Large RNA Segment Research Articles

ANTIGENIC AND GENETIC RELATIONSHIPS AMONG RIFT VALLEY FEVER VIRUS AND OTHER SELECTED MEMBERS OF THE GENUS PHLEBOVIRUS (BUNYAVIRIDAE)

Preliminary serologic data indicated that two South American phleboviruses (Belterra virus [BELTV] and Icoaraci virus [ICOV]) may be related to Rift Valley fever virus (RVFV), an African phlebovirus that causes severe hepatitis and hemorrhagic fever in humans. To further define this relationship and to investigate the underlying genetic basis, comparative serologic and genetic sequence analyses were performed with RVFV and five other New World phleboviruses (ICOV, BELTV, Salobo virus, Joa virus, and Frijoles virus). Serologically, a one-way cross reaction was confirmed between antibodies against these New World viruses and RVFV antigen. In contrast, phylogenetic analysis demonstrated clear separation of these viruses from RVFV, into distinct phylogenies, based on sequences of the small, medium, and large RNA segments.

Sequence determination and functional analysis of the Akabane virus (family Bunyaviridae) L RNA segment

Akabane virus (AKAV) causes epizootic congenital deformities in cattle, sheep, and goats. Due to the lack of a complete genome sequence, the molecular biological properties of this virus are not known. We have cloned and sequenced the functional large (L) RNA segment of AKAV, and shown that it has polymerase activity using a minireplicon system with RNA polymerase I. The complete L RNA segment is 6868 nucleotides long and encodes an L protein of 2251 amino acids, which functions as an RNA-dependent RNA polymerase. A minireplicon reporter plasmid was constructed by flanking either the firefly luciferase or the green fluorescent protein gene in the antisense orientation with the 5'- and 3'-terminal noncoding regions of the small RNA segment. HmLu-1 cells were transfected with the reporter plasmid, and the L protein and nucleoprotein (N protein) expression plasmids. The reporter activity was upregulated in a dose-dependent manner with increasing concentration of either the L or N protein expression plasmid. Furthermore, the reporter activity could be downregulated by the AKAV NSs protein as well as by other orthobunyaviruses. These results show that the AKAV minireplicon system is a powerful tool for studying transcription and for rescuing infectious viruses from cloned cDNAs.

Presence of broadly reactive and group-specific neutralizing epitopes on newly described isolates of Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease in humans with high rates of mortality. The virus has a tripartite genome composed of a small (S), a medium (M) and a large (L) RNA segment; the M segment encodes the two viral glycoproteins, G(N) and G(C). Whilst relatively few full-length M segment sequences are available, it is apparent that both G(N) and G(C) may exhibit significant sequence diversity. It is unknown whether considerable antigenic differences exist between divergent CCHFV strains, or whether there are conserved neutralizing epitopes. The M segments derived from viral isolates of a human case of CCHF in South Africa (SPU 41/84), an infected tick (Hyalomma marginatum) in South Africa (SPU 128/81), a human case in Congo (UG 3010), an infected individual in Uzbekistan (U2-2-002) and an infected tick (Hyalomma asiaticum) in China (Hy13) were sequenced fully, and the glycoproteins were expressed. These novel sequences showed high variability in the N-terminal region of G(N) and more modest differences in the remainder of G(N) and in G(C). Phylogenetic analyses placed these newly identified strains in three of the four previously described M segment groups. Studies with a panel of mAbs specific to G(N) and G(C) indicated that there were significant antigenic differences between the M segment groups, although several neutralizing epitopes in both G(N) and G(C) were conserved among all strains examined. Thus, the genetic diversity exhibited by CCHFV strains results in significant antigenic differences that will need to be taken into consideration for vaccine development.

Co-evolutionary patterns of variation in small and large RNA segments of Crimean-Congo hemorrhagic fever virus

The genus Nairovirus of the family Bunyaviridae includes the Crimean-Congo haemorrhagic fever (CCHF) species group. The species is predominated by the hazard-group 4 pathogens, from which the name and majority of strain entries are derived. Additionally, the species embraces hazard-group 2 viruses that are classified as members by antigenic cross-reactivity. CCHF viruses have a tripartite RNA genome consisting of large (L), medium (M) and small (S) segments. Here, the sequence characterization of previously undescribed L and S segments from novel strains originating in the Middle East and Africa is reported. Further scrutiny of this data with phylogenetic tools, in the context of other publicly available sequence information, reveals analogous grouping patterns between the L and S segments. These groups correlate with the geographical distribution of strain isolation and indicate that the L and S segments of CCHF viruses have evolved together.

Complete L segment coding-region sequences of Crimean Congo hemorrhagic fever virus strains from the Russian Federation and Tajikistan

The large (L) RNA segment of Crimean Congo hemorrhagic fever (CCHF) virus strain AST/TI30908, isolated from pooled Hyalomma marginatum ticks collected in 2002 from the Astrakhan region of European Russia, was amplified piecemeal using reverse-transcription/polymerase chain reaction, followed by direct sequencing of gel-purified amplicons. After removal of 5' and 3' primer-generated termini, the assembled AST/TI30908 L segment sequence is 12112 nucleotides long, with 41.3% G + C content, and is greater than 87% and 96% identical at the nucleotide and translated amino acid levels, respectively, to partial or full-length CCHF virus L segment sequences deposited in GenBank. A complete L segment coding-region sequence for CCHF virus strain TAJ/HU8966, isolated from a patient in Tajikistan in 1990, was determined in a similar fashion. This L segment (12133 nucleotides long, 41.1% G + C content) shares 88% nucleotide identity with the full-length strain Matin from Pakistan, and 97% nucleotide identity with a partial L segment sequence of strain Khodzha from Uzbekistan. Strain TAJ/HU8966 shares at least 96% identity at the translated amino acid level with all other CCHF virus L segment sequences. Although, for the most part, CCHF virus L polyprotein primary sequences are uniformly well conserved, a region of marked variability was identified in the N-terminal half of the RNA-dependent RNA polymerase. This region, approximately 50 amino acids in length, is flanked by previously-reported arenavirus and bunyavirus-conserved regions, and may prove useful in CCHF diagnosis and viral taxonomy.

New insights into the evolutionary relationships between arenaviruses provided by comparative analysis of small and large segment sequences

Arenaviruses are rodent-borne negative-stranded bisegmented RNA viruses. Five arenaviruses are etiologic agents of hemorrhagic fever in humans and are potential agents of bioterrorism. They are classified as Biosafety level 4 agents and listed in the category A of the Pathogen Agents edited by the Center for Disease Control and Prevention. To date, evolution and phylogeny of arenaviruses have been based on the analysis of sequences derived from structural genes (small RNA segment) exclusively, due to the lack of sequences available for the large RNA segment. In this study, partial sequences of the polymerase gene were determined for 18 species of arenaviruses and used to investigate phylogenetic relationships. Comparative analysis of topologies obtained from polymerase and structural gene analyses permitted us to determine the evolutionary origin of the major parent of the North American recombinant arenaviruses, and to investigate the role of genetic exchange (reassortment and recombination) in the evolutionary mechanisms driving the evolution of the genus Arenavirus.

Nucleotide sequence and deduced amino acid sequence of the medium RNA segment of Oropouche, a Simbu serogroup virus: Comparison with the middle RNA of Bunyamwera and California serogroup viruses

The Bunyavirus genus of the family Bunyaviridae contains 18 serogroups. To date nucleotide sequence data has been obtained for three serogroups, Bunyamwera, California and Simbu, based on analysis of the small (S) RNA segment. In comparison, there is only nucleotide sequence data for the large and medium (M) RNA segments for members of the Bunyamwera and California serogroups. In this paper we report the nucleotide sequence of the M RNA of Oropouche (ORO) virus, a member of the Simbu serogroup. The M RNA was 4396 nucleotides in length with G1, G2 and NSm proteins similar in size to those reported for members of the Bunyamwera and California serogroups. However, there was limited nucleotide (50–52%) and amino acid (30–32%) homology between ORO virus M RNA and those of published members of the other two serogroups. The Bunyamwera and California serogroups are more closely related to each other than the Simbu serogroup virus Oropouche. These data were consistent with that previously reported for the S RNA (Saeed et al., 2000. J. Gen. Virol. 81, 743–748). It has been noted previously that three of four potential N-linked glycosylation sites of the Bunayamwera and California serogroups are conserved in G1 and G2 proteins. In contrast, ORO virus was found to have only three potential N-linked glycosylation sites of which only one, in G1, was conserved with members of the other two serogroups. Comparison of M RNA sequences of different strains of ORO virus revealed genetic variation consistent with that reported previously for the S RNA.

Association of RNA polymerase complexes of the parasitic protozoan Cryptosporidium parvum with virus-like particles: heterogeneous system.

RNA polymerase complexes were purified from Cryptosporidium parvum, a parasitic protozoan known to infect many species of mammals including humans. Western blot analysis revealed the association of the complexes with two different proteins, encoded by large and small segments of viral double-stranded RNAs. Each complex was found to contain only double-stranded RNA, both double- and single-stranded RNA, or only single-stranded RNA. Maximum RNA-dependent RNA polymerase activity was observed within the complexes containing both double- and single-stranded RNAs. These complexes possessed both transcriptase and replicase polymerase activities. Virus-like particles with a diameter of 31 nm were copurified with RNA polymerase complexes, and buoyant density and polymerase studies suggest that C. parvum harbors a putative double-stranded RNA virus which separately encapsidates the large and small RNA segments. The mechanism of replication and other characteristics of this virus are similar to those of the viruses of the family Partitiviridae, previously identified only in fungi and plants.

Large RNA segment of Dugbe nairovirus encodes the putative RNA polymerase.

The nucleotide sequence of the large (L) RNA segment of Dugbe (DUG) virus (Nairovirus, Bunyaviridae) was determined, completing the first entire genome sequence of a nairovirus. The L segment comprised 12255 nucleotides, making a total genome size of 18855 nucleotides, and the ends showed identity with the ends of the medium (M) and small (S) genomic segments. A single open reading frame (ORF) was present in the viral complementary strand, sufficient to encode a protein of 459 kDa. The predicted protein sequence showed the core polymerase motifs characteristic of the RNA-dependent RNA polymerases of segmented negative-stranded viruses. Comparison of the conserved motifs with the corresponding region of other segmented negative-strand viruses showed a closer relationship between nairoviruses and phleboviruses than with other Bunyaviridae or with other virus families. However, the core polymerase was the only function that could be assigned to a region of the DUG L gene.

Virus-like particles in Eimeria nieschulzi are associated with multiple RNA segments

RNA preparations from sporulated oocysts of Eimeria nieschulzi were found to contain 2 double-stranded RNA segments of 5.0 kb and 5.7 kb that were not present in other species of Eimeria. Treatment of crude lysates with RNase A revealed that in addition to these two segments, 3 other segments of 0.57 kb, 0.72 kb and 11.5 kb were protected from digestion, suggesting that they were enclosed within particles. Virus-like particles with a diameter of approximately 39 nm were purified by caesium chloride buoyant density centrifugation. Four of the five RNA segments copurified with these particles. In keeping with the nomenclature generally adopted for protozoan viruses, we have named this new isolate ENV 1. The largest RNA segment does not cosediment with ENV 1 particles and may be derived from another RNA-protein complex that is unstable under the conditions used. The particle size and genome structure of ENV 1 both differ from that of the Eimeria stiedae virus (ESV), which is the only other virus to have been isolated from Eimeria to date. Short cDNA clones derived from ENV 1 show significant homology to a region of the Leishmania virus (LRV 1) genome that encodes an RNA-dependent RNA polymerase. The polymerase sequences from ENV 1 and LRV 1 are more closely related to each other than to any other protein sequences in the GenEMBL Database. This raises intriguing questions about the origins of the two viruses, since Eimeria and Leishmania normally infect different hosts and also show different cell tropisms within these hosts.

Molecular determinants of the virulence and infectivity of California serogroup bunyaviruses.

California bunyaviruses cause encephalitis in mammalian hosts after peripheral infection. The virulence of these viruses is determined by their ability to replicate sequentially in striated muscle, cause viremia, and invade and replicate in the central nervous system. These viruses are also able to infect vector mosquitoes following ingestion of a blood meal containing virus. Bunyaviruses are negative stranded RNA viruses with a trisegmented genome, and the large, medium, and small RNA segments encode the polymerase, the glycoproteins, and the nucleoprotein, respectively. Reassortants between virulent and avirulent virus clones have been used to map virulence determinants in mice as well as determinants of infectivity in mosquitoes. Attenuation in mice and infectivity in mosquitoes of some virus clones maps to the medium RNA segment, implying that the virus glycoproteins, which are involved in virus entry, play a role in virulence. Attenuation in mice and mosquito infectivity of other clones maps to the large RNA segment, suggesting that cell-specific differences in the function of the viral polymerase can also determine virulence and host range.

Polygenic control of neuroinvasiveness in California serogroup bunyaviruses

The pathogenesis of the California serogroup bunyaviruses includes both extraneural and intraneural replicative phases that can be separated experimentally. The present study dissects the viral genetic determinants of extraneural replication. We have previously described two attenuated reassortant clones of California serogroup bunyaviruses which exhibit reduced neuroinvasiveness after subcutaneous inoculation into suckling mice. Clone B1-1a bears an attenuated middle RNA segment (neuroinvasiveness phenotype v alpha v), and clone B.5 bears an attenuated large RNA segment (neuroinvasiveness phenotype alpha vv). We prepared reassortant viruses between these two strains and found that the two attenuated gene segments acted independently and additively, since reassortants bearing two attenuated RNA segments were more attenuated than the parental clones. Reassortants bearing no attenuated RNA segments were much more neuroinvasive than either parental clone, indicating that a neuroinvasive strain can be derived from two attenuated clones. Pathogenesis studies demonstrated that after injection of 10(3) PFU, the attenuated reassortant clones did not replicate in peripheral tissue, failed to reach the brain, and did not cause disease. At a dose of 10(6) PFU, attenuated clones failed to replicate to a significant level in peripheral tissue and produced only a minimal passive plasma viremia during the first 24 h but nevertheless reached high titers in the brain and killed mice. Because of this result, we investigated the possibility that neuroinvasion occurs via retrograde axonal transport, by determining whether sciatic nerve sectioning could protect against virus infection after hind leg footpad inoculation. We found that nerve sectioning had no effect on lethality, ruling out this mode of entry and suggesting that passive viremia is likely to be sufficient for invasion of the central nervous system.

Use of Polymerase Chain Reaction for Efficient Cloning of dsRNA Segments of Infectious Bursal Disease Virus

A method is described for efficient cloning of the large RNA segment of infectious bursal disease virus (IBDV) after reverse transcription and cDNA amplification. Complementary DNA segments of IBDV were prepared using reverse transcriptase and specific primers homologous to the conserved region at the 3' end of the IBDV sequence. The resulting cDNA segments were amplified using Taq DNA polymerase and a pair of specific primers. Three separate primer pairs were used for amplification, each yielding a cDNA fragment of the predicted size. The amplified products were directly used for cloning into a cloning vector, pCR1000. Three overlapping cDNA clones, containing the entire coding region of the large RNA segment of IBDV, were obtained. The identity of these clones was confirmed by hybridization with IBDV-specific probe, as well as by sequence analysis. By this method, approximately 3.2 kilobases of the large genome segments of two different strains of IBDV were cloned, and their complete nucleotide sequence was determined.

Neuroattenuation of an avirulent bunyavirus variant maps to the L RNA segment

The derivation and characterization of a neuroattenuated reassortant clone (RFC 25/B.5) of California serogroup bunyavirus was described previously (M. J. Endres, A. Valsamakis, F. Gonzalez-Scarano, and N. Nathanson, J. Virol. 64:1927-1933, 1990). To map the RNA segment responsible for this attenuation, a panel of reassortants was constructed between the attenuated clone B.5 (genotype TLL) and a virulent clone (B1-1a) of reciprocal genotype (LTT). Parent viruses and clones representing all of the six possible reassortants were examined for neurovirulence by intracerebral injection in adult mice. Reassortants bearing the large RNA segment from the virulent parent were almost as virulent as the virulent parent virus, while reassortants bearing the large RNA segment from the avirulent parent virus exhibited low or intermediate virulence. These results indicate that the large RNA segment is the major determinant of neuroattenuation of clone B.5. In addition to its neuroattenuation, clone B.5 was temperature sensitive and exhibited an altered plaque morphology. These phenotypes also segregated with the large RNA segment. The importance of the large RNA segment (which encodes the viral polymerase) in neurovirulence contrasts with prior studies which indicate that the ability to cause lethal encephalitis after peripheral injection of suckling mice (neuroinvasiveness) is primarily determined by the middle-sized RNA segment, which encodes the viral glycoproteins.

Generation of envelope and defective interfering RNA mutants of tomato spotted wilt virus by mechanical passage

During a series of mechanical transfers of tomato spotted wilt virus, two distinct types of mutants were generated. Firstly, a morphologically defective isolate was obtained which had lost the ability to produce the membrane glycoproteins and, as a consequence, was not able to form enveloped particles. Analysis of the genomic RNAs of this isolate suggested that this defect was caused by either point mutations or very small deletions in the medium genomic RNA segment. Secondly, isolates were obtained which had accumulated truncated forms of the large (L) RNA segment. These shortened L RNA molecules most likely represented defective interfering RNAs, since they replicated more rapidly than full-length L RNA and their appearance was often associated with symptom attenuation. Defective L RNAs of different sizes were generated after repeated transfers, and hybridization analysis using L RNA-specific cDNA probes showed that the internal regions deleted varied in length. The presence of defective L RNAs in nucleocapsid fractions as well as in enveloped virus particles indicates that all defective molecules retained the sequences required for replication, encapsidation by nucleocapsid proteins and packaging of the nucleocapsid into virus particles.

Genome cloning and analysis of the large RNA segment (segment A) of a naturally avirulent serotype 2 infectious bursal disease virus

The genome of infectious bursal disease virus (IBDV) of serotype 2 (strain OH) has been cloned, and 3171 nucleotides of genome segment A cDNA sequence have been determined for the first time. Sequence homology of OH-IBDV with the most distant serotype 1 IBDV at the nucleotide level is 83.1%, and the amino acid sequence homology of the polyprotein is 89.6%. Alignment of the polyprotein amino acid sequences showed the hypervariable region in VP2 to be 151–152 amino acid residues long in IBDV. A second variable region, 37 amino acid residues long, was identified in the N-terminal third of the IBDV VP2 molecule. IBDV strains, like the IPNV strains, also contain inverted repeats that may form stem-and-loop structures in the 5′ noncoding sequences. These inverted repeats are variable between the two IBDV serotypes, particularly at the AT basepairs.

Primary structure of the large (L) RNA segment of nephropathia epidemica virus strain Hällnäs B1 coding for the viral RNA polymerase

The L RNA segment of the nephropathia epidemica virus (NEV) strain Hällnäs B1 was characterized by molecular cloning of the corresponding cDNA and subsequent determination of the DNA nucleotide sequence. The L RNA segment is 6550 nucleotides long with complementarity of 20 bases at the 3′ and 5′ termini. The viral messenger sense RNA contains one major open reading frame (ORF) with a coding capacity of 2156 amino acid residues encoding a protein with a calculated molecular weight of 246 kDa and an IEP of pH 7.4. Comparison of the deduced amino acid sequences from NEV hantavirus and Bunyamwera virus (BWV) L segment messenger sense RNAs, revealed a high degree of diversity (overall amino acid identity, 17%). However, three clusters of 30–40% amino acid identity were detected. One of these domains, containing an Asp-Asp motif found in many RNA polymerases, also shares amino acid sequence homology with the PB1 polymerase component of influenza virus type A. These results indicate that the L RNA segment of NEV codes for the viral RNA-dependent RNA polymerase. The data presented here complete our previous studies on the characterization of the NEV genome by cDNA sequencing of the viral M and S RNA segments.

Nucleotide sequence and coding capacity of the large (L) genomic RNA segment of Seoul 80-39 virus, a member of the hantavirus genus

The nucleotide sequence of the large (L) genomic RNA segment of Seoul 80-39 virus was determined from overlapping cDNA clones. The virion L RNA segment is 6530 nucleotides long. The 3' and 5' terminal sequences are inversely complementary for 15 bases. The viral complementary-sense RNA contains a single open reading frame from an AUG codon at nucleotide position 37–39 to a UAA stop codon at nucleotide position 6490–6492. This ORF could encode a polypeptide of 2151 amino acids (246,662 kDa) which likely corresponds to the L protein detected in purified viral particles (Elliott et al., 1984) and is assumed to be an RNA-dependent RNA polymerase molecule (Schmaljohn and Dalrymple, 1983). Comparison of the L protein of the Seoul 80-39 virus with the polymerase proteins encoded by other negative-stranded RNA viruses revealed 44% similarity only with the part of the Bunyamwera virus L protein (Elliott, 1989) and a very weak homology with the PB1 protein of influenza virus.

Sequence of the large double-stranded RNA segment of the N1 strain of infectious pancreatic necrosis virus: a comparison with other Birnaviridae

The cDNA sequence of the large dsRNA segment (segment A) of the N1 strain of infectious pancreatic necrosis virus (IPNV) has been determined. The nucleotide and deduced amino acid sequences were compared to the sequences of segment A of the Jasper strain of IPNV and to the sequences of segments A and B (5' and 3' flanking regions) of the 002-73 strain of infectious bursal disease virus (IBDV). The comparison demonstrated that the precursor protein of the major structural polypeptide, pVP2, is highly conserved at the N and C termini, whereas the amino acid sequence of an internal segment shows greater diversity between the strains. This internal segment probably carries the serotype-specific epitopes of birnaviruses. An alternative open reading frame (ORF) (444 bp) partly overlapping with the large ORF (2916 bp) of segment A was found to be conserved among the IPNV strains and is probably also present in the 002-73 strain of IBDV. This small ORF may encode a novel birnavirus polypeptide with an Mr of 17K. SDS-PAGE of radiolabelled purified IPNV particles revealed a band corresponding to the possible novel 17K polypeptide. Short terminal inverted repeats are found in segment A of the N1 and Jasper strains of IPNV and in segment B of the 002-73 strain of IBDV. Segment A of IPNV and segment B of IBDV also contain adjacent inverted repeats at their 5'-terminal flanking regions.

Nucleotide sequence analysis of the large (L) genomic RNA segment of Bunyamwera virus, the prototype of the family Bunyaviridae

The complete nucleotide sequence of the large (L) genome segment of Bunyamwera virus has been determined from overlapping cDNA clones. The segment is 6875 nucleotides long and has a base composition of 29.8% A, 17.9% C, 15.4% G, and 36.9% U. Eighteen of the terminal 19 nucleotides at the 3′ and 5′ ends are complementary. In the viral-complementary (+ sense) RNA there is a single long open reading frame (ORF) from AUG at bases 51–53 to a UAG stop codon at bases 6765–6767; this ORF encodes a polypeptide of 2238 amino acids (MW 259,000), corresponding to the L protein which has been mapped to the L RNA segment by analysis of reassortants of Bunyamwera, Batai, and Maguari viruses. The amino-terminal 46 amino acids of the L protein show strong homology (63% identity) with the amino-termini of ORFs predicted from limited sequence analysis of the L segments of La Crosse and snowshoe hare bunyaviruses. Comparison with the polymerase proteins encoded by other negative-strand viruses showed weak homology with part of the influenza virus PB1 protein, but no homology was detected with the other influenza virus polymerase proteins nor with the L proteins of arenaviruses, paramyxoviruses, and rhabdoviruses. At the 5′ end of genomic (- sense) RNA there is an AUG-initiated ORF potentially encoding a protein of 14,700; the significance of this ORF is unknown at present.