Usability of Immortalized Porcine Kidney Macrophage Cultures for the Isolation of ASFV without Affecting Virulence.

Immortalized porcine kidney macrophage (IPKM) cells are highly susceptible to major African swine fever virus (ASFV) isolates. To clarify the compatibility of this cell line for ASFV isolation from biomaterials, animal experiments and in vitro isolation were performed. Pork products seized at international airports were subjected to virus inoculation in pigs (in vivo) and IPKM cell cultures (in vitro) to examine the viability and virulence of the contaminating viruses. Moreover, the viruses isolated using IPKM cells were inoculated into pigs to assess the virulence shift from the original materials. All pigs that were inoculated with either homogenate samples of seized pork product or IPKM-isolated ASFVs developed typical symptoms of ASF and died (or were euthanized) within the term of the animal experiments. The success rate of virus isolation in IPKM cells was comparable to that observed in porcine primary alveolar macrophage (PAM) cells. The IPKM cell line would be an ideal tool for the isolation and propagation of live ASFVs with high efficiency and enhanced usability, such as immortal, proliferative, and adhesive properties. The isolated viruses retained biologically similar characteristics to those of the original ones during isolation in vitro.

Establishment of a Direct PCR Assay for Simultaneous Differential Diagnosis of African Swine Fever and Classical Swine Fever Using Crude Tissue Samples.

African swine fever (ASF) and classical swine fever (CSF) are contagious swine diseases that are clinically indistinguishable from each other; hence, reliable test methods for accurate diagnosis and differentiation are highly demanded. By employing a buffer system suitable for crude extraction of nucleic acids together with an impurity-tolerant enzyme, we established a multiplex assay of real-time reverse-transcription polymerase chain reaction (rRT-PCR) for simultaneous detection of ASF virus (ASFV), CSF virus (CSFV) and swine internal control derived genes in a sample without the need for prior purification of viral nucleic acids. We applied this method to test serum and tissue samples of infected pigs and wild boars and compared the statistical sensitivities and specificities with those of standard molecular diagnostic methods. When a serum was used as a test material, the newly established assay showed 94.4% sensitivity for both and 97.9 and 91.9% specificity for ASFV and CSFV detection, respectively. In contrast, the results were 100% identical with those obtained by the standard methods when a crude tissue homogenate was used as a test material. The present data indicate that this new assay offers a practical, quick, and reliable technique for differential diagnosis of ASF and CSF where geographical occurrences are increasingly overlapping.

An immortalized porcine macrophage cell line competent for the isolation of African swine fever virus.

African swine fever virus (ASFV) is the etiological agent of African swine fever (ASF), a fatal hemorrhagic disease of domestic pigs and wild boar. The virus primarily infects macrophage and monocyte host cells, these do not grow in vitro. Many attempts have been made to establish sustainable ASFV-sensitive cell lines, but which supported only low viral replication levels of limited, mostly artificially attenuated strains of ASFV. Here, we examined the competence of a novel cell line of immortalized porcine kidney macrophages (IPKM) for ASFV infection. We demonstrated that IPKM cells can facilitate high levels (> 107.0 TCID50/mL) of viral replication of ASFV, and hemadsorption reactions and cytopathic effects were observed as with porcine alveolar macrophages when inoculated with virulent field isolates: Armenia07, Kenya05/Tk-1, and Espana75. These results suggested that IPKM may be a valuable tool for the isolation, replication, and genetic manipulation of ASFV in both basic and applied ASF research.

Generation of a novel live rabies vaccine strain with a high level of safety by introducing attenuating mutations in the nucleoprotein and glycoprotein.

The current live rabies vaccine SAG2 is attenuated by only one mutation (Arg-to-Glu) at position 333 in the glycoprotein (G333). This fact generates a potential risk of the emergence of a pathogenic revertant by a back mutation at this position during viral propagation in the body. To circumvent this risk, it is desirable to generate a live vaccine strain highly and stably attenuated by multiple mutations. However, the information on attenuating mutations other than that at G333 is very limited. We previously reported that amino acids at positions 273 and 394 in the nucleoprotein (N273/394) (Leu and His, respectively) of fixed rabies virus Ni-CE are responsible for the attenuated phenotype by enhancing interferon (IFN)/chemokine gene expressions in infected neural cells. In this study, we found that amino acid substitutions at N273/394 (Phe-to-Leu and Tyr-to-His, respectively) attenuated the pathogenicity of the oral live vaccine ERA, which has a virulent-type Arg at G333. Then we generated ERA-N273/394-G333 attenuated by the combination of the above attenuating mutations at G333 and N273/394, and checked its safety. Similar to the ERA-G333, which is attenuated by only the mutation at G333, ERA-N273/394-G333 did not cause any symptoms in adult mice after intracerebral inoculation, indicating a low level of residual pathogenicity of ERA-N273/394-G333. Further examination revealed that infection with ERA-N273/394-G333 induces IFN-ß and CXCL10 mRNA expressions more strongly than ERA-G333 infection in a neuroblastoma cell line. Importantly, we found that the ERA-N273/394-G333 stain has a lower risk for emergence of a pathogenic revertant than does the ERA-G333. These results indicate that ERA-N273/394-G333 has a potential to be a promising candidate for a live rabies vaccine strain with a high level of safety.

Defect of rabies virus phosphoprotein in its interferon-antagonist activity negatively affects viral replication in muscle cells.

Attenuated derivative rabies virus Ni-CE replicates in muscle cells less efficiently than does the parental pathogenic strain Nishigahara. To examine the mechanism underlying the less efficient replication of Ni-CE, we compared the activities of Ni-CE and Nishigahara phosphoproteins, viral interferon (IFN) antagonists, to suppress IFN-ß promoter activity in muscle cells and we demonstrated a defect of Ni-CE phosphoprotein in this ability. Treatment with an IFN-ß-neutralizing antibody improved the replication efficiency of Ni-CE in muscle cells, indicating that produced IFN inhibits Ni-CE replication. The results indicate the importance of IFN antagonism of rabies virus phosphoprotein for viral replication in muscle cells.

Roles of the Rabies Virus Phosphoprotein Isoforms in Pathogenesis.

UNLABELLED: Rabies virus (RABV) P gene mRNA encodes five in-frame start codons, resulting in expression of full-length P protein (P1) and N-terminally truncated P proteins (tPs), designated P2, P3, P4, and P5. Despite the fact that some tPs are known as interferon (IFN) antagonists, the importance of tPs in the pathogenesis of RABV is still unclear. In this study, to examine whether tPs contribute to pathogenesis, we exploited a reverse genetics approach to generate CE(NiP)ΔP2-5, a mutant of pathogenic CE(NiP) in which the P gene was mutated by replacing all of the start codons (AUG) for tPs with AUA. We confirmed that while CE(NiP) expresses detectable levels of P2 and P3, CE(NiP)ΔP2-5 has an impaired ability to express these tPs. After intramuscular inoculation, CE(NiP)ΔP2-5 caused significantly lower morbidity and mortality rates in mice than did CE(NiP), indicating that tPs play a critical role in RABV neuroinvasiveness. Further examinations revealed that this less neuroinvasive phenotype of CE(NiP)ΔP2-5 correlates with its impaired ability to replicate in muscle cells, indicative of the importance of tPs in viral replication in muscle cells. We also demonstrated that CE(NiP)ΔP2-5 infection induced a higher level of Ifn-ß gene expression in muscle cells than did CE(NiP) infection, consistent with the results of an IFN-ß promoter reporter assay suggesting that all tPs function to antagonize IFN induction in muscle cells. Taken together, our findings strongly suggest that tPs promote viral replication in muscle cells through their IFN antagonist activities and thereby support infection of peripheral nerves. IMPORTANCE: Despite the fact that previous studies have demonstrated that P2 and P3 of RABV have IFN antagonist activities, the actual importance of tPs in pathogenesis has remained unclear. Here, we provide the first evidence that tPs contribute to the pathogenesis of RABV, especially its neuroinvasiveness. Our results also show the mechanism underlying the neuroinvasiveness driven by tPs, highlighting the importance of their IFN antagonist activities, which support viral replication in muscle cells.

Isolation of a sp. nov. Ljungan virus from wild birds in Japan.

Ljungan virus (LV) has been isolated/detected from rodents in a limited area including European countries and the USA. In this study, we isolated an LV strain from faecal samples of wild birds that had been collected in Japan, and determined the nearly complete sequence of the genome. Sequence analyses showed that the isolate possesses an LV-like genomic organization: 5UTR-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3UTR. Phylogenetic and similarity analyses based on the VP1 region indicated that the strain constitutes a novel genotype within LV. In addition, we identified species origin of the faeces as gull species by using the DNA barcoding technique. These data suggested that the novel LV strain infected a gull species, in which the virus had not been identified. Taken together, this study has provided the first evidence of the presence of a novel LV in Japan, highlighting the possibility of LV infection in birds.

Isolation and characterization of a novel type of rotavirus species A in sugar gliders (Petaurus breviceps).

To estimate the risk of interspecies transmission of rotavirus species A (RVA) from exotic pets to other mammalian species, the prevalence of RVA in sugar gliders (Petaurus breviceps) was investigated. RVAs were detected in 10 of 44 sugar gliders by reverse transcription (RT)-semi-nested PCR. These viruses were classified as G27P[3] and G27P[36] genotypes, with G27 and P[36] being new genotypes as assigned by the Rotavirus Classification Working Group. To characterize sugar glider RVA in detail, one strain, RVA/SugarGlider-tc/JPN/SG385/2012/G27P[36] (SG385-tc), was isolated. All of the genes of the strain were classified as new genotypes (G27-P[36]-I19-R10-C10-M9-A20-N11-T13-E17-H12). The enterotoxin domain in NSP4, which is important for the induction of diarrhoea, was conserved between SG385-tc and previously reported mammalian strains, suggesting the potential of sugar glider RVA to cause diarrhoea in mammalian species. In fact, seven out of nine suckling mice inoculated orally with 3.9 × 104 f.f.u. of strain SG385-tc had diarrhoea and the 50 % diarrhoea-inducing dose (DD50) of strain SG385-tc in suckling mice was 1.2 × 104 f.f.u. Our findings suggest that sugar glider RVA is infective to and possibly pathogenic in other mammalian species.

Genome Sequences of Rotavirus A Strains Ty-1 and Ty-3, Isolated from Turkeys in Ireland in 1979.

To obtain complete genome sequences of turkey rotavirus A strains Ty-1 and Ty-3, we sequenced the gene segments that had not been decoded previously. The genotype constellations of the respective strains were determined to be G17-P[38]-I4-R4-C4-M4-A16-N4-T4-E4-H4 and G7-P[35]-I4-R4-C4-M4-A16-N4-T4-E11-H14. Notably, their VP4 and NSP5 genes were classified into novel genotypes.

Persistence of the rotavirus A genome in mesenteric lymph nodes of cattle raised on farms.

Previous studies revealed that rotavirus A (RVA) is present in not only the small intestine but also various organs. It was reported that RVA persisted in mesenteric lymph nodes (MLNs) in experimental models. However, there have been no reports focused on RVA in MLNs of animals under natural conditions. In this study, in order to investigate the persistence of the RVA genome in MLNs in cattle under natural conditions, reverse transcription-semi-nested PCR was carried out to detect RVA genomes in the MLNs from 17 calves that had been subjected to autopsy examinations. RVA genomes were detected in MLNs from 10 (˜60  %) of the 17 autopsied calves. MLNs from 170 healthy adult cattle that had been slaughtered were also examined; 15 (∼10  %) of the 170 cattle had RVA genomes in their MLNs, indicating that RNA genomes are found frequently in MLNs of cattle under natural conditions. Genetic analyses revealed that RVAs in MLNs were classified as G and/or P genotypes generally prevalent in bovines. Basically, the strains in intestinal contents were genetically identical to those in MLNs from individual cattle, suggesting that bovine RVAs have the ability to spread from the intestine to MLNs. Furthermore, amongst RVA-positive cattle, six of 10 autopsied calves and 12 of 15 healthy adult cattle were negative for the virus in the intestinal contents, indicating that bovine RVA genomes can persist in MLNs after viral clearance in the digestive tract.

Detection of avian-like rotavirus A VP4 from a calf in Japan.

A total of 568 normal feces from calves on a beef farm in Fukui Prefecture, Japan, in 2011-2012 were examined by RT-semi-nested PCR for rotavirus A (RVA) VP4 genes. Through partial sequencing and BLAST analyses of 84 VP4-positive specimens, we identified an avian-like RVA strain, N2342, which shares highest nucleotide identity (80.0%) with known avian-like bovine strain 993/83, in one specimen. Phylogenetic analysis also revealed a close genetic relationship between N2342 and avian RVAs, suggesting bird-to-cattle transmission. We observed frequent contact of wild birds with calves in the farm, suggesting that these birds were the source of the virus.

Involvement of the rabies virus phosphoprotein gene in neuroinvasiveness.

Rabies virus (RABV), which is transmitted via a bite wound caused by a rabid animal, infects peripheral nerves and then spreads to the central nervous system (CNS) before causing severe neurological symptoms and death in the infected individual. Despite the importance of this ability of the virus to spread from a peripheral site to the CNS (neuroinvasiveness) in the pathogenesis of rabies, little is known about the mechanism underlying the neuroinvasiveness of RABV. In this study, to obtain insights into the mechanism, we conducted comparative analysis of two fixed RABV strains, Nishigahara and the derivative strain Ni-CE, which cause lethal and asymptomatic infections, respectively, in mice after intramuscular inoculation. Examination of a series of chimeric viruses harboring the respective genes from Nishigahara in the genetic background of Ni-CE revealed that the Nishigahara phosphoprotein (P) gene plays a major role in the neuroinvasiveness by mediating infection of peripheral nerves. The results obtained from both in vivo and in vitro experiments strongly suggested that the Nishigahara P gene, but not the Ni-CE P gene, is important for stable viral replication in muscle cells. Further investigation based on the previous finding that RABV phosphoprotein counteracts the host interferon (IFN) system demonstrated that the Nishigahara P gene, but not the Ni-CE P gene, functions to suppress expression of the beta interferon (IFN-ß) gene (Ifn-ß) and IFN-stimulated genes in muscle cells. In conclusion, we provide the first data strongly suggesting that RABV phosphoprotein assists viral replication in muscle cells by counteracting the host IFN system and, consequently, enhances infection of peripheral nerves.

Evidence of natural transmission of group A rotavirus between domestic pigs and wild boars (Sus scrofa) in Japan.

Group A rotaviruses (RVAs) are a major cause of acute dehydrating diarrhea in infants and young animals worldwide. RVAs have also been detected in several wild and zoo animals, indicating wide susceptibility of wild animals. However, the role of wild animals in the infection cycle of RVAs is unclear. Wild boars are indigenous in many countries in the world. Japanese wild boars (Sus scrofa leucomystax) have been migrating close to human habitats in Japan, indicating the possibility of natural transmission between domestic animals or humans and wild boars. We investigated infection of RVAs in wild boars in Japan to identify types of RVAs infecting wild animals. We obtained stool samples from 90 wild boars and detected a VP4 gene of RVAs by RT-semi-nested PCR. RVAs were detected in samples from four of the 90 wild boars. Nucleotide analyses of VP7 and VP4 genes revealed that the four strains belong to G9P[23], G4P[23], G9P[13] and G4P[6], suggesting a relation to porcine and human RVAs. We therefore characterized RVAs circulating among domestic pigs living in the same area as the wild boars. We collected stool samples from 82 domestic pigs. RVAs were detected in samples from 49 of the 82 domestic pigs. Phylogenetic and similarity analyses provided evidence for natural transmission between domestic pigs and wild boars. The results also suggested that natural reassortment events occurred before or after transmission between domestic pigs and wild boars. Our findings indicate the possibility that RVAs circulate among wild animals, humans and domestic animals in nature.

Importance of rabies virus nucleoprotein in viral evasion of interferon response in the brain.

By using a cultured neuroblastoma cell line, the present authors recently showed that the N protein of virulent rabies virus fixed strain Nishigahara (Ni), but not that of the attenuated derivative Ni-CE, mediates evasion of induction of type I interferon (IFN). In this study, to determine whether Ni N protein indeed fulfills this function in vivo, the abilities to suppress IFN responses in the mouse brain of Ni-CE and the virulent chimeric virus CE(NiN), which has the N gene from Ni in the genetic background of Ni-CE, were compared. It was demonstrated that CE(NiN) propagates and spreads more efficiently than does Ni-CE in the brain and that IFN response in brains infected with CE(NiN) is weaker than in those infected with Ni-CE. It was also shown that amino acids at positions 273 and 394 in the N protein, which are known as pathogenic determinants, affect the ability of the viruses to suppress IFN response in the brain. These findings strongly suggest that, in the brain, rabies virus N protein plays important roles in evasion of innate immune responses and thereby in efficient propagation and spread of virus leading to lethal outcomes of infection.

Generation of a live rabies vaccine strain attenuated by multiple mutations and evaluation of its safety and efficacy.

An amino acid substitution at position 333 in rabies virus G protein is known to determine the pathogenicity: strains with Arg or Lys at that position kill adult mice after intracerebral inoculation, whereas strains with other amino acids cause non-lethal infection. Based on those findings, attenuated rabies virus strains have been established and used for oral vaccines mainly for wild animals. However, considering the possibility of back-mutation to the virulent phenotype, a strain that is attenuated by multiple mutations not only in the G protein but also in other viral proteins would be more appropriate as a safe live vaccine. We previously demonstrated that the fixed rabies virus Ni-CE strain, which causes only transient body weight loss in adult mice after intracerebral inoculation, is mainly attenuated by mutations in the N, P and M proteins, while this strain has virulent-type Arg at position 333 in the G protein. In this study, to obtain a live vaccine strain that is attenuated by multiple mutations, we generated Ni-CE mutant, Ni-CE(G333Glu) strain, which has an Arg-to-Glu mutation at position 333 in the G protein, and examined its pathogenicity and immunogenicity. We found that, in contrast to Ni-CE strain, Ni-CE(G333Glu) strain did not cause transient body weight loss in adult mice after intracerebral inoculation. The attenuated phenotype of Ni-CE(G333Glu) strain did not change even after 10 serial intracerebral passages in suckling mice. We also demonstrated that inoculation of Ni-CE(G333Glu) strain induced virus-neutralizing antibody in immunized mice and protected the mice from lethal challenge. These results indicate that Ni-CE(G333Glu) strain is a promising candidate for development of a live rabies vaccine with a high safety level.

Amino acid substitution at position 95 in rabies virus matrix protein affects viral pathogenicity.

We previously reported that rabies virus strain CE(NiM), but not the parental Ni-CE strain, killed mice after intracerebral inoculation. CE(NiM) and Ni-CE are genetically identical except for two amino acids at positions 29 and 95 in the M protein. In this study, to identify which residue determines the pathogenicity, we examined pathogenicities of two Ni-CE mutants, CE(NiM29) and CE(NiM95), which were established by replacement of an amino acid residue at position 29 or 95 in the Ni-CE M protein with the corresponding residue of CE(NiM), respectively. We found that CE(NiM95), but not CE(NiM29), killed mice, indicating that the amino acid at position 95 in the M protein is the pathogenic determinant.