of RV-ΔG did not depend on RVG but on RV-ΔG. These findings indicate that RVG variants are an important determinant of rabies monosynaptic tracing.

Sequence analysis of the CXCR4 gene derived from cells surviving feline lentivirus infection.

The feline 3201-S cell line was established from cells that survived productive infection with feline immunodeficiency virus (FIV). We have recently shown that 3201-S cells are free of FIV DNA and are refractory to FIV reinfection. In addition, while the cells express CXCR4, a co-receptor for FIV infection, they are unresponsive to the CXCR4 ligand. In the present study, we show that 3201-S cells encode distinct mutations in the CXCR4 gene. It appears that 3201 cells are heterogeneous, consisting of phenotypically diverse mixed populations resulting from genetic mutations, suggesting that this defect can render the CXCR4 receptor expressed in 3201-S cells biologically dysfunctional.

High mannose-binding antiviral lectin PFL from Pseudomonas fluorescens Pf0-1 promotes cell death of gastric cancer cell MKN28 via interaction with α2-integrin.

Novel anti-HIV lectin family which shows a strict binding specificity for high mannose glycans has been found in lower organisms. The bacterial orthologue has been identified in the genome of Pseudomonas fluorescens Pf0-1 and the gene coding a putative lectin was cloned, expressed in Escherichia coli and purified by one step gel filtration. Glycan array screening of the recombinant lectin, termed PFL, has revealed that PFL preferentially recognizes high mannose glycans with α1-3 Man that was highly exposed at the D2 position. In contrast, masking of this α1-3 Man with α1-2 Man dramatically impaired lectin-carbohydrate interactions. Reducing terminal disaccharide, GlcNAc-GlcNAc of high mannose glycans was also essential for PFL-binding. PFL showed a potent anti-influenza virus activity by inhibiting the virus entry into cells at doses of low nanomolar concentration. At micromolar concentration or higher, PFL showed a cytotoxicity accompanying loss of the cell adhesion against human gastric cancer MKN28 cells. The cell surface molecule to which PFL bound was co-precipitated with biotin-labeled PFL and identified as integrin α2 by peptide mass fingerprinting using MALDI-TOF mass spectrometry. Intriguingly, upon treatment with exogenous PFL, integrin α2 on the cell surface underwent rapid internalization to the cytoplasm and accumulated to perinuclear region, together with the bound PFL. The resulting loss of cell adherence would trigger a signaling pathway that induced anoikis-like cell death. These events were effectively inhibited by pretreatment of PFL with mannnan, indicating the involvement of high mannose glycans on PFL-induced cell death that was triggered by PFL-integrin α2 interactions.

Molecular analysis of the mutational effects of Thai street rabies virus with increased virulence in mice after passages in the BHK cell line.

QS-BHK-P7, street rabies virus, after passages in the BHK cell line, had an in vitro phenotype that distinguished it from its parental virus. Both viruses caused lethal infection in mice by central nervous system inoculation; however, only QS-BHK-P7 killed mice by the intramuscular route. We found four mutations, S23R and H424P in ectodomain of the glycoprotein (G), I1711 V in the polymerase genes, and another at the non-coding region between the phosphoprotein and matrix protein genes of QS-BHK-P7. None of the mutations in the G gene occurred in previously reported pathogenic determinants. The roles of mutations in particular non-coding regions remain to be elucidated.

Serial passage of a street rabies virus in mouse neuroblastoma cells resulted in attenuation: potential role of the additional N-glycosylation of a viral glycoprotein in the reduced pathogenicity of street rabies virus.

Street rabies viruses are field isolates known to be highly neurotropic. However, the viral elements related to their pathogenicity have yet to be identified at the nucleotide or amino acid level. Here, through 30 passages in mouse neuroblastoma NA cells, we have established an attenuated variant of street rabies virus strain 1088, originating from a rabid woodchuck followed by 2 passages in the brains of suckling mice. The variant, 1088-N30, was well adapted to NA cells and highly attenuated in adult mice after intramuscular (i.m.) but not intracerebral (i.c.) inoculations. 1088-N30 had seven nucleotide substitutions, and the R196S mutation of the G protein led to an additional N-glycosylation. Street viruses usually possess one or two N-glycosylation sites on the G protein, 1088 has two, while an additional N-glycosylation site is observed in laboratory-adapted strains. We also established a cloned variant 1088-N4#14 by limiting dilution. Apart from the R196S mutation, 1088-N4#14 possessed only one amino acid substitution in the P protein, which is found in several field isolates. 1088-N4#14 also efficiently replicated in NA cells and was attenuated in adult mice after i.m. inoculations, although it was more pathogenic than 1088-N30. The spread of 1088-N30 in the brain was highly restricted after i.m. inoculations, although the pattern of 1088-N4#14's spread was intermediate between that of the parental 1088 and 1088-N30. Meanwhile, both variants strongly induced humoral immune responses in mice compared to 1088. Our results indicate that the additional N-glycosylation is likely related to the reduced pathogenicity. Taken together, we propose that the number of N-glycosylation sites in the G protein is one of the determinants of the pathogenicity of street rabies viruses.

A unique transcription mode of rabies virus high egg passage-Flury strain detected in infected baby hamster kidney-21 cells.

The transcription mode of rabies virus high egg passage-Flury (HEP) strain was examined and compared with that of Evelyn Rokitniki Abelseth (ERA) strain by northern blot analysis using rabies virus gene-specific probes. The ERA strain was shown to exclusively produce monocistronic mRNAs in transcription. All combinations of multicistronic transcripts, including five monocistronic mRNAs, were detected in the viral RNA transcripts of HEP strain. It was concluded that the unique transcription mode is not due to the nucleotide structure of the genome RNA template, but rather to the viral RNA polymerase of HEP strain. The viral polymerase of HEP strain read through the gene junction at a high frequency. The HEP strain has been passaged many times in chick embryo and cultured cells, and has adapted to propagate well in the baby hamster kidney-21 (BHK-21) cells. Through these passages in various hosts, the HEP strain has acquired a unique transcription mode that might have an advantage in amplification of the virus.