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At least 19 recordsLinked to original sources

PubMed1997

Completion of molecular characterization of Toscana phlebovirus genome: nucleotide sequence, coding strategy of M genomic segment and its amino acid sequence comparison to other phleboviruses.

The M RNA segment of Toscana (TOS) phlebovirus was cloned and the complete nucleotide sequence determined. The M RNA segment is 4215 nucleotides in length, and it contains a single major open reading frame (ORF) in the viral-complementary sequence, between nucleotides 18 and 4034, which can encode for a polyprotein of 1339 amino acids (Mr 149 kDa). The viral segment is expressed via a unique mRNA containing 10-14 non-templated nucleotides at the 5' end and it is truncated at the 3' end by about 140 nucleotides in a purine-rich region. In M predicted amino acid sequences, several hydrophobic regions have been identified. They could function as a signal sequence or a transmembrane region for the different proteins. Comparison of the deduced amino acid sequence of M precursor product revealed 38, 36, and 25% identity and 58, 56, and 47% similarity with those of Rift Valley fever (RVF), Punta Toro (PT) and Unkuniemi (UUK) viruses, respectively. Residues conserved among the proteins are mainly located at the COOH-portion of the precursor, while the major divergence is in the NSm coding regions. Based on sequence comparison and similarity of hydropathic pattern of TOS M segment with other phleboviruses the N-termini of TOS GN and GC glycoproteins were placed at residues 297 and 936 of the precursor.

Amino Acid Sequence↗

PubMed2005

Apoptosis of hepatocytes caused by Punta Toro virus (Bunyaviridae: Phlebovirus) and its implication for Phlebovirus pathogenesis.

Experimental infection of hamsters with Punta Toro virus (PTV) produces a disease with clinical and pathological similarities to the severe human hemorrhagic fever caused by Rift Valley fever virus (RVFV), thus providing an animal model for RVFV pathogenesis. In this model, hepatocytic apoptosis is the main pathological component of liver injuries that are responsible for severe hemorrhagic manifestations. To further elucidate whether viral replication in hepatocytes directly causes apoptosis, we studied the morphological and biochemical changes of apoptosis in HepG2 cells at different time points after PTV infection. Cellular viability began to decrease 12 hours after infection compared with controls. Caspases 3/7 were activated significantly at 48 and 72 hours after infection, and phosphatidylserine translocation and DNA fragmentation were also detected at 48 and 72 hours. Cell cycle analysis by flow cytometry showed that infected HepG2 cells were arrested at G(0)/G(1) phase. Furthermore, virus titer increased with apoptosis progression, suggesting that viral replication is necessary for the apoptotic process. These results indicate that PTV infection alone, without a secondary inflammatory cellular reaction, induces hepatocytic apoptosis and suggest that future therapeutics for RVFV hemorrhagic disease might target inhibition of cellular apoptotic pathways during the acute infection.

Animals↗

PubMed2026

Identification and full genome sequencing of previously unknown sandfly-borne phleboviruses using a newly established capture-based next-generation sequencing approach.

Sandfly-borne phleboviruses cause febrile illness and neuroinvasive disease in humans. While infections are reported in the Mediterranean region, the discovery of previously unknown phleboviruses in sandflies from Kenya suggests a wider geographic distribution. Detection and characterization of novel phleboviruses are often hindered by low-quality and low-viral-load samples. We developed a capture-based target enrichment next-generation sequencing approach that showed a 99%-100% fold enrichment of viral genomes from primary material and provides a robust tool for generating complete genomes of both known and previously unknown viruses. From a collection of 15,652 sandflies in Kenya, we recovered seven complete coding sequences of Embossos, Bogoria, and Kiborgoch viruses, and of two previously unknown phleboviruses, which were named Sosoik and Shable viruses. Sosoik virus shared 83% amino acid identity in its RdRp gene with that of Bogoria virus, while Shable virus shared ca. 88% amino acid identity with viruses of the Salehabad serocomplex. Additionally, a reassortant of Shable virus was detected that possessed an M segment from an undescribed Ponticelli-like virus. DNA barcoding of blood-fed sandflies revealed several potentially novel Sergentomyia species and evidence of host-feeding on humans, livestock, and reptiles, suggesting possibilities for zoonotic transmission. Overall, our findings increase the known genetic diversity of Old World sandfly-borne phlebovirus species from 18 to 25 (by 38.9%), including the detection of viruses from all pathogenic sandfly-borne phlebovirus serocomplexes in East Africa, opening new horizons in disease ecology research.IMPORTANCEKnowledge of the genetic diversity of circulating pathogens is crucial for providing appropriate diagnostics and disease management. This study established a novel capture-based target enrichment next-generation sequencing approach that enabled the near-complete viral genome recovery from primary samples, while native NGS yielded negative or poor-quality results. In addition to the five recently discovered sandfly-borne phleboviruses in Kenya, two previously unknown phleboviruses were detected in sandflies from the same region. The viruses were detected in several sandfly species, which showed diverse host-feeding behaviors, including mixed feeding on humans and chickens. The study significantly advances the understanding of sandfly-borne phleboviruses by uncovering their broader geographic distribution and genetic diversity, particularly in East Africa, highlighting the importance of expanding surveillance efforts beyond traditionally studied regions.

Phlebovirus↗

PubMedDate not supplied

Serological evidence in sheep suggesting phlebovirus circulation in a Rift Valley fever enzootic area in Burkina Faso.

Within the Phlebovirus serogroup, Rift Valley fever (RVF) virus is endemo-enzootic in the African sahelian zone. Recently an RVF epizootic in West Africa prompted a serosurvey in the major sheep and cattle raising areas. Because of the close antigenic relationship between the phleboviruses it appeared of interest to evaluate the prevalence of the other phleboviruses also. In 1987, 482 sheep serum samples were collected in 2 different ecological zones of Burkina Faso and tested for the presence of phlebovirus antibodies. A sensitive but non-specific immunofluorescent antibody test and a specific enzyme-linked immunosorbent assay (ELISA) were used, with the following African phlebovirus antigens: Rift Valley fever (RVF), Arumowot, Gabek Forest, Gordil, Saint Floris and Odrenisrou. A total of 15.8% of the sera sampled had anti-RVF antibody in the ELISA. RVF virus appeared to be more active in drier areas such as the sahelian region, known to be an enzootic area for the disease. Antibodies to other phleboviruses were found in 11.8% of the samples, independent of RVF virus activity. It is assumed that sheep can be infected by different phleboviruses.

Animals↗

PubMedDate not supplied

Infection and coding strategies of arenaviruses, phleboviruses, and nairoviruses.

The infection and coding strategies of three groups of negative-stranded RNA viruses (arena viruses, phleboviruses, and nairoviruses) that include the etiologic agents of hemorrhagic disease in humans have been studied. Arenaviruses have two viral RNA species. The smaller RNA species (S) codes for the viral nucleoprotein (N protein) and for the viral glycoprotein species (G1 and G2, which are derived from a precursor glycoprotein, GPC). The S RNA has an ambisense arrangement. The proteins are translated from subgenomic mRNA species (viz., N protein from a viral-complementary mRNA and glycoprotein from a viral-sense mRNA). The larger arenavirus RNA species (L) is presumed to code for the viral transcriptase/replicase. Phleboviruses and nairoviruses are members of the Bunyaviridae. They both have three species of viral RNA. Other than the sizes of the viral proteins and the viral RNA species, virtually nothing is known about the coding strategy of nairoviruses. Phleboviruses have an ambisense coding arrangement to their smallest (S) RNA species. This S RNA codes for the viral N protein (translated from a viral-complementary mRNA) and a nonstructural protein (translated from a viral-sense mRNA). The middle-size (M) RNA of phleboviruses codes for a precursor to the viral glycoproteins (translated from a viral-complementary mRNA). The largest viral RNA (L) is presumed to code for the viral transcriptase/replicase.

Animals↗

PubMed1990

Uukuniemi virus S RNA segment: ambisense coding strategy, packaging of complementary strands into virions, and homology to members of the genus Phlebovirus.

We determined the complete nucleotide sequence of the small (S) RNA segment of Uukuniemi virus, the prototype of the Uukuvirus genus within the Bunyaviridae family. The RNA, which is 1,720 nucleotides long, contains two nonoverlapping open reading frames. The 5' end of one strand (complementary to the viral strand) encodes the nonstructural protein NSs (273 residues; molecular weight, 32,019), whereas the 5' end of the viral-sense strand encodes the nucleocapsid protein N (254 residues; molecular weight, 28,508). Thus, the S RNA uses an ambisense coding strategy previously described for the S segment of two phleboviruses and the arenaviruses. The localization of the N protein within the S RNA sequence was confirmed by amino-terminal sequence analysis of all five possible cyanogen bromide fragments obtained from purified N protein. Northern (RNA) blot analyses with strand-specific probes showed that the N and NSs proteins are translated from subgenomic mRNAs about 800 and 850 nucleotides long, respectively. These mRNAs are apparently transcribed from full-length S RNAs of opposite polarities. The two mRNA species were also detected in virus-infected cells. Interestingly, highly purified virions contained full-length S RNA copies of both polarities at a ratio of about 10:1. In contrast, virions contained exclusively negative-strand copies of the M RNA segment. The possible significance of these results for viral infection is discussed. The amino acid sequence of the N protein showed 35 and 32% homology (identity) with the N protein of Punta Toro and sandfly fever Sicilian viruses, two members of the Phlebovirus genus. The NSs proteins were much less related (about 15% identity). In addition, the extreme 5' and 3' ends of the S RNA, which are complementary to each other, also showed a high degree of conservation with the two phleboviruses. These results indicate that the uukuviruses and phleboviruses are evolutionarily related and suggest that the two genera could be merged into a single genus within the Bunyaviridae family.

Amino Acid Sequence↗

PubMed1996

Maize stripe tenuivirus RNA2 transcripts in plant and insect hosts and analysis of pvc2, a protein similar to the Phlebovirus virion membrane glycoproteins.

The complete sequence of the maize stripe tenuivirus (MStV) RNA2 was determined (3337 nucleotides). RNA2 contains two large open reading frames (ORFs) arranged in an ambisense orientation and specific RNAs of ca. 700 and 2600 nucleotides corresponding to the ORFs were detected in MStV-infected plants and planthoppers. The deduced amino acid sequence of the 23,500 MW protein (pv2) encoded by viral RNA2 (vRNA2) was similar to proteins encoded by the rice stripe (RStV) and rice hoja blanca tenuiviruses vRNA2. Sequence analysis suggested that pv2 is membrane associated. The 93,900 MW protein (pvc2) encoded by viral complementary MStV RNA2 (vcRNA2) was similar to the 94,000 MW protein of RStV RNA2 and to the virion membrane glycoproteins for Phlebovirus members of the Bunyaviridae. The phlebovirus glycoprotein cleavage site was similar to a region in the MStV and RStV proteins suggesting that the tenuivirus pvc2 may be processed analogous to the phlebovirus glycoproteins.

Blotting, Northern↗

PubMed2003

Phylogenetic relationships among members of the genus Phlebovirus (Bunyaviridae) based on partial M segment sequence analyses.

Viruses in the Phlebovirus genus of the family Bunyaviridae cause clinical syndromes ranging from a short, self-limiting febrile illness to fatal haemorrhagic fever. The genus currently consists of 68 antigenically distinct virus serotypes, most of which have not been genetically characterized. RT-PCR with four 'cocktail' primers was performed to amplify a region of the M segment of the genome of 24 phleboviruses included in the sandfly fever Naples, sandfly fever Sicilian and Punta Toro serocomplexes. Partial M segment sequences were successfully obtained and phylogenetic analysis was performed. The three resultant genotypic lineages were consistent with serological data. The sequence divergences were 27.6 % (nucleotide) and 25.7 % (amino acid) within the Sicilian serocomplex, 33.7 % (nucleotide) and 34.4 % (amino acid) within the Naples serocomplex and 35.6 % (nucleotide) and 37.5 % (amino acid) within the Punta Toro serocomplex. Overall, the diversities among viruses of Sicilian, Naples and Punta Toro serocomplexes were 48.2 % and 57.6 % at the nucleotide and amino acid levels, respectively. This high genetic divergence may explain the difficulties in designing a consensus primer pair for the amplification of all the phleboviruses using RT-PCR. It also suggests that infection with one genotype may not completely immunize against infection with all other genotypes in a given serocomplex. These findings have implications for potential vaccine development and may help explain clinical reports of multiple episodes of sandfly fever in the same individual.

Amino Acid Sequence↗

PubMed1983

Characterization of eight new phlebotomus fever serogroup arboviruses (Bunyaviridae: Phlebovirus) from the Amazon region of Brazil.

Eight new members of the phlebotomus fever arbovirus serogroup (family Bunyaviridae; genus Phlebovirus) from the Amazon region of Brazil are described. One serotype was recovered from a febrile patient, three from small wild animals and four from sand flies. A small serum survey carried out with the human isolate, Alenquer virus, suggests that it rarely infects man. Complement-fixation and plaque reduction neutralization tests were done, comparing the eight new viruses with other members of the phlebotomus fever serogroup. A close antigenic relationship was demonstrated between one of the new agents (Belterra) and Rift Valley fever virus. This finding is of considerable interest and deserves further investigation. Addition of these eight new viruses to the genus Phlebovirus brings to 14 the number of serotypes known to occur in the Amazon region and to 36 the total number reported worldwide. More detailed clinical and epidemiological studies should be conducted in Amazonia in order to define the public health impact caused by phleboviruses.

Animals↗

PubMed1984

Studies on the biology of phleboviruses in sand flies (Diptera: Psychodidae). I. Experimental infection of the vector.

This paper describes a series of experiments which were done to determine the behavior of 14 different phleboviruses in laboratory-reared sand flies (Phlebotomus papatasi, P. perniciosus and Lutzomyia longipalpis) after oral and parenteral infection. Most of the viruses replicated in the sand flies after intrathoracic inoculation; however, the insects were quite refractory to oral infection. Six of 11 phleboviruses tested were transovarially transmitted in one or more sand fly species. The percentage of infected F1 offspring produced by parenterally infected female parents ranged from 1.5-60%, depending on the virus type used. These data support the hypothesis that some of the phleboviruses are maintained in sand flies by transovarial transmission.

Animals↗

PubMed1987

Viremia and immune response with sequential phlebovirus infections.

Four groups of hamsters were infected sequentially with various combinations of Arumowot, Chagres, and Gabek Forest viruses. Following each infection, the survival, level of viremia, and immune response of the animals were monitored. All of the agents produced viremia in the hamsters, regardless of the order of their administration. The antibody response, as measured by plaque reduction neutralization test, was monotypic even after two consecutive phlebovirus infections. Arumowot and Chagres viruses produced nonfatal infections in adult hamsters, which were characterized by viremia of several days duration and subsequent antibody formation. In contrast, Gabek Forest virus produced a fulminating and rapidly fatal disease in phlebovirus nonimmune animals. In hamsters previously infected with Chagres and/or Arumowot viruses, Gabek Forest infection was less severe, indicating some degree of cross-protection. The degree of cross-protection was in part related to the sequence of previous phlebovirus infections. No evidence of immune enhancement or other immunopathologic events were observed in the animals.

Animals↗

PubMed2003

Induction of severe disease in hamsters by two sandfly fever group viruses, Punta toro and Gabek Forest (Phlebovirus, Bunyaviridae), similar to that caused by Rift Valley fever virus.

Adult golden hamsters inoculated subcutaneously with either of two sandfly fever group viruses, Punta Toro and Gabek Forest (Phlebovirus, Bunyaviridae), developed a fulminating fatal illness characterized by hepatic and splenic necrosis and interstitial pneumonitis. Most animals died within three days after infection; this was accompanied by high levels of viremia. Necropsy and histopathologic examination of the infected animals revealed pathologic changes involving multiple organs that resembled those described in Rift Valley fever. These two hamster-phlebovirus systems may serve as alternative animal models for Rift Valley fever and should be useful in studying the pathogenesis of severe phlebovirus infection and for testing potential therapeutic agents.

Animals↗

PubMed2003

Detection and identification of Toscana and other phleboviruses by RT-nested-PCR assays with degenerated primers.

Phleboviruses are a large and widespread group of viruses that are transmitted by arthropods. Toscana virus is one of the principal agents that causes meningitis in humans during the summer in Italy and, possibly, in other Mediterranean countries. Rift Valley Fever virus can cause serious illness in both animals and humans, leading to high morbidity and mortality, and is considered to be a potential agent for epizootics and human epidemics. Since information on this group of viruses is still scant, reliable laboratory tools for diagnosis and epidemiological surveillance must be developed, in order to ascertain their real impact on Public Health. Sequence data obtained from Spanish isolates of Toscana virus and other phleboviruses confirmed that natural genome variability may hamper the diagnosis of these agents by molecular methods, so this must be borne in mind when developing reliable assays. In view of the above, a novel and useful protocol has been developed for the detection and specific identification of every member of the phlebovirus genus present in a sample, including Toscana virus, based on a generic RT-nested-PCR, followed by sequencing of the amplified fragment. A change in this method also allowed specific direct detection and identification of wild isolates of Toscana virus of different geographical origin, using newly designed primers. Testing clinical samples with these assays confirmed the role of Toscana virus as an agent that causes acute aseptic meningitis in the central region of Spain.

Base Sequence↗

PubMed1986

Comparative pathogenicity and antigenic cross-reactivity of Rift Valley fever and other African phleboviruses in sheep.

Homologous and heterologous haemagglutination-inhibition (HAI), complement-fixation (CF), immunodiffusion (ID) and mouse neutralization tests were performed with the Lunyo (LUN) and a Zimbabwean strain of Rift Valley fever (RVF) virus, the prototype and a South African strain of Arumowot (AMT) virus and prototype strains of Gordil (GOR), Saint-Floris (SAF) and Gabek Forest (GF) viruses, using immune mouse ascitic fluids prepared against these viruses. Reactions of identity occurred in all tests between LUN and the Zimbabwean strains of RVF and between the two strains of AMT virus. Otherwise, cross-reactions occurred between all the phleboviruses in HAI tests, while reactions in CF, ID and neutralization tests were monospecific for virus serotypes, except that weak cross-reaction occurred between GOR and SAF viruses in CF and ID tests. Four sheep infected subcutaneously with the Zimbabwean strain of RVF virus developed transient fever, viraemia, leucopaenia, relative thrombocytopaenia, haemoconcentration and raised serum enzyme levels, which indicated that the sheep had developed necrotic hepatitis. Disseminated focal necrotic hepatitis was confirmed in a sheep killed for examination on day 4 post-infection. The other three sheep recovered uneventfully after only mild depression and anorexia. Groups of three sheep infected with SAF, GOR, AMT and GF viruses had no demonstrable viraemia or other sign of infection or illness, except that the sheep infected with AMT developed mild fever lasting less than 24 h. Antibody responses were monitored at intervals over a period of 24 weeks in all sheep by homologous and heterologous HAI, CF and cell culture neutralization (CPENT) tests. Homologous antibody responses were marked in the RVF-infected sheep and their sera cross-reacted strongly in HAI tests with antigens of the other viruses. The sera of the RVF-infected sheep cross-reacted less markedly in CF and CPENT tests. Homologous antibody responses were poor in all the sheep infected with phleboviruses other than RVF, and the cross-reactivity of their sera for RVF antigen or virus was negligible. All sheep were challenged with RVF virus 48 weeks after their initial infection. The sheep which had originally been infected with RVF virus were immune and developed neither fever nor viraemia. All other sheep developed fever, viraemia and antibodies to RVF virus. It was concluded that the African phleboviruses, other than RVF, are unlikely to cause disease in livestock or to induce antibodies which could cause confusion in the diagnosis of RVF.

Animals↗

PubMed1990

Ambisense segment 4 of rice stripe virus: possible evolutionary relationship with phleboviruses and uukuviruses (Bunyaviridae).

The complete nucleotide sequence of segment 4 of the rice stripe virus (RSV) genome was determined from overlapping cDNA clones and by direct RNA sequencing. The segment has two long open reading frames (ORFs). One of the ORFs (534 bases) is in the 5' region of the viral sequence, and the other (858 bases) is in the viral complementary sequence of the viral 3' region. Such ambisense genome organizations have been seen previously in RNAs of phleboviruses, uukuviruses and arenaviruses. The ORF in the viral sequence encodes the major non-structural protein, but the product of the ORF in the viral complementary sequence (Mr 32407) has not yet been identified. The first 18 bases of each of the 5' and 3' ends of segment 4 are complementary in sequence. Among the 3'-terminal 16 bases, 14 were identical between RSV genome segments 3 and 4. These terminal sequences are very similar to those of RNAs of phleboviruses and uukuviruses. These and other characteristics indicate a possible evolutionary relationship between RSV and phleboviruses and/or uukuviruses.

Amino Acid Sequence↗

PubMed1983

Transovarial transmission of Rio Grande virus (Bunyaviridae: Phlebovirus) by the sand fly, Lutzomyia anthophora.

The growth of Rio Grande (RG) virus, the only phlebovirus known to occur in the United States, was studied in Lutzomyia anthophora, its suspected sand fly vector. RG viral titers in infected flies increased more than 10,000-fold within 7 days after intrathoracic inoculation. Experimentally infected female L. anthophora transmitted virus transovarially (vertically) to 54.8% of their F1 progeny. This is the first virologically confirmed demonstration of transovarial transmission of a phlebovirus by sand flies. It indicates one mechanism by which RG and possibly other phleboviruses may be maintained in nature.

Bunyaviridae↗

PubMed1992

Effect of macrophage source and activation on susceptibility in an age-dependent model of murine hepatitis caused by a phlebovirus, Punta Toro.

The Adames strain of a bunyavirus, Punta Toro virus (PTV), is an hepatotrophic virus that has been described to produce an age-dependent lethal hepatic necrosis in 3-4 week old C57BL/6 mice, but 8 week old mice survive with minimal necrosis. The course of PTV infection in vitro in macrophages derived from these mice served as a model to study the pathogenesis of phlebovirus infection. Peripheral blood monocytes, resident or elicited peritoneal macrophages, and Kupffer cell liver macrophages, as well as hepatocytes, were able to support replication of PTV in vitro to a variable extent. Kupffer cells were the only population of macrophages, however, that expressed an age-related ability to affect viral infection and replication in vitro, suggesting that liver macrophages may have a unique modulatory effect on the occurrence and severity of PTV-induced hepatitis in mice. Whereas PTV showed minimal replication in resident peritoneal macrophages, the virus could replicate effectively in peritoneal macrophages elicited by thioglycolate. Activation of peritoneal macrophages with endotoxin resulted in a significant inhibition of intrinsic PTV replication (p less than 0.001), and a modest extrinsic inhibitory effect on PTV replication in cocultured hepatocytes. Both effects persisted in the presence of anti-interferon. These results indicate that the source and state of activation of macrophage/monocyte populations can influence the course of infection in vitro by the phlebovirus, Punta Toro, and can modulate infection in cocultured target cells.

Aging↗

PubMed1992

Analysis of 3' and 5' ends of N and NSs messenger RNAs of Toscana Phlebovirus.

The 5' and 3' ends of N and NSs mRNAs, transcribed from the S segment of Toscana Phlebovirus, were analyzed by oligonucleotide primer extension and S1 nuclease mapping procedures. The results showed that both mRNAs acquired, at their 5' end, approximately 9-15 nucleotides not present in the viral template, suggesting an initiation transcription mechanism similar to the one described for influenza virus. Furthermore, the 3' ends of the two mRNAs were located in a sequence motif conserved in the S segment of two other Phleboviruses, the Rift Valley Fever and Sandfly Fever Sicilian viruses. This finding suggests the possible involvement of this sequence in the mechanism of transcription termination.

Base Sequence↗