Hidetoshi Ikeda

Genetic polymorphism of the nsp2 gene in North American type- Porcine reproductive and respiratory syndrome virus

We determined the complete nucleotide sequence of EDRD-1, a Japanese strain of the North American type-Porcine reproductive and respiratory syndrome virus (PRRSV), and identified a novel 117-base deletion and 108-base insertion previously reported in the nsp2 gene of the SP strain, which contains the largest genome among PRRSV strains. Based on genetic analysis of the partial nsp2 gene in 30 additional Japanese isolates and 50 strains from various countries, we classified North American-type PRRSVs into three nsp2-types, represented by EDRD-1, which contains the 117-base deletion and 108-base insertion; prototypic VR-2332, which does not contain the deletion and insertion; and SP, which contains only the 108-base insertion. The three nsp2-types were phylogenetically separated, suggesting that these structural changes only occurred at earlier stages of viral evolution. In the nsp2 genes, we identified an additional 19 deletions ranging from 3 to 378 bases and 2 insertions of 3 and 21 bases which were not common within each nsp2-type, suggesting that these changes occurred at later stages of viral evolution. In addition, our results suggest that the three nsp2-types can be rapidly differentiated by RT-PCR using their polymorphisms as natural tags.

Genetic variation and geographic distribution of porcine reproductive and respiratory syndrome virus in Japan

Summary.Porcine reproductive and respiratory syndrome virus (PRRSV) has two genotypes, the North American-type (NA-type) and the European-type (EU-type), and each genotype is also genetically diverged. We sequenced the ORF5 gene of 30 PRRSVs isolated from 23 prefectures of Japan during 1992 and 1993 and during 2000 and 2001. All of the isolates were of the NA-type. Phylogenetic analysis of the overall NA-type viruses isolated from around the world identified five major genetic clusters. The 1992–1993 Japanese samples belonged to only two genetic clusters, while the 2000–2001 samples included more diverged ORF5 genomes. One genetic cluster, which included 63% (20/32) of Japanese isolates, one Taiwanese isolate and one Chinese isolate, was mainly found in the eastern part of Japan. Another genetic cluster, which was found in various areas around the world, was distributed in the western part of Japan.

Mutation in the cytoplasmic retrieval signal of porcine epidemic diarrhea virus spike (S) protein is responsible for enhanced fusion activity.

Abstract Murine-adapted porcine epidemic diarrhea virus (PEDV), MK-p10, shows high neurovirulence and increased fusion activity compared with a non-adapted MK strain. MK-p10 S protein had four mutations relative to the original virus S, and one of these (H→R at position 1381, H1381R) in the cytoplasmic tail (CT) was suggested to be responsible for the increased fusion activity. To explore this, we examined fusion activity using recombinant S proteins. We expressed and compared the fusion activity of MK-p10 S, S with the H1381R mutation, S with the three other mutations that were not thought to be involved in high fusion activity, and the original S protein. The MK-p10 and MK-H1381R S proteins induced larger cell fusions than others. We also examined the distribution of these S proteins; the MK-p10 and MK-H1381R S proteins were transported onto the cell surface more efficiently than others. These findings suggest that the H1381R mutation is responsible for enhanced fusion activity, which may be attributed to the efficient transfer of S onto the cell surface. H1381 is a component of the KxHxx motif in the CT region, which is a retrieval signal of the S protein for the endoplasmic reticulum–Golgi intermediate compartment (ERGIC). Loss of this motif could allow for the efficient transfer of S proteins from ERGIC onto the cell surface and subsequent increased fusion activity.

Properties of the Naturally Occurring Soluble Surface Glycoprotein of Ecotropic Murine Leukemia Virus: Binding Specificity and Possible Conformational Change after Binding to Receptor

ABSTRACT Ecotropic murine leukemia virus (MuLV) infection is initiated by the interaction between the surface glycoprotein (SU) of the virus and its cell-surface receptor mCAT-1. We investigated the SU-receptor interaction by using a naturally occurring soluble SU which was encoded by the envelope (env) gene of a defective endogenous MuLV, Fv-4r . Binding of the SU to mCAT-1-positive mouse cells was completed by 1 min at 37°C. The SU could not bind to mouse cells that were persistently infected by ecotropic MuLVs (but not amphotropic or dualtropic MuLVs) or transfected with wild-type ecotropic env genes or a mutantenv gene which can express only precursor Env protein that is restricted to retention in the endoplasmic reticulum. These cells were also resistant to superinfection by ecotropic MuLVs. Thus, superinfection resistance correlated with the lack of SU-binding capacity. After binding to the cells, the SU appeared to undergo some conformational changes within 1 min in a temperature-dependent manner. This was suggested by the different properties of two monoclonal antibodies (MAbs) reactive with the same C-terminal half of theFv-4r SU domain, including a proline-rich motif which was shown to be important for conformation of the SU and interaction between the SU and the transmembrane protein. One MAb reacting with the soluble SU bound to cells was dissociated by a temperature shift from 4 to 37°C. Such dissociation was not observed in cells synthesizing the SU or when another MAb was used, indicating that the dissociation was not due to a temperature-dependent release of the MAb but to possible conformational changes in the SU.

Virological Properties and Nucleotide Sequences of Cas-E-Type Endogenous Ecotropic Murine Leukemia Viruses in South Asian Wild Mice, Mus musculus castaneus

ABSTRACT Two types of endogenous ecotropic murine leukemia viruses (MuLVs), termed AKV- and Cas-E-type MuLVs, differ in nucleotide sequence and distribution in wild mouse subspecies. In contrast to AKV-type MuLV, Cas-E-type MuLV is not carried by common laboratory mice. Wild mice ofMus musculus (M. m.) castaneus carry multiple copies of Cas-E-type endogenous MuLV, including the Fv-4r gene that is a truncated form of integrated MuLV and functions as a hosts resistance gene against ecotropic MuLV infection. Our genetic cross experiments showed that only the Fv-4r gene was associated with resistance to ecotropic F-MuLV infection. Because the spontaneous expression of infectious virus was not detected in M. m. castaneus, we generated mice that did not carry the Fv-4r gene but did carry a single or a few endogenous MuLV loci. In mice not carrying theFv-4r gene, infectious MuLVs were isolated in association with three of six Cas-E-type endogenous MuLV loci. The isolated viruses showed a weak syncytium-forming activity for XC cells, an interfering property of ecotropic MuLV, and a slight antigenic variation. Two genomic DNAs containing endogenous Cas-E-type MuLV were cloned and partially sequenced. All of the Cas-E-type endogenous MuLVs were closely related, hybrid-type viruses with an ecotropicenv gene and a xenotropic long terminal repeat. Duplications and a deletion were found in a restricted region of the hypervariable proline-rich region of Env glycoprotein.

Preferential infection of neuronal and astroglia cells by Akabane virus in primary cultures of fetal bovine brain

Akabane virus is a member of the genus Bunyavirus; it is pathogenic for ruminants and transmitted by arthropod vectors. Infection of adult cattle and sheep causes a transient viremia without obvious clinical signs, while infection of pregnant animals often causes fetal abnormalities including hydranencephaly, poliomyelitis and arthrogryposis. Infectious virus or viral antigens is present in the brain, spinal cord and skeletal muscle of infected fetuses. To understand the interaction between Akabane virus and bovine brain cells, we investigated the viral tropism using primary cultures of fetal bovine brain. The cultured neuronal cells, astroglia cells and microglia cells were distinguished by cell type specific antisera. Akabane virus was found to infect neuronal cells and astroglia cells, which led to degenerative death. No microglia cells were found infected. In some brain cultures, we observed different sensitivities of the cells to two Akabane virus strains: an attenuated strain infected and spread more readily than wild type virus. This difference was not observed in a hamster fibroblast cell line. Both viral and host determinants might be involved in the different susceptibility of brain cells to Akabane virus infection.

Polymerase Chain Reaction–based Genetic Typing of Japanese Porcine Reproductive and Respiratory Syndrome Viruses:

Porcine reproductive and respiratory syndrome viruses (PRRSVs) are classified into 2 distinct genotypes: the North American type and the European type. The Japanese PRRSVs were genotyped by reverse transcriptase–polymerase chain reaction using the reported primer pairs that were either reactive to both types, specific to the North American type or specific to the European type. All the PRRSV genomes from 66 tissue homogenates or sera and 55 infectious viruses were of the North American type, whereas no European-type viral genome was detected. Two PCR primers specific to the North American type showed different detection efficiency. Half of the tissue samples and 15% of the infectious viruses were not detected with one primer pair, although all of them were detected with the other primer pair. Nucleotide sequencing analysis of the forward-and reverse primer–binding sites of the nonreactive viruses indicated that all these viruses had nucleotide mismatches within the 4 bases corresponding to the 3′ end of the reverse primer. These mismatches appeared to be responsible for the nonreactivity of the former primers to these viruses.

Coexistence of multiple strains of porcine parvovirus 2 in pig farms

The porcine parvovirus 2 (PPV2) genome was first identified in 2001 in Myanmar. Recently, the PPV2 genome has been found in several other countries. In this study, the prevalence of PPV2 in Japanese domestic pigs was investigated and found to be 58% (69/120) in healthy domestic pigs and 100% (69/69) in sick domestic pigs. Sequencing and phylogenetic analysis of the PCR products of the VP1 gene and an almost full length PPV2 clone indicated that diverged PPV2 strains exist in Japan. Clearly distinct strains of PPV2 were detected in 7 of the 10 pig farms.

Surveillance of diarrhea-causing pathogens in dairy and beef cows in Yamagata Prefecture, Japan from 2002 to 2011.

The economic consequences of bovine diarrhea are serious. Few long‐term epidemiological data are available concerning the causative pathogens of bovine diarrhea in Japan. From 2002 to 2011, surveillance of enteric pathogens was performed in cows of various breed and age from 302 farms in which diarrhea had occurred in Yamagata Prefecture, Japan. Differences between dairy and beef cows in the number of cases of diarrhea and rates of infection by Salmonella spp. and Eimeria spp. were found. Clinical symptoms (duration of epidemic, hematochezia and complications) caused by bovine rotavirus infection were milder than those caused by bovine coronavirus infection.

Efficiency of neural differentiation of mouse P19 embryonal carcinoma cells is dependent on the seeding density

Serum-free culture conditions for retinoic acid-induced neural differentiation of mouse P19 embryonal carcinoma cells were determined for future ex vivo retroviral gene transfer and brain transplantation studies. Neural differentiation of P19 cells was dependent on the seeding densities, and both neurons and astroglia differentiated efficiently at high seeding densities (2 x 10(4) and 5 x 10(4) cells/cm2) but not at low seeding density (1 x 10(4) cells/cm2). In addition, P19 cells cultured at 5 x 10(4) cells/cm2 showed neural differentiated whether or not they were infected with Friend leukemia virus FrC6-V, which inhibited neural differentiation at 2 x 10(4) cells/cm2. These results indicate that FrC6-V-infected P19 embryonal carcinoma cells should be seeded at high density to achieve efficient neural differentiation in vitro for ex vivo gene transfer with a FrC6-V-derived retroviral vector system.