Factors Affecting MoO4(2-) Inhibitor Release from Zn2Al Based Layered Double Hydroxide and Their Implication in Protecting Hot Dip Galvanized Steel by Means of Organic Coatings.

Zn2Al/-layered double hydroxide (LDH) with intercalated MoO4(2-) was investigated as a potential source of soluble molybdate inhibitor in anticorrosion coatings for hot dip galvanized steel (HDG). The effect of solution pH, soluble chlorides, and carbonates on the release kinetics of the interleaved MoO4(2-) ions from the LDH powder immersed in solutions containing different anions was studied by X-ray diffraction, in situ attenuated total reflectance infrared (ATR-IR) spectroscopy, and inductively coupled plasma atomic emission spectroscopy (ICP-AES). The effect of the solution composition on the total release and the release kinetics was demonstrated. Less than 30% of the total amount of the intercalated MoO4(2-) was released after 24 h of the immersion in neutral 0.005-0.5 M NaCl and 0.1 M NaNO3 solutions whereas the complete release of MoO4(2-) was observed after 1 h in 0.1 M NaHCO3 or Na2SO4 and in alkaline solutions. The in situ ATR-IR experiments and quantification of the released soluble species by ICP-AES demonstrated the release by an anion exchange in neutral solutions and by the dissolution of Zn2Al/-LDH in alkaline solutions. The anion exchange kinetics with monovalent anions was described by the reaction order n = 0.35 ± 0.05 suggesting the diffusion control; for divalent anions, n = 0.70 ± 0.06 suggested the control by a surface reaction. Dissolution of Zn from coated HDG with and without Zn2Al/-MoO4(2-) fillers, leaching of MoO4(2-) from the coating, and the electrochemical impedance spectroscopy response of the coated systems were measured during the immersion in 0.5 M NaCl solutions with and without 0.1 M NaHCO3. Without carbonates, the release of soluble MoO4(2-) was delayed for 24 h with no inhibiting effect whereas with 0.1 M NaHCO3 the immediate release was accompanied by the immediate and strong inhibiting effect on Zn dissolution. The concept of controlling the inhibition performance of LDH hybrid coatings by means of the environment composition is discussed.

Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs.

Rift Valley fever virus (RVFV), a phlebovirus of the family Bunyaviridae, is a major public health threat in Egypt and sub-Saharan Africa. The viral and host cellular factors that contribute to RVFV virulence and pathogenicity are still poorly understood. All pathogenic RVFV strains direct the synthesis of a nonstructural phosphoprotein (NSs) that is encoded by the smallest (S) segment of the tripartite genome and has an undefined accessory function. In this report, we show that MP12 and clone 13, two attenuated RVFV strains with mutations in the NSs gene, were highly virulent in IFNAR(-/-) mice lacking the alpha/beta interferon (IFN-alpha/beta) receptor but remained attenuated in IFN-gamma receptor-deficient mice. Both attenuated strains proved to be excellent inducers of early IFN-alpha/beta production. In contrast, the virulent strain ZH548 failed to induce detectable amounts of IFN-alpha/beta and replicated extensively in both IFN-competent and IFN-deficient mice. Clone 13 has a defective NSs gene with a large in-frame deletion. This defect in the NSs gene results in expression of a truncated protein which is rapidly degraded. To investigate whether the presence of the wild-type NSs gene correlated with inhibition of IFN-alpha/beta production, we infected susceptible IFNAR(-/-) mice with S gene reassortant viruses. When the S segment of ZH548 was replaced by that of clone 13, the resulting reassortants became strong IFN inducers. When the defective S segment of clone 13 was exchanged with the wild-type S segment of ZH548, the reassortant virus lost the capacity to stimulate IFN-alpha/beta production. These results demonstrate that the ability of RVFV to inhibit IFN-alpha/beta production correlates with viral virulence and suggest that the accessory protein NSs is an IFN antagonist.

The S segment of rift valley fever phlebovirus (Bunyaviridae) carries determinants for attenuation and virulence in mice.

Unlike all the other Rift Valley fever virus strains (Bunyaviridae, Phlebovirus) studied so far, clone 13, a naturally attenuated virus, does not form the filaments composed of the NSs nonstructural protein in the nuclei of infected cells (R. Muller, J. F. Saluzzo, N. Lopez, T. Drier, M. Turell, J. Smith, and M. Bouloy, Am. J. Trop. Med. Hyg. 53:405-411, 1995). This defect is correlated with a large in-frame deletion in the NSs coding region of the S segment of the tripartite genome. Here, we show that the truncated NSs protein of clone 13 is expressed and remains in the cytoplasm, where it is degraded rapidly by the proteasome. Through the analysis of reassortants between clone 13 and a virulent strain, we localized the marker(s) of attenuation in the S segment of this attenuated virus. This result raises questions regarding the role of NSs in pathogenesis and highlights, for the first time in the Bunyaviridae family, a major role of the S segment in virulence and attenuation, possibly associated with a defect in the nonstructural protein.

Molecular epidemiology and emergence of Rift Valley fever.

Rift Valley fever (RVF) is a mosquito-borne viral disease which manifested itself during recent epidemics and revealed its significant potential of emergence. Studies on molecular epidemiology undertaken to better understand the factors leading to RVF emergence, have confirmed the mode of circulation of the virus and highlighted probable risks and obstacles for prevention and control. As for several other viral agents, molecular epidemiology is becoming a useful tool in the study of the emergence of RVF as a serious infectious disease.

Molecular epidemiology and emergence of Rift Valley fever

Rift Valley fever (RVF) is a mosquito-borne viral disease which manifested itself during recent epidemics and revealed its significant potential of emergence. Studies on molecular epidemiology undertaken to better understanding the factors leading to RVF emergence, have confirmed the mode of circulation of the virus and highlighted probable risks and obstacles for prevention and control. As for several other viral agents, molecular epidemiology is becoming a useful tool in the study of the emergence of RVF as a serious infectious disease.

Mapping of the mutations present in the genome of the Rift Valley fever virus attenuated MP12 strain and their putative role in attenuation.

The MP12 attenuated strain of Rift Valley fever virus was obtained by 12 serial passages of a virulent isolate ZH548 in the presence of 5-fluorouracil (Caplen et al., 1985. Mutagen-directed attenuation of Rift Valley fever virus as a method for vaccine development. J. Gen. Virol., 66, 2271-2277). The comparison of the M segment of the two strains has already been reported by Takehara et al. (Takehara et al., 1989. Identification of mutations in the M RNA of a candidate vaccine strain of Rift Valley fever virus. Virology 169, 452-457). We have completed the comparison and found that altogether a total of nine, 12 and four nucleotides were changed in the L, M and S segments of the two strains, respectively. Three mutations induced amino acid changes in the L protein but none of them was located in the recognized motifs conserved among RNA dependent polymerases. In the S segment, a single change modified an amino acid in the NSs protein and in the M segment, seven of the mutations resulted in amino acid changes in each of the four encoded G1, G2, 14 kDa and 78 kDa proteins. Characterization of the MP12 virus indicated that determinants for attenuation were present in each segment and that they were introduced progressively during the 12 passages in the presence of the mutagen (Saluzzo and Smith, 1990. Use of reassortant viruses to map attenuating and temperature-sensitive mutations of the Rift Valley fever virus MP-12 vaccine. Vaccine 8, 369-375). Passages 4 and 7-9 were found to be essential for introduction of temperature-sensitive lesions and attenuation. In an attempt to correlate some of the mutations with the attenuated or temperature-sensitive phenotypes, we determined by sequencing the passage level at which the different mutations appeared. This work should help to address the question of the role of the viral gene products in Rift Valley fever pathogenesis.

Persistent infection of mammalian cells by Rift Valley fever virus.

Infection of mammalian cells with Rift Valley fever virus (RVFV) leads generally to the production of virus and cell death. In this paper we examined the fate of Vero cells infected with three strains of RVFV and observed that, while a large proportion of cells exhibited a clear cytopathic effect (CPE), a small but significant fraction did not undergo a lytic infection but was able to proliferate and establish a persistent infection. Several independent RVFV persistently infected cell lines have been established and passaged for more than 1 year after infection with a virulent strain (ZH548) and two attenuated strains (C13 and MP12). Although the viruses used for the primary infection were plaque-purified, we do not know whether defective-interfering particles were responsible for the establishment of the persistent infection. The persistently infected cells became resistant to superinfection with RVFV but not with other viruses and shed low amounts of infectious, lytic and non-lytic virus during a limited number of passages. In all the passages tested, the three genomic segments or related products were synthesized as well as the structural nucleoprotein N and glycoproteins G1 and G2. Abnormal defective RNAs were detected, migrating faster or slower than their respective counterparts. The faster-migrating RNAs were internally deleted, some of them possessing only the very terminal part of the 5' genomic end.

Organization of Germiston bunyavirus M open reading frame and physicochemical properties of the envelope glycoproteins.

We describe the construction of plasmids which express fusion proteins representing various regions of Germiston virus M polyprotein. The fusion proteins were purified and inoculated into rabbits to produce antisera. The N- and C-terminal regions of the polyprotein induced specific antibodies which reacted with glycoproteins G2 and G1, respectively, and the intermediate region induced antibodies against the NSM polypeptide. This enabled us to determine the gene order: G2-NSM-G1. Glycoproteins G1 and G2 form the spikes on the surface of the virion. We attempted to determine the structural organization of the glycoproteins by using a membrane-permeable cross-linking reagent, dimethyl suberimidate, but were unable to demonstrate that G1 and/or G2 form oligomeric structures. We analysed the glycoproteins further and showed that, like peripheral membrane proteins, the G2 and NSM proteins are almost completely extracted into the aqueous phase of detergent Triton X114-treated cellular extracts, whereas glycoprotein G1 is distributed in almost equal proportions between the aqueous and the detergent fractions. This indicates that G1 is a membrane-associated protein, but its presence in the aqueous phase suggests that it is less hydrophobic than a typical membrane protein. We have also characterized the intracellular transport of the envelope glycoproteins from the endoplasmic reticulum to the Golgi complex. Pulse-chase labelling followed by immunoprecipitation and treatment with endoglycosidase H (endo H) showed that both G1 and G2 are transported from the endoplasmic reticulum to the Golgi complex. Conversion to the endo H-resistant form is a rather slow process which takes more than 2 h. The mature G1 and G2 proteins present in the virion particle contain almost completely endo-H-resistant glycans.

Germiston virus transcriptase requires active 40S ribosomal subunits and utilizes capped cellular RNAs.

The transcriptase associated with Germiston virus was assayed in an in vitro reaction in which transcription was coupled to translation by adding reticulocyte lysate under the appropriate salt conditions. When analyzed in polyacrylamide gels, the major transcripts migrated like authentic S mRNAs and possessed 12- to 18-base-long nontemplated 5' extensions similar to the 5' end of viral mRNAs. These transcripts were functional for the synthesis of at least proteins N and NSS. When translation was inhibited by adding protein synthesis inhibitors such as puromycin, cycloheximide, and anisomycin, a drastic inhibitory effect was observed on the synthesis of the complete S mRNA transcripts. However, initiation and part of the elongation process were still active, since short and incomplete RNA molecules with RNA primers at their 5' ends were synthesized. On the other hand, we found that edeine, another inhibitor of protein synthesis, stimulated not only synthesis of S mRNAs but also that of the full-length S cRNAs. Taking into account the mode of action of this antibiotic, we discuss the results, which emphasize the crucial role of active ribosomes during bunyavirus transcription and confirm the observations reported on La Crosse virions. Moreover, we showed that the RNA transcripts synthesized in a transcription-translation reaction were capped and that most of them have acquired the 5' terminal sequences of the alpha- or beta-globin mRNA.

Characterization of the 5' and 3' ends of viral messenger RNAs isolated from BHK21 cells infected with Germiston virus (Bunyavirus).

The 3' ends of the S and M messenger RNAs isolated from BHK21 cells infected with Germiston virus were analyzed by mapping with RNase T2 or nuclease S1. The transcription termination signal was found to be located approximately 115 and 80 nucleotides upstream from the 3' end of the S and M genomic RNA templates, respectively. Both mRNAs were found to possess several adenosine residues at their 3' ends, but were not polyadenylated. They have acquired at their 5' end a heterologous 12- to 18-nucleotide-long sequence, which is not coded for by the virus. Sequencing of the 5' terminal region from single molecules cloned into pBR327 revealed that these primers are rich in C and G residues and possess a U or a C adjacent to the viral sequence.

Nucleotide sequence of the M segment of Germiston virus: comparison of the M gene product of several bunyaviruses.

The complete nucleotide sequence of the M RNA segment of Germiston bunyavirus was determined from plasmids containing overlapping M cDNA inserts. The M segment is 4534 nucleotides long and contains a 50-base-long inverted terminal repeat which can form a stable hydrogen-bonded secondary structure with a delta G of -45.8 kcal/mol. The RNA molecule complementary to viral RNA contains a single large open reading frame that encodes a 1437 amino acid-long protein with hydrophobic amino and carboxy terminal regions, which could represent signal and anchor sequences, respectively. It is presumed that this gene product is the polyprotein precursor to glycoproteins G1 and G2 and to the nonstructural polypeptide NSM. The nucleotide and amino acid sequences of the M RNA of Bunyamwera virus (prototype of the serogroup) and snowshow hare and La Crosse viruses (California serogroup) (Lees et al., 1986; Eshita and Bishop, 1984; Grady et al., 1987) were compared to those of Germiston virus. An overall amino acid sequence homology of 44% was found between Germiston and snowshoe hare viruses and of 61% between Germiston and Bunyamwera viruses. Most of the cysteines, three out of seven of the potential glycosylation sites, as well as the N and C terminal hydrophobic domains, are conserved between the four viruses.

The S segment of the Germiston virus RNA genome can code for three proteins.

The complete sequence of the S segment of Germiston bunyavirus has been determined from plasmids containing S cDNA inserts. The S segment is 980 nucleotides long with the first 15 bases at the 3' end complementary to the first 15 bases at the 5' end. Three overlapping open reading frames (ORF) were identified in the viral complementary RNA strand. The first ORF codes for a polypeptide of 233 amino acids (Mr 26,600) which is the nucleoprotein N. The second ORF codes for a polypeptide of 109 amino acids (Mr 11,800) which corresponds to the NSS protein, also called p12. Following this ORF, in the same frame, a third ORF which could encode a polypeptide of 75 amino acids was identified. Such a polypeptide has not yet been detected in infected cells. The N and NSS proteins of Germiston virus were compared with the corresponding proteins of La Crosse, snowshoe hare, and Aino viruses, and show a high extent of homology.

A transcript from the S segment of the Germiston bunyavirus is uncapped and codes for the nucleoprotein and a nonstructural protein.

Analysis of the RNAs present in BHK-21 cells infected with Germiston virus showed that the transcripts from the L and M segments have a size similar to that of their template, whereas two types of complementary RNA are transcribed from the S segment. One, S1, is a full-length "plus" RNA strand (antigenome), and the other, S2, is an incomplete plus RNA strand which serves as mRNA for at least the synthesis of the N protein and a virus-specific nonstructural polypeptide, p12. The 5' ends of these two transcripts appeared to be identical and complementary to the 3' ends of the viral RNA. Our results suggest that transcription of the S fragment either stops 100 to 150 nucleotides from the 5' end of the template, generating an S2 molecule, or continues, generating an S1 molecule. Neither the S1 antigenome nor the S2 mRNA molecules were polyadenylated at their 3' ends or capped at their 5' ends.

[RNA polymerase activity associated with a bunya virus (Lumbo)]. / Activité RNA polymérasique à un bunyavirus (Lumbo)

RNA dependent RNA polymerase has been demonstrated in purified Lumbo virus (Bunyavirus) which contains a single stranded segmented RNA. Divalent cations (Mn++ and Mg++) are required for optimal in vitro activity. Reaction products can be specifically annealed with the viral genome.