e 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-␤) gene (Ifn-␤) 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. R abies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, infects almost all kinds of mammals, including humans, and causes a severe neurological disease with a high mortality rate of about 100% after a long and inconstant incubation period (usually 20 to 90 days in humans) (reviewed in reference 1). It is estimated that more than 55,000 people die of rabies every year, mainly in Asia and Africa (2), due to the absence of an effective cure and also the complexity and expensiveness of current postexposure prophylaxis, which requires medical treatment (i.e., rabies vaccination) five times over a period of 28 days. In order to develop both therapeutic and novel prophylaxis approaches for rabies, it is necessary to fully understand the pathogenesis of rabies.The pathogenesis of rabies essentially relies on viral spread to and in the nervous system of the infected individual (reviewed in reference 1). RABV secreted into saliva of a rabid animal is generally transmitted via a bite wound caused by the infected animal. After transmission, RABV infects peripheral nerves and then spreads to the central nervous system (CNS) via retrograde axonal transport, followed by active viral replication and spread in the CNS, culminating in severe neurological symptoms and lethal outcome. To date, studies...

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.

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.

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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)⌬P2-5, 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)⌬P2-5 has an impaired ability to express these tPs. After intramuscular inoculation, CE(NiP)⌬P2-5 caused significantly lower morbidity and mortality rates in mice than did CE ( Rabies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, is a zoonotic agent that causes a lethal neurological disease in various mammal species, including humans. After transmission via a bite wound caused by an infected animal, RABV infects peripheral nerves and then spreads to and in the central nervous system (CNS), resulting in severe neurological symptoms with a high case fatality rate of almost 100% (reviewed in reference 1). Due to the absence of an effective cure and insufficient provision of postexposure prophylaxis, approximately 59,000 people die from rabies every year, mainly in developing countries (2). To establish an effective cure and also to develop a novel prophylaxis approach for rabies, it is necessary to understand the molecular mechanisms of the pathogenesis, including immune evasion, of RABV.The phosphoprotein (P protein) of RABV is a multifunctional protein that is indispensable not only for viral replication but also for evasion of host innate immunity. Specifically, this protein plays an essential role in viral RNA synthesis as a cofactor of viral RNA-dependent RNA polymerase (L protein) by bridging nucleoprotein (N protein), which directly binds to viral genomic RNA, and L protein in the ribonucleoprotein complex (reviewed in reference 3). In addition, P protein functions to antagonize the type I interferon (IFN)-mediated antiviral responses by inhibiting both signaling pathways for IFN induction and response (4-12). P protein suppresses activation of interferon regulatory factor 3 (IRF-3), which is an important transcription factor for IFN induction (5, 8). Also, P protein binds to the transcriptional factors signal transducers and activator of transcription 1 (STAT1) and STAT2, which play a key role in the IFN response by activating expression of IFN-stimulated genes (ISGs), and inhibits their nuclear translocation and DNA binding (6,10,11).In RABV-infected cells, mRNA of the P gene is translated from five in-frame start codons by a ribosomal leaky scanning mechanism, resulting in expression of full-length P protein (P1; 297 amino acids) and also less abundant expression of N-terminally truncated P proteins (t...

ABSTRACT. 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.KEY WORDS: matrix protein, pathogenicity, rabies virus.J. Vet. Med. Sci. 73(10): 1363-1366, 2011 Rabies virus (RABV), a member of the genus Lyssavirus of the family Rhabdoviridae, is a highly neurotropic virus that causes encephalomyelitis in mammals including humans with mortality of almost 100%. No effective cure for rabies has so far been established, resulting in estimated 55,000 human deaths every year mainly in Asia and Africa [8]. In order to establish an effective cure, it is important to fully understand the molecular mechanism by which RABV circumvents host immune response and, consequently, causes lethal neurological disease.The genome of RABV is an unsegmented negativestranded RNA of about 12,000 bases that encodes five structural proteins: nucleoprotein (N protein), phosphoprotein (P protein), matrix (M) protein, glycoprotein (G protein) and large (L) protein in that order from the 3' to 5' end of the genome [18]. The N, P and L proteins and the viral genomic RNA compose a ribonucleoprotein complex (RNP). The N protein is responsible for encapsidation of the genomic and antigenomic RNAs, whereas the L protein, in cooperation of the P protein, functions as an RNA-dependent RNA polymerase in infected cells. The M protein is responsible for recruiting RNP to the cell membrane and the budding of enveloped virus particles. The G protein forms spikes that project out from the viral envelope and participate in binding to receptors on host cells.Among these viral proteins, the G protein is known to determine the pathogenicity of RABV. Many studies have shown that pathogenicity in adult mice is altered by amino acid substitutions in the G protein [2,5,13,[15][16][17]. These substitutions affect biological properties of the virus, such as cell-to-cell spread [1,6], membrane fusion [3] and apoptosis-inducing ability [7,12]. While the mechanism by which the G protein determines viral pathogenicity is becoming increasingly clear, little is known about the contribution of viral proteins other than the G protein to pathogenicity.The fixed RABV strain Nishigahara kills adult mice after intracerebral inoculation. In contrast, the Ni-CE strain, which has been established after 100 passages of Nishigahara strain in chicken embryo fibroblast cells, causes nonlethal infection in adult mice. We previously reported t...

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.