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Rabies was eradicated from regions of Switzerland and France where it had been prevalent, through the oral vaccination of foxes. Concern that the vaccine might pose a risk to animals led to the development of an avirulent strain using monoclonal antibodies. The new strain proved as effective as its parent, and posed no risks to rodents used to test it. A second new strain is currently being tested, one which has two mutations where the first had only one and so is doubly avirulent.

Dehydroepiandrosterone (DHEA) and its metabolite androsterone (A) are natural steroids secreted in high quantities in human body. To assess the influence of oral contraceptives, menstrual cycle phase, and also physical exercise (acute and chronic such as training) on these metabolites excretions, a collection of 28 female urine specimens was organized. A three-extraction-step method was developed, and the analyses were performed by gas chromatography–mass spectrometry using deuterated 19-noretiocholanolone as the internal standard. Sample hydration state was found to be of great importance for kinetic studies, as it directly influenced the concentrations. No influence of menstrual cycle and training was found for androsterone and DHEA. However, oral contraceptive intake lowered DHEA excretion in urine and A seems to be slightly affected by exercise. [ABSTRACT FROM AUTHOR]

Over the last decade, fluctuations of retinoids (RETs), also known as vitamin A and derivatives, have proved to be useful biomarkers to assess the environmental chemical pressure on a wide variety of non-target vertebrates. This use of RET-based biomarkers is of particular interest in the non-target sentinel species Gammarus fossarum in which RETs were shown to influence crucial physiological functions. To study and probe this metabolism in this crustacean model, a UHPLC-MS/MS method was developed to 1) identify and 2) monitor several endogenous RETs in unexposed females throughout their reproductive cycle. Then, females were exposed in controlled conditions to exogenous all-trans retinoic acid (atRA) and citral (CIT), a RA synthesis inhibitor, to simulate an excess or deficiency in RA. Perturbation of vitamin A metabolism by pesticides was further studied in response to methoprene (MET), a juvenile hormone analog as well as glyphosate (GLY). The developed method allowed, for the first time in this model, the identification of RA metabolites (all-trans 4-oxo and 13-cis 4-oxo RA), RA isomers (all-trans and 13-cis RA) as well as retinaldehyde (RALD) isomers (all-trans, 11-cis, and 13-cis RALD) and showed two distinct phases in the reproductive cycle. Retinoic acid successfully increased the tissular concentration of both RA isomers and CIT proved to be efficient at perturbating the conversion from RALD to RA. Methoprene perturbed the ratios between RA isomers whereas GLY had no observed effects on the RET system of G. fossarum females. We were able to discriminate different dynamics of RET perturbations by morphogens (atRA or CIT) or MET which highlights the plausible mediation of RETs in MET-induced disorders. Ultimately, our study shows that RETs are influenced by exposure to MET and strengthen their potential to assess aquatic ecosystem chemical status., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Mollusks are very sensitive to aquatic environmental alterations and then, are important bio-indicators for monitoring the contamination of water bodies. Iodinated X-ray contrast media (ICMs) are ubiquitously present in the aquatic environment, primarily due to their high consumption for diagnosis purposes, high injection levels, low biodegradability, and low removal rates by wastewater treatment plants. Although these compounds are assumed to be of low toxicity, aquatic organisms are continuously exposed to these agents, which may result in adverse effects as ICMs can act as iodine source and disrupt the endocrine system. Thus, the evaluation of their environmental risk, especially on aquatic fauna is of great interest. To this end, we first compared the accumulation behavior, based on iodine analysis, of two ICM exhibiting different osmolality, diatrizoic acid and iohexol in Dreissena polymorpha bivalves exposed under laboratory conditions at concentrations of 0, 100, and 1000 μg/L during 4 and 7 days. This study was the first to provide information on iodine concentration in whole soft tissues and several organs in control zebra mussels. Moreover, it showed, after exposure, an increase of iodine content mainly in the digestive glands, followed by gills and gonads, highlighting that ICMs actually enter the organisms. Thus, bioaccumulation of ICMs studies were then performed, by liquid chromatography coupled to tandem mass spectrometry, on entire mollusks and digestive glands of organisms exposed at 0, 10, 100, and 1000 μg/L of both ICMs during 21 days, followed by 4 days of depuration. These first data on ICMs concentrations in zebra mussels, showed a clear accumulation of ICMs in mussels as a function of relative exposure level, as well as a rapid depuration. Osmolality did not seem to have a significant impact on the accumulation level, but a slight difference was observed on the accumulation pattern between both ICMs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: According to European guidelines for cervix cancers, the management of cervical stump cancers should follow recommendations for patients without previous sub-total hysterectomy, but may require adaptation of radiotherapy modalities, especially brachytherapy (BT). However, data with modern BT technics are very scarce., Material and Methods: From May 2013 to March 2020, 6 patients (age range, 71-88 years) with cervical stump squamous cell carcinoma (previously treated by sub-total hysterectomy) have been treated with external beam radiotherapy (intensity-modulated radiation therapy - IMRT, 45 Gy in 25 fractions, 5 patients, or 50.4 Gy in 28 fractions, 1 patient), with concomitant chemotherapy in 2 patients, followed by image-guided adaptive brachytherapy with pulsed-dose-rate in 5 patients or high-dose-rate in 1 patient. We used Utrecht BT applicator (Elekta ® ), with the implant of interstitial needles within the parametrium (unilaterally in 3 and bilaterally in 3 patients), and/or within the residual cervix (3 patients). Dosimetry was based on GEC-ESTRO recommendations., Results: The median high-risk (HR) and intermediate-risk (IR) clinical target volumes (CTV) were 14.5 cc (range, 12-35 cc) and 40 cc (range, 31-62 cc), respectively. The median dose (D 90 ) delivered to HR and IR CTVs were 81.2 (range, 78.2-89.8) and 68.95 (range, 64.3-72.3) Gy EQD 2 , respectively. With a 26.5 months median follow-up (range, 10-92 months), we did not observe any local or distant recurrence. No grade 2 or more acute or late toxicities were reported., Conclusions: Cervix brachytherapy with Utrecht applicator using interstitial needle is safe and effective in cervical stump cancer patients., Competing Interests: The authors report no conflict of interest., (Copyright © 2022 Termedia.)

This study focuses on the preparation of innovative nanocomposite materials based on surface modification of commercial nano-ZrO 2 optimized from Brønsted acid-base surface reactions. This surface modification was carried out by direct grafting of suitable phosphonic acids bearing a vinylic or phenylic substituent in aqueous solution. Different loading quantities of the anchoring organophosphorus compounds were applied for each materials synthesis. The resulting nanohybrids were thoroughly characterized by infrared spectroscopy (DRIFT), solid-state nuclear magnetic resonance (NMR), nitrogen adsorption-desorption (BET), thermogravimetric analysis (TG), and X-ray photoelectron spectroscopy (XPS), demonstrating the reliability and efficient tunability of the surface functionalization based on the starting Zr/P ratio. Our nanocomposite materials exhibited a high specific surface area as well as complex porosity networks with well-defined meso-pore. The as-prepared materials were investigated for the adsorption of a mixture of 16 polycyclic aromatic hydrocarbons (PAHs) at 200 ng·mL -1 in an aqueous solution. Adsorption kinetics experiments of each individual material were carried out on the prepared PAHs standard solution for a contact time of up to 6 h. Pretreatments of the adsorption test samples were performed by solid-phase extraction (SPE), and the resulting samples were analyzed using an ultrasensitive GC-orbitrap-MS system. The pseudo-first-order and the pseudo-second-order models were used to determine the kinetic data. The adsorption kinetics were best described and fitted by the pseudo-second-order kinetic model. The correlation between the nature of the substituent (vinylic or phenylic) and the parameters characterizing the adsorption process were found. In addition, an increase of PAHs adsorption rates with phosphonic acid loading was observed.

One of the most adopted solutions in developed countries to manage stormwater is detention/retention basins which generate large quantities of sediments that have to be removed regularly. In order to manage them properly, accurate data are needed about their physical and chemical characteristics, particularly on micropollutant concentrations and their associated risk. This work consisted in a two-year sampling of dry sediments from a detention-settling basin. Priority substances, including pesticides, polybrominated diphenyl ethers (PBDE), alkylphenols and bisphenol A (BPA), were monitored. Different sites in the basin bottom were sampled in order to investigate spatial distribution of the contamination. Results show that the increase of the sediment thickness in the basin was heterogeneous with a maximum of 15 cm after two years. Pesticides and PBDE were, if detected, mainly found in low concentrations from 2 ng/g to 286 ng/g. Conversely, alkylphenols and bisphenol A were always quantified at concentrations varying from 6 ng/g to 3400 ng/g. These high levels suggest that these sediments should be managed with precautions. Spatial heterogeneity of alkylphenol ethoxylates and BPA concentrations was observed, with higher contamination of alkylphenol ethoxylates in anaerobic zones and BPA levels correlated with total organic carbon and in a lesser extent to fine particles., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Agricultural intensification, and in particular the use of pesticides, leads over the years to a loss of biodiversity and a decline of ecosystem services in cultivated zones and agricultural landscapes. Among the animal communities involved in the functioning of agro-ecosystems, earthworms are ubiquitous and recognized as indicators of land uses and cultural practices. However, little data is available on the levels of pesticides in such organisms in natura, which would allow estimating their actual exposure and the potentially resulting impacts. Thus, the objective of this study was to develop a sensitive analytical methodology to detect and quantify 27 currently used pesticides in earthworms (Allolobophora chlorotica). A modified QuEChERS extraction was implemented on individual earthworms. This step was followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The whole analytical method was validated on spiked earthworm blank samples, with regard to linearity (from 1 to 100 method limit of quantification, r 2  > 0.95), intra-day precision (relative standard deviation (RSD) < 15%), inter-day precision (RSD < 20%), recoveries (mainly in the range 70-110%), and limits of detection and of quantification (inferior to 5 ng/g for most of the pesticides). The developed method was successfully applied to determine the concentrations of pesticides in nine individuals collected in natura. Up to five of the selected pesticides have been detected in one individual. Graphical abstract.

Plant protection spray treatments may expose non-target organisms to pesticides. In the pesticide registration procedure, the honey bee represents one of the non-target model species for which the risk posed by pesticides must be assessed on the basis of the hazard quotient (HQ). The HQ is defined as the ratio between environmental exposure and toxicity. For the honey bee, the HQ calculation is not consistent because it corresponds to the ratio between the pesticide field rate (in mass of pesticide/ha) and LD50 (in mass of pesticide/bee). Thus, in contrast to all other species, the HQ can only be interpreted empirically because it corresponds to a number of bees/ha. This type of HQ calculation is due to the difficulty in transforming pesticide field rates into doses to which bees are exposed. In this study, we used a pragmatic approach to determine the apparent exposure surface area of honey bees submitted to pesticide treatments by spraying with a Potter-type tower. The doses received by the bees were quantified by very efficient chemical analyses, which enabled us to determine an apparent surface area of 1.05 cm(2)/bee. The apparent surface area was used to calculate the exposure levels of bees submitted to pesticide sprays and then to revisit the HQ ratios with a calculation mode similar to that used for all other living species. X-tomography was used to assess the physical surface area of a bee, which was 3.27 cm(2)/bee, and showed that the apparent exposure surface was not overestimated. The control experiments showed that the toxicity induced by doses calculated with the exposure surface area was similar to that induced by treatments according to the European testing procedure. This new approach to measure risk is more accurate and could become a tool to aid the decision-making process in the risk assessment of pesticides.

The use of polymer materials in industry for product packaging is increasing. The presence of additives in the polymer matrix enables the modification or improvement of the properties and performance of the polymer, but these industries are concerned regarding the extractability of these additives. The quantification of these additives is particularly challenging because of the presence of these substances as contaminants in all the analytical equipment and the diversity of their physicochemical properties. In this context, a multi-residue analytical method was developed for the trace analysis of the twenty main additives (and their degradation products) authorized in plastic products such as pharmaceutical packaging (e.g., antioxidants, release agents, and light absorbers). This analytical method consisted of a solid phase extraction (SPE) followed by an analysis using ultra-high performance liquid chromatography coupled to a tandem mass spectrometer (UHPLC-MS/MS). A comparison of two ionization interfaces and the optimization of the extraction procedure were discussed. The influence of the quality of the solvent type (distilled versus not distilled) and the nature of the SPE cartridges (Polypropylene versus Teflon(®)) were demonstrated. The optimized method exhibited a quantification limit lower than 20 ng mL(-1) and recoveries between 70 % and 120 % for all compounds. Finally, the method was validated according to the ICH directive and was subsequently applied to the extraction of polymers under different pH conditions and storage temperatures. To the best of our knowledge, this study presents the first methodology allowing the simultaneous quantification of 24 additives at low ng mL(-1).

Endocrine disrupting compounds (EDCs) are suspected to be responsible for many disorders of the human reproductive system. To establish a causality relationship between exposure to endocrine disruptors and disease, experiments on animals must be performed with improved or new analytical tools. Therefore, a simple, rapid, and effective multi-residue method was developed for the determination of four steroid hormones (i.e., testosterone, androstenedione, estrone, and estradiol), glucuronide and sulfate conjugates of estrone and estradiol and four endocrine disruptors in rat testis (i.e., bisphenol A, atrazine, and active metabolites of methoxychlor and vinclozolin). The sample preparation procedure was based on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) approach. An analytical method was then developed to quantify these compounds at ultra-trace levels by liquid chromatography coupled to tandem mass spectrometry. The QuEChERS extraction was optimized with regard to the acetonitrile/water ratio used in the extraction step, the choice of the cleanup method and the acetonitrile/hexane ratio used in the cleanup step. The optimized extraction method exhibited recoveries between 89% and 108% for all tested compounds except the conjugates (31% to 58%). The detection limits of all compounds were below 20 ng g(-1) of wet weight of testis. The method was subsequently applied to determine the levels of hormones and EDCs in seven rat testis samples.

A simple, rapid, sensitive, and non-consuming solvent method for the determination of cotinine in urine was developed, based on sample preparation by the relatively new technique microextraction in packed sorbent (MEPS) and analysis by GC-MS. This optimized method was compared with conventional solid-phase extraction/liquid-liquid extraction method used as reference. The wide linear range (5-5,000 ng/mL) and high sensitivity of the MEPS method (limit of detection 0.8 ng/mL) allow application to analysis of urine from smokers as well as non-smokers susceptible to passive smoking.

Rabies virus glycoprotein (RVG) is known to be the only factor that mediates rabies infection. The neurotrophin receptor (p75(NTR)), through its cysteine-rich domain 1, is a specific receptor for RVG and neutralizes virus infectivity, but its role in virus infection has remained obscure. We used adult mouse dorsal root ganglion (DRG) neurons as a model to study the role of p75(NTR) in RV infection of primary neurons. We show that RV infects around 20% of DRG neurons, of which more than 80% are p75(NTR) positive, have large diameters, and are capsaicin insensitive. Surprisingly, RV binding and infection are absent in about half of the p75(NTR)-expressing DRG neurons which have small diameters and are often capsaicin sensitive. This indicates that p75(NTR) is not sufficient to mediate RV interaction in sensory neurons. The rate and specificity of neural infection are unchanged in RV-infected p75(NTRExonIV-/-) mice that lack all extracellular receptor domains and in wild-type mice infected with two independent RV mutants that lack p75(NTR) binding. Accordingly, the mortality rate is unchanged in the absence of RV-p75(NTR) interaction. We conclude that although p75(NTR) is a receptor for soluble RVG in transfected cells of heterologous expression systems, an RVG-p75(NTR) interaction is not necessary for RV infection of primary neurons. This means that other receptors are required to mediate RV infection in vivo and in vitro.

During the latent phase of herpes simplex virus type 1 (HSV-1) infection, the latency-associated transcripts (LATs) are the most abundant viral transcripts present in neurons, but some immediate-early viral transcripts, such as those encoding ICP0, have also been reported to be transcribed in latently infected mouse trigeminal ganglia (TG). A murine oro-ocular model of herpetic infection was used to study ICP0 gene expression in the major anatomical sites of HSV-1 latency, including the TG, superior cervical ganglion, spinal cord, and hypothalamus. An HSV-1 recombinant strain, SC16 110LacZ, revealed ICP0 promoter activity in several neurons in latently infected ganglia, and following infection with wild-type HSV-1 strain SC16, in situ hybridization analyses identified ICP0 transcripts in the nuclei of neurons at times consistent with the establishment of latency. Reverse transcription (RT)-PCR assays performed on RNA extracted from latently infected tissues indicated that ICP0 transcripts were detected in all anatomical sites of viral latency. Furthermore, quantitative real-time RT-PCR showed that neurons differentially expressed the LATs and ICP0 transcripts, with splicing of ICP0 transcripts being dependent on the anatomical location of latency. Finally, TG neurons were characterized by high-level expression of LATs and detection of abundant unspliced ICP0 transcripts, a pattern markedly different from those of other anatomical sites of HSV-1 latency. These results suggest that LATs might be involved in the maintenance of HSV-1 latency through the posttranscriptional regulation of ICP0 in order to inhibit expression of this potent activator of gene expression during latency.

Purpose: Between December 1998 and October 2001, patients <19 years old were treated for standard-risk medulloblastoma according to the Medulloblastome-Société Française d'Oncologie Pédiatrique 1998 (M-SFOP 98) protocol. Patients received hyperfractionated radiotherapy (36 Gy in 36 fractions) to the craniospinal axis, a boost with conformal therapy restricted to the tumor bed (to a total dose of 68 Gy in 68 fractions), and no chemotherapy. Records of craniospinal irradiation were reviewed before treatment start., Results: A total of 48 patients were considered assessable. With a median follow-up of 45.7 months, the overall survival and progression-free survival rate at 3 years was 89% and 81%, respectively. Fourteen major deviations were detected and eight were corrected. No relapses occurred in the frontal region and none occurred in the posterior fossa outside the boost volume. Nine patients were available for volume calculation without reduction of the volume irradiated. We observed a reduction in the subtentorial volume irradiated to >60 Gy, but a slight increase in the volume irradiated to 40 Gy. No decrease in intelligence was observed in the 22 children tested during the first 2 years., Conclusion: This hyperfractionated radiotherapy protocol with a reduced boost volume and without chemotherapy was not associated with early relapses in children. Moreover, intellectual function seemed to be preserved. These results are promising.

Purpose: To localize the sites of HSV1 latency in mice after a primary infection induced by injection into the lip and to assess their connection to the eye., Methods: The SC16 strain of HSV1, or a recombinant virus containing the HSV1 latency-associated transcript (LAT)-promoter driving expression of the LacZ reporter gene, were injected into the left upper lip. Tissues from animals killed at 6, 28, 180, and 720 days postinoculation (dpi) were analyzed for LATs, either by in situ hybridization (ISH) or by identifying LAT-promoter-driven transgene expression. HSV1 antigens were detected by immunochemistry., Results: At 28 dpi, all the neurologic structures that were acutely infected at 6 dpi exhibited a pattern of virus gene expression consistent with HSV1 latency--that is, LATs with no detectable HSV1 antigens. LAT staining differed among structures: intense and widespread within trigeminal neurons, intermediate within the sympathetic intermediolateral cell group of the spinal cord and the facial motor nucleus, and weak in other sites. Long-term expression of LATs (positive at 180 and 720 days) was observed only in tissues where the staining was intense or intermediate at 28 dpi., Conclusions: After inoculation into the upper lip of mice, HSV1 established latency in several nervous system structures that have direct or indirect connections with ocular tissues. These results suggest that after an oral primary infection, the most frequent in humans, HSV1 may establish latency in several sites connected to the eye and may finally result in herpetic ocular disease involving the cornea, the iris, or even the retina.

Purpose: To identify possible neuronal pathways leading to herpetic ocular disease after primary oral infection in mice., Methods: The SC16 strain of herpes simplex virus (HSV)-1 (10(6) plaque-forming units) was injected into the mucocutaneous border of the left upper lip. Animals were killed 2 to 10 days postinoculation (DPI). Spread of the virus in neural structures was studied by immunochemistry., Results: HSV1 first replicated at the site of inoculation and then at the superior cervical ganglion (at 2 DPI). The trigeminal ganglion and the facial nerve fibers were infected by 4 DPI. Infection of the ciliary body and iris occurred at 6 DPI, together with several brain stem nuclei belonging to the autonomic or sensory pathways. Between 8 and 10 DPI, the neural infection gradually cleared up, except for the ipsilateral sympathetic ganglion, and ipsilateral keratitis appeared in some animals., Conclusions: The pattern of viral dissemination in this mouse model suggests that infection of iris and ciliary body results from transfer of virus in the superior cervical ganglion from sympathetic neurons innervating the lip to neighboring neurons innervating the anterior uvea. Later, zosteriform spread of virus from the trigeminal system may have contributed to the clinical and histologic findings.

Herpetic retinitis in humans is characterized by a high frequency of bilateral localization. In order to determine the possible mechanisms leading to bilateral retinitis, we studied the pathways by which herpes simplex virus type 1 (HSV-1) is propagated from one retina to the other after intravitreal injection in mice. HSV-1 strain SC16 (90 p.f.u.) was injected into the vitreous body of the left eye of BALB/c mice. Animals were sacrificed 1, 2, 3, 4 and 5 days post-inoculation (p.i.). Histological sections were studied by immunochemical staining. Primary retinitis in the inoculated eye (beginning 1 day p.i.) was followed by contralateral retinitis (in the uninoculated eye) starting at 3 days p.i. Infected neurons of central visual pathway nuclei (lateral geniculate nuclei, suprachiasmatic nuclei and pretectal areas) were detected at 4 days p.i. Iris and ciliary body infection was minimal early on, but became extensive thereafter and was accompanied by the infection of connected sympathetic and parasympathetic pathways. The pattern of virus propagation over time suggests that the onset of contralateral retinitis was mediated by local (non-synaptic) transfer in the optic chiasm from infected to uninfected axons of the optic nerves. Later, retinopetal transneuronal propagation of the virus from visual pathways may have contributed to increase the severity of contralateral retinitis.

Around 15% of our anti-glycoprotein monoclonal antibodies (MAbs) failed to neutralize the infectivity of the rabies virus during a 1-hr incubation at room temperature. In previous studies, we have demonstrated that it is possible to induce a massive conformational change of the glycoprotein population by incubating the virus at acidic pH. The conformational change is reversible and consequently viral infectivity is not affected by transient exposure at acidic pH. The proportion of glycoproteins in acidic or neutral configuration depends on the pH which means that even at neutral pH some glycoproteins transiently adopt the acidic configuration and vice versa. Here we report that some of our nonneutralizing MAbs recognize the acidic form of the glycoprotein at the virion surface. After incubation of the virus at pH 6.4, most glycoproteins are in the acidic configuration. Further 1-hr incubation with these MAbs at the same pH resulted in more immunoglobulins being attached to the virus and consequently neutralization was induced. It was also possible to induce neutralization with the same MAbs by incubation at neutral pH for a longer period or at a higher temperature. Mutants resistant to neutralization by these MAbs could be selected. Mutations confering resistance to neutralization were not localized in previously described antigenic sites and did not modify these sites at distance. They had no effect on the pathogenic power of the virus. Either they are situated in the epitope or they modify the epitope, so that it is no longer recognized by the antibody on the acidic configuration of the protein. Alternatively, these mutations may stabilize the protein in its neutral configuration. In addition, these experiments confirm our previous finding that neutralization requires the fixation of a large number of immunoglobulins on the virus, irrespective of the region of the protein recognized by the antibody.

All five major viral proteins were synthesized in chicken embryo cells infected with vesicular stomatitis virus temperature-sensitive (ts) mutants of complementation groups III and V and maintained at the nonpermissive temperature. The distribution of these proteins among cytoplasmic cellular fractions separated on discontinuous sucrose gradients was identical for wild-type and tsIII-infected cells. Strikingly different patterns were observed for the G protein in gradients from cells infected by tsV mutants; very little, if any, G protein was found in the lightest fraction. Pulse and chase experiments with wild-type, virus-infected cells showed that protein G moves from the heaviest to the lightest fraction before being incorporated into the virion. After shift down to the permissive temperature (30 C), G protein synthesized at 39.6 C in tsV-infected cells became associated with the lightest cellular fraction and later with the released virions. In contrast, M protein, synthesized at 39.6 C in tsIII-infected cells, was not incorporated into the virions after shift down. These data strongly suggest, first, that M protein is encoded by the vesicular stomatitis gene III, and second, that incorporation of G protein in the lightest cellular fraction is a necessary step of vesicular stomatitis maturation. This step is impaired by tsV mutations.