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.

Differences in gait parameters at a preferred walking speed in healthy subjects due to age, height and body weight.

The objective of our cross-sectional study was to investigate the changes associated with age and gender in walking speed, stride length and cadence of healthy women and men over the adult age range, and establish the effects of anthropometric indices such as height and body weight. We examined 118 women and 121 men (age range, 19-90 years). Subjects walked at their preferred speed over a 12-meter walkway crossing two Kistler force plates: cadence was calculated from heel strike times recorded from the Kistler force plates; walking speed was measured using an infrared reflecting system; and stride length was calculated from the walking speed and cadence. Older healthy subjects had lower values for walking speed and stride length than younger subjects. While there is little difference in the percentage reduction between women and men over the adult age range. the absolute values for walking speed are lower in women than men at all ages. In women, the percentage of explained variance for decline in walking speed was 30%, and for decline in stride length 400%. If body weight was also taken into account, the percentage of explained variance for walking speed was 37%, and for stride length 59%. A similar calculation for men yields 34% for decline in walking speed, and 42% for decline in stride length. Cadence was not associated with age, height and body weight. The standard errors for the estimates of walking speed in both women and men, respectively, are reduced by 8% and 3% using the multiple regression technique. The corresponding standard errors for stride length were reduced by, respectively, 19% and 13% if height in either sexes, or height and body weight in women, were taken into account. In conclusion, preferred walking speed and stride length decline with age in healthy people. Lower values found in old healthy subjects partly contributed to the difference in height and body weight between old and young subjects. Cadence was not correlated with age, height and body weight.

Characterisation and expression of pbs25, a sexual and sporogonic stage specific protein of Plasmodium berghei.

Following gametogenesis and fertilisation in the bloodmeal within the mosquito midgut, the newly formed zygotes of the malaria parasite develop into motile invasive ookinetes. During this development, surface molecules are synthesised de novo including molecules of 21-28 kDa from the zygote-ookinete stages. An antiserum recognising a 26 kDa protein of Plasmodium berghei was used to clone the corresponding gene from a cDNA library, which was shown to be identical to the reported Pbs25 gene sequence. We show here that Pbs25 was detectable in preparations of gametes 30 min post-gametocyte activation, expression continued on zygotes, ookinetes and oocysts indicating there is a significant overlap of expression of the two immunogenic zygote-ookinete proteins belonging to the P25/28 protein family of sexual stage antigens. Biochemical analysis of Pbs25 demonstrates the presence of a malaria-specific glycosylphosphatidylinositol (GPI) anchor. Antibodies recognising Pbs25 impaired parasite development in the mosquito.

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.

Relationships between physical performance measures, age, height and body weight in healthy adults.

OBJECTIVE: we measured muscle strength and functional mobility in healthy men and women over the adult age range to investigate the changes with age and sex, and to establish the effects of the anthropometric indices height and weight. DESIGN: cross-sectional study. SUBJECTS AND METHODS: we recruited 74 healthy women (mean age 49.0, range 20-90) and 81 healthy men (mean age 51.6, range 20-90). We measured maximum isometric knee extension strength, handgrip strength and explosive leg extensor power. We assessed functional mobility quantitatively with the timed 'get up and go' test and the modified Cooper test. RESULTS: older subjects had lower values for muscle strength and muscle power than young subjects. Times for the timed 'get up and go' test were longer and distances in the modified Cooper test shorter. At about the age of 55, women showed an acceleration in the decline of isometric knee extension strength and handgrip strength (between 20 and 55 years, knee strength decreased by 10.3% and handgrip strength decreased by 8.2%, between 55 and 80 years the decreases were 40.2% and 28% respectively). Men showed a more gradual declines over the adult age range, with decreases in knee and handgrip strength of 24% and 19.6% between 20 and 55 years, and 23% and 17.4% between 55 and 80 years. The age-related decline is partly associated with differences in height and body weight. Women had higher correlations between muscle strength and functional mobility tests than men. CONCLUSIONS: muscle strength and functional mobility decline with age in healthy people; in women we observed an accelerated decrement in muscle strength above the age of 55. Lower values in healthy old subjects are partly associated with differences in height and body weight.

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.

Are arm span measurements useful in the prediction of osteoporosis in postmenopausal women?

A consensus report on osteoporosis in The Netherlands recommends general practitioners to use an arm span-height difference of at least 3 cm as one of the criteria for suspecting osteoporosis. In this study it was investigated how well this criterion discriminates between patients with established osteoporosis and controls in a group of postmenopausal women. When the Dutch general practitioners criterion of arm span-height difference exceeding 3 cm was applied in a logistic regression analysis to predict the probability of having osteoporosis, a resultant sensitivity of 58% and a specificity of 56% were found. In order to improve the predicted probability of osteoporosis, two more predictors in addition to the arm span-height difference were introduced in the logistic regression model. The first additional predictor related to information whether subject's age was below or above 70 years, and the second whether the arm span was below or above 160 cm. All three predictors appeared to be highly significant. It was shown that the predicted probability of osteoporosis by this model could be considerably improved when the age category and the arm span category were also taken into account, leading to a sensitivity of 81% while the specificity amounts to 64%. This seems quite satisfactory for such a simple method and clearly improves on the original single criterion of arm span-height difference.

Spatial-temporal analysis of mobility over the adult age range using the Postural-Locomotor-Manual Test.

BACKGROUND: Mobility measurements were performed on healthy adult subjects to investigate changes over the adult age range and to obtain reference values. METHODS: Ninety-five males (19-90 years, mean 58.2) and 122 females (19-90 years, mean 51.8) were measured using the commercially available Postural-Locomotor-Manual Test (PLM-test). Subjects are required to perform a standard maneuver that is recorded using an optoelectronic technique. RESULTS: Older subjects carried out all phases of the maneuver more slowly than younger subjects. At about the age of 50 years, females showed a more rapid slowing down. Males showed a more gradual decrease over the adult age range. CONCLUSION: The PLM-test provides a fairly simple noninvasive method of assessing motor performance. However, it is important to separate male and female data in the determination of reference values.

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.