Genomic relationship of porcine hemagglutinating encephalomyelitis virus to bovine coronavirus and human coronavirus OC43 as studied by the use of bovine coronavirus S gene-specific probes

The genomic relationship of porcine hemagglutinating encephalomyelitis virus (HEV) to bovine coronavirus (BCV) and human coronavirus (HCV) strain OC43 was examined by dot blot hybridization assays. Two BCV S gene-specific probes were generated by polymerase chain reaction from the avirulent L9-strain of BCV. Probes were located in the S1 and the S2 region of the peplomeric (S) glycoprotein gene. The S1 probe (726 bp) hybridized with BCV and HCV-OC43, but not with HEV under moderate stringency hybridization conditions (50 degrees C). Only slight signals were present with mouse hepatitis virus (MHV) and no signals were observed with feline infectious peritonitis virus (FIPV) or canine coronavirus (CCV). At high stringency conditions (60 degrees C) the S1 probe hybridized with BCV only. Using the S2 probe (680 bp) under moderate stringency conditions, hybridization signals were obtained with BCV, HCV-OC43 and HEV (strains 67N, NT9, VW572). The signals obtained by the three HEV strains were altogether weaker than with BCV and HCV-OC43. The S2 probe did not react with MHV, FIPV and CCV. At high stringency the S2-specific probe hybridized with BCV and HCV-OC43 but did not hybridize with HEV. Nucleotide sequence analysis of the region covering the S2 probe in HEV revealed 92.6% nucleotide sequence homology to BCV and 91.9% to HCV-OC43. In contrast, the region covering the S1 probe in HEV could not be amplified using the BCV S1-specific primers. The hybridization and sequencing results thus indicate a closer genomic relationship between BCV and HCV-OC43 than there is between HEV and BCV or HCV-OC43 respectively.     

Utilization of chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus to study the molecular basis of virulence

Chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus (HAV) were constructed to evaluate the effect of the 2C gene of AGM-27 on HAV replication in cell culture and virulence in tamarins (Saguinus mystax) and chimpanzees (Pan troglodytes). Kinetic studies and radioimmunofocus assays demonstrated that replacement of the 2C gene of HAV/7, a cell culture-adapted strain of HM-175, with that of AGM-27 drastically reduced the ability of the virus to replicate in cultured cells. Intragenic chimeras containing AGM-27 sequences in either the 5' or 3' half of the 2C gene replicated in cell culture at an intermediate level. Whereas HAV/7 is attenuated for tamarins, a chimera containing the simian virus 2C gene in the HAV/7 background was virulent in tamarins, demonstrating that the simian virus 2C gene alone can confer the phenotype of virulence to an otherwise attenuated virus. Clusters of AGM-27-specific residues near both ends of the 2C protein were required for virulence since a chimera containing AGM-27 sequences in the carboxy-terminal half of 2C was partially attenuated for tamarins while one containing AGM-27 sequences only in the amino-terminal half of 2C was even more attenuated. Chimeras containing either the entire or only the 3' half of the simian virus 2C gene in the HAV/7 background were attenuated for chimpanzees.     

Sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to the human immunodeficiency virus type 1 Vpu protein

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus receptor for infection of human host cells. We have recently demonstrated that Vpu, a human immunodeficiency virus type 1-encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. Using an in vitro model system, we demonstrated that Vpu targets specific sequences in the cytoplasmic domain of CD4 to promote its degradation. In this report, we have further delineated regions within CD4 which are required for susceptibility to Vpu. Transfer of the CD4 cytoplasmic region into a heterologous protein, CD8, rendered the chimeric protein sensitive to Vpu-dependent degradation. In contrast, substitution of the CD8 transmembrane domain with the analogous region from CD4 did not confer sensitivity to Vpu. Finally, mutant forms of the CD4 protein containing the extracellular region alone or the extracellular and transmembrane regions linked to a heterologous cytoplasmic domain were not targeted by Vpu. Thus, sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to Vpu.     

Channel catfish virus: comparative replication and sensitivity of cell lines from channel catfish ovary and the brown bullhead

A cell line derived from channel catfish ovary tissue was compared with the brown bullhead (BB) cell line for their respective abilities to replicate and detect channel catfish virus (CCV). The channel catfish ovary cell line (CCO) produced cytopathic effects (CPE) more rapidly and detected CCV at higher dilutions than did the BB cell line. Production of CCV was more rapid in CCO cells than in BB cells, but the peak titers of the two lines were not significantly different. The CCO cell line was shown to be the more sensitive cell line for CCV research and diagnostics.     

Mapping human interferon-alpha (IFN-alpha 2) binding determinants of the type I interferon receptor subunit IFNAR-1 with human/bovine IFNAR-1 chimeras

Type I interferons bind to a common receptor (IFNAR), composed of two transmembrane polypeptides, IFNAR-1 and IFNAR-2. Although human IFNAR-1 has a weak intrinsic affinity for human Type I interferons (IFNs), bovine IFNAR-1 binds human Type I IFNs with moderate (nM) affinity, and can be conveniently used to investigate the regions of IFNAR-1 involved in ligand binding. We have constructed 14 bovine/human IFNAR-1 chimeras by exchanging homologous subdomains in the extracellular portion of the receptor. These chimeras were expressed at very high levels on COS cells, and their ability to bind HuIFN-alpha2 was measured. No single bovine subdomain substituted into human IFNAR-1 could confer moderate-affinity ligand binding on the resulting chimera. Simultaneous substitution of bovine IFNAR-1 subdomains 2 and 3 for the homologous human subdomains resulted in a dramatic increase in the binding of IFN-alpha2, suggesting that critical determinants for moderate-affinity ligand binding by BoIFNAR-1 reside in these two subdomains. Bovine subdomains 1 and/or 4 each further enhanced IFN-alpha2 binding in the presence of bovine subdomains 2 and 3. Thus, the binding interactions of BoIFNAR-1 with IFNs appears to be more complex than that of other class II cytokine receptors with their ligands.     

Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor

An immunoscreening strategy was used to isolate a cDNA clone encoding the binding domain for the external glycoprotein gp51 of the bovine leukemia virus (BLV). Three recombinant phages demonstrating BLV binding activity and containing 2.3-kbp cDNA inserts with identical nucleotide sequences were isolated from a lambda gt11 cDNA library of bovine kidney cells (MDBK). One clone, BLVRcp1, hybridized with a 4.8-kb mRNA from cells of bovine origin and was also found to be conserved as a single-copy gene in murine, bovine, ovine, primate, canine, feline, and porcine DNAs. The same gene is amplified in caprine DNA isolated from a BLV-induced tumor. The longest open reading frame of BLVRcp1 encodes a protein fragment of 729 amino acids with a putative receptor structure. BLVRcp1 cDNA was cloned in the eucaryotic expression vector pXT-1 and transfected into murine NIH 3T3 and human HEp-2 cells. Cells expressing BLVRcp1 mRNA became susceptible to BLV infection. BLVRcp1 has no known physiological function and has no significant homology with sequences registered in the GenBank and EMBL data libraries (31 July 1992). Expression of deleted constructs of BLVRcp1 indicates that the BLV binding region is encoded at the 5' side of the receptor clone.     

Cytokines and therapeutic oligonucleotides

Non-infectious UV-inactivated transmissible gastroenteritis virus (TGEV) was previously shown to induce interferon alpha (IFN alpha) secretion following in vitro incubation with blood mononuclear cells. In this study, pig foetuses at different stages of gestation were injected in utero with (a) partially UV-inactivated wild TGEV or (b) fully UV-inactivated wild or dm49-4 mutant TGEV coronavirus. Nucleated cells from foetal liver, bone marrow, spleen and blood were isolated 10 or 20 h after injection and assayed ex vivo for IFN alpha secretion by ELISPOT and ELISA techniques. The administration of TGEV induced IFN alpha-secreting cells in foetal lymphohaematopoietic organs at mid-gestation. In contrast, IFN alpha was not detected in control sham-operated foetuses. A specific point mutation in the amino acid sequence of the viral membrane glycoprotein M of TGEV mutant dm49-4 was associated with lower or absent IFN alpha in utero inducibility by mutant virus as compared with wild virus. Flow cytometry analysis did not show differences in leukocyte surface marker expression between control and TGEV- or between dm49-4 and wild virus-treated foetus cells, with the exception of a reduction in percentages of polymorphonuclear cells in TGEV-treated lymphohaematopoietic tissues, which is probably due to IFN alpha secretion. The present data provided in vivo evidence of IFN alpha secretion at the cell level in foetal lymphohaematopoietic organs. Such IFN alpha-secreting cells in lymphohaematopoietic tissues may be the source of IFN alpha detected during foetal infections.     

Enzyme-linked immunosorbent assay, using staphylococcal protein A for detecting virus antibodies

A modification of the indirect enzyme-linked immunosorbent assay (ELISA) was developed which used staphylococcal protein A linked to horseradish peroxidase. Virus antibodies in equine, bovine, porcine, feline, canine, lagomorphic (rabbit), and human sera were detected, using the indirect ELISA in which the antiglobulin enzyme conjugate was replaced by protein A linked to horseradish peroxidase. Results of the ELISA were compared with the results of the serum-virus neutralization test. The application of the test in laboratories performing serologic assays with sera from diverse animal species is discussed.     

Contribution of passive immunity to porcine respiratory coronavirus to protection against transmissible gastroenteritis virus challenge exposure in suckling pigs

OBJECTIVE: To determine the ability of porcine respiratory coronavirus (PRCV) infections to induce passive immunity in suckling pigs to transmissible gastroenteritis virus (TGEV) challenge exposure. DESIGN AND ANIMALS: 4 TGEV seronegative sows and their litters (group A) served as controls, whereas 2 other groups (B and C) of sows (also TGEV seronegative) were oronasally inoculated with live PRCV during 1 or 2 subsequent pregnancies, respectively. PROCEDURE: Effectiveness of passive immunity provided to pigs via colostrum and milk was assessed after TGEV challenge exposure, and TGEV antibody responses in colostrum and milk were analyzed. RESULTS: Mortality in the 3 groups of young pigs correlated with severity of clinical signs of TGEV infection and was highest in control litters (86% in group-A pigs) and lowest in litters of sows inoculated with PRCV in 2 subsequent pregnancies (14% in group-C pigs). Virus-neutralization and IgA and IgG TGEV antibody titers of milk collected from sows at challenge exposure had significant positive correlation with litter survival. Significantly higher numbers of TGEV-specific IgA and IgG antibody-secreting cells were found in group-A pigs than in group-C pigs, suggesting that high titer of maternal antibodies (induced in group-C sows multiply exposed to PRCV) may interfere with active antibody responses. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that, in PRCV-infected pig herds, multiple exposures of pregnant sows are associated with higher IgA and IgG antibody titers to TGEV in milk, and these titers contribute to protection against TGEV infection.     

A murine and a porcine coronavirus are released from opposite surfaces of the same epithelial cells

Epithelial cells are important target cells for coronavirus infection. Earlier we have shown that transmissible gastroenteritis coronavirus (TGEV) and mouse hepatitis coronavirus (MHV) are released from different sides of porcine and murine epithelial cells, respectively. To study the release of these viruses from the same cells, we constructed a porcine LLC-PK1 cell line stably expressing the recombinant MHV receptor cDNA (LMR cells). The MHV and TGEV receptor glycoproteins were shown by immunofluorescence to appear at the surface of the cells and to be functional so that the cells were susceptible to both MHV and TGEV infection. Both coronaviruses entered polarized LMR cells only through the apical surface. Remarkably, while the cells remained susceptible to TGEV for long periods, infectability by MHV decreased with time after plating of the cells onto filters. This was not due to a lack of expression of the MHV receptor, since this glycoprotein was still abundant on the apical surface of these cells. TGEV and MHV appeared to exit LMR cells from opposite sides. Whereas TGEV was released preferentially at the apical membrane, MHV was released preferentially at the basolateral surface. These results show that vesicles containing the two coronaviruses are targeted differently in LMR cells. We propose that the viruses are sorted at the Golgi complex into different transport vesicles that carry information directing them to one of the two surface domains. The apical release of TGEV and the basolateral release of MHV might be factors contributing to the difference in virus spread found between TGEV and MHV in their respective natural hosts, the former causing mainly a localized enteric infection, the latter spreading through the body to other organs.     

Discrimination between transmissible gastroenteritis virus isolates

Twenty TGEV isolates were compared by sequencing a 393-414 nucleotide stretch near the 5' end of the S gene, after amplification by RT-PCR. This part of the S gene is known to show considerable variation between porcine, canine and feline coronaviruses and is completely deleted from porcine respiratory coronaviruses. The discrimination achieved by nucleotide sequence analysis was compared with that obtained by monoclonal antibody typing. The viruses could be split into several clusters, and recent isolates of TGEV from England, The Netherlands and Belgium showed the greatest differences compared to earlier reference types. However, not all viruses with unique isolation histories were distinct, suggesting either genetic stability over many years, laboratory cross-contaminations or repeated introductions of similar viruses into the field. Firm conclusions on evolutionary trends cannot be drawn without obtaining a larger number of isolates, preferably from outbreaks with known epidemiological links. The sequences of some field isolates from the 1980s contained both nucleotide deletions and insertions. The latter included a short sequence of fourteen nucleotides with identity to a region of the TGEV polymerase gene.     

Inhibition of influenza virus hemagglutinin-mediated membrane fusion by a compound related to podocarpic acid

Entry of influenza virus into the host cell is dependent on the fusion of the viral envelope with the endosomal membrane and is mediated by a low-pH-induced change of the viral hemagglutinin (HA) to a conformation that is fusogenic. A compound related to podocarpic acid (180299) was identified that inhibits multicycle replication of influenza A/Kawasaki/86 (H1N1) virus in culture. Treatment of Madin-Darby canine kidney (MDCK) cells with 180299 at 1 h before infection resulted in the inhibition of viral protein synthesis. Addition of 20 microgram of 180299/ml at 1 h p.i. had no effect, indicating that 180299 affects an early step of the influenza viral replication cycle. Genetic analysis of reassortants between sensitive and resistant viruses demonstrated that hemagglutinin (HA) conferred the 180299-resistant (180299(r)) phenotype. Twelve independent isolates of influenza A/Kawasaki/86 were selected for resistance to 180299, and sequence analysis revealed that each of these viruses contained amino acid substitutions in the HA. These mutations are dispersed throughout the HA primary amino acid sequence and cluster in one of two regions: the interface between HA1 and HA2 and in a region near the fusion domain of HA2. When compared with the parent virus, the pH-of-inactivation of the resistant mutants was increased by 0.3 to 0.6 pH unit, suggesting that the mutant HAs undergo the conformational change at an elevated pH. Fusion of human erythrocytes to MDCK cells infected with parent influenza A/Kawasaki/86 was inhibited by 180299 (0.1-10 microgram/ml) in a concentration-dependent manner, whereas fusion of erythrocytes to MDCK cells infected with 180299(r) mutants was not affected. These results suggest that 180299 interacts with the neutral pH conformation of influenza A HA and prevents the low-pH-induced change of HA to its fusogenic conformation.     

Sorting of two polytopic proteins, the gamma-aminobutyric acid and betaine transporters, in polarized epithelial cells

The gamma-aminobutyric acid transporter (GAT-1) isoform of the gamma-aminobutyric acid and the betaine (BGT) transporters exhibit distinct apical and basolateral distributions when introduced into Madin-Darby canine kidney cells (Pietrini, G., Suh, Y. J., Edelman, L., Rudnick, G., and Caplan, M. J. (1994) J. Biol. Chem. 269, 4668-4674). We have investigated the presence of sorting signals in their COOH-terminal cytosolic domains by expression in Madin-Darby canine kidney cells of mutated and chimeric transporters. Whereas truncated GAT-1 (DeltaC-GAT) maintained the original functional activity and apical localization, either the removal (DeltaC-myc BGT) or the substitution (BGS chimera) of the cytosolic tail of BGT generated proteins that accumulated in the endoplasmic reticulum. Moreover, we have found that the cytosolic tail of BGT redirected apical proteins, the polytopic GAT-1 (GBS chimera) and the monotopic human nerve growth factor receptor, to the basolateral surface. These results suggest the presence of basolateral sorting information in the cytosolic tail of BGT. We have further shown that information necessary for the exit of BGT from the endoplasmic reticulum and for the basolateral localization of the GBS chimera is contained in a short segment, rich in basic residues, within the cytosolic tail of BGT.     

The ectodomain of a novel member of the immunoglobulin subfamily related to the poliovirus receptor has the attributes of a bona fide receptor for herpes simplex virus types 1 and 2 in human cells

We report on the functional cloning of a hitherto unknown member of the immunoglobulin (Ig) superfamily selected for its ability to confer susceptibility to herpes simplex virus (HSV) infection on a highly resistant cell line (J1.1-2 cells), derived by exposure of BHKtk- cells to a recombinant HSV-1 expressing tumor necrosis factor alpha (TNF-alpha). The sequence of herpesvirus Ig-like receptor (HIgR) predicts a transmembrane protein with an ectodomain consisting of three cysteine-bracketed domains, one V-like and two C-like. HIgR shares its ectodomain with and appears to be an alternative splice variant of the previously described protein PRR-1 (poliovirus receptor-related protein). Both HIgR and PRR-1 conferred on J1.1-2 cells susceptibility to HSV-1, HSV-2, and bovine herpesvirus 1. The viral ligand of HIgR and PRR-1 is glycoprotein D, a constituent of the virion envelope long known to mediate viral entry into cells through interaction with cellular receptor molecules. Recently, PRR-1, renamed HveC (herpesvirus entry mediator C), and the related PRR-2, renamed HveB, were reported to mediate the entry of HSV-1, HSV-2, and bovine herpesvirus 1, and the homologous poliovirus receptor was reported to mediate the entry of pseudorabies virus (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618-1620, 1998; M. S. Warner, R. J. Geraghty, W. M. Martinez, R. I. Montgomery, J. C. Whitbeck, R. Xu, R. J. Eisenberg, G. H. Cohen, and P. G. Spear, Virology 246:179-189, 1998). Here we further show that HIgR or PRR-1 proteins detected by using a monoclonal antibody to PRR-1 are widely distributed among human cell lines susceptible to HSV infection and commonly used for HSV studies. The monoclonal antibody neutralized virion infectivity in cells transfected with HIgR or PRR-1 cDNA, as well as in the human cell lines, indicating a direct interaction of virions with the receptor molecule, and preliminarily mapping this function to the ectodomain of HIgR and PRR-1. Northern blot analysis showed that HIgR or PRR-1 mRNAs were expressed in human tissues, with the highest expression being detected in nervous system samples. HIgR adds a novel member to the cluster of Ig superfamily members able to mediate the entry of alphaherpesviruses into cells. The wide distribution of HIgR or PRR-1 proteins among human cell lines susceptible to HSV infection, coupled with the neutralizing activity of the antibody in the same cells, provides direct demonstration of the actual use of this cluster of molecules as HSV-1 and HSV-2 entry receptors in human cell lines. The high level of expression in samples from nervous system makes the use of these proteins in human tissues very likely. This cluster of molecules may therefore be considered to constitute bona fide receptors for HSV-1 and HSV-2.     

Replication of bovine respiratory syncytial virus in bovine and ovine peripheral blood lymphocytes and monocytes and monocytic cell lines

The present study compared the replication of bovine respiratory syncytial virus (BRSV) in bovine and ovine peripheral blood mononuclear cells, ovine and bovine monocytic cell lines and ovine alveolar macrophages. Low titres of virus were detected in ovine and bovine lymphocytes and monocytes 24-96 h post-exposure to the virus but there was no apparent replication of the virus in ovine alveolar macrophages during the culture period. The virus replicated to higher but statistically insignificant titres in ovine and bovine peripheral blood monocytes than in lymphocytes, with lymphocytes yielding peak titres significantly earlier. The secondary cell lines obtained from ovine liver and bone marrow also supported the replication of BRSV to high titres. The titres of BRSV in ovine and bovine lymphocytes and monocytes were significantly lower than in secondary cell lines. The addition of human recombinant tumour necrosis factor alpha after exposure to the virus or pre-incubation of ovine or bovine monocytic cells with either human recombinant interleukin 2 or phorbol myristate acetate before exposure to BRSV, did not significantly affect virus titre. Pre-incubation of cells with indomethacin or actinomycin significantly lowered virus titre (p < 0.05).     

A single amino acid in the PB2 gene of influenza A virus is a determinant of host range

The single gene reassortant virus that derives its PB2 gene from the avian influenza A/Mallard/NY/78 virus and remaining genes from the human influenza A/Los Angeles/2/87 virus exhibits a host range restriction (hr) phenotype characterized by efficient replication in avian tissue and failure to produce plaques in mammalian Madin-Darby canine kidney cells. The hr phenotype is associated with restriction of viral replication in the respiratory tract of squirrel monkeys and humans. To identify the genetic basis of the hr phenotype, we isolated four phenotypic hr mutant viruses that acquired the ability to replicate efficiently in mammalian tissue. Segregational analysis indicated that the loss of the hr phenotype was due to a mutation in the PB2 gene itself. The nucleotide sequences of the PB2 gene of each of the four hr mutants revealed that a single amino acid substitution at position 627 (Glu-->Lys) was responsible for the restoration of the ability of the PB2 single gene reassortant to replicate in Madin-Darby canine kidney cells. Interestingly, the amino acid at position 627 in every avian influenza A virus PB2 protein analyzed to date is glutamic acid, and in every human influenza A virus PB2 protein, it is lysine. Thus, the amino acid at residue 627 of PB2 is an important determinant of host range of influenza A viruses.     

Is the screening patch test tray still worth using?

A small DNA virus was newly isolated from the small intestines of a pig with diarrhea in 1987, in Japan. Concerning the physicochemical properties, hemagglutination and susceptibility of cell culture to the virus, the virus was identical to a formerly isolated small DNA virus, the H-45 strain and also physicochemically similar to the parvovirus group. In a serological test however, the virus was distinctly, antigenically different from the H-45 strain as well as each of porcine, bovine and canine parvoviruses.     

Sequence and characterisation of a ribosomal RNA operon from Agrobacterium vitis

Because certain antiganglioside monoclonal antibodies can facilitate antibody-dependent cellular cytotoxicity against GD2+ ganglioside-bearing human and canine tumor cells, we wished to determine if clinically relevant antiganglioside monoclonal antibodies (Mabs) could also fix canine complement to lyse tumor cells in vitro. Using flow cytometry, human tumor cell lines (M21 melanoma and OHS osteosarcoma) were shown to highly express ganglioside GD2 and, to a lesser degree, GD3. In 51Cr release assays, M21 cells were lysed with canine serum, as a source of complement, plus either Mab 14.G2a or its mouse-human chimera, ch 14.18, specific for GD2. Heating canine serum abrogated its lytic activity and addition of rabbit complement reconstituted M21 lysis. Similar results were obtained with M21 cells when Mab R24 (against GD3) and canine serum were used. OHS cells were also lysed with canine serum plus Mab 14.G2a and lytic activity was abolished by heating canine serum but reconstituted with rabbit complement. Alone, canine serum or Mabs were not lytic to M21 or OHS cells. Conversely, human neuroblastoma (LAN-5) and K562 erythroleukemia cells were lysed by canine serum alone which was shown by flow cytometry to contain naturally occurring canine IgM antibodies that bound LAN-5 and K562 cells. The lytic activity of canine serum for LAN-5 or K562 cells was abolished by heating and restored by addition of either human or rabbit complement. Thus, human tumor cell lines can be lysed with antiganglioside Mabs through fixation and activation of canine complement-dependent lytic pathways. Canine xenoantibodies also mediate complement-dependent cytotoxicity of some human tumor cell lines. Together, these results are significant because they demonstrate an antitumor effect of the canine immune system which is of potential importance for cancer immunotherapy in a promising animal model.