Turkeys possess diverse Siaα2-3Gal glycans that facilitate their dual susceptibility to avian influenza viruses isolated from ducks and chickens.

Avian influenza viruses (AIVs) circulating in wild ducks are rarely transmitted directly to chickens. Previous studies demonstrated that chickens possess fucosylated and/or sulfated α2,3 sialosides on their tracheal epithelia, whereas intestinal epithelia of ducks express canonical α2,3 sialosides. Turkeys, the third major poultry species in the world, are known to show broad susceptibility to various avian influenza viruses.

Sulfated glycans containing NeuAcα2-3Gal facilitate the propagation of human H1N1 influenza A viruses in eggs.

When human influenza viruses are isolated and passaged in chicken embryos, variants with amino acid substitutions around the receptor binding site of hemagglutinin (HA) are selected; however, the mechanisms that underlie this phenomenon have yet to be elucidated. Here, we analyzed the receptor structures that contributed to propagation of egg-passaged human H1N1 viruses. The analysis included seasonal and 2009 pandemic strains, both of which have amino acid substitutions of HA found in strains isolated or passaged in eggs.

E190V substitution of H6 hemagglutinin is one of key factors for binding to sulfated sialylated glycan receptor and infection to chickens.

Avian influenza viruses (AIVs) recognize sialic acid linked α2,3 to galactose (SAα2,3Gal) glycans as receptors. In this study, the interactions between hemagglutinins (HAs) of AIVs and sulfated SAα2,3Gal glycans were analyzed to clarify the molecular basis of interspecies transmission of AIVs from ducks to chickens. It was revealed that E190V and N192D substitutions of the HA increased the recovery of viruses derived from an H6 duck virus isolate, A/duck/Hong Kong/960/1980 (H6N2), in chickens.

Optimization of Haemophilus influenzae adhesin transmembrane domain expression in Escherichia coli.

To obtain a high yield of the transmembrane domain of Haemophilus influenzae adhesin (HiaTD) in Escherichia coli, we attempted to express the HiaTD with and without a signal sequence using a T7 expression system. The expression level of HiaTD after induction was followed by quantification of the purified HiaTD, flow cytometric analysis of the outer membrane integrated HiaTD, and immunoblotting assay of fractionated cell lysate. In the expression system with a signal sequence, although the amount of cell-surface-expressed HiaTD increased over time, the number of HiaTD-expressing cells decreased, probably because of plasmid instability.

Splicing of Friend Murine Leukemia Virus env-mRNA Enhances Its Ability to Form Polysomes.

Friend murine leukemia virus (MLV) belongs to the gamma retroviruses of the Retroviridae family. The positive-sense RNA of its genome contains a 5' long terminal repeat (LTR), 5' leader sequence, gag, pol, env, and 3' LTR. Transcription from proviral DNA begins from the R region of the 5' LTR and ends at the polyadenylation signal located at the R region of the other end of the 3' LTR.

Polyadenylation of Friend murine leukemia virus env-mRNA is affected by its splicing.

As splicing was previously found to be important for increasing Friend murine leukemia virus env-mRNA stability and translation, we investigated whether splicing of env-mRNA affected the poly(A) tail length using env expression vectors that yielded unspliced or spliced env-mRNA. Incomplete polyadenylation was detected in a fraction of the unspliced env-mRNA products in an env gene-dependent manner, showing that splicing of Friend murine leukemia virus plays an important role in the efficiency of complete polyadenylation of env-mRNA. These results suggested that the promotion of complete polyadenylation of env-mRNA by splicing might partially explain up-regulation of Env protein expression as a result of splicing.

A chicken influenza virus recognizes fucosylated α2,3 sialoglycan receptors on the epithelial cells lining upper respiratory tracts of chickens.

Influenza viruses recognize sialoglycans as receptors. Although viruses isolated form chickens preferentially bind to sialic acid α2,3 galactose (SAα2,3Gal) glycans as do those of ducks, chickens were not experimentally infected with viruses isolated from ducks. A chicken influenza virus, A/chicken/Ibaraki/1/2005 (H5N2) (Ck/IBR) bound to fucose-branched SAα2,3Gal glycans, whereas the binding towards linear SAα2,3Gal glycans was weak.

A 38 nt region and its flanking sequences within gag of Friend murine leukemia virus are crucial for splicing at the correct 5' and 3' splice sites.

The genome of the Friend murine leukemia virus (Fr-MLV) contains a 5' splice site (5'ss) located at 205 nt and a 3'ss located at 5489 nt. In our previous studies, it was shown that if the HindIII-BglII (879-1904 bp) fragment within gag is deleted from the proA8m1 vector, which carries the entire Fr-MLV sequence, then cryptic splicing of env-mRNA occurs. Here, attempts were made to identify the genomic segment(s) in this region that is/are essential to correct splicing.

Frequent glycan structure mining of influenza virus data revealed a sulfated glycan motif that increased viral infection.

Motivation: It is well known influenza viruses recognize and bind terminal sialic acid (SA) on glycans that are found on the cell surface. In this work, we used a data mining technique to analyze the glycan array data of influenza viruses to find novel glycan structures other than SA that may be involved in viral infection.

Results: In addition to SA structures noted previously, we noted the sulfated structures in the mining results.

Kinetic studies of the effect of a 17-nucleotide difference in the 0.3-kb region containing the R-U5-5' leader sequence of Friend murine leukemia virus on viral gene expression.

In addition to the env gene, a 0.3-kb fragment containing the R-U5-5' leader sequence is essential for the induction of spongiform neurodegeneration by Friend murine leukemia virus (Fr-MLV) clone A8 and it also influences expression of the Env protein. Kinetic studies were carried out using two recombinant viruses, R7f, carrying the A8 0.

O-sulfate groups of heparin are critical for inhibition of ecotropic murine leukemia virus infection by heparin.

There is increasing evidence that soluble glycosaminoglycans such as heparin can interfere with the infectivity of various viruses, including ecotropic murine leukemia viruses (MLVs). The ecotropic MLV, Friend MLV (F-MLV) and the neuropathogenic variants A8 MLV and PVC-211 MLV, were susceptible to heparin-mediated inhibition of infection of NIH 3T3 cells. To investigate the interaction between the envelope glycoprotein (Env) of MLV and heparin, we prepared vesicular stomatitis virus-based pseudotyped viruses carrying the Env of F-, A8, or PVC-211 MLVs.

Narrowing down the critical region within env gene for determining neuropathogenicity of murine leukemia virus A8.

Friend murine leukemia virus clone A8 causes spongiform neurodegeneration in the rat brain, and the env gene of A8 is a primary determinant of neuropathogenicity. In order to narrow down the critical region within the env gene that determines neuropathogenicity, we constructed chimeric viruses having chimeric env between A8 and non-neuropathogenic 57 on the background of A8 virus. After replacement of the BamHI (at nucleotide 5715)-AgeI (at nucleotide 6322) fragment of A8 virus with the corresponding fragment of 57, neuropathogenicity was lost.

Analysis of cis-regulatory elements in the 5' untranslated region of murine leukemia virus controlling protein expression.

It has previously been reported by us that high-level expression of the Env protein of Fr-MLV clone A8 in brains is crucial for induction of spongiform neurodegeneration, and that the 0.3-kb fragment containing the R, U5, and the 5' leader sequence of A8 is responsible for neuropathogenicity. In the present study, the role of the 5' untranslated region in protein expression was investigated.

Friend murine leukemia virus A8 regulates Env protein expression through an intron sequence.

Friend murine leukemia virus (Fr-MLV) produces unspliced mRNAs, as well as singly-spliced mRNAs by which the Env protein is generated. We investigated the role of the intron within the Fr-MLV gene in Env expression using vectors with serially truncated introns. The up-regulatory regions, the 361-878 and the 5135-5399 fragments, and the down-regulatory regions, the 1904-2849 and the 3995-4287 fragments, were found to influence the splicing efficiency.

Neuropathology induced by infection with Friend murine leukemia viral clone A8-V depends upon the level of viral antigen expression.

A8-V is a neuropathogenic clone isolated from the Friend murine leukemia virus which causes spongiosis in the rat brain after infection at birth. Serial studies using chimeric viruses derived from the A8-V and the 57 virus (57-V), which is a non-neuropathogenic strain of Friend murine leukemia virus, proved that the long terminal repeat (LTR) and 5' leader (LTR-leader/A8) derived from A8-V, in addition to the env gene (env/A8) of A8-V, are necessary for the neuropathogenesis of A8-V. The enhancer element within the LTR of A8-V (LTR/A8) has been supposed to contribute to the severe manifestation of spongiosis by inducing high levels of viral production in the brain after A8-V infection.

Cerebral metabolism in brains of rats infected with neuropathogenic murine leukemia viruses.

Friend murine leukemia virus A8 and PVC211 cause spongiform neurodegeneration in rat brains. Glutamate is an important neurotransmitter synthesized from alpha-ketoglutaric acid, an intermediate product of the citric acid cycle, and glutamine is synthesized from glutamate. To examine the brain metabolism of rats infected with neuropathogenic viruses, the amount of glutamate and glutamine in the brains of rats infected with A8, PVC211, and non-neuropathogenic 57 was measured using high performance liquid chromatography, and the (13)C-label incorporation into the C4 position of glutamate and glutamine from [1-(13)C] glucose was measured with (13)C nuclear magnetic resonance.

A viral non-coding region determining neuropathogenicity of murine leukemia virus A8 is responsible for envelope protein expression in the rat brain.

Friend murine leukemia virus clone A8 causes spongiform neurodegeneration in the rat brain. A 0.3-kb fragment containing the R-U5-5' leader sequence of A8 is required in addition to the A8-env gene to induce spongiosis.

Interruption of env gene expression depending on the length of the SV40 early region used for the polyA signal.

In order to invent a screening system to check in vivo gene function and the efficiency of gene transfer mediated by a retroviral vector system, we established a novel packaging cell, PacNIH/A8, based on the neuropathogenic retrovirus A8-V. To construct the expression vector, pA8(Psi-), which expresses the genes gag, pol and env derived from A8-V, the SV40 early region was used for the polyadenylation signal (polyA signal). When a 0.

Analysis of the distribution of neuropathogenic retroviral antigens following PVC211 or A8-V infection.

A8-V and PVC211 are neuropathogenic strains of the Friend murine leukemia virus (Fr-MLV) that cause spongiosis in the rat brain after infection at birth. PVC211 exhibited stronger neuropathogenicity than A8-V, and induced more severe neurological symptoms such as hind-leg paralysis. These symptoms correlated with the neuropathological spread and intensity, which were more severe in the spinal cord of rats infected with PVC211 than in those infected with A8-V, without exhibiting neuropathological differences in other areas of the CNS.

Increased expression of c-myc is associated with thymoma in rats infected with murine leukemia virus A8.

Infection of rats with Friend murine leukemia virus (Fr-MLV) clone A8 causes thymoma in all the animals within 7 weeks. The rapid induction of thymoma is associated with a unique enhancer structure in the U3 region of the A8-LTR. Our Southern blot analyses showed that the thymomas were oligo clonal.

A 0.3-kb fragment containing the R-U5-5' leader sequence is essential for the induction of spongiform neurodegeneration by A8 murine leukemia virus.

Friend murine leukemia virus (Fr-MLV) clone A8 causes spongiform neurodegeneration in the rat brain. The A8-env gene is a primary determinant of neuropathogenicity, and the 1.5-kb ClaI-HindIII fragment containing the LTR and 5' leader from A8 are additionally required for spongiosis.

Unique three-repeat sequences containing FVa, LVb/C4, and CORE motifs in LTR-U3 of Friend murine leukemia virus clone A8 accelerate the induction of thymoma in rat.

Friend murine leukemia virus (Fr-MLV) clone A8 causes thymoma 7 weeks postinfection in rats with a more rapid progression than clone 57. The U3 region of A8-LTR contains a unique structure of enhancer motifs consisting of three repeats of a 38-bp sequence containing FVa, LVb/C4, and CORE motifs. Replacement or deletion of the 38-bp sequence in the A8-U3 resulted in a marked reduction in tumorigenicity.

Identification of genetic determinants that regulate tumorigenicity of Friend murine leukemia virus in rats.

A neuropathogenic variant of Friend murine leukemia virus (FrMLV), clone A8, has been shown to cause thymoma and infiltration of leukemic cells to organs at 7-8 weeks post-infection in rats with a more rapid progression than clone 57. We have previously reported that the determinant for induction of aggressive leukemia in rats is located in the ClaI-AatII fragment containing the long terminal repeat (LTR) and the 5' half of the 5' leader sequence of A8 virus. Further studies of chimeric viruses restricted the determinant for the induction of thymoma to only the 0.

Neuropathology of experimental autoimmune encephalomyelitis modified by retroviral infection.

The A8 virus is a molecular clone of the neuropathogenic FrC6 virus derived from the Friend murine leukemia virus (F-MuLV). To elucidate the effects of A8 virus-infection on immune-mediated diseases in the central nervous system, we investigated the development of acute and monophasic experimental autoimmune encephalomyelitis (EAE) in A8 virus-infected Lewis rats. In EAE rats after A8 virus infection (A8-EAE), many inflammatory cells were found in the gray matter including the frontal lobe, where almost no inflammatory cells were found in rats with EAE alone.