Biomedical subjects

P A Nuttall

Publications and source records attributed to P A Nuttall.

At least 19 recordsLinked to original sources

PubMed1992

Modification of the skin feeding site by tick saliva mediates virus transmission.

A tick vector of Thogoto (THO) virus was shown to secrete a factor in saliva which potentiates the transmission of THO virus to uninfected ticks feeding on an apparently non-viraemic host. The effect of the saliva activated transmission (SAT) factor on the virus occurred at the site of inoculation in the skin and was apparent even when the virus was introduced 3 days after the SAT factor. The results suggest that tick saliva can play an important role in disease transmission by virtue of host modification at the site of feeding.

Animals↗

PubMed1992

Saliva-activated transmission (SAT) of Thogoto virus: dynamics of SAT factor activity in the salivary glands of Rhipicephalus appendiculatus, Amblyomma variegatum, and Boophilus microplus ticks.

Thogoto (THO) virus is transmitted from infected to uninfected ticks when co-feeding on uninfected guinea-pigs, even though the guinea-pigs do not develop a detectable viraemia. This form of non-viraemic transmission is potentiated by a factor(s) secreted by the saliva of ticks and hence has been termed saliva-activated transmission (SAT). The synthesis of the SAT factor by the salivary glands of three ixodid tick species was determined by placing uninfected nymphal ticks on guinea-pigs that were subsequently inoculated with a mixture of THO virus and salivary gland extract (SGE) derived from one of the tick species. SAT factor activity was measured by determining the number of nymphs that acquired THO virus. For the three-host ixodid species, Rhipicephalus appendiculatus and Amblyomma variegatum, maximum enhancement of THO virus transmission was observed when salivary glands were derived from uninfected female ticks that had fed for a period of 6 or 8 days, respectively. In contrast, when salivary glands were derived form uninfected female Boophilus microplus, a one-host ixodid tick species, enhancement of THO virus transmission was observed throughout the tick feeding period. Thus, the natural feeding behaviour of ticks appears to be an important factor in determining the relative importance of these vectors in mediating SAT.

Animals↗

PubMed1992

The glycoprotein of Thogoto virus (a tick-borne orthomyxo-like virus) is related to the baculovirus glycoprotein GP64.

Thogoto (THO) virus is a tick-transmitted virus which shares morphological and biochemical characteristics with members of the Orthomyxoviridae. The genome of Thogoto virus comprises six segments of single-stranded, negative sense RNA. The complete nucleotide sequence of the fourth largest RNA segment of THO virus has been determined from cDNA analyses. This RNA segment is 1574 nt long and has a coding capacity for a glycoprotein of 512 amino acids with a predicted molecular weight of 57,550 Da. The sequence of this protein has extensive homology with the putative envelope protein of Dhori (DHO) virus, the only other recorded tick-borne orthomyxo-like virus, but not with the envelope glycoproteins of the influenza viruses. A search of the translation of the available nucleotide database has produced evidence for a relationship between the glycoproteins of THO and DHO viruses and the gp64 glycoprotein of two DNA-containing insect baculoviruses, Autographa californica nuclear polyhedrosis virus and Orgyia pseudotsugata nuclear polyhedrosis virus. The baculovirus gp64 protein is a membrane protein implicated in endocytotic fusion events during infection. A multiple alignment between these four glycoproteins gave significance scores of greater than 28 standard deviations, indicating that the homologies between them are highly significant. The distribution of cysteine residues is conserved between all four proteins which also have similar hydropathy profiles, suggestive of a type I membrane protein topology.

Amino Acid Sequence↗

PubMed1992

Enhanced neurovirulence of tick-borne orbiviruses resulting from genetic modulation.

The genome of orbiviruses (Reoviridae family) comprises 10 segments of double-stranded RNA. The fourth largest segment of the tick-borne Kemerovo (KEM) group orbiviruses is the genetic determinant of neurovirulence in experimentally infected mice, and segment 6 determines serotype. Reassortant viruses derived from a cross between two KEM-related viruses, Great Island (GI) and Wexford (WEX), that had the heterotypic gene combination W4G6 (segment 4 of WEX virus and segment 6 from GI virus) were nonpathogenic in mice. This apparent genetic modulation of neurovirulence may have resulted from steric interaction between the two outer capsid proteins of nonpathogenic reassortants. Further data are consistent with this hypothesis. Reassortants generated from additional KEM group viruses showed various degrees of enhanced neurovirulence in terms of their PFU/LD50 (ratio of infectivity in cell culture and in mice) and ASTmax (the average survival time at the highest virus dilution resulting in 100% mortality). Some reassortants were more pathogenic than either of their parental viruses. The results indicate that the gene determining neurovirulence dictates ASTmax, and the PFU/LD50 is a measure of the interaction between the products of the gene determining neurovirulence and that determining serotype. The nonpathogenic phenotype of a low passage isolate (St. Abb's 84-34 virus), derived from a single tick, generated neurovirulent reassortants. This result indicates that genetic modulation of KEM group viruses may occur in nature.

Animals↗

PubMed1992

Comparison of the nonstructural protein, NS3, of tick-borne and insect-borne orbiviruses.

The complete nucleotide sequence of the smallest RNA segment (segment 10) of Broadhaven (BRD) virus, a tick-borne orbivirus, was determined from a full-length cDNA clone. The genome segment is 702 nucleotides in length and has a coding capacity for two proteins of either 205 or 199 amino acids, having net charges of +16.5 and +17.5, respectively, at neutral pH. Comparison of the sequence of RNA segment 10 of BRD, bluetongue, African horse sickness, and Palyam viruses revealed amino acid homology of 20 to 30% between the four orbiviruses, with one conserved region of 40 to 50% homology which, in segment 10 of BRD virus, is found between residues 26 and 71.

Amino Acid Sequence↗

PubMed1992

Comparison of the S RNA segments and nucleoprotein sequences of Crimean-Congo hemorrhagic fever, Hazara, and Dugbe viruses.

The S RNA segments of the nairoviruses Crimean-Congo hemorrhagic fever (CCHF) virus (Chinese isolate) and Hazara (HAZ) virus were cloned and sequenced from PCR products. The RNAs comprise 1672 and 1677 nucleotides, respectively, and each encodes a protein in the viral complementary strand (54.0 and 54.2 kDa, respectively). The deduced protein sequences show homology to each other and to the nucleoprotein of Dugbe (DUG) nairovirus, although both the CCHF and HAZ viral proteins are larger. Alignment of the nucleoprotein sequences of CCHF, HAZ, and DUG viruses show that the CCHF and HAZ sequences are somewhat more closely related to each other (60.0% identity) than either is to the DUG sequence (55.4 and 53.0% identity, respectively); 39.5% of residues are identical across all three proteins. The carboxyl-terminus of DUG N protein shows a 40-residue deletion relative to the N proteins of the other two viruses.

Amino Acid Sequence↗

PubMed1992

Dugbe Nairovirus M RNA: nucleotide sequence and coding strategy.

The coding assignments of the medium-sized (M) RNA segment of the Dugbe (DUG) virus (Nairovirus, Bunyaviridae) were investigated. The complete nucleotide sequence of 4888 nucleotides (nt) contained one long open reading frame in the viral complementary RNA, extending from an AUG start codon at nt 48-50 to a stop codon at nt 4701-4703 (numbered from the 5' terminus of vcRNA). Comparison of the terminal sequences with the ends of the DUG S segment revealed sequence identity between the first nine nucleotides of both segments. No sequence homologies were found with the M segments of other members of the Bunyaviridae, or with their polypeptide products. Expression of portions of the DUG M open reading frame in Escherichia coli demonstrated the carboxyl terminal region of the M open reading frame codes for the G1 structural glycoprotein, which is the target for neutralising antibodies. Confirmation of this assignment was obtained by sequencing the amino terminus of the G1 protein. Two nonstructural glycoproteins which share epitopes with G1 were identified in virus-infected cells, one of which (85 kDa) is processed over a period of several hours to produce G1. The G2 coding region was located upstream of the G1 sequence. The region between the carboxyl terminus of G2 and the 5' end of the long open reading frame apparently encodes a nonstructural protein of about 70 kDa, which is a precursor of the G2 protein.

Amino Acid Sequence↗

PubMed1992

Expression of the nucleocapsid protein of Dugbe virus and antigenic cross-reactions with other nairoviruses.

The small (S) RNA segment of Dugbe (DUG) virus (Nairovirus, Bunyaviridae) encodes a single protein, the nucleocapsid (N) protein, of M(r) 49.4 kDa. cDNA derived from the complete coding region for the N protein was cloned into Autographa californica nuclear polyhedrosis virus (AcNPV) under control of the polyhedrin promoter and used to infect Spodoptera frugiperda insect cells. Western blotting analysis using monoclonal antibodies demonstrated the production of DUG N protein in the infected cells. Monoclonal and polyclonal antibodies to the N protein of Crimean-Congo haemorrhagic fever (CCHF) virus were found to cross-react weakly with the baculovirus expressed DUG N protein by Western blotting. When used in an enzyme linked immunoassay (ELISA), the DUG N protein reacted with polyclonal mouse immune ascitic fluids raised against either CCHF or Hazara viruses (both members of the CCHF serogroup of nairoviruses). Cross-reactions between DUG virus (Nairobi sheep disease serogroup) and members of other nairovirus serogroups were not detected.

Antigens, Viral↗

PubMed1992

Comparison of the major structural core proteins of tick-borne and Culicoides-borne orbiviruses.

Comparison of sequence data for Broadhaven (BRD) virus, a tick-borne orbivirus, and bluetongue virus (BTV), the type species of the genus, indicated that RNA segments 2 and 7 of BRD virus encode the two structural core proteins, VP2 and VP7, respectively. Segment 2 is 2792 nucleotides in length with a coding capacity for a protein (VP2) of 908 amino acids and a net charge of +8.5 at neutral pH. Segment 7 is 1174 nucleotides in length with a coding capacity for a protein (VP7) of 356 amino acids and a net charge of +11.5 at neutral pH. Comparison of the two sequences with BTV serotype 10 revealed amino acid identity of 35% between the product of segment 2 and BTV VP3, and 21% between the product of segment 7 and BTV VP7. The core proteins therefore show evidence of significant evolutionary divergence compared with that shown between different insect-borne orbiviruses. In particular, the amino terminus of BRD virus VP7 differed markedly from the equivalent region in VP7 of BTV and African horse sickness virus. This region is thought to interact with the outer capsid layer of insect-borne orbiviruses.

Amino Acid Sequence↗

PubMed1992

Saliva activated transmission (SAT) of Thogoto virus: relationship with vector potential of different haematophagous arthropods.

Tick saliva (or salivary gland extract) potentiates the transmission of Thogoto (THO) virus to uninfected ticks feeding on a non-viraemic guinea-pig. This phenomenon has been named saliva activated transmission (SAT). To investigate the potential of different haematophagous arthropods to mediate SAT, guinea-pigs were infested with uninfected R.appendiculatus Neumann nymphs and inoculated with THO virus and salivary gland extract (SGE) derived from a range of ixodid (metastriate and prostriate) or argasid ticks, or mosquitoes; control guinea-pigs were inoculated with virus alone. Enhancement of THO virus transmission was observed only when SGE was derived from metastriate ticks. Comparison with the vector potential of these various arthropod species revealed that enhancement of THO virus transmission was specific for ticks which were competent vectors of the virus. The data indicate a correlation between vector competence and the ability of haematophagous arthropods to mediate SAT of THO virus.

Aedes↗

PubMed1991

Structure and morphogenesis of Dugbe virus (Bunyaviridae, Nairovirus) studied by immunogold electron microscopy of ultrathin cryosections.

We have studied the structure and morphogenesis of Dugbe (DUG) virus (Bunyaviridae, Nairovirus) in cultured porcine kidney (PS) cells and a tick cell line (Ra 243) using immunogold electron microscopy. DUG virus is a tickborne arbovirus, considered to be a low health hazard, that is antigenically and genetically related to Crimean Congo haemorrhagic fever (CCHF) virus (Marriott et al., 1990). We have investigated the maturation and intracellular transport of DUG virus particles as a model for other more pathogenic nairoviruses using monoclonal antibodies for immunogold labelling of ultrathin cryosections and immunofluorescence techniques. The spherical DUG virus particle measures about 90 nm in diameter, with a 5 nm thick membrane covered by 5-7 nm long projections or "spikes". These projections form hollow cylindrical morphological units, about 5 nm in diameter. DUG virus infection caused only a slight cytopathogenic effect in mammalian cells and none in tick cells. DUG virus particles assembled by budding from the Golgi complex, where the DUG virus glycoprotein G1 accumulated in vesicles originating from Golgi cisternae. The nucleocapsid protein N accumulated in scattered foci throughout the cytoplasm, and this appears to be related to the limited maturation of DUG virus particles that occurred. The reduced number of budding virus particles observed in tick cells was correlated with the reduced cytopathology observed.

Animals↗

PubMed1991

Dissemination, replication, and trans-stadial persistence of Dugbe virus (Nairovirus, Bunyaviridae) in the tick vector Amblyomma variegatum.

The dissemination and replication of Dugbe (DUG) virus and its tissue tropisms in the tick vector Amblyomma variegatum were examined by immunohistochemical analysis using specific antibody, in situ hybridization with a viral-complementary riboprobe, and infectivity assays of dissected tissues. Dugbe virus was localized in both unfed and feeding adults inoculated as nymphs or orally infected by capillary feeding, and in nymphs infected by capillary feeding. In non-feeding ticks, the main sites of DUG virus replication were the epidermis, hemocytes associated with loose connective tissue, and a small number of phagocytic digestive cells in the gut lumen. Virus infectivity in the hemolymph was associated entirely with hemocytes. Dugbe viral antigen or infectivity was not detected in the salivary glands until after the start of feeding. Viral titers in the salivary glands of feeding ticks were about ten-fold higher than in gut, ovary, or loose connective tissue. The level of infection decreased during molting and increased during feeding. Viral particles and pathologic effects were not detected in infected ticks. The primary site of trans-stadial persistence of DUG virus is the hemocytes. Tick hemocytes and other motile cells may be important in the transmission of persistent virus infection from one cell or organ to another by diapedesis.

Animals↗

PubMed1990

Genetic determinants modulating the pathogenic phenotype of tick-borne orbiviruses.

Genetic studies have been carried out on orbiviruses in the Great Island (GI) antigenic subgroup of the Kemerovo serogroup (Orbivrus, Reoviridae) to elucidate the functions of the 10 genomic double-stranded RNA segments. Such studies have shown that segment 4 is the major genetic determinant of neurovirulence (P.A. Nuttall, S.R. Moss, L.D. Jones, and D. Carey, 1989, Virology 172, 428-434), whereas segment 5 of Wexford (WEX) virus and segment 6 of GI virus are the major determinants of serotype specificity (S.R. Moss, C.M. Ayres, and P.A. Nuttall, 1987, Virology 157, 137-144; S.R. Moss, C.M. Ayres, and P.A. Nuttall, 1988, J. Gen. Virol. 69, 2721-2727). In studies with reassortants isolated following dual infection of cell cultures with WEX and GI viruses, the gene combination W4G6 (i.e., viruses deriving segment 4 from WEX virus and segment 6 from GI virus) resulted in nonpathogenic reassortants. Unlike the parental viruses, the avirulent reassortants did not produce clinical evidence of infection in inoculated 2-day-old mice although, suprisingly, they replicated in the brains of the mice. The alternate heterotypic gene combination, G4W5, resulted in typical neurovirulent reassortants. The results indicate that segment 6 of GI virus is able to modulate the phenotypic expression of segment 4 of WEX virus, but not vice versa. Modulation probably results from interactions between the products of these two genomic segments, possibly at the level of virion structure.

Animals↗

PubMed1990

RNA segment 5 of broadhaven virus, a tick-borne orbivirus, shows sequence homology with segment 5 of bluetongue virus.

The sequence of Broadhaven (BRD) virus segment 5, the major genetic determinant of serotype, is 1658 nucleotides in length and contains a single open reading frame (ORF) having the coding capacity for a protein of Mr 52.5K. Comparison of the ORF of segment 5 of BRD virus with published sequences of bluetongue virus (BTV) revealed 30% nucleotide homology and 31% amino acid homology with the protein encoded by segment 5 of BTV serotype 10. Significant homology was not shown with segment 2 of BTV, the major genetic determinant of the BTV serotype. The sequences at the 3' and 5' ends determined for BRD segment 5 were similar to the respective 3' and 5' regions of BTV. The sequence data provide evidence of an evolutionary relationship between two ecologically distinct groups of orbiviruses and demonstrate changes that have occurred in the functions of genetically related genomic segments.

Amino Acid Sequence↗

PubMed1990

Coding strategy of the S RNA segment of Dugbe virus (Nairovirus; Bunyaviridae).

The S RNA segment of Dugbe (DUG) virus (Nairovirus; Bunyaviridae) was sequenced from three overlapping cDNA clones and by primer extension. The S RNA is 1712 nucleotides in length and contains one large open reading frame (ORF) of 1326 nucleotides coding for a 49.4-kDa protein on viral complementary (vc) RNA. This protein in size corresponds to the DUG nucleocapsid (N) protein (P. Cash, 1985, J. Gen. Virol. 66, 141-148). The 49.4-kDa product was expressed as a fusion protein with beta-galactosidase in Escherichia coli cells and confirmed as DUG N protein by Western blotting with DUG N-specific monoclonal antibody. An additional ORF of 150 nucleotides coding for a possible 5.9-kDa protein is present in the +1 reading frame, 3' to the N protein ORF on vcRNA. DUG S segment mRNA was found to be essentially full length. No evidence was obtained for the existence of a smaller mRNA species that could code for a 5.9-kDa protein. Comparisons of the DUG S RNA sequence and predicted N protein amino acid sequence, with the respective sequences of snowshoe hare, La Crosse (bunyaviruses), Punta Toro, Sandfly fever Sicilian (phleboviruses), and Hantaan (hantavirus) viruses, failed to detect any sequence similarity, although the genomic structure of DUG S RNA is similar to that of the S RNA segment of Hantaan (HTN) virus.

Amino Acid Sequence↗

PubMed1990

Detection of an arbovirus in an invertebrate and a vertebrate host using the polymerase chain reaction.

The ability of the polymerase chain reaction (PCR) to diagnose an arboviral infection in an arthropod vector or a mammalian host was examined. Dugbe (DUG) viral RNA was detected in RNA extracts from infected tissue samples by reverse transcription and enzymatic amplification of the resulting cDNA using Taq DNA polymerase, followed by characterisation of the amplified product by agarose gel electrophoresis or dot-blot hybridisation. Viral RNA was detected in the organs and haemolymph of infected Amblyomma variegatum ticks, and in the brain and blood of infected mice. The PCR technique was found to be as sensitive as a plaque assay for detecting DUG virus, but not as sensitive as intracerebral inoculation of mice. The sensitivity of the technique was greatest using crude RNA extracts combined with dot-blot analysis of the resulting PCR products using a DUG specific cDNA probe. A result was obtained within 48 h using PCR whereas biological assays took at least 8 days to diagnose the virus infection.

Animals↗

PubMed1990

RNA probes detect nucleotide sequence homology between members of two different nairovirus serogroups.

Cloned cDNA derived from the small (S) and medium (M) genomic RNA segments of Dugbe (DUG) virus, isolate ArD44313, a member of the Nairobi sheep disease (NSD) serogroup of nairoviruses (family, Bunyaviridae) was used to prepare 32P-labelled DNA and RNA probes. The S and M segments of six isolates of DUG virus all hybridised to both DNA and RNA probes, although the M segment of isolate KT281/75 reacted only weakly. Of nine other nairoviruses tested, representing all the six other serogroups within the Nairovirus genus, none hybridised to the DNA probes. However, under conditions of low stringency, the DUG S and M RNA probes hybridised to the respective S and M segments of Ganjam (GAN) virus (another member of the NSD serogroup). The DUG S RNA probe also hybridised to the S segments of Crimean-Congo haemorrhagic fever (CCHF) virus and Hazara (HAZ) virus (members of the CCHF serogroup). The indicated sequence relationships between DUG, GAN, CCHF and HAZ viruses show that the NSD serogroup is more closely related to members of the CCHF serogroup than it is to nairoviruses of the other five serogroups.

Animals↗

PubMed1990

The effect of host resistance to tick infestation on the transmission of Thogoto virus by ticks.

Tick-borne virus transmission was examined using guinea-pigs and hamsters previously infested with ticks. Guinea-pigs developed immunity to Rhipicephalus appendiculatus after a single exposure to the ticks. Nymphal and adult stages that fed on resistant guinea-pigs had increased mortality during feeding, and reduced engorged weights. Egg production from female ticks fed on resistant hosts fell by at least 50%. Guinea-pigs maintained high levels of immunity to tick infestation for at least 210 days after the initial exposure. In contrast, hamsters did not develop resistance to ticks even after three or four infestations. R. appendiculatus adults infected with Thogoto (THO) virus (donors) were allowed to co-feed with uninfected nymphs (recipients) on either resistant or naive guinea-pigs. The number of recipient ticks that acquired virus was significantly reduced on resistant guinea-pigs. In contrast, feeding on pre-infested hamsters did not affect tick-borne transmission of THO virus. Host resistance to tick infestation, if prevalent in nature, may severely limit the spread of tick-borne viruses. Such an effect could result directly from a reduction in the number of ticks that acquire virus, or indirectly from poor egg production (in the case of viruses maintained in ticks by vertical transmission) and reduced survival of ticks fed on resistant hosts.

Animals↗