The Cellular Characterization of SARS-CoV-2 Spike Protein in Virus-Infected Cells Using the Receptor Binding Domain Binding Specific Human Monoclonal Antibodies.

A human monoclonal antibody panel (PD4, PD5, PD7, SC23, and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2-infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 spike (S) protein, only PD5, PD7, and SC23 were able to bind to the receptor binding domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localized within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD-binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognized the uncleaved S protein, indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining were demonstrated for each isolate, SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection. IMPORTANCE The SARS-CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS-CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection, and the S protein RBD is therefore a focus of therapeutic and vaccine interventions. However, new virus variants have emerged with altered biological properties in the RBD that can potentially negate these interventions. Therefore, an improved understanding of the biological properties of the RBD in virus-infected cells may offer future therapeutic strategies to mitigate SARS- CoV-2 infection. We used physiologically relevant antibodies that were isolated from the B cells of convalescent COVID-19 patients to monitor the RBD in cells infected with SARS-CoV-2 clinical isolates. These immunological reagents specifically recognize the correctly folded RBD and were used to monitor the appearance of the RBD in SARS-CoV-2-infected cells and identified the site where the RBD first appears.

Characteristics of Effective Auditory Training: Implications From Two Training Programs That Successfully Trained Nonnative Cantonese Tone Identification in Monolingual Mandarin and Bilingual Mandarin-Taiwanese Tone Speakers.

Purpose Auditory training is important in pedagogical and clinical settings. In search of a more effective perceptual program for training new suprasegmental categories, this study examined the effect of two auditory programs that incorporated five elements that have previously been identified to be effective for training nonnative segmental and suprasegmental speech sounds on the identification of a complex foreign lexical tone system (Cantonese) that contrasts both pitch shapes and pitch heights. To investigate the training outcomes in learners with different tonal systems, monolingual Mandarin-speaking learners who have a smaller native tonal system that contrasts pitch shapes only and bilingual Mandarin-Taiwanese-speaking learners who have a larger native tonal system that contrasts both pitch shapes and pitch heights were recruited for training. Method Thirty Mandarin-speaking monolinguals and 33 Mandarin-Taiwanese-speaking bilinguals in Taiwan were randomly assigned to two training programs, one with different tones and the other with the same tone preceding the target words in the same training block, and received six 90-min training sessions within 2 weeks. They took a Cantonese Tone Identification Test before training and after each training session. Twenty Cantonese native speakers in Hong Kong served as the reference group and took the same Cantonese Tone Identification Test. Results The two training programs were equally effective. Before training, the monolinguals performed poorer than the bilinguals. After training, the monolinguals and bilinguals in both training programs identified the six Cantonese tones in new words, new utterances, and novel speakers with comparable results, and their overall accuracy did not differ from that of the Cantonese native speakers. Conclusions Though learners with a larger and more complex native tonal system have initial advantage in learning nonnative tones, the intensive high-variability full-set training programs that provide explicit phonetic instruction and contrastive feedback of nonnative tones effectively promote nonnative tone acquisition in learners of different tone languages. The findings revealed factors affecting nonnative tone acquisition in tone speakers. The design of the two programs can be adopted in future programs for effective auditory training of segmental and suprasegmental speech sounds.

On the Relationship Between General Auditory Sensitivity and Speech Perception: An Examination of Pitch and Lexical Tone Perception in 4- to 6-Year-Old Children.

Purpose Theoretical models and substantial research have proposed that general auditory sensitivity is a developmental foundation for speech perception and language acquisition. Nonetheless, controversies exist about the effectiveness of general auditory training in improving speech and language skills. This research investigated the relationships among general auditory sensitivity, phonemic speech perception, and word-level speech perception via the examination of pitch and lexical tone perception in children. Method Forty-eight typically developing 4- to 6-year-old Cantonese-speaking children were tested on the discrimination of the pitch patterns of lexical tones in synthetic stimuli, discrimination of naturally produced lexical tones, and identification of lexical tone in familiar words. Results The findings revealed that accurate lexical tone discrimination and identification did not necessarily entail the accurate discrimination of nonlinguistic stimuli that followed the pitch levels and pitch shapes of lexical tones. Although pitch discrimination and tone discrimination abilities were strongly correlated, accuracy in pitch discrimination was lower than that in tone discrimination, and nonspeech pitch discrimination ability did not precede linguistic tone discrimination in the developmental trajectory. Conclusions Contradicting the theoretical models, the findings of this study suggest that general auditory sensitivity and speech perception may not be causally or hierarchically related. The finding that accuracy in pitch discrimination is lower than that in tone discrimination suggests that comparable nonlinguistic auditory perceptual ability may not be necessary for accurate speech perception and language learning. The results cast doubt on the use of nonlinguistic auditory perceptual training to improve children's speech, language, and literacy abilities.

Detection and characterization of a novel marine birnavirus isolated from Asian seabass in Singapore.

BACKGROUND: Lates calcarifer, known as seabass in Asia and barramundi in Australia, is a widely farmed species internationally and in Southeast Asia and any disease outbreak will have a great economic impact on the aquaculture industry. Through disease investigation of Asian seabass from a coastal fish farm in 2015 in Singapore, a novel birnavirus named Lates calcarifer Birnavirus (LCBV) was detected and we sought to isolate and characterize the virus through molecular and biochemical methods. METHODS: In order to propagate the novel birnavirus LCBV, the virus was inoculated into the Bluegill Fry (BF-2) cell line and similar clinical signs of disease were reproduced in an experimental fish challenge study using the virus isolate. Virus morphology was visualized using transmission electron microscopy (TEM). Biochemical analysis using chloroform and 5-Bromo-2'-deoxyuridine (BUDR) sensitivity assays were employed to characterize the virus. Next-Generation Sequencing (NGS) was also used to obtain the virus genome for genetic and phylogenetic analyses. RESULTS: The LCBV-infected BF-2 cell line showed cytopathic effects such as rounding and granulation of cells, localized cell death and detachment of cells observed at 3 to 5 days' post-infection. The propagated virus, when injected intra-peritoneally into naïve Asian seabass under experimental conditions, induced lesions similar to fish naturally infected with LCBV. Morphology of LCBV, visualized under TEM, revealed icosahedral particles around 50 nm in diameter. Chloroform and BUDR sensitivity assays confirmed the virus to be a non-enveloped RNA virus. Further genome analysis using NGS identified the virus to be a birnavirus with two genome segments. Phylogenetic analyses revealed that LCBV is more closely related to the Blosnavirus genus than to the Aquabirnavirus genus within the Birnaviridae family. CONCLUSIONS: These findings revealed the presence of a novel birnavirus that could be linked to the disease observed in the Asian seabass from the coastal fish farms in Singapore. This calls for more studies on disease transmission and enhanced surveillance programs to be carried out to understand pathogenicity and epidemiology of this novel virus. The gene sequences data obtained from the study can also pave way to the development of PCR-based diagnostic test methods that will enable quick and specific identification of the virus in future disease investigations.

A System Based-Approach to Examine Cytokine Response in Poxvirus-Infected Macrophages.

The poxviruses are large, linear, double-stranded DNA viruses about 130 to 230 kbp, that have an animal origin and evolved to infect a wide host range. Variola virus (VARV), the causative agent of smallpox, is a poxvirus that infects only humans, but other poxviruses such as monkey poxvirus and cowpox virus (CPXV) have crossed over from animals to infect humans. Therefore understanding the biology of poxviruses can devise antiviral strategies to prevent these human infections. In this study we used a system-based approach to examine the host responses to three orthopoxviruses, CPXV, vaccinia virus (VACV), and ectromelia virus (ECTV) in the murine macrophage RAW 264.7 cell line. Overall, we observed a significant down-regulation of gene expressions for pro-inflammatory cytokines, chemokines, and related receptors. There were also common and virus-specific changes in the immune-regulated gene expressions for each poxvirus-infected RAW cells. Collectively our results showed that the murine macrophage RAW 264.7 cell line is a suitable cell-based model system to study poxvirus host response.

Cantonese Tone Identification in Three Temporal Cues in Quiet, Speech-Shaped Noise and Two-Talker Babble.

Purpose: Cochlear implant processors deliver mostly temporal envelope information and limited fundamental frequency (F0) information to the users, which make pitch and lexical tone perception challenging for cochlear implantees. Different factors have been found to affect Mandarin tone perception in temporal cues but the most effective temporal cues for lexical tone identification across different backgrounds remained unclear because no study has comprehensively examined the effects and interactions of these factors, particularly, in languages that use both pitch heights and pitch shapes to differentiate lexical meanings. The present study compared identification of Cantonese tones in naturally produced stimuli, and in three temporal cues, namely the amplitude contour cue (TE50), the periodicity cue (TE500), and the temporal fine structure cue (TFS), in three different numbers of frequency bands (B04, B08, B16) in quiet and two types of noise (two male talker-babble and speech-shaped noise). Method: Naturally produced Cantonese tones and synthetic tones that combined different acoustic cues and different number of frequency bands were presented to 18 young native Cantonese speakers for tone identification in quiet and noise. Results: Among the three temporal cues, TFS was the most effective for Cantonese tone identification in quiet and noise, except for T4 (LF) identification. Its effect was even stronger when the tones were presented in 4 or 8 bands rather than 16 bands. Neither TE500 nor TE50 was effective for Cantonese tone identification in quiet or noise. In noise, most tones in TE500 and TE50 were misheard as T4 (LF), demonstrating errors in both tone shapes and tone heights. Types of noise had limited effect on tone identification. Conclusions: Findings on Mandarin tone perception in temporal cues may not be applicable to other tone languages with more complex tonal systems. TFS presented in four bands was the most effective temporal cue for Cantonese tone identification in quiet and noise. Temporal envelope cues were not effective for tone, tone shape or tone height identification in Cantonese. These findings have implications for future design of cochlear implants for tone speakers who use pitch heights or a combination of pitch heights and pitch shapes to differentiate meanings.

Testing the Hyperarticulation and Prosodic Hypotheses of Child-Directed Speech: Insights From the Perceptual and Acoustic Characteristics of Child-Directed Cantonese Tones.

Purpose: The function of child-directed speech has been debated for decades. This study examined the perceptual and acoustic characteristics of child- and adult-directed Cantonese tones to test the hyperarticulation and prosodic hypotheses that have been proposed to account for the acoustic modifications in child-directed speech. Method: Sixty-two mother-child dyads participated in the study. The mothers verbally labeled 30 pictures in monosyllabic isolated words and in the final position of a carrier sentence to the experimenter and their 1- to 5-year-old children. The 8,634 adult- and child-directed productions were low-pass filtered to eliminate lexical information and presented to 5 judges for tone identification. Acoustic analysis was performed on the productions. Results: Acoustically, child-directed tones were produced with an elevated pitch, and the pitch level decreased as the child's age increased. Acoustic contrasts between phonetically similar and more confusing tones were not enhanced in child-directed speech, and unexpectedly, child-directed tones were identified with a lower accuracy than adult-directed tones. The perceptual errors of child-directed tones mirrored the errors found in identifying tones excised from sentence-final position, which had a pitch-lowering effect on the tones. The lower perceptual accuracy, the lack of enhanced acoustic contrasts in confusing tone pairs, and the similarities in the error patterns in identifying tones in child-directed speech and tones in utterance-final position suggest that the acoustic modifications in child-directed tones are prosodic effects serving pragmatic purposes. Conclusion: The findings reject the hyperarticulation hypothesis and support the prosodic hypothesis of child-directed speech.

Mothers do not enhance tonal contrasts in child-directed speech: Perceptual and acoustic evidence from child-directed Mandarin lexical tones.

Prosodically, child-directed speech typically has a higher pitch and more varied pitch contours. Studies that have examined acoustic differences between child-directed and adult-directed vowels and consonants have reported mixed results and proposed two hypotheses explaining the function of the acoustic modifications in child-directed speech. The hyperarticulation hypothesis suggests that mothers enhance the phonemic contrasts in child-directed speech to facilitate speech and language acquisition in children. The pragmatic hypothesis claims that the acoustic differences between child-directed and adult-directed speech result from mothers' expression of affective emotions towards young children. In tone languages, pitch is used at the syllable level to make lexical contrasts and at the utterance level to serve pragmatic functions. This study compared the perceptual clarity and acoustic characteristics of adult-directed and child-directed Mandarin tones to test the two hypotheses. 1648 child-directed and adult-directed tones produced by 20 mothers in monosyllabic and disyllabic words were low-pass filtered to eliminate segmental information and presented to five judges for tone identification. Child-directed tones were identified with poorer accuracy than adult-directed tones. Acoustic analysis revealed that child-directed tones, regardless of tone type, were produced with higher pitch and more positive slopes than adult-directed tones. The findings did not support the hyperarticulation hypothesis.

Suprasegmental Features Are Not Acquired Early: Perception and Production of Monosyllabic Cantonese Lexical Tones in 4- to 6-Year-Old Preschool Children.

Purpose: Previous studies reported that children acquire Cantonese tones before 3 years of age, supporting the assumption in models of phonological development that suprasegmental features are acquired rapidly and early in children. Yet, recent research found a large disparity in the age of Cantonese tone acquisition. This study investigated Cantonese tone development in 4- to 6-year-old children. Method: Forty-eight 4- to 6-year-old Cantonese-speaking children and 28 mothers of the children labeled 30 pictures representing familiar words in the 6 tones in a picture-naming task and identified pictures representing words in different Cantonese tones in a picture-pointing task. To control for lexical biases in tone assessment, tone productions were low-pass filtered to eliminate lexical information. Five judges categorized the tones in filtered stimuli. Tone production accuracy, tone perception accuracy, and correlation between tone production and perception accuracy were examined. Results: Children did not start to produce adultlike tones until 5 and 6 years of age. Four-year-olds produced none of the tones with adultlike accuracy. Five- and 6-year-olds attained adultlike productions in 2 (T5 and T6) to 3 (T4, T5, and T6) tones, respectively. Children made better progress in tone perception and achieved higher accuracy in perception than in production. However, children in all age groups perceived none of the tones as accurately as adults, except that T1 was perceived with adultlike accuracy by 6-year-olds. Only weak association was found between children's tone perception and production accuracy. Conclusions: Contradicting to the long-held assumption that children acquire lexical tone rapidly and early before the mastery of segmentals, this study found that 4- to 6-year-old children have not mastered the perception or production of the full set of Cantonese tones in familiar monosyllabic words. Larger development was found in children's tone perception than tone production. The higher tone perception accuracy but weak correlation between tone perception and production abilities in children suggested that tone perception accuracy is not sufficient for children's tone production accuracy. The findings have clinical and theoretical implications.

Acoustic characteristics of highly distinguishable Cantonese entering and non-entering tones.

Cantonese has one of the most complex tone systems. Few studies have thoroughly examined or compared the acoustic properties of the full set of Cantonese tones, particularly the entering tones, compromising deeper understanding of Cantonese tone difficulties in various clinical populations. This study (1) describes a theory-driven method for acoustic analysis of tones that successfully normalized the intrinsic pitch of male and female speakers, (2) provides detailed acoustic data on distinctly enunciated Cantonese tones, (3) examines the acoustic similarities and differences between the entering and non-entering tones, and (4) compares the acoustic properties of three easily confused tone pairs. Seventeen male and female native speakers produced 1802 Cantonese tones that were correctly identified by five judges in filtered stimuli. Counter to the established notion that the entering tones are shorter versions of the three level tones, the results revealed that the entering tones have falling contours, suggesting that the entering and non-entering tones should be examined separately in research and clinical settings. The detailed description of the acoustic properties of the nine tones and the acoustic contrasts of the entering and non-entering tones and the three easily confused tone pairs provides references for future Cantonese tone studies with different populations.

Cantonese-Speaking Children Do Not Acquire Tone Perception before Tone Production-A Perceptual and Acoustic Study of Three-Year-Olds' Monosyllabic Tones.

Models of phonological development assume that speech perception precedes speech production and that children acquire suprasegmental features earlier than segmental features. Studies of Chinese-speaking children challenge these assumptions. For example, Chinese-speaking children can produce tones before two-and-a-half years but are not able to discriminate the same tones until after 6 years of age. This study compared the perception and production of monosyllabic Cantonese tones directly in 3 -year-old children. Twenty children and their mothers identified Cantonese tones in a picture identification test and produced monosyllabic tones in a picture labeling task. To control for lexical biases on tone ratings, the mother- and child-productions were low-pass filtered to eliminate lexical information and were presented to five judges for tone classification. Detailed acoustic analysis was performed. Contrary to the view that children master lexical tones earlier than segmental phonemes, results showed that 3-year-old children could not perceive or produce any Cantonese tone with adult-like proficiency and incorrect tone productions were acoustically different from criterion. In contrast to previous findings that Cantonese-speaking children mastered tone production before tone perception, we observed more accuracy during speech perception than production. Findings from Cantonese-speaking children challenge some of the established tenets in theories of phonological development that have been tested mostly with native English speakers.

Phonetic complexity affects children's Mandarin tone production accuracy in disyllabic words: A perceptual study.

This is the first study to examine the effect of phonetic contexts on children's lexical tone production. Mandarin tones in disyllabic words produced by forty-four 2- to 6-year-old children and twelve mothers were low-pass filtered to eliminate lexical information. Native Mandarin-speaking adults categorized the tones based on the pitch information in the filtered stimuli. All mothers' tones were categorized with ceiling accuracy. Counter to the findings in most previous studies on children's tone acquisition and the prevailing assumption in models of speech development that children acquire suprasegmental features much earlier than segmental features, this study found that children as old as six years of age have not mastered the production of Mandarin tones. Children's tones were judged with significantly lower accuracy than mothers' productions. Tone accuracy improved, while cross subject variability in tone accuracy decreased, with age. Children's tone accuracy was affected by the articulatory complexity of phonetic contexts. Children made more errors in tone combinations with more complex fundamental frequency (F0) contours than tone sequences with simpler F0 changes. When producing disyllabic tone sequences with complex F0 contours, children tended to shift the F0 contour of the first tone to reduce the F0 change, resulting in more tone errors in the first syllable than in the second syllable and showing substantially more anticipatory coarticulation than adults. The results provide further evidence that acquisition of lexical tones is a protracted process in children. Tones produced accurately by children in one phonetic context may not be produced correctly in another phonetic context. Children demonstrate more anticipatory coarticulation in their disyllabic productions than adults, which may be attributed to children's immature speech motor control in tone production, and is presumably a by-product of their inability to accomplish complex F0 changes within the syllable time-frame.

Hepatitis E in Singapore: A Case-Series and Viral Phylodynamics Study.

AbstractThe incidence of hepatitis E in Singapore appears to be increasing. A retrospective case-series study of patients diagnosed with hepatitis E in a tertiary hospital from 2009 to 2013 was conducted. Of 16 cases, eight (50%) were solid-organ transplant recipients (SOTRs), and 14 (88%) were found infected by genotype 3 hepatitis E virus (HEV-3). Bayesian inferences based on HEV subgenomic sequences from seven cases suggest that HEV-3 strains were introduced to Singapore as two principal lineages. Within limitations of the study, it can be inferred that one lineage, in the 3efg clade, emerged about 83 years ago, probably originating from Japan, whereas the other, in the 3abchij clade, emerged about 40 years ago, from the United States. Establishment and subsequent transmissions of strains from these two lineages likely contribute to the current endemicity of hepatitis E in Singapore.

Molecular and biochemical characterization of the NS1 protein of non-cultured influenza B virus strains circulating in Singapore.

In this study we compared the NS1 protein of Influenza B/Lee/40 and several non-cultured Influenza B virus clinical strains detected in Singapore. In B/Lee/40 virus-infected cells and in cells expressing the recombinant B/Lee/40 NS1 protein a full-length 35 kDa NS1 protein and a 23 kDa NS1 protein species (p23) were detected. Mutational analysis of the NS1 gene indicated that p23 was generated by a novel cleavage event within the linker domain between an aspartic acid and proline at amino acid residues at positions 92 and 93 respectively (DP92-93), and that p23 contained the first 92 amino acids of the NS1 protein. Sequence analysis of the Singapore strains indicated the presence of either DP92-93 or NP92-93 in the NS1 protein, but protein expression analysis showed that p23 was only detected in NS1 proteins with DP92-93.. An additional adjacent proline residue at position 94 (P94) was present in some strains and correlated with increased p23 levels, suggesting that P94 has a synergistic effect on the cleavage of the NS1 protein. The first 145 amino acids of the NS1 protein are required for inhibition of ISG15-mediated ubiquitination, and our analysis showed that Influenza B viruses circulating in Singapore with DP92-93 expressed truncated NS1 proteins and may differ in their capacity to inhibit ISG15 activity. Thus, DP92-93 in the NS1 protein may confer a disadvantage to Influenza B viruses circulating in the human population and interestingly the low frequency of DP92-93detection in the NS1 protein since 2004 is consistent with this suggestion.

Morphogenesis of respiratory syncytial virus in human primary nasal ciliated epithelial cells occurs at surface membrane microdomains that are distinct from cilia.

The distribution of cilia and the respiratory syncytial virus (RSV) nucleocapsid (N) protein, fusion (F) protein, attachment (G) protein, and M2-1 protein in human ciliated nasal epithelial cells was examined at between 1 and 5 days post-infection (dpi). All virus structural proteins were localized at cell surface projections that were distinct from cilia. The F protein was also trafficked into the cilia, and while its presence increased as the infection proceeded, the N protein was not detected in the cilia at any time of infection. The presence of the F protein in the cilia correlated with cellular changes in the cilia and reduced cilia function. At 5dpi extensive cilia loss and further reduced cilia function was noted. These data suggested that although RSV morphogenesis occurs at non-cilia locations on ciliated nasal epithelial cells, RSV infection induces changes in the cilia body that leads to extensive cilia loss.

Imaging analysis of human metapneumovirus-infected cells provides evidence for the involvement of F-actin and the raft-lipid microdomains in virus morphogenesis.

BACKGOUND: Due to difficulties of culturing Human metapneumovirus (HMPV) much of the current understanding of HMPV replication can be inferred from other closely related viruses. The slow rates of virus replication prevent many biochemical analyses of HMPV particles. In this study imaging was used to examine the process of HMPV morphogenesis in individually infected LLC-MK2 cells, and to better characterise the sites of HMPV assembly. This strategy has circumvented the problems associated with slow replication rates and allowed us to characterise both the HMPV particles and the sites of HMPV morphogenesis. METHODS: HMPV-infected LLC-MK2 cells were stained with antibodies that recognised the HMPV fusion protein (F protein), attachment protein (G protein) and matrix protein (M protein), and fluorescent probes that detect GM1 within lipid-raft membranes (CTX-B-AF488) and F-actin (Phalloidin-FITC). The stained cells were examined by confocal microscopy, which allowed imaging of F-actin, GM1 and virus particles in HMPV-infected cells. Cells co-expressing recombinant HMPV G and F proteins formed virus-like particles and were co-stained with antibodies that recognise the recombinant G and F proteins and phalloidin-FITC and CTX-B-AF594, and the distribution of the G and F proteins, GM1 and F-actin determined. RESULTS: HMPV-infected cells stained with anti-F, anti-G or anti-M revealed a filamentous staining pattern, indicating that the HMPV particles have a filamentous morphology. Staining of HMPV-infected cells with anti-G and either phalloidin-FITC or CTX-B-AF488 exhibited extensive co-localisation of these cellular probes within the HMPV filaments. This suggested that lipid-raft membrane domains and F-actin structures are present at the site of the virus morphogenesis, and are subsequently incorporated into the HMPV filaments. Furthermore, the filamentous virus-like particles that form in cells expressing the G protein formed in cellular structures containing GM1 and F-actin, suggesting the G protein contains intrinsic targeting signals to the sites of virus assembly. CONCLUSIONS: These data suggest that HMPV matures as filamentous particles and that virus morphogenesis occurs within lipid-raft microdomains containing localized concentrations of F-actin. The similarity between HMPV morphogenesis and the closely related human respiratory syncytial virus suggests that involvement of F-actin and lipid-raft microdomains in virus morphogenesis may be a common feature of the Pneumovirinae.

Perceptual evidence for protracted development in monosyllabic Mandarin lexical tone production in preschool children in Taiwan.

This study used the same methodology in Wong [J. Speech Lang. Hear. Res. 55, 1423-1437 (2012b)] to examine the perceived accuracy of monosyllabic Mandarin tones produced by 4- and 5-year-old Mandarin-speaking children growing up in Taiwan and combined the findings with those of 3-year-olds reported in Wong [J. Speech Lang. Hear. Res. 55, 1423-1437 (2012b)] to track the development of monosyllabic tone production in preschool children. Tone productions of adults and children were collected in a picture naming task and low-pass filtered to remove lexical information and reserve tone information. Five native-speakers categorized the target tones in the filtered productions. Children's tone accuracy was compared to adults' to determine mastery and developmental changes. The results showed that preschool children in Taiwan have not fully mastered the production of monosyllabic Mandarin tones. None of the tones produced by the children in the three age groups reached adult-like accuracy. Little developmental change was found in children's tone accuracy during the preschool years. A similar order of accuracy of the tones was observed across the three age groups and the order appeared to follow the order of articulatory complexity in producing the tones. The findings suggest a protracted course of development in children's acquisition of Mandarin tones and that tone development may be constrained by physiological factors.

Monosyllabic Mandarin tone productions by 3-year-olds growing up in Taiwan and in the United States: interjudge reliability and perceptual results.

PURPOSE: The author compared monosyllabic Mandarin lexical tones produced by 3-year-old Mandarin-speaking children growing up in Taiwan and in the United States. METHOD: Following the procedures in Wong, Schwartz, and Jenkins (2005), the author collected monosyllabic tone productions from 3-year-old Mandarin-speaking children in Taiwan and low-pass filtered them to eliminate lexical information but retain tone information. Five Mandarin-speaking adults residing in Taiwan categorized these filtered tones and those produced by the Mandarin-speaking children growing up in the United States, the latter of which was reported in Wong et al. (2005). Agreements on tone categorization by judges residing in Taiwan and in the United States were evaluated. Tone accuracy of children growing up in Taiwan and the United States were examined and compared. RESULTS: The Mandarin-speaking judges residing in the United States and in Taiwan showed high agreements on tone categorization. None of the 4 tones produced by the Mandarin-speaking children growing up in the United States and in Taiwan was adultlike.Children in Taiwan made more errors in Tone 2 and Tone 4 than did Mandarin-speaking children growing up in the United States. Accuracy rates of Tone 1 and Tone 3 were comparable in the 2 groups of children. CONCLUSION: Mandarin tone acquisition is a protracted process. Three-year-old Mandarin-speaking children growing up in Taiwan and the United States show similar developmental patterns and have not yet produced monosyllabic tones with adultlike accuracy.

Perception and production of lexical tones by 3-year-old, Mandarin-speaking children.

The present study investigated 3-year-old children's perception and production of Mandarin lexical tones in monosyllabic words. Thirteen 3-year-old, Mandarin-speaking children participated in the study. Tone perception was examined by a picture-pointing task, and tone production was investigated by picture naming. To compare children's productions with the adult forms, 4 mothers of the children were asked to say the same set of words to their children in a picture-reading activity. The children's and mothers' productions were low-pass filtered at 500 Hz and 400 Hz, respectively, to eliminate segmental information. Ten Mandarin-speaking judges identified the productions of tones from the filtered speech. Adult productions were more accurately identified than productions of the children. The children perceived the level, rising, and falling tones with relatively high accuracy. The dipping tone posed the greatest difficulty for the children in both perception and production.