Host range recombinants of fowl plague (influenza A) virus

Recombinants between the influenza virus strains fowl plague virus (FPV, Hav1N1) and Hong Kong (H3N2) have been isolated which form plaques on MDCK cells but not on chick embryo cells, although they carry the hemagglutinin of FPV. These host range recombinants have been characterized and one of them has been used for a second recombination with virus N (Hav2Neq1) or equi 2 (Heq2Neq2) on chick embryo cells. In this way, recombinants were obtained with a mixed genome which have regained the natural host range of FPV and some pathogenic properties for chicken. The results are discussed as a possible mechanism for a pandemic influenza strain to survive in an animal reservoir by changing its host range by recombination, and to regain the original host range by a second recombination but always keeping the same hemagglutinin.     

Host range selection of transformation-defective hr-t mutants of polyoma virus.

Nineteen independent mutants have been isolated by host range selection using polyoma transformed 3T3 cells as a permissive host and normal 3T3 cells as a nonpermissive host. All nineteen mutants fail to transform rat and hamster fibroblasts. Complementation experiments indicate that these mutants belong to a single group. This group is designated “hr-t,” indicating defects in host range and transformation. In both productive and nonproductive infections, hr-t mutants induce the synthesis of polyoma-specific T antigen(s), and are therefore not blocked prior to uncoating. Some of the mutants bear small deletions in their DNAs, notably in the proximal (5′) part of the early region. The ensemble of results to date shows a uniform biological behavior for all mutants isolated by this procedure, and suggests that single mutations are responsible for the reduced host range and the inability to induce cell transformation. These and earlier results are discussed in terms of a model in which the hr-t viral function acts to alter the expression of cellular genes.     

Temperature-sensitive mutants of influenza virus

Summary Six temperature-sensitive (ts) mutants were isolated from the progeny of wildtype influenza A virus grown in the presence of 5-fluorouracil. All mutants had the efficiency of plating lower than 10^–4 at 38° C compared with 31° C. One mutant (ts- 5) failed to produce hemagglutinin at the nonpermissive temperature. Temperature-shift experiments revealed that the temperature-sensitive step ofts-5 resides in the process normally taking place at 4–6 hours post infection. After mixed infection ofts-5 andts-9, different from each other in physiological defect, the occurrence of both complementation and recombination was demonstrated, confirming that they carry genetic defects at different genes.     

Host range control of cauliflower mosaic virus

Studies with recombinant genomes of cauliflower mosaic virus (CaMV) strains D4, CM1841, and Cabb-B have shown that a host range determinant of CaMV is encoded within the first half of region VI, a gene which codes for P62, an inclusion body protein. In order to further study the host specificity of CaMV, a fourth CaMV strain, W260, was chosen that has a host range that is intermediate between D4 and CM1841. To determine which portion of the W260 genome controls systemic spread, recombinant viruses made between this strain and CM1841 and D4 were tested for their ability to systemically infect several solanaceous plants (Datura stramonium, Nicotiana edwardsonii, and Nicotiana bigelovii). The first half of gene VI specified the type of local lesions and systemic spread of recombinant strains in D. stramonium. In N. edwardsonii, it was found that the first half of gene VI controlled the type of local lesion formed but systemic spread was dependent on the whole of gene VI. In N. bigelovii the number of genes that determined systemic spread of CaMV varied with the strain of CaMV. Systemic spread of D4 in N. bigelovii was dependent on the first half of gene VI. In contrast, systemic spread of W260 in the same host was dependent on the whole of gene VI and another locus which mapped within genes I-V. Consequently, it appears that other viral proteins may interact with P62 or that P62 may function well in some hosts only in compatible forms of other viral proteins.     

Host range temperature-conditional mutants in the adenovirus DNA binding protein are defective in the assembly of infectious virus

R(ts107)202 is a host range temperature-conditional mutant of adenovirus type 5. This mutant is temperature sensitive for replication and plaquing in 293 cells but is temperature independent for growth and plaquing in HeLa cells (J.C. Nicolas, F. Suarez, A. J. Levine, and M. Girard (1981)Virology108, 521–524). The mutant was isolated in HeLa cells as a temperature-independent revertant of the H5ts107 temperature-sensitive mutant that maps in the adenovirus DNA binding protein (DBP). The reasons for the temperature conditional phenotype of this mutant in 293 cells were investigated. The mutant synthesized an unstable DBP in both HeLa and 293 cells at 39°. In 293 cells at 39°, about two- to threefold less viral DNA was synthesized by r(ts107)202 as compared to Ad5wt. R(ts107)202 infected cells at 39° produced normal (wild-type) amounts of all detectable late viral structural proteins. The mutant failed, however, to produce infectious virus or assemble virus particles in 293 cells at 39°. The altered DBP may therefore play a role in the assembly of virus particles, either directly or indirectly via an altered DNA structure. The failure of r(ts107)202 to assemble virion particles in 293 cells at 39° furthermore suggests that virus assembly is dependent upon cellular factors that differ in HeLa and 293 cell.     

Cold-adapted vaccine strains of influenza A virus act as dominant negative mutants in mixed infections with wild-type influenza A virus

The cold-adapted reassortant of influenza A, which is a candidate live virus vaccine, interfered with the replication of parental wild-type virus in mixed infections of either MDCK cells or embryonated eggs. The interference occurred at either the permissive or nonpermissive temperature for the cold-adapted virus. In doubly infected cells, the yield of the wild-type virus was reduced by as much as 3000-fold and the protein synthesis phenotype expressed was that of the cold-adapted virus. The interference was detected even when infection with wild-type virus was carried out at a 9-fold excess or 2 hr before infection with the cold-adapted virus. As well as interfering with its wild-type parental virus, the cold-adapted virus also inhibited the replication of a heterologous influenza A subtype. In addition to its immunogenic potential, the ability to interfere with the replication of wild-type viruses is a desirable trait for any live, attenuated virus vaccine.     

Temperature-sensitive mutants of influenza A/Udorn/72 (H3N2) virus. III. Genetic analysis of temperature-dependent host range mutants

One hundred thirty-three ts mutants of influenza A/Udorn/72 virus were arranged into eight complementation groups, A-H, on Madin-Darby canine kidney (MDCK) monolayer cultures at the restrictive temperature of 40 degrees. The eight complementation groups, A-H, on MDCK cells corresponded to the eight recombination groups, A-H, on rhesus monkey kidney (RMK) cells, respectively, and this suggested that each MDCK complementation group represented one of the eight influenza A RNA gene segments. These ts viruses were used to identify the locus of the ts mutation in temperature-dependent host range (td-hr) mutants of the A/Udorn/72 virus. Sixteen of the 133 ts mutants exhibited distinct host (MDCK)-dependent restriction of plaque formation at 40 degrees but not at 34 degrees and were referred to as td-hr mutants. These 16 td-hr mutants were ts+ (not ts) on RMK cells but ts on MDCK cells. The td-hr mutants did not share a common lesion and the ts lesions were distributed among the eight complementation groups, A-H, when tested on MDCK cells. An analysis of one of the td-hr mutants indicated that an extrageneic RMK-dependent suppressor mutation did not account for the td-hr phenotype. These data suggested that a host-dependent ts mutation was responsible for the td-hr restriction of this mutant. Representation of td-hr mutations in each of the eight complementation groups indicates that the influenza A virus genome can undergo mutation leading to an altered host range in any of its eight RNA segments.     

Emergence of HA mutants during influenza virus pneumonia

During the influenza pandemic of 2009, the number of viral pneumonia cases showed a marked increase in comparison with seasonal influenza viruses. Mutations at amino acid 222 (D222G mutations) in the virus hemagglutinin (HA) molecule, known to alter the receptor-recognition properties of the virus, were detected in a number of the more severely-affected patients in the early phases of the pandemic. To understand the background for the emergence of the mutant amino acid D222G in human lungs, we conducted histological examinations on lung specimens of patients from Mexico who had succumbed in the pandemic. Prominent regenerative and hyperplastic changes in the alveolar type II pneumocytes, which express avian-type sialoglycan receptors in the respiratory tract of severely affected individuals, were observed in the Mexican patients. An infection model utilizing guinea pigs, which was chosen in order to best simulate the sialic acid distribution of severe pneumonia in human patients, demonstrated an increase of D222G mutants and a delay in the diminution of mutants in the lower respiratory tract in comparison to the upper respiratory tract. Our data suggests that the predominance of avian-type sialoglycan receptors in the pneumonic lungs may contribute to the emergence of viral HA mutants. This data comprehensively illustrates the mechanisms for the emergence of mutants in the clinical samples.     

Host proteins in purified concentrations of the influenza virus

A comparative study of three methods for purification and concentration of influenza virus (adsorption on and elution from formalin-treated erythrocytes, sorption method, and purification on nuclear filters) demonstrated a significant decreased in ovalbumin content. By this criterion, all the three methods of preliminary purification yield the final preparation with a similar ovalbumin content. A more detailed study of the protein composition of influenza virus concentrates showed purification by elution from formalin-treated erythrocytes to remove greated amounts of protein admixtures. Electrophoregrams of virus concentrates produced by the adsorption method using macropore glass 8000 revealed a protein which passed into the virus suspension in sufficiently large amounts. This protein was identified as conalbumin.     

Temperature-sensitive mutants of influenza virus: A mutation in the hemagglutinin gene

A mutant (ts-61S) belonging to a single recombination-complementation group (Group VI) was obtained by segregation of an influenza virus WSN (HON1) temperature-sensitive double mutant (ts-61) that possessed mutational lesions characteristic of Groups V and VI. The segregant retained the thermolabile hemagglutinating activity of the parental mutant, ts-61, but lost the defectiveness in virion RNA synthesis manifested by the parent at the nonpermissive temperature. No hemagglutinating activity developed in cells infected with ts-61S at the nonpermissive temperature. In rescue experiments all HO-serotype progeny from the cross between ts-61S (HO-serotype) and temperature-resistant H3-serotype virus were temperature-sensitive, localizing the ts defect in the hemagglutinin gene. No glycosylated hemagglutinin polypeptide was detected in the polyacrylamide gel electropherogram of cells infected with ts-61S at the nonpermissive temperature, whereas the synthesis of neuraminidase (the other virion glycoprotein) proceeded normally at both permissive and nonpermissive temperatures. The results indicate that the Group VI mutation is in the gene coding for the viral hemagglutinin.     

Structure-based design of NS2 mutants for attenuated influenza A virus vaccines

We previously characterised the matrix 1 (M1)-binding domain of the influenza A virus NS2/nuclear export protein (NEP), reporting a critical role for the tryptophan (W78) residue that is surrounded by a cluster of glutamate residues in the C-terminal region that interacts with the M1 protein (Akarsu et al., 2003). To gain further insight into the functional role of this interaction, here we used reverse genetics to generate a series of A/WSN/33 (H1N1)-based NS2/NEP mutants for W78 or the C-terminal glutamate residues and assessed their effect on virus growth. We found that simultaneous mutations at three positions (E67S/E74S/E75S) of NS2/NEP were important for inhibition of influenza viral polymerase activity, although the W78S mutant and other glutamate mutants with single substitutions were not. In addition, double and triple substitutions in the NS2/NEP glutamine residues, which resulted in the addition of seven amino acids to the C-terminus of NS1 due to gene overlapping, resulted in virus attenuation in mice. Animal studies with this mutant suggest a potential benefit to incorporating these NS mutations into live vaccines.     

Receptor specificity of H5 influenza virus escape mutants

The binding of viruses to synthetic polyacrylamide (PAA)-based sialylglycoconjugates was used to characterize the receptor specificities of antibody escape mutants of the influenza virus A/Mallard/Pennsylvania/10218/84 (H5N2). The sialylglycoconjugates that were used carried identical terminal Neu5Ac2–3Gal moieties but differed in the structure of the next saccharide residue(s). Our data show that mutations in the vicinity of the haemagglutinin (HA) receptor-binding site (RBS) effect the recognition of the third saccharide residue and change the affinity pattern of binding. The affinity of the majority of the escape mutants for sialyl receptors increased compared to the parental strain.     

Host defense mechanisms against influenza virus: interaction of influenza virus with murine macrophages in vitro.

The interaction of mouse macrophages with influenza virus was examined as part of a study into the defense mechanisms against influenza infection. Macrophages exposed to A/Port Chalmers/1/73 virus produced infectious foci on susceptible indicator cell monolayers. Sampling of supernatant fluids and cells from infected macrophage cultures showed release of virus adsorbed to the cell surface. Active virus replication in macrophages could not be demonstrated. Exposing macrophages to specific antibody before or after virus infection resulted in a significant decrease in the number of infectious macrophages. The results suggest that although macrophages are not the source of replicating influenza virus, they are able to spread the infection by having virus attaching to their surface. This activity is interfered with by the presence of specific antibody.     

Characterization of an influenza A virus from seals

An influenza A virus antigenically similar to A/FPV/Dutch/27 (Hav1Neq1) [H7N7] was isolated from harbor seals (Phoca vitulina) that had died of acute hemorrhagic pneumonia on Cape Cod Peninsula, beginning in the winter of 1979–1980. High titers of virus were obtained from the lungs and lower titers from the brains of the seals. Although antigenic analyses and characterization of the RNAs show that all of the genes and gene products are closely related to different avian influenza viruses, biologically the virus behaves more like a mammalian strain. The seal virus replicated and produced pneumonia in experimentally infected harbor seals, but the clinical course and pathology were less severe than in the natural infection; the virus also replicated in ferrets, cats, and pigs but produced no disease. In avian species, the seal influenza virus replicated poorly, produced no disease signs, and was not shed in the feces. Although the seal influenza virus can cause conjunctivitis in humans who have known contamination of the eyes from infected animals, serological studies detected no evidence of seroconversion among persons working with infected seals or with the virus. Preliminary studies detected antibodies to this virus in harbor seals on the New England coast but not in harbor seals, gray seals, or fur seals from other areas, suggesting that this virus may be a new introduction to this species. An Hav1Neq1 [H7N7] virus was also isolated from feral ducks in Iceland in 1980, but the two viruses could be distinguished by analysis of their RNAs and host range. The A/Seal/Mass/1/80 influenza virus provides the first evidence suggesting that a strain deriving all of its genes from one or more avian influenza viruses can be associated with severe disease in a mammalian population in nature. Whether this breach of species specificity represents a unique event in influenza evolution remains to be determined, but raises the possibility that human or animal influenza viruses may be derived directly from avian strains.     

Host range conversion of murine leukemia virus resulting from recombination with endogenous virus

Summary. Ecotropic murine leukemia viruses (MuLVs) are classified into B-, N-, or NB-tropic MuLV by their host range determined by the Fv-1 gene product. B-tropic MuLV is restricted in N-type mouse cells (Fv-1^n/n) and N-tropic MuLV is restricted in B-type mouse cells (FV-1^b/b). Although forced passages in a restrictive host grant a wider host range (NB-tropism), we show here a host range conversion from B to N tropism. The conversion was most likely a result of recombination between the exogenously infected B-tropic MuLV and an endogenously expressed N-tropic MuLV in a C57BL/6 mouse cell line, YH-7. Received June 19, 1996 Accepted August 24, 1996