Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice.

The natural reservoir for influenza viruses is waterfowl, and from there they succeeded in crossing the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precision-cut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1 to P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized α2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with α2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190+HA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. In contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs.IMPORTANCE Swine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but have not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by passaging the virus three times in differentiated porcine respiratory epithelial cells. Among the four mutations detected, the two HA mutations were analyzed by generating recombinant viruses. Depending on the infection system used, the mutations differed in their phenotypic expression, e.g., sialic acid binding activity, replication kinetics, plaque size, and pathogenicity in inbred mice. However, none of the mutations affected the ciliary activity which serves as a virulence marker. Thus, early adaptive mutation enhances the replication kinetics, but more mutations are required for IAV of the H9N2 subtype to become virulent.

Dynamic Virus-Bacterium Interactions in a Porcine Precision-Cut Lung Slice Coinfection Model: Swine Influenza Virus Paves the Way for Streptococcus suis Infection in a Two-Step Process.

Swine influenza virus (SIV) and Streptococcus suis are common pathogens of the respiratory tract in pigs, with both being associated with pneumonia. The interactions of both pathogens and their contribution to copathogenesis are only poorly understood. In the present study, we established a porcine precision-cut lung slice (PCLS) coinfection model and analyzed the effects of a primary SIV infection on secondary infection by S. suis at different time points. We found that SIV promoted adherence, colonization, and invasion of S. suis in a two-step process. First, in the initial stages, these effects were dependent on bacterial encapsulation, as shown by selective adherence of encapsulated, but not unencapsulated, S. suis to SIV-infected cells. Second, at a later stage of infection, SIV promoted S. suis adherence and invasion of deeper tissues by damaging ciliated epithelial cells. This effect was seen with a highly virulent SIV subtype H3N2 strain but not with a low-virulence subtype H1N1 strain, and it was independent of the bacterial capsule, since an unencapsulated S. suis mutant behaved in a way similar to that of the encapsulated wild-type strain. In conclusion, the PCLS coinfection model established here revealed novel insights into the dynamic interactions between SIV and S. suis during infection of the respiratory tract. It showed that at least two different mechanisms contribute to the beneficial effects of SIV for S. suis, including capsule-mediated bacterial attachment to SIV-infected cells and capsule-independent effects involving virus-mediated damage of ciliated epithelial cells.

Viral infectivity and intracellular distribution of matrix (M) protein of canine distemper virus are affected by actin filaments.

To investigate the role of cytoskeletal components in canine distemper virus (CDV) replication, various agents were used that interfere with turnover of actin filaments and microtubules. Only inhibition of actin filaments significantly reduced viral infectivity. Analysis of the intracellular localization of the viral matrix (M) protein revealed that it aligned along actin filaments. Treatment with actin filament-disrupting drugs led to a marked intracellular redistribution of M protein during infection as well as transfection. In contrast, the localization of the CDV fusion (F) protein was not significantly changed during transfection. Thus, a M protein-actin filament interaction appears to be important for generation of infectious CDV.

Comparative analysis of the sialic acid binding activity and the tropism for the respiratory epithelium of four different strains of avian infectious bronchitis virus.

Avian infectious bronchitis virus (IBV) is a major pathogen in commercial poultry flocks. We recently demonstrated that sialic acid serves as a receptor determinant for IBV on the tracheal epithelium. Here we compared the IBV strains Beaudette, 4/91, Italy02, and QX for their sialic acid-binding properties. We demonstrate that sialic acid binding is important for the infection of primary chicken kidney cells and the tracheal epithelium by all four strains. There were only slight differences between the four strains, indicating the universal usage of sialic acids as receptor determinants by IBV. In addition, we analysed the primary target cells in the respiratory epithelium of the four different strains and found that all of them infected ciliated cells and goblet cells.

Bovine respiratory syncytial virus lacking the virokinin or with a mutation in furin cleavage site RA(R/K)R109 induces less pulmonary inflammation without impeding the induction of protective immunity in calves.

The BRSV fusion (F) protein is cleaved at two furin consensus sequence sites, resulting in the generation of disulphide-linked F1 and F2 subunits and the release of an intervening peptide of 27 amino acids (pep27), which is converted into a biologically active tachykinin (virokinin). The role of the virokinin and the importance of one of the furin cleavage sites, FCS-2 [RA(R/K)R109], in the pathogenesis of BRSV infection and in the subsequent development of immunity was studied in gnotobiotic calves infected with a recombinant BRSV (rBRSV) lacking pep27 (rBRSVdelta p27) or with rBRSV108/109, which contains two amino acid substitutions in FCS-2 (RANN109). Although replication of the mutant viruses and the parental wild-type (WT) rBRSV in the lungs was similar, the extent of gross and microscopic lesions induced by the mutant viruses was less than that induced by WT rBRSV. Furthermore, the numbers of eosinophils in the lungs of calves infected with the mutant viruses were significantly less than that in calves infected with WT virus. These observations suggest a role for the virokinin in the pathogenesis of BRSV infection. Following mucosal immunization with rBRSVdelta p27, the levels of BRSV-specific serum antibodies were similar to those induced by WT virus. In contrast, the level of neutralizing antibodies induced by rBRSV108/109 was 10-fold lower than that induced by WT virus. Nevertheless, resistance to BRSV challenge induced by the mutant and WT viruses was similar, suggesting that neither pep27 nor FCS-2 plays a major role in the induction of protective immunity.

Transmissible gastroenteritis virus infection: a vanishing specter.

About twenty years ago, a new coronavirus, porcine respiratory coronavirus (PRCoV), was detected in swine herds. This virus is related to transmissible gastroenteritis virus (TGEV); however, it is not enteropathogenic but causes only minor respiratory symptoms. As PRCoV shares some epitopes for neutralizing antibodies with TGEV, it acts like a nature-made vaccine against TGEV resulting in a drastic reduction of TGE outbreaks in Europe. A major difference between the two porcine coronaviruses is a large deletion in the surface protein S gene of PRCoV. Because of this structural difference, TGEV but not PRCoV has a sialic acid binding activity that allows the attachment to mucins and mucin-type glycoproteins. The sialic acid binding activity may allow TGEV to overcome the mucus barrier in the gut and to get access to the intestinal epithelium for initiation of infection.

The hemagglutinin of canine distemper virus determines tropism and cytopathogenicity.

Canine distemper virus (CDV) and measles virus (MV) cause severe illnesses in their respective hosts. The viruses display a characteristic cytopathic effect by forming syncytia in susceptible cells. For CDV, the proficiency of syncytium formation varies among different strains and correlates with the degree of viral attenuation. In this study, we examined the determinants for the differential fusogenicity of the wild-type CDV isolate 5804Han89 (CDV(5804)), the small- and large-plaque-forming variants of the CDV vaccine strain Onderstepoort (CDV(OS) and CDV(OL), respectively), and the MV vaccine strain Edmonston B (MV(Edm)). The cotransfection of different combinations of fusion (F) and hemagglutinin (H) genes in Vero cells indicated that the H protein is the main determinant of fusion efficiency. To verify the significance of this observation in the viral context, a reverse genetic system to generate recombinant CDVs was established. This system is based on a plasmid containing the full-length antigenomic sequence of CDV(OS). The coding regions of the H proteins of all CDV strains and MV(Edm) were introduced into the CDV and MV genetic backgrounds, and recombinant viruses rCDV-H(5804), rCDV-H(OL), rCDV-H(Edm), rMV-H(5804), rMV-H(OL), and rMV-H(OS) were recovered. Thus, the H proteins of the two morbilliviruses are interchangeable and fully functional in a heterologous complex. This is in contrast with the glycoproteins of other members of the family Paramyxoviridae, which do not function efficiently with heterologous partners. The fusogenicity, growth characteristics, and tropism of the recombinant viruses were examined and compared with those of the parental strains. All these characteristics were found to be predominantly mediated by the H protein regardless of the viral backbone used.

Proteolytic activation of respiratory syncytial virus fusion protein. Cleavage at two furin consensus sequences.

The F (fusion) protein of the respiratory syncytial viruses is synthesized as an inactive precursor F(0) that is proteolytically processed at the multibasic sequence KKRKRR(136) into the subunits F(1) and F(2) by the cellular protease furin. This maturation process is essential for the F protein to gain fusion competence. We observed that proteolytic cleavage additionally occurs at another basic motif, RARR(109), that also meets the requirements for furin recognition. Cleavage at both sites leads to the removal from the polypeptide chain of a glycosylated peptide of 27 amino acids. When the sequence RARR(109) was changed to NANR(109) or to RANN(109) by site-directed mutagenesis, cleavage by furin was completely prevented. Although the mutants were still processed at position Arg(136), they did not show any syncytia formation. Proteolytic cleavage of the modified motifs was achieved by treatment of transfected cells with trypsin converting the F mutants into their fusogenic forms. Our findings indicate that both furin consensus sequences have to be cleaved in order to activate the fusion protein.

A single amino acid change in the cytoplasmic domains of measles virus glycoproteins H and F alters targeting, endocytosis, and cell fusion in polarized Madin-Darby canine kidney cells.

As we have shown previously, release of measles virus (MV) from polarized epithelial cells is not determined by the viral envelope proteins H and F. Although virus budding is restricted to the apical surfaces, both proteins were abundantly expressed on the basolateral surface of Madin-Darby canine kidney cells. In this report, we provide evidence that the basolateral expression of the viral proteins is of biological importance for the MV infection of polarized epithelial cells. We demonstrate that both MV glycoproteins possess a basolateral targeting signal that is dependent upon the unique tyrosine in the cytoplasmic tails. These tyrosines are shown to be also part of an endocytosis signal. In MV-infected cells, internalization of the glycoproteins was not observed, indicating that recognition of the endocytosis signals is disturbed by viral factors. In contrast, basolateral transport was not substantially hindered, resulting in efficient cell-to-cell fusion of polarized Madin-Darby canine kidney cells. Thus, recognition of the signals for endocytosis and polarized transport is differently regulated in infected cells. Mutation of the basolateral sorting signal in one of the MV glycoproteins prevented fusion of polarized cells. These results suggest that basolateral expression of the MV glycoproteins favors virus spread in epithelia.

N-glycans of F protein differentially affect fusion activity of human respiratory syncytial virus.

The human respiratory syncytial virus (Long strain) fusion protein contains six potential N-glycosylation sites: N27, N70, N116, N120, N126, and N500. Site-directed mutagenesis of these positions revealed that the mature fusion protein contains three N-linked oligosaccharides, attached to N27, N70, and N500. By introducing these mutations into the F gene in different combinations, four more mutants were generated. All mutants, including a triple mutant devoid of any N-linked oligosaccharide, were efficiently transported to the plasma membrane, as determined by flow cytometry and cell surface biotinylation. None of the glycosylation mutations interfered with proteolytic activation of the fusion protein. Despite similar levels of cell surface expression, the glycosylation mutants affected fusion activity in different ways. While the N27Q mutation did not have an effect on syncytium formation, loss of the N70-glycan caused a fusion activity increase of 40%. Elimination of both N-glycans (N27/70Q mutant) reduced the fusion activity by about 50%. A more pronounced reduction of the fusion activity of about 90% was observed with the mutants N500Q, N27/500Q, and N70/500Q. Almost no fusion activity was detected with the triple mutant N27/70/500Q. These data indicate that N-glycosylation of the F2 subunit at N27 and N70 is of minor importance for the fusion activity of the F protein. The single N-glycan of the F1 subunit attached to N500, however, is required for efficient syncytium formation.

Comparison of the sialic acid binding activity of transmissible gastroenteritis coronavirus and E. coli K99.

Transmissible gastroenteritis coronavirus (TGEV) and Escherichia coli K99 are both enteropathogenic for pigs with infections being most severe in neonate animals. For both microorganisms, a sialic acid binding activity has been shown to be an essential pathogenicity factor. Here we demonstrate with haemagglutination and haemagglutination-inhibition assays that TGEV and E. coli K99 differ in their sialic acid binding activities with respect to the type and amount of sialic acid residues required on the erythrocytes surface as well as with respect to the type of sialoglycoconjugate preferentially recognized. Intestinal mucins from piglets (12-14 days old) and adult animals were shown to inhibit TGEV to the same extent. From our results we conclude that E. coli K99 and TGEV interact with different sialoglycoconjugates to establish an intestinal infection. The implications for the enteropathogenicity of TGEV are discussed.

Sialic acid binding activity of transmissible gastroenteritis coronavirus affects sedimentation behavior of virions and solubilized glycoproteins.

The sedimentation behavior of transmissible gastroenteritis coronavirus (TGEV) was analyzed. Upon sucrose gradient centrifugation, the major virus band was found at a density of 1.20 to 1.22 g/cm(3). This high density was observed only when TGEV with a functional sialic acid binding activity was analyzed. Mutants of TGEV that lacked sialic acid binding activity due to a point mutation in the sialic acid binding site of the S protein were mainly recovered at a lower-density position on the sucrose gradient (1.18 to 1.19 g/cm(3)). Neuraminidase treatment of purified virions resulted in a shift of the sedimentation value from the higher to the lower density. These results suggest that binding of sialoglycoproteins to the virion surface is responsible for the sedimentation behavior of TGEV. When purified virions were treated with octylglucoside to solubilize viral glycoproteins, ultracentrifugation resulted in sedimentation of the S protein of TGEV. However, when neuraminidase-treated virions or mutants with a defective sialic acid binding activity were analyzed, the S protein remained in the supernatant rather than in the pellet fraction. These results indicate that the interaction of the surface protein S with sialoglycoconjugates is maintained after solubilization of this viral glycoprotein by detergent treatment.

Recombinant measles virus requiring an exogenous protease for activation of infectivity.

Proteolytic cleavage of the fusion protein (F) is an important control mechanism of the biological activity of paramyxoviruses. The sequence R-R-H-K-R(112) at the cleavage site of the F protein of measles virus (MV) was altered by site-directed mutagenesis to R-N-H-N-R(112), which is not recognized by the ubiquitous cellular protease furin. When transiently expressed in cell cultures standard F protein was cleaved, whereas the mutant remained in the uncleaved form. Syncytium formation by the mutant that was analysed after coexpression with haemagglutinin protein depended on the presence of trypsin. Recombinant MV containing the mutation required trypsin activation for fusion and infectivity in cell culture. Intranasal infection of transgenic mice susceptible to MV infection (Ifnar(tm)-CD46Ge) resulted in a moderately productive infection and inflammation of the lung. In contrast to parental virus, intracerebral inoculation did not induce neural disease. The possible effects of the change in cleavage activation on tissue tropism and pathogenicity are discussed.

Characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by analysis of haemagglutination-deficient mutants.

Transmissible gastroenteritis coronavirus (TGEV) agglutinates erythrocytes of several species by virtue of sialic acid binding activity of the surface protein S. We have isolated and characterized five haemagglutination-defective (HAD) mutants. In contrast to the parental virus, the mutants were unable to bind to porcine submandibulary mucin, a substrate rich in sialic acid. Each of the mutants was found to contain a single point mutation in the S protein (Cys155Phe, Met195Val, Arg196Ser, Asp208Asn or Leu209Pro), indicating that these amino acids are affecting the sialic acid binding site. In four of the HAD mutants a nearby antigenic site is affected in addition to the sialic acid binding site, as indicated by reactivity with monoclonal antibodies. The parental virus was found to have an increased resistance to the detergent octylglucoside compared to the HAD mutants. This effect depended on cellular sialoglycoconjugates bound to the virion. If the binding of sialylated macromolecules was prevented by neuraminidase treatment, the parental virus was as sensitive to octylglucoside as were the HAD mutants. We discuss the possibility that the sialic acid binding activity helps TGEV to resist detergent-like substances encountered during the gastrointestinal passage and thus facilitates the infection of the intestinal epithelium. An alternative function of the sialic acid binding activity - accessory binding to intestinal tissues - is also discussed.

Cloning and characterization of gp36, a human mucin-type glycoprotein preferentially expressed in vascular endothelium.

A mucin-type glycoprotein has been described in murine, rat and canine tissues as a differentiation antigen and influenza-virus receptor. We have cloned a cDNA from human placenta RNA encoding the corresponding human protein, a type-I integral membrane protein of 162 amino acids. Madin-Darby canine kidney cells transfected with the cDNA clone directed the cell-surface expression of a 36-kDa O-glycosylated sialoglycoprotein, gp36, and two minor isoforms of 28 and 70 kDa. gp36 has a broad tissue distribution with strong expression in lung, placenta and skeletal muscle, as shown by PCR screening of different cDNA libraries. Immunohistochemical detection of gp36 in cryo-sections of human placenta, kidney, lung and nasal polyps showed that the glycoprotein is expressed at the apical plasma membrane of vascular endothelial cells. Expression of gp36 was not restricted to endothelial cells, as alveolar epithelial cells were found to express gp36 as well.

Importance of the carboxyl-terminal FTSL motif of membrane cofactor protein for basolateral sorting and endocytosis. Positive and negative modulation by signals inside and outside the cytoplasmic tail.

Membrane cofactor protein (MCP), a widely distributed complement regulatory protein, is expressed on the basolateral surface of polarized epithelial cells, and it is not endocytosed. The carboxyl-terminal tetrapeptide (FTSL) is required for polarized surface expression. The ability of this tetrapeptide to serve as an autonomous sorting signal has been analyzed by adding this sequence motif to the C terminus of an apical membrane protein, the influenza A virus hemagglutinin (HA). The recombinant protein HA-FTSL retained the apical localization of the parental HA protein. Substitution of the complete cytoplasmic tail of MCP for the cytoplasmic tail of HA resulted in the targeting of the chimeric protein (HA/MCP) to the basolateral surface suggesting that the carboxyl-terminal FTSL motif is a weak sorting signal that requires additional targeting information from the membrane-proximal part of the cytoplasmic tail of MCP for redirecting an apical protein to the basolateral membrane domain. In contrast to the native HA, the HA-FTSL protein was subject to endocytosis. The basolateral HA/MCP was also found to be internalized and thus differed from the basolateral MCP. This result suggests that the carboxyl-terminal FTSL motif serves as an internalization signal and that in native MCP sorting information outside the cytoplasmic tail counteracts this endocytosis signal. Substitution of a tyrosine for the phenylalanine dramatically increased the internalization with most of the HA-YTSL protein being present intracellularly. Our results are consistent with the view that the interplay of multiple sorting signals and the modification of a well known targeting signal (YTSL) by amino acid exchange (FTSL) determine the constitutive expression of MCP on the basolateral surface of polarized epithelial cells.

The cytoplasmic tail of the influenza C virus glycoprotein HEF negatively affects transport to the cell surface.

The surface glycoprotein, HEF, of influenza C virus (C/Johannesburg/1/66) has been shown to undergo a post-translation conformational change that is evident in a dramatic change of electrophoretic mobility. If the corresponding gene is expressed in the absence of other viral proteins, this folding process does not occur at all or only very inefficiently. A chimaeric protein, HEF-HA(Tail), in which the short cytoplasmic tail (Arg-Thr-Lys) of HEF was replaced by the cytoplasmic tail of the haemagglutinin of an influenza A virus (fowl plague virus) was constructed. In contrast to the wild-type protein, the chimaeric protein was detected on the cell surface. No further improvement of the surface expression was observed when both the transmembrane domain and the cytoplasmic tail were replaced by the corresponding domains of either the influenza A haemagglutinin or gp40, an endogenous protein of MDCK cells. For the HEF-HA(Tail) construct this study shows that a substantial amount of the protein is converted to the 100 kDa mature form that is observed in virus-infected cells. The HEF-HA expressed on the cell surface reacted positively in esterase and haemadsorption assays, indicating that it was present in a biologically active form. The results show that the short cytoplasmic tail of HEF has a negative effect on the folding and surface transport of this protein. How this effect may be prevented during a virus infection is discussed.

Is the sialic acid binding activity of the S protein involved in the enteropathogenicity of transmissible gastroenteritis virus?

Transmissible gastroenteritis virus (TGEV) is able to recognize sialic acid on sialo-glycoconjugates. Analysis of mutants indicated that single point mutations in the S protein (around amino acids 145-155) of TGEV may result both in the loss of the sialic acid binding activity and in a drastic reduction of the enteropathogenicity. From this observation we conclude that the sialic acid binding activity is involved in the enteropathogenicity of TGEV. On the basis of our recent results we propose that binding of sialylated macromolecules to the virions surface may increase virus stability. This in turn would explain how TGEV as an enveloped virus can survive the gastrointestinal passage and cause intestinal infections.

Isolation of hemagglutination-defective mutants for the analysis of the sialic acid binding activity of transmissible gastroenteritis virus.

The surface protein S of transmissible gastroenteritis virus (TGEV) has a sialic acid binding activity that enables the virus to agglutinate erythrocytes. A protocol is described that has been successfully applied to the isolation of hemgglutination-defective mutants. The potential of these mutants for the characterization of the sialic acid-binding site and the function of the binding activity is discussed.