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Host range determination and functional mapping of the nucleoprotein and matrix genes of influenza viruses using monoclonal antibodies.

Construction and comparison of phylogenetic trees, the standard approach to determining the host-specific lineage of influenza A virus genes is tedious and expensive. In this study, panels of monoclonal antibodies (Mabs) produced against the matrix proteins (M1) of A/WSN and A/PR/8/34 and the nucleoprotein (NP) of A/WSN were assessed for their value in identifying the hosts of origin of the M1 and NP genes in influenza virus isolates and in mapping the proteins' functional domains. Using ELISA against a broad spectrum of reference viruses, we found two Mabs against the NP (150/4 and 469/6) to be useful in determining host-specific lineage. Comparative sequence analysis placed five amino acids within the antigenic domains recognized by Mab 150/4 and two amino acids within the domains recognized by 469/6. One Mab against the NP (5/1) recognized a conserved epitope that is present on each of the 36 influenza A viruses tested. This epitope may be a type-specific determinant for influenza A viruses and an RNA binding site. Monoclonal antibodies to M1 did not discriminate among species, but they did contribute information to the construction of a functional map of M1. These results demonstrate that Mabs to defined protein epitopes can provide useful information on the molecular epidemiology of influenza viruses.

Animals↗

Host range, amplification and arboviral disease emergence.

Etiologic agents of arboviral diseases are primarily zoonotic pathogens that are maintained in nature in cycles involving arthropod transmission among a variety of susceptible reservoir hosts. In the simplest form of human exposure, spillover occurs from the enzootic cycle when humans enter zoonotic foci and/or enzootic amplification increases circulation near humans. Examples include Eastern (EEEV) and Western equine encephalitis viruses (WEEV), as well as West Nile (WNV), St. Louis encephalitis (SLEV) and Yellow fever viruses. Spillover can involve direct transmission to humans by primary enzootic vectors (e.g. WNV, SLEV and WEEV) and/or bridge vectors with more catholic feeding preferences that include humans (e.g. EEEV). Some viruses, such as Rift Valley fever, Japanese encephalitis and Venezuelan equine encephalitis viruses (VEEV) undergo secondary amplification involving replication in livestock animals, resulting in greater levels of spillover to humans in rural settings. In the case of VEEV, secondary amplification involves equines and requires adaptive mutations in enzootic strains that allow for efficient viremia production. Two of the most important human arboviral pathogens, Yellow fever and dengue viruses (DENV), have gone one step further and adopted humans as their amplification hosts, allowing for urban disease. The ancestral forms of DENV, sylvatic viruses transmitted among nonhuman primate reservoir hosts by arboreal mosquitoes, adapted to efficiently infect the urban mosquito vectors Aedes aegypti and Ae. albopictus during the past few thousand years as civilizations arose. Comparative studies of the sylvatic and urban forms of DENV may elucidate the evolution of arboviral virulence and the prospects for DENV eradication should effective vaccines be implemented.

Alphavirus↗

Host range comparisons of five serotypes of caliciviruses.

Two new serotypes of San Miguel sea lion virus (SMSV), designated SMSV-4 and SMSV-5, were studied in vivo and in vitro. The host cell spectrums were compared with SMSV-1, SMSV-2, and vesicular exanthema of swine virus type A-48. Based on the result of these broad host spectrums, a numerical scoring system was devised for ranking each virus on the basis of its potential for infecting terrestrial mammals, including the important domestic species.

Animals↗

Broad host range of streptococcal macrolide resistance plasmids.

Four macrolide-lincosamide-streptogramin B resistance plasmids transferred into 13 recipients belonging to Streptococcus, Staphylococcus, and Listeria genera. The plasmids were stably maintained in all new hosts except Streptococcus sanguis, Streptococcus pneumoniae, Staphylococcus aureus, and Listeria innocua and were identical to those found in the corresponding donor strains.

Anti-Bacterial Agents↗

Sleeping Beauty, a wide host-range transposon vector for genetic transformation in vertebrates.

Sleeping Beauty (SB), a member of the Tc1/mariner superfamily of transposable elements, is the only active DNA-based transposon system of vertebrate origin that is available for experimental manipulation. We have been using the SB element as a research tool to investigate some of the cis and trans-requirements of element mobilization, and mechanisms that regulate transposition in vertebrate species. In contrast to mariner transposons, which are regulated by overexpression inhibition, the frequency of SB transposition was found to be roughly proportional to the amount of transposase present in cells. Unlike Tc1 and mariner elements, SB contains two binding sites within each of its terminal inverted repeats, and we found that the presence of both of these sites is a strict requirement for mobilization. In addition to the size of the transposon itself, the length as well as sequence of the DNA outside the transposon have significant effects on transposition. As a general rule, the closer the transposon ends are, the more efficient transposition is from a donor molecule. We have found that SB can transform a wide range of vertebrate cells from fish to human. However, the efficiency and precision of transposition varied significantly among cell lines, suggesting potential involvement of host factors in SB transposition. A positive-negative selection assay was devised to enrich populations of cells harboring inserted transposons in their chromosomes. Using this assay, of the order of 10,000 independent transposon insertions can be generated in human cells in a single transfection experiment. Sleeping Beauty can be a powerful alternative to other vectors that are currently used for the production of transgenic animals and for human gene therapy.

Animals↗

Endophytic Phomopsis species: host range and implications for diversity estimates.

Foliar endophyte assemblages of teak trees growing in dry deciduous and moist deciduous forests of Nilgiri Biosphere Reserve were compared. A species of Phomopsis dominated the endophyte assemblages of teak, irrespective of the location of the host trees. Internal transcribed spacer sequence analysis of 11 different Phomopsis isolates (ten from teak and one from Cassia fistula) showed that they fall into two groups, which are separated by a relatively long branch that is strongly supported. The results showed that this fungus is not host restricted and that it continues to survive as a saprotroph in teak leaf, possibly by exploiting senescent leaves as well as the litter. Although the endophyte assemblage of a teak tree growing about 500 km from the forests was also dominated by a Phomopsis sp., it separated into a different group based on internal transcribed spacer sequence analysis. Our results with an endophytic Phomopsis sp. reinforce the earlier conclusions reached by others for pathogenic Phomopsis sp., i.e., that this fungus is not host specific, and the species concept of Phomopsis needs to be redefined.

Ascomycota↗

Sialic acid species as a determinant of the host range of influenza A viruses.

The distribution of sialic acid (SA) species varies among animal species, but the biological role of this variation is largely unknown. Influenza viruses differ in their ability to recognize SA-galactose (Gal) linkages, depending on the animal hosts from which they are isolated. For example, human viruses preferentially recognize SA linked to Gal by the alpha2,6(SAalpha2,6Gal) linkage, while equine viruses favor SAalpha2,3Gal. However, whether a difference in relative abundance of specific SA species (N-acetylneuraminic acid [NeuAc] and N-glycolylneuraminic acid [NeuGc]) among different animals affects the replicative potential of influenza viruses is uncertain. We therefore examined the requirement for the hemagglutinin (HA) for support of viral replication in horses, using viruses whose HAs differ in receptor specificity. A virus with an HA recognizing NeuAcalpha2,6Gal but not NeuAcalpha2,3Gal or NeuGcalpha2,3Gal failed to replicate in horses, while one with an HA recognizing the NeuGcalpha2,3Gal moiety replicated in horses. Furthermore, biochemical and immunohistochemical analyses and a lectin-binding assay demonstrated the abundance of the NeuGcalpha2,3Gal moiety in epithelial cells of horse trachea, indicating that recognition of this moiety is critical for viral replication in horses. Thus, these results provide evidence of a biological effect of different SA species in different animals.

Animals↗

The enhanced tumor selectivity of an oncolytic vaccinia lacking the host range and antiapoptosis genes SPI-1 and SPI-2.

The ability of cancer cells to evade apoptosis may permit survival of a recombinant vaccinia lacking antiapoptotic genes in cancer cells compared with normal cells. We have explored the deletion of two vaccinia virus host range/antiapoptosis genes, SPI-1 and SPI-2, for their effects on the viral replication and their ability to induce cell death in infected normal and transformed cells in vitro. Indeed, in three paired normal and transformed cell types, the SPI-1 and SPI-2 gene-deleted virus (vSP) preferentially replicates in transformed cells or p53-null cells when compared with their normal counterparts. This selectivity may be derived from the fact that vSP-infected normal cells died faster than infected cancer cells. A fraction of infected cells died with evidence of necrosis as shown by both flow cytometry and detection of high-mobility group B1 protein released from necrotic cells into the culture supernatant. When administered to animals, vSP retains full ability to replicate in tumor tissues, whereas replication in normal tissues is greatly diminished. In a model of viral pathogenesis, mice treated with vSP survived substantially longer when compared with mice treated with the wild-type virus. The mutant virus vSP displayed significant antitumoral effects in an MC38 s.c. tumor model in both nude (P < 0.001) and immunocompetent mice (P < 0.05). We conclude that this recombinant vaccinia vSP shows promise for oncolytic virus therapy. Given its enhanced tumor selectivity, improved safety profile, and substantial oncolytic effects following systemic delivery in murine models, it should also serve as a useful vector for tumor-directed gene therapy.

Animals↗

TOL is a broad-host-range plasmid.

We readily isolated insertions of the carbenicillin resistance element Tn401 into the TOL plasmid in Pseudomonas putida. Hybrid TOL::Tn401 plasmids stably express the Cbr phenotype in Pseudomonas aeruginosa and Escherichia coli. Whereas the replicative and conjugative functions are expressed in both hosts, the ability to grow on m-toluate is only expressed in the Pseudomonas species.

Conjugation, Genetic↗

Alteration in tRNA methyltransferase activity in mengovirus infection: host range specificity.

The tRNA methyltransferase activity in mengovirus-infected L cells, HeLa cells, and Maden Derby bovine kidney cells has been examined during the course of infection. The first two cell lines yield a productive infection, but have different kinetics of inhibition of host RNA synthesis, whereas the bovine kidney cells are a restrictive host. In infected L cells the enzymes show altered capacity and base specificity throughout the infection. In infected HeLa cells and in infected bovine kidney cells less marked changes were seen. No inhibitors or activators of the enzymes were detected in any of the infected cell lines. Labeling experiments in infected cells indicated that in infected L cells synthesis of RNA was inhibited to a greater degree than was methylation of RNA. The consequence of this would be a hypermethylation of RNA. The methylated derivatives synthesized in infected L cells showed changes in relative proportions. Infection of HeLa cells and bovine kidney cells did not show such marked effects on methylation of RNA.

Animals↗

The feeding habits of the tsetse, Glossina pallidipes Austen on the south Kenya coast, in the context of its host range and trypanosome infection rates in other parts of east Africa.

The results of blood-meal identifications for 651 Glossina pallidipes from 5 subpopulations near the Kenya coast south of Mombasa, and one, 70 km inland, are presented. Bushpigs and/or warthogs were important hosts for G. pallidipes at all sites. Other major hosts included elephant, buffalo and bushbuck where they were present, and on a dairy ranch nearly 30% of feeds were taken from cattle. There was a general relationship between the numbers and diversity of wild herbivores and the abundance of G. pallidipes. These results are discussed in relation to published data on feeding patterns and trypanosome infection rates for G. pallidipes from other parts of East Africa. Overall, there are significant correlations between the proportions of bovid feeds and T. vivax infections. Bovid-feeding G. pallidipes populations with high T. vivax infection rates in south-east Uganda and western Kenya contrast with the coastal, suid-feeding populations with low T. vivax rates. These characteristics are presented as clines extending across East Africa.

Africa, Eastern↗