The nucleoprotein and the viral RNA of infectious salmon anemia virus (ISAV) are localized in the nucleolus of infected cells

The infectious salmon anemia virus (ISAV), which belongs to the new genus Isavirus of the Orthomyxoviridae family, is an important pathogen of the salmon farming industry. Indirect immunofluorescence assays carried out with monoclonal antibodies specific for the nucleoprotein (NP) reveal differential staining of sub-cellular compartments in infected cells. Particularly interesting was the staining of the nucleolus, which showed co-localization with nucleolin in CHSE-214, EPC and SHK-1 cells infected with ISAV. These results were confirmed by co-immunoprecipitation studies showing an interaction between NP and nucleolin. In addition, in situ hybridization carried out with probes specific for each of the 8 RNA segments of ISAV showed that the genomic as well as the anti-genomic strands were also localized in the nucleolus. These results suggest a role of the nucleolus in the replication and/or in the packaging of the ISAV genome.     

Infectious salmon anemia virus (ISAV) replication is transiently inhibited by Atlantic salmon type I interferon in cell culture

Infectious salmon anemia virus (ISAV) is a piscine orthomyxovirus, which causes multisystemic disease in farmed Atlantic salmon that may result in large losses. Previous work has suggested that ISAV is able to resist the antiviral state induced in cells by type I interferon (IFN). These studies were, however, mainly based on cytopathic effect (CPE) reduction assays. Here we have investigated the antiviral activity of Atlantic salmon IFNa1, IFNb and IFNc against ISAV using quantitative PCR (qPCR) of segment 6, Western blot analysis of ISAV proteins and viral yield reduction assays, in addition to CPE reduction assays. Antiviral effects of IFNs were tested against the high virulent strain ISAV4 and the low virulent strain ISAV7 both at the optimum growth temperature 15 °C and at 20 °C. As expected, IFNa1 showed little protection against CPE development in cells after infection with both strains at 15 °C. However, the qPCR and Western blot analysis clearly showed strong inhibition of replication of the virus strains by IFNa1 between 24 and 72 h after infection. The inhibitory effect declined four to five days post-infection, which explains the low protection against CPE development 7–10 days later. At 20 °C, IFNa1 showed strong protection against CPE development, probably due to slower virus growth. IFNc showed similar antiviral activity as IFNa1 against ISAV4 while IFNb showed lower activity. There were observed differences between ISAV4 and ISAV7 both with respect inhibition by IFNa1 and ability to induce the two IFN-inducible antiviral effector proteins, Mx and ISG15, which may be related to differences in virulence properties and/or adaption to growth in cell culture.     

Molecular and functional characterization of two infectious salmon anaemia virus (ISAV) proteins with type I interferon antagonizing activity

In this study we characterize two proteins encoded by the two smallest genomic segments of the piscine orthomyxovirus infectious salmon anaemia virus (ISAV). Both proteins, encoded by the un-spliced ORF from genomic segment 7 (s7ORF1) and the larger ORF from segment 8 (s8ORF2), are involved in modulation of the type I interferon (IFN) response. The data suggests that the s7ORF1 protein is collinearly encoded, non-structural, contains no nuclear localisation signals, localises mainly to the cytoplasmic perinuclear area and does not bind single- or double-stranded RNA. On the other hand, genomic segment 8 uses a bicistronic coding strategy and the encoded s8ORF2 protein is a structural component of the viral particle. This protein contains two nuclear localisation signals, has a predominantly nuclear localisation, binds both double-stranded RNA and poly-A tailed single-stranded RNA, but not double-stranded DNA. In poly I:C stimulated salmon cells both ISAV proteins independently down-regulate the type I IFN promoter activity. Thus, ISAV counteracts the type I IFN response by the action of at least two of its gene products, rather than just one, as appears to be the case for other known members of the Orthomyxoviridae.     

Characterization of the Lassa virus matrix protein Z: electron microscopic study of virus-like particles and interaction with the nucleoprotein (NP)

Lassa virus is the causative agent of a hemorrhagic fever endemic in west Africa. The RNA genome of Lassa virus encodes the glycoprotein precursor GP-C, a nucleoprotein (NP), the viral polymerase L and a small protein Z (11 kDa). Here, we analyze the role of Z protein for virus maturation. We have recently shown that expression of Z protein in the absence of other viral proteins is sufficient for the release of enveloped Z-containing particles. In this study, we examined particles secreted into the supernatant of a stably Z protein-expressing CHO cell line by electron microscopy. The observed Z-induced virus-like particles did not significantly differ in their morphology and size from Lassa virus particles. Mutation of two proline-rich domains within Z which are known to drastically reduce the release of virus-like particles, had no effect on the cellular localization of the protein nor on its membrane-association. Furthermore, we present evidence that Z interacts with the NP. We assume that Z recruits NP to cellular membranes where virus assembly takes place. We conclude from our data that Lassa virus Z protein plays an essential role in Lassa virus maturation.     

Nucleotide sequence analysis of two nuclear inclusion body and coat protein genes of a sweet potato feathery mottle virus severe strain (SPFMV-S) genomic RNA

Summary Recombinant DNA molecules containing cDNA of a sweet potato feathery mottle virus severe strain (SPFMV-S) RNA genome were constructed and the partial nucleotide sequences were determined for three DNA inserts, which cover 4.2 kb from the 3-terminus excluding the poly (A) tail. This region of the genome consists of an open reading frame of 1340 amino acids (a.a.) and a 3-non-translated region of 224 nucleotides. The protein products expected were 6K₂ (53 a.a.) NIa, (435 a.a.), NIb (521 a.a) and CP (315 a.a.). Among NIa, NIb and coat proteins, the NIb protein was found to be the most conserved (59–68%) when compared to the corresponding proteins of other distinct potyviruses.     

The non-structural 3 (NS3) protein of dengue virus type 2 interacts with human nuclear receptor binding protein and is associated with alterations in membrane structure

Flaviviral infections produce a distinct array of virus-induced intracellular membrane alterations that are associated with the flaviviral replication machinery. Currently, it is still unknown which flaviviral protein(s) is/are responsible for this induction. Using yeast two-hybrid and co-immunoprecipitation analyses, we demonstrated that the NS3 protein of dengue virus type 2 interacted specifically with nuclear receptor binding protein (NRBP), a host cellular protein that influences trafficking between the endoplasmic reticulum (ER) and Golgi, and that interacts with Rac3, a member of the Rho-GTPase family. Co-expression of NS3 and NRBP in baby hamster kidney cells exhibited significant subcellular co-localization, and revealed the redistribution of NRBP from the cytoplasm to the perinuclear region. Furthermore, a set of membrane structures affiliated with the rough ER at the perinuclear region was induced in cells transfected with NS3. These structures are reminiscent of the virus-induced convoluted membranes previously observed in flavivirus-infected cells. This interaction between dengue viral and host cell proteins as well as the formation of the NS3-induced membrane structures suggest that NS3 may subvert the role of NRBP in ER-Golgi trafficking.     

Conservation of the influenza virus membrane protein (M1) amino acid sequence and an open reading frame of RNA segment 7 encoding a second protein (M2) in H1N1 and H3N2 strains

The complete sequence of a full-length cloned DNA copy of the influenza virus A/Udorn/72 (H3N2) RNA segment 7 has been determined. A second open reading frame has been found which overlaps the membrane protein (M₁) sequence by 68 nucleotides. This second reading frame could code, in the +1 reading frame, for a protein (M₂) with a maximum of 97 amino acids depending on whether there is splicing of the mRNA and the methionine residue used for initiation of protein synthesis. Comparison of the present H3N2 sequence with the previously published sequence (18., 19.) of RNA segment 7 of A/PR/8/34 (H1N1), a strain isolated 38 years earlier, has shown that the amino acid sequence for the M₁ protein has been greatly conserved. In a total of 252 amino acids, only seven amino acid changes have occurred, of which only one results in a change in charge. In the amino acid sequences coded by the second open reading frame, changes were more common between strains, 11 out of a total of 96 amino acids.     

Simultaneous trisomy 9q3 and monosomy 5p in two children with der(5),t(5;9)(p15.1;q34.13): report of an extended family.

We present a family segregating for t(5;9)(p15.1;q34.13). Two cases with der(5),t(5;9), resulting in a partial duplication 9q34.13----qter and partial deletion of 5p15.12----pter, were ascertained. The phenotypes were consistent with features of both the cri du chat and trisomy 9q3 syndromes.     

Identification of hepatitis C virus (HCV) subtype 1b strains that are highly,or only weakly,associated with hepatocellular carcinoma on the basis of the secondary structure of an amino-terminal portion of the HCV NS3 protein

The NS3 protein of hepatitis C virus subtype 1b (HCV-1b) isolates obtained from 89 patients with hepatocellular carcinoma (HCC) and 78 patients without HCC were analyzed. On the basis of the secondary structure of the amino-terminal 120 residues of NS3, HCV-1b isolates were classified into group A, group B, and an indeterminate group, each of which was further divided into a number of subgroups, such as A1-1, A1-2, A2-1, A2-2, B1-1, B1-2, B2-1, B2-2, C-1, C-2, and C-3. HCV-1b isolates of subgroup B1-1 were found in 53 (59.6%) of 89 patients with HCC and 19 (24.4%) of 78 patients without HCC, with the difference between the two patient groups being statistically significant (P < 0.00001). Although the number of isolates was small, subgroup B2-1 was also highly associated with HCC, with all five isolates in that subgroup being found in patients with HCC (P < 0.05). On the other hand, HCV-1b isolates of subgroup A1-1 were associated only weakly with HCC; they were found in 6 (6.7%) of 89 patients with HCC and in 25 (32.1%) of 78 patients without HCC, with the difference between the two patient groups being statistically significant (P < 0.0001). The other subgroups, such as A1-2, A2-1, B1-2, C-1, C-2, and C-3, were moderately associated with HCC; their distribution patterns among patients with HCC did not differ significantly from those among patients without HCC. Taken together, our results suggest that HCV-1b isolates of subgroups B1-1 and B2-1 are highly associated with HCC and that this secondary structure analysis may be useful for predicting the relative risk of developing HCC.