Lower leg morphology in runners: forefoot strikers have longer heels but not bigger muscles than rearfoot strikers.

Foot strike pattern used during running may relate to lower leg morphology. We tested the hypotheses that forefoot strike (FFS) runners have longer plantarflexor moment arms (r) and larger plantarflexor muscles than rearfoot strike (RFS) runners. FFS runners had 17% longer r than RFS runners, but all runners had similarly sized medial and lateral gastrocnemius (MG and LG) muscles. Because muscle size also depends on activation pattern ( Ahn et al., 2011), we compared MG:LG activation bias during walking in 24 runners and 23 sedentary subjects. Half of all subjects activated their MG and LG muscles equally ('unbiased') while walking, while the other half activated their MG more strongly than their LG muscles ('MG-biased'). Unbiased sedentary subjects had 16-23% smaller MG muscles compared with MG-biased sedentary subjects, unbiased runners and MG-biased runners. Muscle contraction dynamics during FFS running may balance the effects of longer plantarflexor moment arms in determining MG and LG muscle size.

Different Segments within Vertebrate Muscles Can Operate on Different Regions of Their Force-Length Relationships.

To relate in vivo behavior of fascicle segments within a muscle to their in vitro force-length relationships, we examined the strain behavior of paired segments within each of three vertebrate muscles. After determining in vivo muscle activity patterns and length changes of in-series segments within the semimembranosus muscle (SM) in the American Toad (Bufo americanus) during hopping and within the sternohyoid (SH) muscle in the rat (Rattus rattus) during swallowing, and of spatially separated fascicles within the medial gastrocnemius (MG) muscle in the rat during trotting, we measured their corresponding in vitro (toad) or in situ (rat) force-length relationships (FLRs). For all three muscles, in vivo strain heterogeneity lasted for about 36-57% of the behavior cycle, during which one segment or fascicle shortened while the other segment or fascicle simultaneously lengthened. In the toad SM, the proximal segment shortened from the descending limb across the plateau of its FLR from 1.12 to 0.91 of its optimal length (Lo), while the distal segment lengthened (by 0.04 ± 0.04 Lo) before shortening down the ascending limb from 0.94 to 0.83 Lo. In the rat SH muscle, the proximal segment tended to shorten on its ascending limb from 0.90 to 0.85 Lo while the distal segment tended to lengthen across Lo (0.96-1.12 Lo). In the rat MG muscle, in vivo strains of proximal fascicles ranged from 0.72 to 1.02 Lo, while the distal fascicles ranged from 0.88 to 1.11 Lo. Even though the timing of muscle activation patterns were similar between segments, the heterogeneous strain patterns of fascicle segments measured in vivo coincided with different operating ranges across their FLRs simultaneously, implying differences in force-velocity behavior as well. The three vertebrate skeletal muscles represent a diversity of fiber architectures and functions and suggest that patterns of in vivo contractile strain and the operating range over the FLR in one muscle region does not necessarily represent other regions within the same muscle.

Association of Condylar Bone Quality with TMJ Osteoarthritis.

The etiology and treatment of temporomandibular joint (TMJ) osteoarthritis (TMJOA) remain complex and unclear. Based on clinical observations, we hypothesized that low condylar bone quality is significantly correlated with TMJOA and explored this association in a cross-sectional study with human patients. A total of 254 postmenopausal female participants were included in this study. Radiographic findings from cone beam computed tomography (CBCT) and clinical symptoms were used to classify each TMJ data sample as healthy control ( n = 124) or TMJOA ( n = 130). Condylar bone mineral density (BMD) (computed tomography Hounsfield unit [CT HU]) and bone volume fraction (BV/TV) were measured and modeled as predictors of healthy control versus TMJOA status in multilevel logistic regression analyses. Both CT HU (adjusted odds ratio [AOR] = 0.9989, interquartile odds ratio [IOR] = 0.4206) and BV/TV (AOR= 0.8096, IOR = 0.1769) were negatively associated with TMJOA ( P = 0.049, 0.011, respectively). To assess the diagnostic performance of CT HU and BV/TV for identification of TMJOA, receiver operating characteristic (ROC) curves were plotted. The estimated areas under the curve (AUC) were 0.6622 for BV/TV alone, 0.6074 for CT HU alone, and 0.7136 for CT HU and BV/TV together. The model incorporating CT HU and BV/TV together had a significantly higher AUC than the models using BV/TV alone ( P = 0.038) or HU alone ( P = 0.021). In conclusion, we found that low condylar bone quality was significantly correlated with TMJOA development and that condylar CT HU and BV/TV can be used together as a potential diagnostic tool for TMJOA. Careful clinical evaluation of the condyle coupled with appropriate radiographic interpretation would thus be critical for the early detection of TMJOA.

A Simulated Heat Wave Has Diverse Effects on Immune Function and Oxidative Physiology in the Corn Snake (Pantherophis guttatus).

Animals will continue to encounter increasingly warm environments, including more frequent and intense heat waves. Yet the physiological consequences of heat waves remain equivocal, potentially because of variation in adaptive plasticity (reversible acclimation) and/or aspects of experimental design. Thus, we measured a suite of physiological variables in the corn snake (Pantherophis guttatus) after exposure to field-parameterized, fluctuating temperature regimes (moderate temperature and heat wave treatments) to address two hypotheses: (1) a heat wave causes physiological stress, and (2) thermal performance of immune function exhibits adaptive plasticity in response to a heat wave. We found little support for our first hypothesis because a simulated heat wave had a negative effect on body mass, but it also reduced oxidative damage and did not affect peak performance of three immune metrics. Likewise, we found only partial support for our second hypothesis. After exposure to a simulated heat wave, P. guttatus exhibited greater performance breadth and reduced temperature specialization (the standardized difference between peak performance and performance breadth) for only one of three immune metrics and did so in a sex-dependent manner. Further, a simulated heat wave did not elicit greater performance of any immune metric at higher temperatures. Yet a heat wave likely reduced innate immune function in P. guttatus because each metric of innate immune performance in this species (as in most vertebrates) was lower at elevated temperatures. Together with previous research, our study indicates that a heat wave may have complex, modest, and even positive physiological effects in some taxa.

Effect of temperature on leg kinematics in sprinting tarantulas (Aphonopelma hentzi): high speed may limit hydraulic joint actuation.

Tarantulas extend the femur-patella (proximal) and tibia-metatarsal (distal) joints of their legs hydraulically. Because these two hydraulically actuated joints are positioned in series, hemolymph flow within each leg is expected to mechanically couple the movement of the joints. In the current study, we tested two hypotheses: (1) at lower temperatures, movement of the two in-series hydraulic joints within a leg will be less coupled because of increased hemolymph viscosity slowing hemolymph flow; and (2) at higher temperatures, movement of the two in-series hydraulic joints will be less coupled because the higher stride frequencies limit the time available for hemolymph flow. We elicited maximal running speeds at four ecologically relevant temperatures (15, 24, 31 and 40°C) in Texas Brown tarantulas (Aphonopelma hentzi). The spiders increased sprint speed 2.5-fold over the temperature range by changing their stride frequency but not stride length. The coefficient of determination for linear regression (R(2)) of the proximal and distal joint angles was used as the measure of the degree of coupling between the two joints. This coupling coefficient between the proximal and distal joint angles, for both forelegs and hind-legs, was significantly lowest at the highest temperature at which the animals ran the fastest with the highest stride frequencies. The coordination of multiple, in-series hydraulically actuated joints may be limited by operating speed.

Mild closed head injury promotes a selective trigeminal hypernociception: implications for the acute emergence of post-traumatic headache.

BACKGROUND: Headache is one of the most common symptoms following traumatic head injury. The mechanisms underlying the emergence of such post-traumatic headache (PTH) remain unknown but may be related to injury of deep cranial tissues or damage to central pain processing pathways, as a result of brain injury. METHODS: A mild closed head injury in mice combined with the administration of cranial or hindpaw formalin tests was used to examine post-traumatic changes in the nociceptive processing from deep cranial tissues or the hindpaw. Histological analysis was used to examine post-traumatic pro-inflammatory changes in the calvarial periosteum, a deep cranial tissue. RESULTS: At 48 h after head injury, mice demonstrated enhanced nociceptive responses following injection of formalin into the calvarial periosteum, a deep cranial tissue, but no facilitation of the nociceptive responses following injection of formalin into an extracranial tissue, the hindpaw. Mice also showed an increase in the number of activated periosteal mast cells 48 h following mild head trauma, suggesting an inflammatory response. CONCLUSION: Our study demonstrates that mild closed head injury is associated with enhanced processing of nociceptive information emanating from trigeminal-innervated deep cranial tissues, but not from non-cranial tissues. Based on these finding as well as the demonstration of head injury-evoked degranulation of calvarial periosteal mast cells, we propose that inflammatory-evoked enhancement of peripheral cranial nociception, rather than changes in supraspinal pain mechanisms play a role in the initial emergence of PTH. Peripheral targeting of nociceptors that innervate the calvaria may be used to ameliorate PTH pain.

Variability of neural activation during walking in humans: short heels and big calves.

People come in different shapes and sizes. In particular, calf muscle size in humans varies considerably. One possible cause for the different shapes of calf muscles is the inherent difference in neural signals sent to these muscles during walking. In sedentary adults, the variability in neural control of the calf muscles was examined with muscle size, walking kinematics and limb morphometrics. Half the subjects walked while activating their medial gastrocnemius (MG) muscles more strongly than their lateral gastrocnemius (LG) muscles during most walking speeds ('MG-biased'). The other subjects walked while activating their MG and LG muscles nearly equally ('unbiased'). Those who walked with an MG-biased recruitment pattern also had thicker MG muscles and shorter heel lengths, or MG muscle moment arms, than unbiased walkers, but were similar in height, weight, lower limb length, foot length, and exhibited similar walking kinematics. The relatively less plastic skeletal system may drive calf muscle size and motor recruitment patterns of walking in humans.

Kinematic evidence for superfast locomotory muscle in two species of teneriffiid mites.

Locomotory muscles typically operate over a narrow range of contraction frequencies, characterized by the predominant fiber types and functional roles. The highest documented frequencies in the synchronous sound-producing muscles of insects (550 Hz) and toadfish (200 Hz) far exceed the contraction frequencies observed in weight-bearing locomotory muscles, which have maximum documented frequencies below 15-30 Hz. Laws of scaling, however, predict that smaller arthropods may employ stride frequencies exceeding this range. In this study we measured running speed and stride frequency in two undescribed species of teneriffiid mites from the coastal sage scrub of southern California. Relative speeds of both species [129-133 body lengths (BL)s(-1)] are among the fastest documented for any animal. Measured stride frequencies for both species far exceed those documented for any weight-bearing locomotory muscle, with measured values for one species ranging from 93 Hz at 25 degrees C to 111 Hz at 45 degrees C. Stride frequencies either closely approximate or, for one species, exceed predicted values based on an interspecific scaling of frequency and animal mass. Consequently, while the ultra-high frequencies of these muscles must depend on appropriately scaled kinetics of the calcium transient and contraction-relaxation cycle, these do not appear to limit the operating frequencies during running. The predicted low muscle forces operating at these very high frequencies evidently suffice for locomotion, probably because of the larger relative muscle force generated by smaller animals.

Sumatriptan alleviates nitroglycerin-induced mechanical and thermal allodynia in mice.

The association between the clinical use of nitroglycerin (NTG) and headache has led to the examination of NTG as a model trigger for migraine and related headache disorders, both in humans and laboratory animals. In this study in mice, we hypothesized that NTG could trigger behavioural and physiological responses that resemble a common manifestation of migraine in humans. We report that animals exhibit a dose-dependent and prolonged NTG-induced thermal and mechanical allodynia, starting 30-60 min after intraperitoneal injection of NTG at 5-10 mg/kg. NTG administration also induced Fos expression, an anatomical marker of neuronal activity in neurons of the trigeminal nucleus caudalis and cervical spinal cord dorsal horn, suggesting that enhanced nociceptive processing within the spinal cord contributes to the increased nociceptive behaviour. Moreover, sumatriptan, a drug with relative specificity for migraine, alleviated the NTG-induced allodynia. We also tested whether NTG reduces the threshold for cortical spreading depression (CSD), an event considered to be the physiological substrate of the migraine aura. We found that the threshold of CSD was unaffected by NTG, suggesting that NTG stimulates migraine mechanisms that are independent of the regulation of cortical excitability.

Animal models of migraine: looking at the component parts of a complex disorder.

Animal models of human disease have been extremely helpful both in advancing the understanding of brain disorders and in developing new therapeutic approaches. Models for studying headache mechanisms, particularly those directed at migraine, have been developed and exploited efficiently in the last decade, leading to better understanding of the potential mechanisms of the disorder and of the action for antimigraine treatments. Model systems employed have focused on the pain-producing cranial structures, the large vessels and dura mater, in order to provide reproducible physiological measures that could be subject to pharmacological exploration. A wide range of methods using both in vivo and in vitro approaches are now employed; these range from manipulation of the mouse genome in order to produce animals with human disease-producing mutations, through sensitive immunohistochemical methods to vascular, neurovascular and electrophysiological studies. No one model system in experimental animals can explain all the features of migraine; however, the systems available have begun to offer ways to dissect migraine's component parts to allow a better understanding of the problem and the development of new treatment strategies.

In situ muscle power differs without varying in vitro mechanical properties in two insect leg muscles innervated by the same motor neuron.

The mechanical behavior of muscle during locomotion is often predicted by its anatomy, kinematics, activation pattern and contractile properties. The neuromuscular design of the cockroach leg provides a model system to examine these assumptions, because a single motor neuron innervates two extensor muscles operating at a single joint. Comparisons of the in situ measurements under in vivo running conditions of muscle 178 to a previously examined muscle (179) demonstrate that the same inputs (e.g. neural signal and kinematics) can result in different mechanical outputs. The same neural signal and kinematics, as determined during running, can result in different mechanical functions, even when the two anatomically similar muscles possess the same contraction kinetics, force-velocity properties and tetanic force-length properties. Although active shortening greatly depressed force under in vivo-like strain and stimulation conditions, force depression was similarly proportional to strain, similarly inversely proportional to stimulation level, and similarly independent of initial length and shortening velocity between the two muscles. Lastly, passive pre-stretch enhanced force similarly between the two muscles. The forces generated by the two muscles when stimulated with their in vivo pattern at lengths equal to or shorter than rest length differed, however. Overall, differences between the two muscles in their submaximal force-length relationships can account for up to 75% of the difference between the two muscles in peak force generated at short lengths observed during oscillatory contractions. Despite the fact that these muscles act at the same joint, are stimulated by the same motor neuron with an identical pattern, and possess many of the same in vitro mechanical properties, the mechanical outputs of two leg extensor muscles can be vastly different.

Walking and running in the red-legged running frog, Kassina maculata.

Although most frog species are specialized for jumping or swimming, Kassina maculata (red-legged running frog) primarily uses a third type of locomotion during which the hindlimbs alternate. In the present study, we examined Kassina's distinct locomotory mode to determine whether these frogs walk or run and how their gait may change with speed. We used multiple methods to distinguish between terrestrial gaits: the existence or absence of an aerial phase, duty factor, relative footfall patterns and the mechanics of the animal's center of mass (COM). To measure kinematic and kinetic variables, we recorded digital video as the animals moved over a miniature force platform (N=12 individuals). With respect to footfall patterns, the frogs used a single gait and walked at all speeds examined. Duty factor always exceeded 0.59. Based on COM mechanics, however, the frogs used both walking and running gaits. At slower speeds, the fluctuations in the horizontal kinetic energy (E(k)) and gravitational potential energy (E(p)) of the COM were largely out of phase, indicating a vaulting or walking gait. In most of the trials, Kassina used a combined gait at intermediate speeds, unlike cursorial animals with distinct gait transitions. This combined gait, much like a mammalian gallop, exhibited the mechanics of both vaulting and bouncing gaits. At faster speeds, the E(k) and E(p) of Kassina's COM were more in phase, indicating the use of a bouncing or running gait. Depending on the definition used to distinguish between walking and running, Kassina either only used a walking gait at all speeds or used a walking gait at slower speeds but then switched to a running gait as speed increased.

In vivo and in vitro heterogeneity of segment length changes in the semimembranosus muscle of the toad.

Many studies examine sarcomere dynamics in single fibres or length-tension dynamics in whole muscles in vivo or in vitro, but few studies link the various levels of organisation. To relate data addressing in vitro muscle segment behaviour with in vivo whole muscle behaviour during locomotion, we measured in vivo strain patterns of muscle segments using three sonomicrometry crystals implanted along a fascicle of the semimembranosus muscle in the American toad (Bufo americanus; n = 6) during hopping. The centre crystal emitted an ultrasonic signal, while the outer crystals received the signal allowing the instantaneous measurement of lengths from two adjacent muscle segments. On the first day, we recorded from the central and distal segments. On the second day of recordings, the most distal crystal was moved to a proximal position to record from a proximal segment and the same central segment. When the toads hopped a distance of two body lengths, the proximal and central segments strained -15.1 +/- 6.1 and -14.0 +/- 4.9 % (i.e. shortening), respectively. Strain of the distal segment, however, was significantly lower and more variable in pattern, often lengthening before shortening during a hop. From rest length, the distal segment initially lengthened by 2.6 +/- 2.0 % before shortening by 6.5 +/- 3.2 % at the same hop distance. Under in vitro conditions, the central segment always shortened more than the distal segment, except when passively cycled, during which the segments strained similarly. When the whole muscle was cycled sinusoidally and stimulated phasically in vitro, the two adjacent segments strained in opposite directions over much (up to 34 %) of the cycle. These differences in strain amplitude and direction imply that two adjacent segments can not only produce and/or absorb varying amounts of mechanical energy, but can also operate on different regions of their force-length and force-velocity relationships when activated by the same neural signal. Understanding regional differences in contractile dynamics within muscles is therefore important to linking our understanding of sarcomere behaviour with whole muscle behaviour during locomotion.

A motor and a brake: two leg extensor muscles acting at the same joint manage energy differently in a running insect.

The individual muscles of a multiple muscle group at a given joint are often assumed to function synergistically to share the load during locomotion. We examined two leg extensors of a running cockroach to test the hypothesis that leg muscles within an anatomical muscle group necessarily manage (i.e. produce, store, transmit or absorb) energy similarly during running. Using electromyographic and video motion-analysis techniques, we determined that muscles 177c and 179 are both active during the first half of the stance period during muscle shortening. Using the in vivo strain and stimulation patterns determined during running, we measured muscle power output. Although both muscles were stimulated during the first half of shortening, muscle 177c generated mechanical energy (28 W x kg(-1)) like a motor, while muscle 179 absorbed energy (-19 W x kg(-1)) like a brake. Both muscles exhibited nearly identical intrinsic characteristics including similar twitch kinetics and force-velocity relationships. Differences in the extrinsic factors of activation and relative shortening velocity caused the muscles to operate very differently during running. Presumed redundancy in a multiple muscle group may, therefore, represent diversity in muscle function. Discovering how muscles manage energy during behavior requires the measurement of a large number of dynamically interacting variables.

Idiopathic adhesive capsulitis. A prospective functional outcome study of nonoperative treatment.

BACKGROUND: Idiopathic adhesive capsulitis is a commonly recognized but poorly understood cause of a painful and stiff shoulder. Although most orthopaedic literature supports treatment with physical therapy and stretching exercises, some studies have demonstrated late pain and functional deficits. The purpose of this study was to evaluate the outcome of patients with idiopathic adhesive capsulitis who were treated with a stretching-exercise program. METHODS: Seventy-five consecutive patients (seventy-seven shoulders) with phase-II idiopathic adhesive capsulitis were treated with use of a specific four-direction shoulder-stretching exercise program and evaluated prospectively. The initial evaluation included the recording of a detailed medical and orthopaedic history and assessment of pain, range of motion, and function. The outcome evaluation included assessment of pain, range of motion, and function; completion of the Disabilities of the Arm, Shoulder, and Hand (DASH) Questionnaire; and completion of the Short Form-36 (SF-36) Health Survey. The mean duration of follow-up was twenty-two months (range, twelve to forty-one months). One patient died prior to the final evaluation, and three patients were lost to follow-up. RESULTS: Sixty-four (90 percent) of the patients reported a satisfactory outcome. Seven (10 percent) were not satisfied with the outcome, and five (7 percent) underwent manipulation and/or arthroscopic capsular release. The outcomes of the patients who did not have manipulation or capsular release were evaluated. There were significant improvements in the scores for pain at rest (from a mean of 1.57 points before treatment to a mean of 1.16 points at the final evaluation; p < 0.001) and pain with activity (from a mean of 4.12 points before treatment to a mean of 1.33 points at the final evaluation; p < 0.0001). On the average, active forward elevation increased 43 degrees, active external rotation increased 25 degrees, passive internal rotation increased eight vertebral levels, and the glenohumeral rotation arc at 90 degrees of abduction increased 72 degrees (p < 0.00001). The number of "yes" responses to the Simple Shoulder Test increased from a mean of 4.1 (of a possible twelve) to a mean of 10.75 (p < 0.00001). Despite the significant improvements and the high rate of patient satisfaction, there were still significant differences in the pain and motion of the affected shoulder when compared with those of the unaffected, contralateral shoulder (p < 0.00001). At the final outcome evaluation, the DASH scores demonstrated limitations when compared with known population norms, whereas the profiles of the SF-36 were comparable with those of age and gender-matched control populations. Prior treatment with physical therapy and a Workers' Compensation claim or pending litigation were the only variables that were associated with the eventual need for manipulation or capsular release. Male gender and diabetes mellitus were associated with worse motion at the final evaluation. Patients with a greater severity of pain with activity at the initial evaluation had significantly lower DASH scores at the final evaluation, and patients with lower initial scores on the Simple Shoulder Test had comparatively lower scores on the Simple Shoulder Test at the outcome evaluation. CONCLUSIONS: The vast majority of patients who have phase-II idiopathic adhesive capsulitis can be successfully treated with a specific four-direction shoulder-stretching exercise program. Although measurable limitations and deficiencies were noted at the outcome evaluation, these appeared to be acceptable to most of the patients and did not affect their general health status. Patients with more severe pain and functional limitations before treatment had relatively worse outcomes. More aggressive treatment such as manipulation or capsular release was rarely necessary, and the efficacy of early use of these treatments should be further studied.

Formation and characterization of the trimeric form of the fusion protein of Semliki Forest Virus.

Enveloped animal viruses infect cells via fusion of the viral membrane with a host cell membrane. Fusion is mediated by a viral envelope glycoprotein, which for a number of enveloped animal viruses rearranges itself during fusion to form a trimeric alpha-helical coiled-coil structure. This conformational change from the metastable, nonfusogenic form of the spike protein to the highly stable form involved in fusion can be induced by physiological activators of virus fusion and also by a variety of destabilizing conditions. The E1 spike protein subunit of Semliki Forest virus (SFV) triggers membrane fusion upon exposure to mildly acidic pH and forms a homotrimer that appears necessary for fusion. We have here demonstrated that formation of the E1 homotrimer was efficiently triggered under low-pH conditions but not by perturbants such as heat or urea, despite their induction of generalized conformational changes in the E1 and E2 subunits and partial exposure of an acid-specific E1 epitope. We used a sensitive fluorescence assay to show that neither heat nor urea treatment triggered SFV-liposome fusion at neutral pH, although either treatment inactivated subsequent low-pH-triggered fusion activity. Once formed, the low-pH-induced E1 homotrimer was very stable and was only dissociated under harsh conditions such as heating in sodium dodecyl sulfate. Taken together, these data, as well as protein structure predictions, suggest a model in which the less stable native E1 subunit specifically responds to low pH to form the more stable E1 homotrimer via conformational changes different from those of the coiled-coil type of fusion proteins.

An epitope of the Semliki Forest virus fusion protein exposed during virus-membrane fusion.

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.

Growth and stability of a cholesterol-independent Semliki Forest virus mutant in mosquitoes.

Semliki Forest virus (SFV) is an enveloped alphavirus that is transmitted in the wild by mosquito vectors. In tissue culture cells, SFV requires cholesterol in the cell membrane both for virus membrane fusion and for the efficient exit of progeny virus from the cell. A previously isolated SFV mutant, srf-3, is strikingly less cholesterol-dependent for virus fusion, exit, and growth due to a single amino acid change in the E1 spike protein subunit, proline 226 to serine. Here we show that when mosquitoes were infected by intrathoracic injection at a range of virus multiplicities, the growth of srf-3 was significantly more rapid than that of wild-type virus, particularly at low multiplicity infection. The differential cholesterol requirements for wild-type and srf-3 infection were maintained during virus passage through mosquitoes. The presence or absence of cholesterol in the srf-3 virus membrane did not affect its infection properties in mosquitoes. Thus the srf-3 mutation causes a growth advantage in the tissues of the mosquito host.