Viral diseases of the giant fresh water prawn Macrobrachium rosenbergii: a review.

The giant freshwater prawn Macrobrachium rosenbergii is cultivated essentially in Southern and South-eastern Asian countries such as continental China, India, Thailand and Taiwan. To date, only two viral agents have been reported from this prawn. The first (HPV-type virus) was observed by chance 25 years ago in hypertrophied nuclei of hepatopancreatic epithelial cells and is closely related to members of the Parvoviridae family. The second, a nodavirus named MrNV, is always associated with a non-autonomous satellite-like virus (XSV), and is the origin of so-called white tail disease (WTD) responsible for mass mortalities and important economic losses in hatcheries and farms for over a decade. After isolation and purification of these two particles, they were physico-chemically characterized and their genome sequenced. The MrNV genome is formed with two single linear ss-RNA molecules, 3202 and 1250 nucleotides long, respectively. Each RNA segment contains only one ORF, ORF1 coding for the RNA-dependant RNA polymerase located on the long segment and ORF2 coding for the structural protein CP-43 located on the small one. The XSV genome (linear ss-RNA), 796 nucleotides long, contains a single ORF coding for the XSV coat protein CP-17. The XSV does not contain any RdRp gene and consequently needs the MrNV polymerase to replicate.

Adeno-associated virus type 5 exploits two different entry pathways in human embryo fibroblasts.

The helper-dependent adeno-associated viruses (AAVs) have attracted great interest as vectors for gene therapy. Uptake and intracellular trafficking pathways of AAV are of importance, since they are often rate-limiting steps in infection. Here, we have investigated the entry of AAV type 5 (AAV5) in primary human embryo fibroblasts. At low binding temperatures, numerous virions are concentrated between cells, at contact points between cells and cellular protrusions, and at filopodia. When the temperature is raised to 37 degrees C, uptake of AAV5 takes place but up to 80 % of the bound virions dissociate from the cells. Uptake is achieved by cellular structures that are part of at least two different entry pathways. In addition to the common clathrin-dependent route, caveolar endocytosis and caveosome-like organelles are involved in a second pathway not yet described for parvoviruses. Both pathways can be used in parallel to enter an individual cell.

Virus respiratorios emergentes / Emerging respiratory viruses

Con el desarrollo de técnicas de biología molecular se han descrito nuevos agentes respiratorios, algunos de ellos emergentes y otros en circulación desde hace años. La mayoría afecta a lactantes y producen cuadros respiratorios que requieren hospitalización. El espectro clínico aún no se encuentra completamente definido. Estos nuevos agentes deben incluirse en el diagnóstico de las infecciones respiratorias en Pediatría.

A parvo-like virus persistently infecting a C6/36 clone of Aedes albopictus mosquito cell line and pathogenic for Aedes aegypti larvae.

We have isolated and partially characterized from an apparently healthy C6/36 subclone of Aedes albopictus cell line a small icosahedral non-enveloped DNA virus, designated AaPV. This virus proved to be highly pathogenic for Aedes aegypti neonate larvae. Viral infection persisted for over 4 years in the cell culture without any cytopathic effect. Attempts to infect suckling mice, Drosophila melanogaster adults and Spodoptera littoralis larvae with AaPV were unsuccessful. Similarly, the AaPV failed to replicate in vertebrate and Drosophila cell lines. Virions, about 22 nm in diameter, had a buoyant density of 1.43 g/cm3 and contained three capsid polypeptides with molecular weights of 53, 41 and 40 kDa. A preliminary study of the viral genome indicated the presence of single-stranded DNA. By its biophysical and biochemical properties, this virus appears to be related to the genus Densovirus within the family Parvoviridae, but lacks serological relationships with the other members of this genus.

Modification of some biological properties of HeLa cells containing adeno-associated virus DNA integrated into chromosome 17.

Parvoviruses are known to interfere with cellular transformation and carcinogenesis. Since infecting adeno-associated virus (AAV) frequently integrates its DNA into the cellular genome, we analyzed whether this integration influences the transformed phenotype of the human tumor cell line HeLa. Analysis of three independent HeLa cell clones with integrated AAV DNA (HA-3x, HA-16, and HA-28) revealed the following phenotypic changes of these cells: (i) reduced growth rate, (ii) increased serum requirement, (iii) reduced capacity for colony formation in soft agar, (iv) reduced cloning efficiency on plastic, (v) elevated sensitivity to genotoxic agents (N-methyl-N'-nitro-N-nitrosoguanidine, 7,12-dimethylbenz[a]anthracene, human tumor necrosis factor alpha, UV irradiation [256 nm], and heat [42 degrees C]), and (vi) reduced sensitivity to the cytolytic effect of parvovirus H-1. Reduced growth rate and enhanced sensitivity to gamma irradiation were also observed in vivo when tumors from AAV DNA-containing HeLa cells were transplanted into nude mice. This alteration of the biological properties of HeLa cells was independent of the number of AAV genomes integrated, the physical structure of integrated AAV DNA, and the transcription of AAV genes. Integration of AAV DNA was found to occur preferentially on the long arm of chromosome 17 in the three HeLa cell clones analyzed. These findings demonstrate that genomic integration of AAV DNA can alter the biological properties of human tumor cells.

Susceptibility of human cells to killing by the parvoviruses H-1 and minute virus of mice correlates with viral transcription.

Human fibroblasts and epithelial cells differing in their susceptibility to killing by the autonomous parvoviruses H-1 and minute virus of mice were compared for their capacity to express viral mRNAs and proteins. The transition from a parvovirus-resistant to a parvovirus-sensitive phenotype correlated with a proportional increase in the production of the three major viral transcripts and of structural and nonstructural proteins. In contrast, cell sensitization to parvovirus could not be correlated with detectable changes in virus uptake, intracellular localization of gene products, stability of viral mRNAs, or phosphorylation of viral nonstructural polypeptides. Moreover, the H-1 virus-sensitive keratinocyte line studied did not sustain a greater level of viral DNA amplification than its resistant derivative. Therefore, the differential susceptibility of the human cells tested to parvovirus infection appears to be mainly controlled at the level of transcription of the viral genome. Parvoviral gene expression could not be elevated by increasing the input multiplicity of infection in either of the cell systems analyzed. Together, these data suggest that a cellular factor(s) regulating parvoviral transcription may be modulated by oncogenic transformation or by differentiation, as both features have been shown to affect cell susceptibility to parvoviruses.

NS-1 and NS-2 proteins may act synergistically in the cytopathogenicity of parvovirus MVMp.

The interaction of parvovirus minute virus of mice (prototype strain, MVMp) with simian virus 40 (SV40)-transformed human cells (NB-E) was investigated by means of transfection with MVMp molecular clones derived from the infectious recombinant plasmid (pMM984). pMM984 inhibits stable transformation of NB-E cells to geneticin resistance (G418R) upon cotransfection with the selectable pSV2neo plasmid. We show here that this inhibition is not merely caused by a repression of marker gene expression from the SV40 early region promoter in pSV2neo and rather is likely to reflect the cytotoxic action of the parvovirus. Starting from plasmid pMM984, defined mutations were introduced into the genome of MVMp and more particularly into sequences coding for the NS-1 and/or NS-2 nonstructural proteins. In this way we could show that the NS-1 protein is necessary for the inhibition of transformation to G418R and that the NS-2 protein acts synergistically to enhance this effect. Moreover, results obtained with different viral mutants indicate that the inhibitory action of NS-1 on stable transformation can be dissociated from the ability of this protein both to transactivate the parvoviral p39 promoter of the capsid protein-encoding region and to drive parvoviral DNA amplification. Altogether these data point to a probable direct toxicity of MVMp nonstructural proteins for permissive host cells.

Susceptibility of human erythropoietic cells to B19 parvovirus in vitro increases with differentiation.

B19 human parvovirus is the etiologic agent of transient aplastic crisis. To better understand B19 virus-induced hematopoietic suppression, we studied the host cell range of the virus using in vitro bone marrow cultures. First, B19 virus replication was examined in the presence of various purified cytokines using DNA dot blot analysis. Replication was detected only in erythropoietin-containing cultures. The other cytokines (granulocyte/macrophage colony-stimulating factor [GM-CSF], G-CSF, M-CSF, interleukin-1 [IL-1], IL-2, IL-3, and IL-6) did not support virus replication, indicating the restriction of B19 virus replication to the erythroid cell lineage. Second, hematopoietic progenitor cells were serially assayed in B19-infected and uninfected bone marrow cultures. At initiation, B19 virus infection caused marked and moderate reduction in colony-forming unit erythroid (CFU-E) and burst-forming unit erythroid (BFU-E) numbers, respectively, without affecting CFU-Mix and CFU-GM numbers. Interestingly, the recovery of the erythroid progenitor numbers was observed at a late stage of cultures despite the sustained reduction in erythroblasts. The cells in the bursts derived from such reappearing BFU-E did not contain the virus genome. Although infectious virus was detected in the culture supernatants, the cultured CFU-E harvested at day 5 was relatively resistant to B19 virus infection compared with the CFU-E in fresh bone marrow. These findings suggest that pluripotent stem cells escaped B19 virus infection and restored the erythroid progenitor cells later in infected cultures. We conclude that the target cells of B19 virus are in the erythroid lineage from BFU-E to erythroblasts, with susceptibility to the virus increasing along with differentiation. Furthermore, the suppression of erythropoiesis and the subsequent recovery of the erythroid progenitor numbers in B19-infected liquid cultures may be analogous in part to the clinical features of B19 virus-induced transient aplastic crisis.

Replication of two porcine parvovirus isolates at non-permissive temperatures.

Previous studies have shown that replication in vitro of the porcine parvovirus (PPV) isolate, KBSH, was restricted at 39 degrees C but not at 37 degrees C. In contrast, replication of the Kresse isolate was restricted at 37 degrees C but not at 39 degrees C. In this study, Kresse and KBSH isolates were passaged up to ten times in swine testicle (ST) cells at non-permissive temperatures, and at subsequent passage viral protein synthesis, viral DNA synthesis, and progeny virus were evaluated. KBSH became adapted for replication at 39 degrees C upon serial passages, displaying an appreciable increase in viral progeny, viral polypeptides, and viral DNA concentration. This finding was also observed with Kresse virus isolate continuously passaged at 37 degrees C. Neither isolate became adapted for replication at 32 degrees C. In an attempt to examine the effect of in vitro passage at non-permissive temperatures on pathogenicity in swine, KBSH passaged 10 times either at 37 degrees C or 39 degrees C was inoculated into swine fetuses. Two of four fetuses inoculated with 39 degrees C-passaged KBSH were dead and hemorrhagic or mummified. All four fetuses inoculated with 39 degrees C-KBSH contained viral antigen and viral DNA. In contrast, fetuses inoculated with 37 degrees C-passaged KBSH, or with cell culture fluid were normal in appearance. Viral antigen and viral DNA were not demonstrated in fetuses inoculated with 37 degrees C-KBSH or cell culture fluids. These findings suggest the possibility that the ability to replicate at 39 degrees C is associated with virulence in swine fetuses.

Sensitization of transformed rat cells to parvovirus MVMp is restricted to specific oncogenes.

The rat cell line FR3T3 was transformed with the retroviral oncogenes v-myc or v-src, with the DNA tumor viruses SV40 or bovine papilloma virus strain 1 (BPV-1) or with the 69% transforming region of BPV-1. The transformants were compared with the uncloned parental line for their susceptibility to the lytic effect and to the replication of MVMp, an autonomous parvovirus. Expression of v-myc and v-src proteins and of SV40 large T antigen correlated with a greater cell susceptibility to MVMp-induced killing. Thus, the expression of both cytoplasmic and nuclear oncogene products may sensitize rat fibroblasts to MVMp. In contrast, cell lines transformed by BPV-1, including highly tumorigenic and tumor-derived clones, were on the average as resistant as the parental cell line to MVMp infection. A similar resistance to MVMp-induced killing was displayed by BPV-1-transformed NIH3T3 cells. However, supertransformation of one of the BPV-1-transformants by the human EJ-Harvey ras-1 oncogene, known to sensitize FR3T3 and NIH3T3 cells, correlated with an increase in susceptibility to MVMp. Therefore, the failure of BPV-1 transformation to sensitize murine cells to parvoviral attack may be ascribed to the tumor virus rather than to the cells undergoing transformation. Hence, cell sensitization to MVMp appears to be oncogene-specific and cannot be taken as an absolute correlative with neoplastic transformation.

[Viruses and malignant hemopathies]. / Virus et hémopathies malignes.

Review on general problems raised by the role of some viruses: parvoviruses, herpes viruses, retroviruses, in the physiopathology of human malignant blood diseases.

Sensitization of human keratinocytes to killing by parvovirus H-1 takes place during their malignant transformation but does not require them to be tumorigenic.

To investigate the antineoplastic activity of parvoviruses, proliferating normal human epidermal cells and a series of established keratinocyte cell lines derived from squamous cell carcinomas or transformed in vitro, were compared for the outcome of H-1 virus infection. All established keratinocyte cell lines were more sensitive to killing by H-1 virus than normal epidermal cells, although to varying extents. Using a step-wise procedure for malignant transformation in vitro, we found that sensitization of transformed epidermal cells to H-1 virus can be dissociated from the acquisition of a tumorigenic phenotype. Thus, spontaneously- or SV40-immortalized human keratinocytes were moderately and highly sensitive to H-1 virus, respectively, and could be made tumorigenic by Harvey-ras oncogene transfection without a major change in their susceptibility to the virus. The capacity of human keratinocytes for replicating and expressing H-1 virus DNA appears to be a revealer of cellular alterations that take place in at least some pathways to malignant transformation but that may be insufficient to confer a tumorigenic potential.

The gene encoding the nonstructural protein of B19 (human) parvovirus may be lethal in transfected cells.

The B19 parvovirus is a cause of bone marrow failure in humans. B19 is toxic to erythroid progenitor cells in vitro. Viral products possibly responsible for toxicity were explored by transfection of cloned B19 genome into HeLa cells. The nonstructural (NS) protein was detected in cells 30 h after transfection. Plasmids containing the B19 genome were transfected with selectable marker genes in stable transformation assays. Plasmids that contained the left side of the B19 genome, which encodes the NS protein of the virus, inhibited antibiotic-resistant colony formation. Transformation occurred when NS protein expression was blocked by mutation. Suppression of transformation by NS protein was not tissue specific, suggesting a role for NS protein in toxicity for nonpermissive cells without parvovirus replication or virion accumulation.

Novel transcription map for the B19 (human) pathogenic parvovirus.

The B19 parvovirus, a small single-stranded DNA virus of 5.4 kilobases, is pathogenic in humans. B19 has remarkable specificity for erythroid progenitor cells and has been propagated in vitro only with human erythroid bone marrow. Replication of viral DNA and the viral protein products of B19 appear similar to those of other animal parvoviruses. However, B19 transcription had unusual features in comparison with that in other animal parvoviruses. At least nine overlapping poly(A)+ transcripts were identified in infected cells; all but one contained large introns. B19 differed from other parvoviruses in the initiation of all transcripts at a strong left side promoter (p6) and the absence of a functional internal promoter; the presence of short 5' leader sequences of about 60 bases and very large introns for RNAs encoded by the right side of the genome; two separate transcription termination sites, in contrast to cotermination at the far right side of the genome for other parvoviruses; the probable utilization by three transcripts of a variant polyadenylation signal (ATTAAA or AATAAC) in the middle of the genome; and the abundance of two unique transcripts from the middle of the genome which did not code for capsid proteins. The unusual transcription map of B19 suggests that regulation of the relative abundance of transcripts occurs by splicing and termination-polyadenylation events rather than by promoter strength. In combination with the published nucleotide sequence, the novel transcription map separated the pathogenic B19 virus at a molecular level from other animal parvoviruses and human adeno-associated virus.

KBSH parvovirus: comparison with porcine parvovirus.

We compared the molecular, antigenic, and pathogenic properties of KBSH parvovirus to those of porcine parvovirus (PPV) isolate NADL-8. KBSH, propagated in swine testes cells in culture, possessed two major capsid polypeptides of 83 and 64 kilodaltons that were similar in size to those of PPV. KBSH-infected cells also contained an 86-kilodalton nonstructural polypeptide that was identical in size to the PPV nonstructural polypeptide (NS-1). The KBSH polypeptides were structurally similar but not identical to the corresponding PPV polypeptides, as revealed by partial proteolysis mapping. Viral replicative-form DNA from KBSH-infected cells was similar in size to PPV replicative-form DNA and exhibited similar but not identical restriction endonuclease cleavage patterns to that of PPV replicative-form DNA. Antigenically, the two viruses were also very closely related. By using heterologous and homologous antisera, the two viruses were indistinguishable in hemagglutination inhibition and immunoprecipitation assays. However, pathogenically these viruses were dramatically different. NADL-8 caused fetal death when injected into swine fetuses in utero and viremia and high persisting antibody titers when administered orally to weaning-age swine. KBSH-inoculated fetuses were normal in appearance, and pigs orally exposed to KBSH failed to establish viremia and demonstrated only transient antibody titers. Thus, KBSH appears to be a PPV that is very closely related to a highly pathogenic PPV isolate, yet is itself nonpathogenic in swine. This reduced pathogenic potential of KBSH may be attributable to its poor ability to replicate in swine.

[Transformation by SV 40 virus sensitizes fibroblasts of human skin to the lytic action of H-1 parvovirus]. / La transformation par le virus SV40 sensibilise les fibroblastes de peau humaine à l'action lytique du parvovirus H-1.

Human skin fibroblasts which are naturally resistant to Parvovirus H-1 can be lysed by this virus after SV40 transformation. This observation raises the possibility that oncosuppression by Parvovirus involves a direct oncolytic effect.

Speculations on the viral etiology of acquired immune deficiency syndrome and Kaposi's sarcoma.

The acquired immune deficiency syndrome (AIDS) appeared in the United States in late 1978 and has spread at an epidemic rate through the four major coastal cities of this country. The disease appears to show the same epidemiologic distribution as hepatitis B virus infection, and for this reason, most investigators feel that this new disease is caused by a blood-borne sexually transmitted virus. A number of viral agents have been suggested as the cause of AIDS, but to date, no virus has been consistently isolated. The most likely candidate is a retrovirus that has recently been introduced into the human population and has found its way into two extremely high-risk groups, namely, promiscuous male homosexuals and intravenous drug abusers. The relationship between Kaposi's sarcoma and cytomegalovirus is still unclear, but evidence is mounting that cytomegalovirus may be the agent that initiates this multifocal malignancy. Multiple factors must be involved in this process. It is known that some immunosuppressed individuals develop Kaposi's sarcoma, which completely resolves when the immunosuppression is reversed; however, in individuals with classical Kaposi's sarcoma, the profound degree of helper T-cell depression that characterizes the acquired immune deficiency syndrome is not seen.

Mutagenesis of intact parvovirus H-1 is expressed co-ordinately with enhanced reactivation of ultraviolet irradiated virus in human and rat cells treated with 2-nitronaphthofurans.

The exposure of human or rat cells to non-toxic concentrations of two 2-nitronaphthofuran derivatives activated co-ordinately the transient expression of mutator and repair activities. These activities gave rise to both an increase in the mutagenesis (enhanced mutagenesis, EM) and survival (enhanced reactivation, ER) of unirradiated and u.v.-irradiated parvovirus H-1 used as respective probes. The kinetics of expression was the same for mutator and repair activities and for the two chemicals studied. The dose-responses of these activities were also parallel for a given chemical, but one of the furan derivatives exerted its inducing effect at concentrations 20-25 times lower than the other derivative. Both EM and ER were depressed by cycloheximide, and inhibitor of de novo protein synthesis. This is the first report which shows that chemicals can enhance the mutagenesis of undamaged DNA by activating the expression of mutator functions in mammalian, including human, cells. The ability of the two 2-nitronaphthofuran derivatives to trigger EM and ER was found to correlate with their reported mutagenicity in a conventional bacterial test system.