Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes.

The influenza A virus pandemic of 1918-1919 resulted in an estimated 20-40 million deaths worldwide. The hemagglutinin and neuraminidase sequences of the 1918 virus were previously determined. We here report the sequence of the A/Brevig Mission/1/18 (H1N1) virus nonstructural (NS) segment encoding two proteins, NS1 and nuclear export protein. Phylogenetically, these genes appear to be close to the common ancestor of subsequent human and classical swine strain NS genes. Recently, the influenza A virus NS1 protein was shown to be a type I IFN antagonist that plays an important role in viral pathogenesis. By using the recently developed technique of generating influenza A viruses entirely from cloned cDNAs, the hypothesis that the 1918 virus NS1 gene played a role in virulence was tested in a mouse model. In a BSL3+ laboratory, viruses were generated that possessed either the 1918 NS1 gene alone or the entire 1918 NS segment in a background of influenza A/WSN/33 (H1N1), a mouse-adapted virus derived from a human influenza strain first isolated in 1933. These 1918 NS viruses replicated well in tissue culture but were attenuated in mice as compared with the isogenic control viruses. This attenuation in mice may be related to the human origin of the 1918 NS1 gene. These results suggest that interaction of the NS1 protein with host-cell factors plays a significant role in viral pathogenesis.

Influenza virus (A/HK/156/97) hemagglutinin expressed by an alphavirus replicon system protects chickens against lethal infection with Hong Kong-origin H5N1 viruses.

Venezuelan equine encephalitis virus replicon particles (VRP) containing the gene expressing hemagglutinin (HA) from the human Hong Kong Influenza A isolate (A/HK/156/97) were evaluated as vaccines in chicken embryos and young chicks. Expressed HA was readily detected in bird-tissue staining with anti-H5 HA antibody and in chicken cells infected with the replicon preparations following immunoprecipitation with monoclonal antibody. Birds challenged with a dose of the lethal parent virus were protected to different extents depending on the age of the bird. In ovo and 1-day-old inoculated animals that received no boost with the VRP were partially protected; birds 2 weeks of age were completely protected with a single dose of VRP.

Distinct pathogenesis of hong kong-origin H5N1 viruses in mice compared to that of other highly pathogenic H5 avian influenza viruses.

In 1997, an outbreak of virulent H5N1 avian influenza virus occurred in poultry in Hong Kong (HK) and was linked to a direct transmission to humans. The factors associated with transmission of avian influenza virus to mammals are not fully understood, and the potential risk of other highly virulent avian influenza A viruses infecting and causing disease in mammals is not known. In this study, two avian and one human HK-origin H5N1 virus along with four additional highly pathogenic H5 avian influenza viruses were analyzed for their pathogenicity in 6- to 8-week-old BALB/c mice. Both the avian and human HK H5 influenza virus isolates caused severe disease in mice, characterized by induced hypothermia, clinical signs, rapid weight loss, and 75 to 100% mortality by 6 to 8 days postinfection. Three of the non-HK-origin isolates caused no detectable clinical signs. One isolate, A/tk/England/91 (H5N1), induced measurable disease, and all but one of the animals recovered. Infections resulted in mild to severe lesions in both the upper and lower respiratory tracts. Most consistently, the viruses caused necrosis in respiratory epithelium of the nasal cavity, trachea, bronchi, and bronchioles with accompanying inflammation. The most severe and widespread lesions were observed in the lungs of HK avian influenza virus-infected mice, while no lesions or only mild lesions were evident with A/ck/Scotland/59 (H5N1) and A/ck/Queretaro/95 (H5N2). The A/ck/Italy/97 (H5N2) and the A/tk/England/91 (H5N1) viruses exhibited intermediate pathogenicity, producing mild to moderate respiratory tract lesions. In addition, infection by the different isolates could be further distinguished by the mouse immune response. The non-HK-origin isolates all induced production of increased levels of active transforming growth factor beta following infection, while the HK-origin isolates did not.

Role of endogenous interleukin-18 in resolving wild-type and attenuated Salmonella typhimurium infections.

The stimulation of gamma interferon (IFN-gamma) has been shown to be essential in resolving infections by intracellular pathogens. As such, several different cytokines including, interleukin-12 (IL-12) and IL-18, can induce IFN-gamma. To resolve Salmonella infections, the stimulation of IL-12 and IFN-gamma are important for mediating its clearance. In this present study, the relevance of IL-18 in protection against oral challenge with Salmonella typhimurium was investigated to determine the role of this IFN-gamma-promoting cytokine. Rabbit anti-murine IL-18 antisera was generated and administered prior to the oral challenge of BALB/c and IL-12p40-deficient knockout (IL-12KO) mice with a wild-type S. typhimurium strain. The median survival time was reduced by 2 days for the anti-IL-18-treated BALB/c mice, while no significant reduction in survival rate for the anti-IL-18-treated IL-12KO mice was observed compared to vehicle-treated mice. To investigate the contribution of IL-18 to resolving Salmonella infections, an attenuated aro-negative mutant (H647) was orally administered to BALB/c mice. This Salmonella infection induced both IL-12 and IFN-gamma in both the Peyer's patches and the spleens. In vehicle-treated mice, Peyer's patch IL-12 peaked by 24 h, while IL-18 levels peaked at 3 days, suggesting sequential support by these cytokines for IFN-gamma. Anti-IL-18 treatment exerted its greatest effect upon the mucosal compartment, limiting early IFN-gamma production. However, anti-IL-18 treatment had little effect upon splenic IFN-gamma levels until late in the response. Infection of IL-12KO mice with H647 strain induced IFN-gamma, but it was not supported by IL-18, although IL-18 levels were reduced by this treatment. These results suggest that IL-18 does contribute to the clearance of S. typhimurium and that endogenously induced IL-18 could not substitute for IL-12.

Antigenic and immunogenic properties of baculovirus-expressed infectious bursal disease viral proteins.

Genomic segment A of the variant Md infectious bursal disease virus (IBDV) was amplified by reverse transcriptase/polymerase chain reaction, cloned, and expressed in the baculovirus expression system. Three different baculovirus recombinants expressing the genes encoding VP2, VP2/VP4, and the complete polyprotein (VP2/VP4/VP3) were prepared. The three antigens were used in separate enzyme-linked immunosorbent assays, and each detected antibodies against the variant IBDV strains Md, Del-A, Del-E, and GLS and the classic IBDV strain D78 throughout a 14-wk period following vaccination. Eight-week-old specific-pathogen-free (SPF) chickens were inoculated subcutaneously using the VP2/VP4 or VP2/VP4/VP3 baculovirus recombinant or wild-type baculovirus-infected insect cell lysates. Virus-neutralizing antibodies were detected in the VP2/VP4 and VP2/VP4/VP3 baculovirus-inoculated birds at 13 days postinoculation (PI) and at 43 days PI when titrated against Md IBDV. No virus-neutralizing antibody titer was observed in the negative controls or wild-type baculovirus-inoculated birds. One-week-old SPF chickens were inoculated with the VP2, VP2/VP4/VP3 baculovirus recombinants or wild-type baculovirus-infected insect cell lysates. The birds were boosted 2 wk later and challenged at 4 wk of age with 0.5 ml/bird classic STC IBDV (10(2) median embryo infective dose [EID50]/ml) or variant Md IBDV (10(5) EID50/ml) via the oral/nasal route. The VP2 and VP2/VP4/VP3 baculovirus-inoculated birds were partially protected against challenge with the classic STC IBDV. The birds were protected against clinical disease and death but not bursal damage, whereas the wild-type baculovirus-inoculated birds exhibited clinical signs of disease, 13% mortality, and bursal damage. When challenged with the variant Md IBDV, neither the VP2, VP2/VP4/VP3 baculovirus recombinants nor the wild-type baculovirus elicited protection against bursal damage and atrophy.

Expression of MD infectious bursal disease viral proteins in baculovirus.

Genomic segment A of the variant infectious bursal disease virus (IBDV) strain MD was amplified by reverse transcriptase/polymerase chain reaction and further characterized by baculovirus expression. Three different baculovirus clones were constructed containing the genes encoding VP2, VP2/VP4, and the complete polyprotein cloned into the baculovirus transfer vector pVL1392. Baculovirus recombinants were identified by dot blot hybridization and were plaque purified three times. Baculovirus expression of the recombinants produced IBDV-specific proteins that were comparable in molecular size to native MD IBDV viral proteins VPX (48 kD), VP2 (45 kD), VP3 (32 kD), and VP4 (28 kD) as determined by sodium dodecyl sulfare-polyacrylamide gel electrophoresis and western immunoblot analysis. All three recombinants produced a 48-kD protein that possibly represents VPX, the precursor product of VP2. In addition to the 48-kD protein, the VP2/VP4 recombinant produced an IBDV-specific protein corresponding to the 28-kD VP4. The baculovirus-expressed polyprotein gene produced, in addition to the 48-kD protein, a 32-kD (VP3) IBDV-specific protein and a 29-kD protein that migrated slightly slower than MD VP4. The baculovirus-expressed proteins were used as antigens in an indirect enzyme-linked immunosorbent assay (ELISA). The ELISAs detected antibodies against the variant IBDV strains MD, GLS, and IN and the classic IBDV strains SAL and STC but did not detect antibodies against the variant Del-A and classic IBDV strain BVM or the serotype 2 IBDV strain OH.

Restriction analysis of the MD infectious bursal disease virus strain.

The variant MD strain of infectious bursal disease virus (IBDV) was examined using restriction fragment length polymorphism (RFLP) and the molecular variation was compared with published sequences of both variant and classic IBDV strains. Viral RNA was transcribed into cDNA using reverse transcriptase and then amplified by the polymerase chain reaction (PCR). Three separate but overlapping fragments of 1603 bp, 1496 bp, and 1066 bp containing the entire coding region for VP2, VP4, and VP3, respectively, were amplified. These amplified products were initially cloned using the TA Cloning kit and further subcloned into the pGEM-3Zf and pUC19. Eight restriction enzymes, PstI, BamHI, AvaI, EcoRI, HindII, XmaI, SmaI, and XbaI, were tested for their ability to digest the MD PCR products. The resulting RFLP was analyzed and compared with the published genome segment A sequences of the classic IBDV strain STC and the variant IBDV strain GLS. Differences in restriction fragment profiles were observed after digestion with BamHI, EcoRI, XmaI, and SmaI, with the absence of the EcoRI site in both variant strains, GLS and MD. The MD PCR products lacked both XmaI and SmaI sites, which were present in both STC and GLS. The MD PCR products contained a BamHI site within the hypervariable region of VP2, which is present in STC but not in the variant GLS. An additional BamHI site was identified in the VP4 gene of MD and GLS but not in STC.

Cryogenic solvents inhibit the binding of the Escherichia coli heat-stable enterotoxin to intestinal brush border membranes.

The cryogenic solvents ethylene glycol, glycerol, dimethyl sulfoxide (Me2SO) and dimethylformamide (Me2FM), with increasing potency, produced concentration-dependent inhibition of the binding of the Escherichia coli heat-stable enterotoxin (STa) to pig intestinal brush border membranes. Inhibition increased with time, and both Me2SO and Me2FM appeared to decrease both the affinity of the STa receptor and the effective receptor number. Both solvents stimulated the release of previously bound 125I-STa (Me2FM > Me2SO), 3 M Me2FM inducing 93% release by 120 min. These effects were reversible, and preincubation of membranes with up to 3 M Me2SO or Me2FM at 37 degrees C for 30 min, followed by washing, did not alter subsequent 125I-STa binding. Also, 125I-STa released from membranes by 3 M Me2FM was shown to rebind to the membranes after 10-fold dilution of Me2FM. Since pretreating membranes with the thiol reagent p-chloromercuribenzenesulfonate had no effect on the release of bound 125I-STa by Me2SO or Me2FM, and since neither of these can reduce disulfide bonds, the formation of mixed disulfides between STa and receptor is unlikely. Me2SO inhibition of 125I-STa binding was greater with membranes than with a partially purified receptor preparation, which may result from the substitution of detergent for the phospholipid normally associated with the receptor(s).

Nucleotide regulation of heat-stable enterotoxin receptor binding and of guanylate cyclase activation.

Certain nucleotides were found to regulate the binding of the Escherichia coli heat-stable enterotoxin (STa) to its receptor in pig intestinal brush border membranes. ATP and adenine nucleotide analogues inhibited 125I-STa binding, while guanine nucleotide analogues stimulated binding, with maximal effects at 0.5-1.0 mM. The strongest inhibitors were adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) (36%) and adenosine 5'-[beta-thio]diphosphate (ADP[S]) (41%). Inhibition did not require Mg2+, and was blocked by p-chloromercuribenzenesulphonate (PCMBS). Stimulation of binding required Mg2+, was not prevented by PCMBS and was maximal with GDP[S] (41%). While App[NH]p and MgGDP[S] appeared to be acting at different sites, they also interfered with each other. These nucleotides exerted only inhibitory effects on STa-stimulated guanylate cyclase activity, in contrast with the stimulatory effects of adenine nucleotides on atrial natriuretic peptide (ANP)-stimulated guanylate cyclase. Inhibition by low concentrations of MgApp[NH]p and MgATP was weaker above 0.1 mM, while MgGDP[S] and magnesium guanosine 5'-[gamma-thio]triphosphate (MgGTP[S]) inhibited in a single phase. Inhibition by MgApp[NH]p, at all concentrations, was competitive with the substrate (MgGTP), as was that by MgGDP[S] and MgGTP[S]. Whereas membrane guanylate cyclases usually show positively co-operative kinetics with respect to the substrate, STa-stimulated activity exhibited Michaelis-Menten kinetics with respect to MgGTP. This changed to positive co-operativity when Lubrol PX was the activator, or when the substrate was MnGTP. These results suggest the presence of both a regulatory and a catalytic nucleotide-binding site, which do not interact co-operatively with STa activation.

Solubilization and reprecipitation from intestinal brush border membranes of a complex containing guanylate cyclase activatable by the heat-stable enterotoxin.

Extraction of pig intestinal brush border membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (Chaps) in the presence of 0.5 M KCl yielded a solution which contained 60-70% of the receptor for the Escherichia coli heat-stable enterotoxin (STa) and of the Lubrol PX-activated guanylate cyclase activity present in the membrane. When the supernatant solution was diluted fivefold with 10 mM Hepes buffer (pH 7.4) and kept at 4 degrees C overnight, a precipitate formed. Centrifugation yielded a pellet (P2) which contained 25-30% of both the cyclase and the receptor in the original membranes, with a 2.5- to 3-fold enrichment of both. The process could be repeated for further enrichment (P4). The addition of MgCl2 to the diluted extract affected both basal and STa-stimulated activity of P2; 1 mM was optimal. P2 resembled membranes with respect to competitive inhibition of 125I-STa binding by STa, and the concentration-dependent activation of cyclase by STa. Guanylate cyclase in resolubilized P2 was also activated by STa. Most of the enzymes interfering with guanylate cyclase determinations were removed, as were the brush border marker enzymes sucrase and gamma-glutamyltransferase, and a GTP-binding protein that is a pertussis toxin substrate. Specific cross-linking of 125I-STa to receptors in the membrane was preserved in P2 and P4, the three proteins showing the strongest radioactivity having relative molecular masses of 55,000-60,000, 70,000-80,000, and 135,000-140,000. P2 and P4 appear to contain a complex of membrane proteins with certain functional properties intact.

Genome cloning and analysis of the large RNA segment (segment A) of a naturally avirulent serotype 2 infectious bursal disease virus.

The genome of infectious bursal disease virus (IBDV) of serotype 2 (strain OH) has been cloned, and 3171 nucleotides of genome segment A cDNA sequence have been determined for the first time. Sequence homology of OH-IBDV with the most distant serotype 1 IBDV at the nucleotide level is 83.1%, and the amino acid sequence homology of the polyprotein is 89.6%. Alignment of the polyprotein amino acid sequences showed the hypervariable region in VP2 to be 151-152 amino acid residues long in IBDV. A second variable region, 37 amino acid residues long, was identified in the N-terminal third of the IBDV VP2 molecule. IBDV strains, like the IPNV strains, also contain inverted repeats that may form stem-and-loop structures in the 5' noncoding sequences. These inverted repeats are variable between the two IBDV serotypes, particularly at the AT basepairs.