Species-specific alterations in Anopheles mosquito olfactory responses caused by Plasmodium infection.

Mosquitoes infected with malaria parasites have demonstrated altered behaviour that may increase the probability of parasite transmission. Here, we examine the responses of the olfactory system in Plasmodium falciparum infected Anopheles gambiae, Plasmodium berghei infected Anopheles stephensi, and P. berghei infected An. gambiae. Infected and uninfected mosquitoes showed differential responses to compounds in human odour using electroantennography coupled with gas chromatography (GC-EAG), with 16 peaks triggering responses only in malaria-infected mosquitoes (at oocyst, sporozoite or both stages). A selection of key compounds were examined with EAG, and responses showed differences in the detection thresholds of infected and uninfected mosquitoes to compounds including lactic acid, tetradecanoic acid and benzothiazole, suggesting that the changes in sensitivity may be the reason for differential attraction and biting at the oocyst and sporozoite stages. Importantly, the different cross-species comparisons showed varying sensitivities to compounds, with P. falciparum infected An. gambiae differing from P. berghei infected An. stephensi, and P. berghei infected An. gambiae more similar to the P. berghei infected An. stephensi. These differences in sensitivity may reflect long-standing evolutionary relationships between specific Plasmodium and Anopheles species combinations. This highlights the importance of examining different species interactions in depth to fully understand the impact of malaria infection on mosquito olfactory behaviour.

Detection of malaria sporozoites expelled during mosquito sugar feeding.

Malaria is a severe disease of global importance transmitted by mosquitoes of the genus Anopheles. The ability to rapidly detect the presence of infectious mosquitoes able to transmit malaria is of vital importance for surveillance, control and elimination efforts. Current methods principally rely on large-scale mosquito collections followed by labour-intensive salivary gland dissections or enzyme-linked immunosorbent (ELISA) methods to detect sporozoites. Using forced salivation, we demonstrate here that Anopheles mosquitoes infected with Plasmodium expel sporozoites during sugar feeding. Expelled sporozoites can be detected on two sugar-soaked substrates, cotton wool and Whatman FTA cards, and sporozoite DNA is detectable using real-time PCR. These results demonstrate a simple and rapid methodology for detecting the presence of infectious mosquitoes with sporozoites and highlight potential laboratory applications for investigating mosquito-malaria interactions. Our results indicate that FTA cards could be used as a simple, effective and economical tool in enhancing field surveillance activities for malaria.

Knockout of the rodent malaria parasite chitinase pbCHT1 reduces infectivity to mosquitoes.

During mosquito transmission, malaria ookinetes must cross a chitin-containing structure known as the peritrophic matrix (PM), which surrounds the infected blood meal in the mosquito midgut. In turn, ookinetes produce multiple chitinase activities presumably aimed at disrupting this physical barrier to allow ookinete invasion of the midgut epithelium. Plasmodium chitinase activities are demonstrated targets for human and avian malaria transmission blockade with the chitinase inhibitor allosamidin. Here, we identify and characterize the first chitinase gene of a rodent malaria parasite, Plasmodium berghei. We show that the gene, named PbCHT1, is a structural ortholog of PgCHT1 of the avian malaria parasite Plasmodium gallinaceum and a paralog of PfCHT1 of the human malaria parasite Plasmodium falciparum. Targeted disruption of PbCHT1 reduced parasite infectivity in Anopheles stephensi mosquitoes by up to 90%. Reductions in infectivity were also observed in ookinete feeds-an artificial situation where midgut invasion occurs before PM formation-suggesting that PbCHT1 plays a role other than PM disruption. PbCHT1 null mutants had no residual ookinete-derived chitinase activity in vitro, suggesting that P. berghei ookinetes express only one chitinase gene. Moreover, PbCHT1 activity appeared insensitive to allosamidin inhibition, an observation that raises questions about the use of allosamidin and components like it as potential malaria transmission-blocking drugs. Taken together, these findings suggest a fundamental divergence among rodent, avian, and human malaria parasite chitinases, with implications for the evolution of Plasmodium-mosquito interactions.

Identification of differentially regulated genes of Plasmodium by suppression subtractive hybridization.

Plasmodium, the causative agent of malaria, has many morphologically and functionally distinct developmental stages. In the mosquito host alone, there are five transitions during the development of a gametocyte into a sporozoite. Determining which genes are expressed at the different developmental stages is vital to our understanding of the parasite. There are a growing number of techniques designed to study gene expression, including microarray. Here, Johannes Dessens, Gabrielle Margos, Maria del Carmen Rodriguez and Robert Sinden describe a novel method: suppression subtractive hybridization (SSH) and its successful application in obtaining mosquito midgut stage-specific genes of Plasmodium.

CTRP is essential for mosquito infection by malaria ookinetes.

The malaria parasite suffers severe population losses as it passes through its mosquito vector. Contributing factors are the essential but highly constrained developmental transitions that the parasite undergoes in the mosquito midgut, combined with the invasion of the midgut epithelium by the malaria ookinete (recently described as a principal elicitor of the innate immune response in the Plasmodium-infected insect). Little is known about the molecular organization of these midgut-stage parasites and their critical interactions with the blood meal and the mosquito vector. Elucidation of these molecules and interactions will open up new avenues for chemotherapeutic and immunological attack of parasite development. Here, using the rodent malaria parasite Plasmodium berghei, we identify and characterize the first microneme protein of the ookinete: circumsporozoite- and TRAP-related protein (CTRP). We show that transgenic parasites in which the CTRP gene is disrupted form ookinetes that have reduced motility, fail to invade the midgut epithelium, do not trigger the mosquito immune response, and do not develop further into oocysts. Thus, CTRP is the first molecule shown to be essential for ookinete infectivity and, consequently, mosquito transmission of malaria.

Mx1-based resistance to thogoto virus in A2G mice is bypassed in tick-mediated virus delivery.

The interferon-induced mouse Mx1 protein has intrinsic antiviral activity against orthomyxoviruses, including Thogoto virus. Thus, Mx1(+) A2G mice are apparently resistant to infection following needle- or tick-borne virus challenge. However, tick-borne challenge and, to a lesser degree, injection of virus mixed with tick salivary gland extract resulted in virus transmission to uninfected ticks feeding on the A2G mice. The data indicate that immunomodulatory components in tick saliva can overcome a natural antiviral mechanism.

An endonuclease switching mechanism in the virion RNA and cRNA promoters of Thogoto orthomyxovirus.

An in vitro assay was developed to investigate endonuclease activity of Thogoto virus, a tick-borne orthomyxovirus. Endonuclease activity relied on an interaction between the 3' and 5' termini of virion RNA (vRNA) and not those of cRNA. Evidence was obtained that cap structures are cleaved directly from cap donors and that cleavage does not occur after pyrimidines. A 5' hook structure, present in the vRNA promoter but not the cRNA promoter, was introduced into cRNA promoter mutants. These mutants stimulated endonuclease activity, although at levels slightly lower than that of vRNA. The ability of the cRNA promoter to stimulate endonuclease activity when mutated to contain a 5' hook structure indicates that this structure constitutes a switching mechanism for endonuclease activity between the vRNA and cRNA promoters.

The Thogoto orthomyxovirus cRNA promoter functions as a panhandle but does not stimulate cap snatching in vitro.

The cRNA promoter of Thogoto virus, a tick-borne orthomyxovirus, was investigated using an in vitro polymerase assay based on purified viral cores and synthetic oligoribonucleotides corresponding to the 3' and 5' ends of cRNA. In vitro polymerase activity relied on an interaction between the 3' and 5' ends of cRNA and was ApG primer-dependent. Mutational analysis of the promoter showed that interstrand base-pairing of residues 11 and 12 of the 3' promoter arm with residues 10 and 11 of the 5' promoter arm, respectively, was essential for polymerase activity. These data provide the first clear evidence for a cRNA panhandle in an orthomyxovirus. No evidence was obtained for the presence of a 5' or 3' hook structure in the cRNA promoter, and transcription could not be primed with rabbit globin mRNA or synthetic cap analogues. This demonstrates that cap snatching activity relies on the presence of the vRNA terminal sequences.

In vivo reconstitution of active Thogoto virus polymerase: assays for the compatibility with other orthomyxovirus core proteins and template RNAs.

Tick-borne Thogoto virus (THOV), the prototype of a new genus in the Orthomyxoviridae family, contains six single-stranded RNA segments of negative polarity. Four of them encode gene products that correspond to the influenza virus PB1, PB2, PA and NP core proteins. Here we describe an in vivo system in which the expression of a THOV model RNA is driven by THOV core proteins synthesized from cloned cDNAs. Our results demonstrated the biological activity of our cloned genes and showed that the three polymerase subunits and the NP are required for gene expression. For comparison, we also used the in vivo reconstituted systems of the influenza A and B viruses. None of the polymerase or NP proteins was active in a heterologous orthomyxovirus core, indicating a high specificity in core assembly and/or function. Interestingly, the THOV polymerase did not recognize the influenza A virus promoter and vice versa.

Asymmetrical Distribution of Barley Yellow Dwarf Virus PAV Variants Between Host Plant Species.

ABSTRACT A large epidemiological study of the genetic variation of barley yellow dwarf virus (BYDV) serotype PAV involving different host plant species was conducted. French BYDV PAV isolates were collected from barley and ryegrass, and their capsid protein gene sequences characterized using restriction fragment length polymorphism, single-strand conformation polymorphism, and sequence analyses. The data show that BYDV PAV isolates from five different continents are separated into two distinct groups named cpA and cpB, which are distributed irrespective of geographical location. Amino acid identity of the capsid proteins ranged from 93 to 99.5% in group cpA and from 95 to 99.5% in group cpB, while this value was only from 82 to 88% between the groups. Moreover, isolates from each group were found preferentially (up to 98%) in one of the two plant species examined. These results show that host plant species play a role in isolate selection and maintenance and that they contribute to the genetic diversity of BYDV PAV.

35S promoter-driven cDNAs of barley mild mosaic virus RNA1 and RNA2 are infectious on barley plants.

Full-length cDNAs of barley mild mosaic bymovirus RNA1 and RNA2 were cloned downstream of a modified cauliflower mosaic virus 35S promoter with double enhancer. Mechanical inoculation of barley seedlings with a mixture of both cDNAs resulted in systemic mosaic symptoms, typical of barley mild mosaic virus infection. The presence of both RNA species and their gene products in the systemically infected leaves was demonstrated by RT-PCR and Western blot analyses, respectively. Virions were detected by immunogold labelling, demonstrating that the RNAs are encapsidated. This is the first report of the 35S promoter used in successfully infecting a monocot plant host with cDNA from a strictly monocot plant RNA virus.

In vitro polymerase activity of Thogoto virus: evidence for a unique cap-snatching mechanism in a tick-borne orthomyxovirus.

The tick-borne Thogoto virus (THOV) is the type species of a new genus in the family Orthomyxoviridae. Its genome comprises six segments of single-stranded, negative-sense RNA. Each segment possesses conserved regions of semicomplementary nucleotides at the 3' and 5' termini which strongly resemble those of influenza virus. An in vitro polymerase assay based on reconstituted THOV viral cores was developed, and activity was shown to rely on an interaction between the conserved 3'- and 5'-terminal sequences and to be primer dependent. Addition of globin mRNA primed transcription, catalyzing the addition of an extra nucleotide to the transcripts, corresponding to the 5'-terminal m7G cap residue. Priming with various cap analogs suggested that THOV transcription is initiated preferentially with m7GpppAm and involves base pairing. This is the first experimental evidence of endonuclease activity in THOV as part of a unique cap-snatching mechanism.

Striking conformational similarities between the transcription promoters of Thogoto and influenza A viruses: evidence for intrastrand base pairing in the 5' promoter arm.

In the accompanying report, we describe an in vitro polymerase assay based on reconstituted Thogoto virus (THOV) cores which provided evidence of a double-stranded vRNA promoter consisting of both the 3' and 5' sequences of vRNA (M. B. Leahy, J. T. Dessens, and P. A. Nuttall, J. Virol. 71:8347-8351, 1997). This system was used to investigate further the THOV vRNA promoter structure by using short, synthetic vRNA promoters. The results obtained show that interstrand base pairing between residues 10 and 11 of the 3' promoter arm with residues 11 and 12 of the 5' promoter arm, respectively, is important for promoter activity. In addition, intrastrand base pairing between residues 2 and 3 with residues 9 and 8 of the 5' promoter arm, respectively, was shown to be involved in promoter activity, while no evidence of intrastrand base pairing between residues 2 and 9 of the 3' promoter arm was obtained. These observations are consistent with a hook-like structure in the 5' promoter arm of the THOV promoter. The THOV cores were able to transcribe an influenza A virus (FLUA) vRNA-like promoter, as well as hybrid THOV-FLUA promoters. Hence, the THOV and FLUA vRNA promoters appear to be both structurally and functionally similar.

The fourth genus in the Orthomyxoviridae: sequence analyses of two Thogoto virus polymerase proteins and comparison with influenza viruses.

The tick-borne Thogoto virus (THOV) is the type species of a newly recognized fourth genus, Thogotovirus, in the family Orthomyxoviridae. Because of the distant relationship of THOV with the influenza viruses, determination of its genomic information can potentially be used to identify important domains in influenza virus proteins. We have determined the complete nucleotide sequence of the second longest RNA segment of THOV. The molecule comprises 2212 nucleotides with a single large open reading frame encoding a protein of 710 amino acids, estimated Mr 81,284. The protein shares 77% amino acid similarity with the PB1-like protein of Dhori virus, a related tick-borne virus, and 50-53% with the PB1 polymerase proteins of influenza virus A, B and C. All the motifs characteristic of RNA-dependent polymerases were identified including the SSDD motif common to all RNA-dependent RNA polymerases, indicating that the THOV protein is functionally analogous to the influenza virus PB1 proteins and involved in chain elongation. We also report the corrected sequence of the third longest RNA segment of THOV, encoding a protein which shares 44-47% amino acid similarity with the PA-like polymerase proteins of influenza virus A, B and C. The biological significance of conserved domains in these orthomyxovirid proteins is discussed.

The 24 kDa proteinases of comoviruses are virus-specific in cis as well as in trans.

To investigate the specificity of comoviral 24 kDa ('24K') proteinases, a full-length cDNA copy of red clover mottle virus (RCMV) RNA 1 has been cloned downstream of a T7 promoter. Translation in rabbit reticulocyte lysates of in vitro transcripts from this clone resulted in the synthesis of a 200K protein which was processed in a manner similar to that of the equivalent protein from cowpea mosaic virus (CPMV). Full-length cDNA clones of the RNA 1 molecules of RCMV and CPMV were used to create hybrid RNA 1 molecules. RNA transcribed in vitro from these hybrids was translated in vitro and the ability of the 24K proteinase from one comovirus to cleave the 32K/170K processing site from the other assessed. The results of the experiments show that the 24K proteinases are virus-specific in cis.

The complete nucleotide sequence of barley mild mosaic virus RNA1 and its relationship with other members of the Potyviridae.

The complete nucleotide sequence of RNA1 of a French barley mild mosaic bymovirus isolate has been determined. The molecule is composed of 7261 nucleotides and contains a single large open reading frame encoding a protein of 2258 amino acids with a calculated Mr of 256,375. Amino acid sequence comparison with poty- and rymoviruses reveals eight domains corresponding to the potyviral P3, 6K1, Cl, 6K2, Nla-VPg, Nla-Pro, Nlb and capsid proteins, respectively. Seven putative cleavage sites, similar to those mediated by potyvirus Nla proteinases, were identified. The presence of two, so far undescribed putative cleavage sites (6K2/Nla-VPg and Nla-VPg/Nla-Pro) in the polyproteins encoded by RNA1 of barley yellow mosaic virus and wheat spindle streak mosaic virus, was predicted. These data indicate that the bymovirus RNA1 fits the consensus potyviral genetic map downstream of the helper component gene.

Identification of structural similarities between putative transmission proteins of Polymyxa and Spongospora transmitted bymoviruses and furoviruses.

Comparison of amino acid sequence and hydropathy profiles shows conserved, structural similarities between the capsid readthrough protein of potato mop top virus (transmitted by Spongospora subterranea) and furovirus and bymovirus proteins implicated in transmission by Polymyxa spp. This suggests that these proteins have a common ancestry and are involved in a common biological process: virus transmission by plasmodiophorid fungi.

Characterization of fungally and mechanically transmitted isolates of barley mild mosaic virus: two strains in competition.

The virus populations of two French barley mild mosaic virus isolates have been characterized using Western blot, sequence, PCR, and RFLP analyses. One of the isolates (M) was obtained by mechanical transmission of the other, fungally transmitted isolate (P). The results obtained show that the two isolates contain distinct RNA1 and RNA2 molecules with respect to their nucleotide sequences, and hence contain distinct barley mild mosaic virus strains. One strain is present mainly in isolate P and closely resembles a Japanese and two German isolates. It contains an RNA2 of approximately 3.5 kb. The other strain is present mainly in isolate M and closely resembles a UK isolate. This strain contains a smaller RNA2 of approximately 2.4 kb. The results show that mechanical transmission causes a shift in the virus populations in favor of the mutant strain.

Primary structure and sequence analysis of RNA2 of a mechanically transmitted barley mild mosaic virus isolate: an evolutionary relationship between bymo- and furoviruses.

The RNA2 nucleotide sequence of a mechanically transmitted isolate of barley mild mosaic virus (BaMMV) has been determined. A combination of Northern blot and sequence analysis indicates that this RNA2 lacks approximately 1000 nucleotides of its C-terminal protein (P2) gene, with respect to Polymyxa graminis transmitted BaMMV. This is confirmed by sequence comparison with RNA2 of a fungus transmitted BaMMV isolate, which reveals the presence of a single deletion located in the 3'-terminal part of the P2 gene. As a consequence, this RNA2 codes for a P2 protein of only 34 K. Sequence homology between the bymovirus P2 protein and the capsid readthrough protein of beet necrotic yellow vein virus and soil-borne wheat mosaic virus suggests an evolutionary relationship between bymo- and furoviruses.

Cauliflower mosaic virus 35S promoter-controlled DNA copies of cowpea mosaic virus RNAs are infectious on plants.

Clones have been constructed that contain full-length cDNA copies of cowpea mosaic virus RNA1 and RNA2, downstream of the cauliflower mosaic virus 35S promoter. The clones, when linearized downstream of the viral sequences, give rise to cowpea mosaic virus-like symptoms when inoculated onto cowpea plants. Viral RNA and virions can be detected in the inoculated plants, demonstrating that the clones are directly infectious.