DNA sequence of the filamentous bacteriophage Pf1.

The genome of the class II filamentous bacteriophage Pf1 has been sequenced by a combination of the chain termination and chemical degradation methods. It consists of 7349 nucleotides in a closed, circular loop of single-stranded DNA. The size and position of its open reading frames (ORFs) in general resemble those of other filamentous bacteriophage genomes. The size and position of the spaces between the ORFs have not been conserved, however, and six short reading frames (2 of which overlap) occupy a region corresponding to that filled by genes 2 and 10 in the Ff genome. Most of the ORFs are preceded by sequences resembling ribosome binding sites from the phage's host. Pseudomonas aeruginosa, that appear to differ somewhat from their counterparts in Escherichia coli. A search for sequences related to known pseudomonad promoters suggests that the promoters in this bacteriophage may well be ntr-dependent, with the two strongest preceding the gene for the major coat protein (gene 8) and another ORF (430). Gene 8 is followed by a sequence with the properties of a rho-independent terminator of transcription, like that at the same position in the genome of Ff. The Pf1 genome contains no collection of potential stem-and-loop structures corresponding to those that initiate replication of Ff DNA and assembly of the Ff virion, although isolated structures of this kind are present. The available evidence suggests that at least 13 of the 14 major ORFs are expressed. Overall, the organization of the Pf1 genome differs from that of the other class II filamentous phage whose genome has been sequenced, Pf3, as much as it does from that of the class I phages Ff and IKe.

Development rates, fecundity and survival of developmental stages of the ticks Rhipicephalus appendiculatus, Boophilus decoloratus and B. microplus under field conditions in Zimbabwe.

To determine development rates, fecundity and survival of Rhipicephalus appendiculatus, Boophilus decoloratus and B. microplus, a study was carried out in long and short grass in the highveld of Zimbabwe. Engorged adult females of the three species and engorged larvae and nymphs of R. appendiculatus were buried beneath the soil in small cages in the rainy, cool and hot seasons in 1980 and 1981. Half the number of cages were examined regularly to determine development rates and half were left undisturbed to determine survival rates and the fecundity of engorged females. Development was most rapid during warm conditions and slowest during cool conditions, but high temperatures appeared to prolong the preoviposition periods of all species. The relationship between fluctuating temperatures and rate of development in the field was defined using a least-squares procedure. Survival of engorged females was usually high, but was reduced by predation when they were not protected. Fecundity was reduced in long grass during the cool season and in short grass during the hot season. A higher percentage of eggs hatched in the rainy season than in the cool or dry seasons. The survival of engorged larvae and nymphs was usually high in all seasons. Engorged nymphs were the hardiest stage and eggs the most suceptible stage to adverse microclimatic conditions.

Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of Escherichia coli.

Nucleotide sequence analysis of the Escherichia coli chromosomal DNA inserted in the plasmid pLC33-5 of the Clarke and Carbon library [Clarke & Carbon (1976) Cell 9, 91-99] revealed the existence of the gene, fda, encoding the Class II (metal-dependent) fructose 1,6-bisphosphate aldolase of E. coli. The primary structure of the polypeptide chain inferred from the DNA sequence of the fda gene comprises 359 amino acids, including the initiating methionine residue, from which an Mr of 39,146 could be calculated. This value is in good agreement with that of 40,000 estimated from sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified dimeric enzyme. The amino acid sequence of the Class II aldolase from E. coli showed no homology with the known amino acid sequences of Class I (imine-forming) fructose 1,6-bisphosphate aldolases from a wide variety of sources. On the other hand, there was obvious homology with the N-terminal sequence of 40 residues already established for the Class II fructose 1,6-bisphosphate aldolase of Saccharomyces cerevisiae. These Class II aldolases, one from a prokaryote and one from a eukaryote, evidently are structurally and evolutionarily related. A 1029 bp-fragment of DNA incorporating the fda gene was excised from plasmid pLC33-5 by digestion with restriction endonuclease HaeIII and subcloned into the expression plasmid pKK223-3, where the gene came under the control of the tac promoter. When grown in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside, E. coli JM101 cells transformed with this recombinant expression plasmid generated the Class II fructose 1,6-bisphosphate aldolase as approx. 70% of their soluble protein. This unusually high expression of an E. coli gene should greatly facilitate purification of the enzyme for any future structural or mechanistic studies.

Characterization of Barmah forest virus: an alphavirus with some unusual properties.

Barmah Forest virus has been characterized in a number of ways including electron microscopy of infected cells; physical studies of the virion, its RNA, and associated proteins; N-terminal sequence analysis of the two envelope glycoproteins; studies of macromolecular species present in infected cells; and serological cross-reactions with alphaviruses and bunyaviruses. From these results Barmah Forest virus is clearly an alphavirus since the structure of the virion, the mode of replication, and the macromolecular species present in infected cells are typical of alphaviruses. The N-terminal regions of the two glycoproteins E1 and E2 show extensive sequence homology (approximately 50%) with those of other alphaviruses. Barmah Forest virus cross-reacts in hemagglutination inhibition tests, although not in complement fixation tests or infectivity neutralization tests, with other alphaviruses. In some of its properties Barmah Forest virus is unusual, however. It cross-reacts in complement fixation and hemagglutination inhibition tests with Umbre virus, a bunyavirus, which originally led it to be classified as a bunyavirus; the glycosylation pattern of E2 of Barmah Forest virus appears to differ from that of other alphaviruses; and the sedimentation coefficient of the virion appears to be slightly less than that of other alphaviruses.

The Mapputta group of arboviruses: ultrastructural and molecular studies which place the group in the bunyavirus genus of the family Bunyaviridae.

We have characterized members of the Mapputta group of 'bunyavirus-like' viruses in terms of morphology, structure, ultrastructural development and virus-directed RNA and protein synthesis. Our primary study has been with Maprik virus (MPK) as a representative of the group. The MPK virion is uniformly spherical (congruent to 90 nm diameter) and possesses a membrane envelope. Virus maturation is by budding into small vesicles in the perinuclear region. During infection of BHK cells which is cytopathic, maximum viral RNA synthesis is congruent to 20% of RNA synthesis in uninfected cells without actinomycin D. In Aedes albopictus cells, where high initial titres are followed by a persistent infection, maximum viral RNA synthesis is only 1.5% of levels in control cells. MPK virions contain three RNA species. MPK generates four virus-specific RNAs (L, M, S1 and S2) and three nucleocapsid species in infected BHK cells. Six virus-specific polypeptides are present in infected cells; the four largest correspond to L (a probable transcriptase component), G1 and G2 (envelope proteins) and N (the nucleocapsid protein), respectively. These results and complementary studies with Trubanaman and Gan Gan viruses indicate that the members of the Mapputta serogroup can be placed in the bunyavirus genus of the family Bunyaviridae.

Seven infection-specific polypeptides in BHK cells infected with Bunyamwera virus.

Virus-specific polypeptide synthesis was examined in BHK cells and Vero cells infected with Bunyamwera virus. In BHK cells, in addition to the four previously reported virus-coded proteins (L, G1, G2, and N), three other infection-specific proteins were detected. These proteins, of nominal molecular weight 50,000 (p50), 16,000 (p16), and 13,000 (p13), were not labeled in mock-infected cells, were first synthesized between 4 and 8 h after infection, and were relatively prominent among the limited number of proteins generated late in infection. In preparations of purified Bunyamwera virus from BHK cell supernatants, p16 was detected but not p50 or p13. In Vero cells infected with Bunyamwera virus, both p50 and p13 were labeled strongly. Maprik virus, a member of the Mapputta group of arboviruses, is a member of the Bunyavirus genus (S.E. Newton, unpublished data). Maprik virus did not induce the synthesis of p50, p16, or p13; however, two smaller proteins (p17 and p15) which may correspond to p16 and p13 were labeled late in Maprik infection. Our data argue that p16 is a virus-coded component of the Bunyamwera virus particle and that p50 and p13 are virus-coded, nonstructural proteins.

The DNA-binding protein of Pf1 filamentous bacteriophage: amino-acid sequence and structure of the gene.

The amino-acid sequence of the single-stranded DNA-binding protein of bacteriophage Pf1 and the nucleotide sequence of the corresponding gene have been determined. The protein has 144 amino acids and a molecular weight of 15 400; the gene consists of 435 nucleotides. The amino-acid sequence was determined by Edman degradation, carboxypeptidase A, B, and P digestion of intact protein and of peptides derived by chymotrypsin, Staphylococcus aureus V8 protease, and trypsin digestion. The nucleotide sequence was determined by the dideoxy method after random cloning of fragments of Pf1 DNA into M13. No sequence homology could be established between the amino-acid sequence of the DNA-binding protein of Pseudomonas aeruginosa-specific bacteriophage Pf1 and bacteriophage fd of Escherichia coli.

Bunyamwera virus replication in cultured Aedes albopictus (mosquito) cells: establishment of a persistent viral infection.

Bunyamwera virus replication was examined in Aedes albopictus (mosquito) cell cultures in which a persistent infection is established and in cytopathically infected BHK cells. During primary infection of A. albopictus cells, Bunyamwera virus reached relatively high titers ( approximately 10(7) PFU/ml), and autointerference was not observed. Three virus-specific RNAs (L, M, and S) and two virion proteins (N and G1) were detected in infected cells. Maximum rates of viral RNA synthesis and viral protein synthesis were extremely low, corresponding to <2% of the synthetic capacities of uninfected control cells. Viral protein synthesis was maximal at 12 h postinfection and was shut down to barely detectable levels at 24 h postinfection. Virus-specific RNA and nucleocapsid syntheses showed similar patterns of change, but later in infection. The proportions of cells able to release a single PFU at 3, 6, and 54 days postinfection were 100, 50, and 1.5%, respectively. Titers fell to 10(3) to 10(5) PFU/ml in carrier cultures. Persistently infected cultures were resistant to superinfection with homologous virus but not with heterologous virus. No changes in host cell protein synthesis or other cytopathic effects were observed at any stage of infection. Small-plaque variants of Bunyamwera virus appeared at approximately 7 days postinfection and increased gradually until they were 75 to 95% of the total infectious virus at 66 days postinfection. Temperature-sensitive mutants appeared between 23 and 49 days postinfection. No antiviral activity similar to that reported in A. albopictus cell cultures persistently infected with Sindbis virus (R. Riedel and D. T. Brown, J. Virol. 29: 51-60, 1979) was detected in culture fluids by 3 months after infection. Bunyamwera virus replicated more rapidly in BHK cells than in mosquito cells but reached lower titers. Autointerference occurred at multiplicities of infection of approximately 10. Virus-specific RNA and protein syntheses were at least 20% of the levels in uninfected control cells. Host cell protein synthesis was completely shut down, and nucleocapsid protein accumulated until it was 4% of the total cell protein. We discuss these results in relation to possible mechanisms involved in determining the outcome of arbovirus infection of vertebrate and mosquito cells.

Regulation of seasonal occurrence in the tick Rhipicephalus appendiculatus Neumann, 1901.

Published data on the seasonal occurrence of Rhipicephalus appendiculatus at 8 localities in east, central and southern Africa has been compared and an analysis of the regulatory roles of different climatic factors has been undertaken. It has been shown that the pattern of seasonal occurrence is largely dependent on the timing of the activity period of the adult stage and this is regulated by the combined influences of humidity, temperatures and daylength. By use of a simple model it is shown how the adult activity period in a given climatic regimen can be predicted on the basis of easily obtainable meteorological data. The value of the model in planning tick and disease control programmes is discussed.

The seasonal activity of Rhipicephalus appendiculatus Neumann 1901 (Acarina: Ixodidae) in the highveld of Zimbabwe Rhodesia.

Rhipicephalus appendiculatus passes through one generation per annum in the highveld of Zimbabwe, Rhodesia and shows a well-defined pattern of seasonal activity. Peak adult activity occurs in the rainy season, whereas peak larval and nymphal activity occurs in the dry season. Adult activity is regulated by the combined influences of temperature, humidity, and day length. Climatic factors have little or no direct influence on the activity of larvae and nymphs. The occurrence of the larval and nymphal activity peaks is determined by the timing of the adult activity peak and the duration of the preceeding developmental periods, which are temperature dependent. In the early rainy season, unfed adults climb to the tips of the grass and enter a period of quiescence prior to becoming active.