Ebola and Marburg virus diseases in Africa: Increased risk of outbreaks in previously unaffected areas?

Filoviral hemorrhagic fever (FHF) is caused by ebolaviruses and marburgviruses, which both belong to the family Filoviridae. Egyptian fruit bats (Rousettus aegyptiacus) are the most likely natural reservoir for marburgviruses and entry into caves and mines that they stay in has often been associated with outbreaks of MVD. On the other hand, the natural reservoir for ebola viruses remains elusive; however, handling of wild animal carcasses has been associated with some outbreaks of EVD. In the last two decades, there has been an increase in the incidence of FHF outbreaks in Africa, some being caused by a newly found virus and some occurring in previously unaffected areas such as Guinea, Liberia and Sierra Leone, in which the most recent EVD outbreak occurred in 2014. Indeed, the predicted geographic distribution of filoviruses and their potential reservoirs in Africa includes many countries in which FHF has not been reported. To minimize the risk of virus dissemination in previously unaffected areas, there is a need for increased investment in health infrastructure in African countries, policies to facilitate collaboration between health authorities from different countries, implementation of outbreak control measures by relevant multi‐disciplinary teams and education of the populations at risk.     

HIV Restriction by APOBEC3 in Humanized Mice

Innate immune restriction factors represent important specialized barriers to zoonotic transmission of viruses. Significant consideration has been given to their possible use for therapeutic benefit. The apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3) family of cytidine deaminases are potent immune defense molecules capable of efficiently restricting endogenous retroelements as well as a broad range of viruses including Human Immunodeficiency virus (HIV), Hepatitis B virus (HBV), Human Papilloma virus (HPV), and Human T Cell Leukemia virus (HTLV). The best characterized members of this family are APOBEC3G (A3G) and APOBEC3F (A3F) and their restriction of HIV. HIV has evolved to counteract these powerful restriction factors by encoding an accessory gene designated viral infectivity factor (vif). Here we demonstrate that APOBEC3 efficiently restricts CCR5-tropic HIV in the absence of Vif. However, our results also show that CXCR4-tropic HIV can escape from APOBEC3 restriction and replicate in vivo independent of Vif. Molecular analysis identified thymocytes as cells with reduced A3G and A3F expression. Direct injection of vif-defective HIV into the thymus resulted in viral replication and dissemination detected by plasma viral load analysis; however, vif-defective viruses remained sensitive to APOBEC3 restriction as extensive G to A mutation was observed in proviral DNA recovered from other organs. Remarkably, HIV replication persisted despite the inability of HIV to develop resistance to APOBEC3 in the absence of Vif. Our results provide novel insight into a highly specific subset of cells that potentially circumvent the action of APOBEC3; however our results also demonstrate the massive inactivation of CCR5-tropic HIV in the absence of Vif.     

Molecular characterization of the duck enteritis virus <Emphasis Type="Italic">UL4</Emphasis> gene

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious and fatal disease. In the present article, the DEV UL4 gene was cloned and sequenced from a vaccine virus. A degenerate oligonucleotide primer for the consensus site of herpesvirus UL3 gene and a specific primer located in UL5 were used in the polymerase chain reaction (PCR) to amplify a DNA product 2 086 bp in size. DNA sequence analysis revealed that a 714 bp open reading frame (ORF) of DEV encoding a 237 amino acid polypeptide is homologous to the family of herpesvirus UL4 proteins and therefore has been characterized as a DEV UL4 gene. Alignment of the DEV UL4 protein sequence with those of other alphaherpesviruses showed that 10 amino acid residues are completely conserved. Phylogenetic tree analysis showed that the seventeen alphaherpesviruses viruses analyzed were classified into four large groups, and the duck enteritis virus branched separately, closely related to the Mardiviruses group comprising Gallid herpesvirus 2 (GaHV-2), Gallid herpesvirus 3 (GaHV-3) and Meleagrid herpesvirus 1 (MeHV-1). The present study showed that the evolutionary relationship of the UL4 protein could be used for classification of alphaherpesviruses.      

Phylogenetic Analysis of theArenaviridae:Patterns of Virus Evolution and Evidence for Cospeciation between Arenaviruses and Their Rodent Hosts

Viruses of theArenaviridaecause hemorrhagic fevers and neurologic disease in humans. Historically, the arenaviruses have been divided into two complexes (LASV-LCMV, Tacaribe) through the use of antigenic typing. The phylogeny of theArenaviridaeas a whole has not been estimated previously due to a lack of sequence data for all members of the family. In this study, nucleocapsid protein gene sequence data were obtained for all currently known arenaviruses and used to estimate, for the first time, a phylogeny of the entire virus family. The LCMV-LASV complex arenaviruses are monophyletic and comprise three distinct lineages. The Tacaribe complex viruses also are monophyletic and occupy three distinct lineages. Comparisons of arenavirus phylogeny with rodent host phylogeny and taxonomic relationships provide several examples in which virus–host cospeciation is potentially occurring. The pathogenic arenaviruses do not appear to be monophyletic, suggesting that the pathogenic phenotype has arisen in multiple independent events during virus evolution.     

Identification of MBF2 family genes in Bombyx mori and their expression in different tissues and stages and in response to Bacillus bombysepticus infection and starvation

The Multiprotein bridge factor 2 (MBF2) gene was first identified as a co‐activator involved in BmFTZ‐F1‐mediated activation of the Fushi tarazu gene. Herein, nine homologous genes of MBF2 gene are identified. Evolutionary analysis showed that this gene family is insect‐specific and that the family members are closely related to response to pathogens (REPAT) genes. Tissue distribution analysis revealed that these genes could be expressed in a tissue‐specific manner. Developmental profiles analysis showed that the MBF2 gene family members were highly expressed in the different stages. Analysis of the expression patterns of nine MBF2 family genes showed that Bacillus bombysepticus treatment induced the up‐regulation of several MBF2 family genes, including MBF2‐4, ‐7, ‐9, ‐8. Furthermore, we found the MBF2 family genes were modulated by starvation and the expression of these genes recovered upon re‐feeding, except for MBF2‐5, ‐9. These findings suggested roles for these proteins in insect defense against pathogens and nutrient metabolism, which has an important guiding significance for designing pest control strategies.     

Molecular characterization of Fasciola gigantica from Mauritania based on mitochondrial and nuclear ribosomal DNA sequences

Fasciolosis caused by Fasciola hepatica and Fasciola gigantica (Platyhelminthes: Trematoda: Digenea) is considered the most important helminth infection of ruminants in tropical countries, causing considerable socioeconomic problems. From Africa, F. gigantica has been previously characterized from Burkina Faso, Senegal, Kenya, Zambia and Mali, while F. hepatica has been reported from Morocco and Tunisia, and both species have been observed from Ethiopia and Egypt on the basis of morphometric differences, while the use of molecular markers is necessary to distinguish exactly between species. Samples identified morphologically as F. gigantica (n = 60) from sheep and cattle from different geographical localities of Mauritania were genetically characterized by sequences of the first (ITS-1), the 5.8S, and second (ITS-2) Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA (rDNA) genes and the mitochondrial Cytochrome c Oxidase I (COI) gene.Comparison of the sequences of the Mauritanian samples with sequences of Fasciola spp. from GenBank confirmed that all samples belong to the species F. gigantica. The nucleotide sequencing of ITS rDNA of F. gigantica showed no nucleotide variation in the ITS-1, 5.8S, and ITS-2 rDNA sequences among all samples examined and those from Burkina Faso, Kenya, Egypt and Iran. The phylogenetic trees based on the ITS-1 and ITS-2 sequences showed a close relationship of the Mauritanian samples with isolates of F. gigantica from different localities of Africa and Asia. The COI genotypes of the Mauritanian specimens of F. gigantica had a high level of diversity, and they belonged to the F. gigantica phylogenically distinguishable clade. The present study is the first molecular characterization of F. gigantica in sheep and cattle from Mauritania, allowing a reliable approach for the genetic differentiation of Fasciola spp. and providing basis for further studies on liver flukes in the African countries.     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

Construction and characterization of a full-length infectious clone of Getah virus in vivo

Getah virus (GETV) is a mosquito-borne virus of the genus Alphavirus in the family Togaviridae and, in recent years, it has caused several outbreaks in animals. The molecular basis for GETV pathogenicity is not well understood. Therefore, a reverse genetic system of GETV is needed to produce genetically modified viruses for the study of the viral replication and its pathogenic mechanism. Here, we generated a CMV-driven infectious cDNA clone based on a previously isolated GETV strain, GX201808 (pGETV-GX). Transfection of pGETV-GX into BHK- 21 cells resulted in the recovery of a recombinant virus (rGETV-GX) which showed similar growth characteristics to its parental virus. Then three-day-old mice were experimentally infected with either the parental or recombinant virus. The recombinant virus showed milder pathogenicity than the parental virus in the mice. Based on the established CMV-driven cDNA clone, subgenomic promoter and two restriction enzyme sites (BamHI and EcoRI) were introduced into the region between E1 protein and 30UTR. Then the green fluorescent protein (GFP), red fluorescent protein (RFP) and improved light-oxygen-voltage (iLOV) genes were inserted into the restriction enzyme sites. Transfection of the constructs carrying the reporter genes into BHK-21 cells proved the rescue of the recombinant reporter viruses. Taken together, the establishment of a reverse genetic system for GETV provides a valuable tool for the study of the virus life cycle, and to aid the development of genetically engineered GETVs as vectors for foreign gene expression.     

High-resolution crystal structure of FKBP12 from Aedes aegypti

Dengue is one of the most infectious viral diseases prevalent mainly in tropical countries. The virus is transmitted by Aedes species of mosquito, primarily Aedes aegypti. Dengue remains a challenging drug target for years as the virus eludes the immune responses. Currently, no vaccines or antiviral drugs are available for dengue prevention. Previous studies suggested that the immunosuppressive drug FK506 shows antimalarial activity, and its molecular target, FK506‐binding protein (FKBP), was identified in the Plasmodium parasite. Likewise, a FKBP family protein has been identified in A. aegypti (AaFKBP12) in which AaFKBP12 is assumed to play a similar role in its life cycle. FKBPs belong to a highly conserved class of proteins and are considered as an attractive pharmacological target. Herein, we present a high‐resolution crystal structure of AaFKBP12 at 1.3 Å resolution and discuss its structural features throwing light in facilitating the design of potential antagonists against the dengue‐transmitting mosquito.     

Marker-assisted combination of major genes for pathogen resistance in potato

Closely linked PCR-based markers facilitate the tracing and combining of resistance factors that have been introgressed previously into cultivated potato from different sources. Crosses were performed to combine the Ry _adg gene for extreme resistance to Potato virus Y (PVY) with the Gro1 gene for resistance to the root cyst nematode Globodera rostochiensis and the Rx1 gene for extreme resistance to Potato virus X (PVX), or with resistance to potato wart (Synchytrium endobioticum). Marker-assisted selection (MAS) using four PCR-based diagnostic assays was applied to 110 F1 hybrids resulting from four 2× by 4× cross-combinations. Thirty tetraploid plants having the appropriate marker combinations were selected and tested for presence of the corresponding resistance traits. All plants tested showed the expected resistant phenotype. Unexpectedly, the plants segregated for additional resistance to pathotypes 1, 2 and 6 of S. endobioticum, which was subsequently shown to be inherited from the PVY resistant parents of the crosses. The selected plants can be used as sources of multiple resistance traits in pedigree breeding and are available from a potato germplasm bank.     

The Beta vulgaris-derived resistance gene Rz2 confers broad-spectrum resistance against soilborne sugar beet-infecting viruses from different families by recognizing triple gene block protein 1

Sugar beet cultivation is dependent on an effective control of beet necrotic yellow vein virus (BNYVV, family Benyviridae), which causes tremendous economic losses in sugar production. As the virus is transmitted by a soilborne protist, the use of resistant cultivars is currently the only way to control the disease. The Rz2 gene product belongs to a family of proteins conferring resistance towards diverse pathogens in plants. These proteins contain coiled-coil and leucine-rich repeat domains. After artificial inoculation of homozygous Rz2 resistant sugar beet lines, BNYVV and beet soilborne mosaic virus (BSBMV, family Benyviridae) were not detected. Analysis of the expression of Rz2 in naturally infected plants indicated constitutive expression in the root system. In a transient assay, coexpression of Rz2 and the individual BNYVV-encoded proteins revealed that only the combination of Rz2 and triple gene block protein 1 (TGB1) resulted in a hypersensitive reaction (HR)-like response. Furthermore, HR was also triggered by the TGB1 homologues from BSBMV as well as from the more distantly related beet soilborne virus (family Virgaviridae). This is the first report of an R gene providing resistance across different plant virus families.     

Nitric oxide reduction, the last step in denitrification by Fusarium oxysporum, is obligatorily mediated by cytochrome P450nor

The involvement of cytochrome P450nor (P450nor) is the most striking feature of the fungal denitrifying system, and has never been shown in bacterial systems. To establish the physiological significance of the P450nor, we constructed and investigated mutants of Fusarium oxysporum that lacked the gene for P450nor. We mutated the gene by targeted integration of a disrupted gene into the chromosome of F. oxysporum. The mutants were shown to contain neither P450nor protein nor nitric oxide (NO) reductase (Nor) activity, implying that they are indeed deficient in P450nor. These mutants had apparently lost the denitrifying activity and failed to evolve nitrous oxide (N₂O) upon incubation under oxygen-limiting conditions in the presence of nitrate. Their mycelia exhibited normal levels of dissimilatory nitrite reductase (Nir) activity and were able to evolve NO under these conditions. The promoter region of the P450nor gene was fused to lacZ and introduced into the wild-type strain of F. oxysporum. The transformed strain produced β-galactosidase under denitrifying conditions as efficiently as the wild type does P450nor. These results represent unequivocal genetic evidence that P450nor is essential for the reduction of NO to N₂O, the last step in denitrification by F. oxysporum. Received: 28 June 1999 / Accepted: 22 December 1999     

A novel tomato spotted wilt virus isolate encoding a noncanonical NSm C118F substitution associated with Sw-5 tomato gene resistance breaking

The nonstructural protein NSm of tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant of the tomato single dominant Sw-5 resistance gene. Although Sw-5 effectiveness has been shown for most TSWV isolates, the emergence of resistance-breaking (RB) isolates has been observed. It is strongly associated with two point mutations (C118Y or T120N) in the NSm viral protein. TSWV-like symptoms were observed in tomato crop cultivars (+Sw-5) in the Baja California peninsula, Mexico, and molecular methods confirmed the presence of TSWV. Sequence analysis of the NSm 118–120 motif and three-dimensional protein modelling exhibited a noncanonical C118F substitution in seven isolates, suggesting that this substitution could emulate the C118Y-related RB phenotype. Furthermore, phylogenetic and molecular analysis of the full-length genome (TSWV-MX) revealed its reassortment-related evolution and confirmed that putative RB-related features are restricted to the NSm protein. Biological and mutational NSm 118 residue assays in tomato (+Sw-5) confirmed the RB nature of TSWV-MX isolate, and the F118 residue plays a critical role in the RB phenotype. The discovery of a novel TSWV-RB Mexican isolate with the presence of C118F substitution highlights a not previously described viral adaptation in the genus Orthotospovirus, and hence, the necessity of further crop monitoring to alert the establishment of novel RB isolates in cultivated tomatoes.     

Organization of the lux genes of photobacterium phosphoreum

The lux genes from Photobacterium phosphoreum (NCMB844) have been cloned into Escherichia coli in a plasmid containing the T7-bacteriophage promoter. By specific expression in vivo under the T7 promoter, five structural genes (luxA-E) coding for the fatty acid reductase and luciferase polypeptides were identified as well as a new gene, designated as luxF, which codes for a 26kDa polypeptide. This new gene is located between luxB and luxE and thus disrupts the structural gene order of luxCDABE found in the Vibrio genus. The luxF gene and the protein it codes for have recently been identified in other Photobacterium species and so appears to be widely distributed within this genus. Nucleotide sequencing of the luxF gene has shown it to code for a protein homologous to the luciferase subunits, coded by the luxA and luxB genes. Although this gene is not necessary for light emission in all luminescent bacteria, it must play an essential role in the biochemistry, physiology, or ecology of the luminescent system in species of the Photobacterium genus.     

Myxoma Virus Encodes an α2,3-Sialyltransferase That Enhances Virulence

A 4.7-kb region of DNA sequence contained at the right end of the myxoma virus EcoRI-G2 fragment located 24 kb from the right end of the 163-kb genome has been determined. This region of the myxoma virus genome encodes homologs of the vaccinia virus genes A51R, A52R, A55R, A56R, and B1R; the myxoma virus gene equivalents have been given the prefix M. The MA55 gene encodes a protein belonging to the kelch family of actin-binding proteins, while the MA56 gene encodes a member of the immunoglobulin superfamily related to a variety of cellular receptors and adhesion molecules. A novel myxoma virus early gene, MST3N, is a member of the eukaryotic sialyltransferase gene family located between genes MA51 and MA52. Detergent lysates prepared from myxoma virus-infected cell cultures contained a virally encoded sialyltransferase activity that catalyzed the transfer of sialic acid (Sia) from CMP-Sia to an asialofetuin glycoprotein acceptor. Analysis of the in vitro-sialylated glycoprotein acceptor by digestion with N-glycosidase F and by lectin binding suggested that the MST3N gene encodes an enzyme with Galβ1,3(4)GlcNAc α2,3-sialyltransferase specificity for the N-linked oligosaccharide of glycoprotein. Lectin binding assays demonstrated that α2,3-sialyltransferase activity is expressed by several known leporipoxviruses that naturally infect Sylvilagus rabbits. The sialyltransferase is nonessential for myxoma virus replication in cell culture; however, disruption of the MST3N gene caused attenuation in vivo. The possible implications of the myxoma virus-expressed sialyltransferase in terms of the host’s defenses against infection are discussed.     

Evaluation of M307 of <Emphasis Type="Italic">FUT1</Emphasis> gene as a genetic marker for disease resistance breeding of sutai pigs

Alpha (1,2) fucosyltransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of the receptor for ETEC F18. The genetic variations in the position of M307 nucleotide in open reading frame of FUT1 have been proposed as a marker for selecting ETEC F18 resistant pigs. The polymorphisms of M307 in FUT1 of breeding base group for ETEC F18 resistance of Sutai pigs (Duroc × Meishan) was detected and their correlations to some immune indexes, growth and development ability, carcass traits and meat quality were also analyzed, which aimed to investigate feasibility of further breeding for diseases resistance based on M307 of FUT1 for Sutai pigs. After digested by Hin6 I, M307 of FUT1 gene could be divided into three kinds of genotypes, AA, AG, and GG. The frequencies were 0.235, 0.609, and 0.156, respectively. The results indicated that Sutai pigs with the AA genotype in M307 of FUT1 gene not only have relatively strong general disease resistance ability in piglets, but also have higher growth and development ability and stable carcass traits and meat quality. It is entirely feasible to raise the new strains of Sutai pigs resistant to Escherichia coli F18 based on genetic marker of the M307 position in FUT1gene.     

Mutation of the TYTLE Motif in the Cytoplasmic Tail of the Sendai Virus Fusion Protein Deeply Affects Viral Assembly and Particle Production

Enveloped viruses contain glycoproteins protruding from the viral membrane. These proteins play a crucial role in the extra-cellular steps of the virus life cycle, namely attachment to and entry into cells. Their role during the intracellular late phase of virus multiplication has been less appreciated, overlooked by the documented central organizer role of the matrix M protein. Sendai virus, a member of the Paramyxoviridae family, expresses two trans-membrane proteins on its surface, HN and F. In previous work, we have shown that suppression of F in the context of an infection, results in about 70% reduction of virus particle production, a reduction similar to that observed upon suppression of the matrix M protein. Moreover, a TYTLE motif present in F cytoplasmic tail has been proposed essential for virus particle production. In the present work, using original alternate conditional siRNA suppression systems, we generated a double F gene recombinant Sendai virus expressing wt-F and a nonviable mutated TYTLE/5A F protein (F_5A). Suppression of the wild type F gene expression in cells infected with this virus allowed the analysis of F_5A properties in the context of the infection. Coupling confocal imaging analysis to biochemical characterization, we found that F_5A i) was not expressed at the cell surface but restricted to the endoplasmic reticulum, ii) was still capable of interaction with M and iii) had profound effect on M and HN cellular distribution. On the basis of these data, we propose a model for SeV particle formation based on an M/F complex that would serve as nucleation site for virus particle assembly at the cell surface.     

Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phages

The filamentous phage protein pIV is required for assembly and secretion of the virus and possesses regions homologous to those found in a number of Gram-negative bacterial proteins that are essential components of a widely distributed extracellular protein-export system. These proteins form multimers that may constitute an outer membrane channel that allows phage/protein egress. Three sets of f1 gene IV mutants were isolated at positions that are absolutely (G355 and P375) or largely (F381) conserved amongst the 16 currently known family members. The G355 mutants were non-functional, interfered with assembly of plV⁺ phage, and made Escherichia coli highly sensitive to deoxycholate. The P375 mutants were non-functional and defective in multimerization. Many of the F381 mutants retained substantial function, and even those in which charged residues had been introduced supported some phage assembly. Some inferences about the roles of these conserved amino acids are made from the mutant phenotypes.     

Drosophila genes which intervene in multiplication of sigma virus (author''s transl)]

Summary Distinction between Drosophila strains, differing their capacity for supporting multiplication of sigma virus, arises essentially from comparison of the incubation time after inoculation of a viral suspension. This is the most general and the most useful characteristic. By this mean five allelic differences with the reference Drosophila strain Oregon have been found. Corresponding genes, ref(1)H, ref(2)M, ref(2)P, ref(3)O and ref(3)D are located all over Drosophila chromosomes. The specific spectra of viral strains sensitive to the one or the other allele was determined for each gene.Some characteristic properties of flies in which the virus has been brought by injection or heredity were compared between heterozygotes and homozygotes for the permissive and for the non permissive allele:time of incubation as a function of the size of the inoculum,probability of initiating infection,kinetics of the virus multiplication in inoculated fly,efficiency of a viral genome brought by a spermatozoa in infecting an egg,perpetuation of the carrier state of sigma virus in germ line cells of stabilized females or males and in somatic cells.The properties concerning the perpetuation of sigma virus carrier state allow to distinguish two classes of viral functions in which the considered ref gene product can intervene: 1) functions necessary for viral genome replication and, of course, for perpetuation of carrier state, 2) other functions, (late functions — necessary for maturation - and functions necessary for cell penetration of inoculated virus).Homozygotes for each of the two alleles of a gene which acts on incubation time can show no difference in one property which is specific of a differenciated cell type only because the considered gene is not expressed in the cell type involved. Conversely genes can exist which act on such a property and which have no action on incubation time. Probably such a gene has been discovered; it intervenes in the transmission of sigma virus by stabilized males; this gene is named ref(3)V.Discussion of all the properties of flies homozygotes for each allele permits us to conclude that ref(1)H, ref(2)M, ref(2)P, ref(3)D and possibly ref(3)V genes (if this last gene intervenes directly in sigma's physiology) are involved in a function necessary for replication. No conclusive evidence has been found for ref(3)O, still it seems to intervene in a late function. Problems of functional interactions between products of the first five ref genes have been mentioned.