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SUMMARYThe SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localises to mitochondria during infection, where it inhibits innate immunity by restricting IFN-β production, but not NF-κB activation or JAK-STAT signalling downstream of type I IFN stimulation. We find that ORF3c acts after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterised mechanism of innate immune evasion by this important human pathogen.

Despite being the target of extensive research efforts due to the COVID-19 (coronavirus disease 2019) pandemic, relatively little is known about the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication within cells. We investigate and characterize the tightly orchestrated virus assembly by visualizing the spatiotemporal dynamics of the four structural SARS-CoV-2 proteins at high resolution. The nucleoprotein is expressed first and accumulates around folded endoplasmic reticulum (ER) membranes in convoluted layers that contain viral RNA replication foci. We find that, of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ER-to-Golgi intermediate compartment before the spike and envelope proteins. Relocation of a lysosome marker toward the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide insights into the spatiotemporal regulation of SARS-CoV-2 assembly and refine the current understanding of SARS-CoV-2 replication.

Fragile X-associated premature ovarian insufficiency (FXPOI) is caused by expansion of a CGG repeat sequence located in the 5’ untranslated region of the FMR1 gene. Women with FXPOI have a depleted ovarian reserve, resulting in amenorrhea, hypoestrogenism, and loss of fertility before the age of 40. FXPOI is caused by CGG sequence expansions to lengths between 55 and 200 repeats, known as a FMRI premutation, however the mechanism by which the premutation drives disease pathogenesis remains unclear. Two main hypotheses exist, which describe an mRNA toxic gain-of-function mechanism or that repeat-associated non-AUG (RAN) translation results in the production of an abnormal protein, called FMRpolyG. We have developed an in vitro granulosa cell model of the FMR1 premutation by ectopically expressing CGG-repeat RNA and FMRpolyG protein. We show that expanded CGG-repeat RNA accumulated in intranuclear RNA structures, and these aggregates were able to cause significant granulosa cell death independent of FMRpolyG expression. Furthermore, using an innovative RNA pulldown, mass spectrometry-based approach we have identified proteins that bind CGG-repeat RNA in granulosa cells in vitro, and thus may be deregulated as consequence of this interaction. Collectively, these data provide evidence for the contribution of an mRNA gain-of-function mechanism to FXPOI disease biology.

B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3’ of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern.

Despite being the target of extensive research efforts due to the COVID-19 pandemic, relatively little is known about the dynamics of SARS-CoV-2 replication within cells. We investigate and characterise the tightly orchestrated sequence of events during different stages of the infection cycle by visualising the spatiotemporal dynamics of the four structural proteins of SARS-CoV-2 at high resolution. The nucleoprotein is expressed first and accumulates around folded ER membranes in convoluted layers that connect to viral RNA replication foci. We find that of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ERGIC before the spike and envelope proteins. Relocation of the lysosome marker LAMP1 towards the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide new insights into the spatiotemporal regulation of SARS-CoV-2 assembly, and refine current understanding of SARS-CoV-2 replication.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including theCoronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrated that SARS-CoV-2 infection causes tetherin downregulation, and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigated the potential viral proteins involved in abrogating tetherin function and found that SARS- CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles via reduced retrograde recycling and increases tetherin localisation to late endocytic organelles. By removing tetherin from the Coronavirus budding compartments, ORF3a enhances virus release. We also found expression of Spike protein caused a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.Author SummarySince it was identified in 2019, SARS-CoV-2 has displayed voracious transmissibility which has resulted in rapid spread of the virus and a global pandemic. SARS-CoV-2 is a member of theCoronaviridaefamily whose members are encapsulated by a host-derived protective membrane shell. Whilst the viral envelope may provide protection for the virus, it also provides an opportunity for the host cell to restrict the virus and stop it spreading. The anti-viral restriction factor, tetherin, acts to crosslink viruses to the surface of infected cells and prevent their spread to uninfected cells. Here, we demonstrate that SARS-CoV-2 undergoes viral restriction by tetherin, and that SARS-CoV-2 moves tetherin away from the site of Coronavirus budding to enhance its ability to escape and infect naïve cells. Tetherin depletion from cells enhanced SARS-CoV-2 viral release and increased propagation of the virus. We found that the SARS-CoV-2 protein, ORF3a, redirects tetherin away from the biosynthetic organelles where tetherin would become incorporated to newly forming SARS-CoV-2 virions – and instead relocalises tetherin to late endocytic organelles. We also found that SARS-CoV-2 Spike downregulates tetherin. These two mechanisms, in addition to the well described antagonism of interferon and subsequent ISGs highlight the multiple mechanisms by which SARS-CoV-2 abrogates tetherin function. Our study provides new insights into how SARS-CoV-2 subverts human antiviral responses and escapes from infected cells.

Equine hepacivirus (EHcV) (now also classified as hepacivirus A) is the closest genetic relative to hepatitis C virus (HCV) and is proposed to have diverged from HCV within the last 1000 years. The 5′ untranslated regions (UTRs) of both HCV and EHcV exhibit internal ribosome entry site (IRES) activity, allowing cap-independent translational initiation, yet only the HCV 5′UTR has been systematically analysed. Here, we report a detailed structural and functional analysis of the EHcV 5′UTR. The secondary structure was determined using selective 2′ hydroxyl acylation analysed by primer extension (SHAPE), revealing four stem–loops, termed SLI, SLIA, SLII and SLIII, by analogy to HCV. This guided a mutational analysis of the EHcV 5′UTR, allowing us to investigate the roles of the stem–loops in IRES function. This approach revealed that SLI was not required for EHcV IRES-mediated translation. Conversely, SLIII was essential, specifically SLIIIb, SLIIId and a GGG motif that is conserved across the Hepaciviridae. Further SHAPE analysis provided evidence that this GGG motif mediated interaction with the 40S ribosomal subunit, whilst a CUU sequence in the apical loop of SLIIIb mediated an interaction with eIF3. In addition, we showed that a microRNA122 target sequence located between SLIA and SLII mediated an enhancement of translation in the context of a subgenomic replicon. Taken together, these results highlight the conservation of hepaciviral translation mechanisms, despite divergent primary sequences.

As an infant grows, human milk may become the substrate for cariogenic bacteria. Here's what to watch for and how to counsel parents.

The substitution rates of viral polymerases have been studied extensively. However less is known about the tendency of these enzymes to ‘slip’ during RNA synthesis to produce progeny RNAs with nucleotide insertions or deletions. We recently described the functional utilization of programmed polymerase slippage in the family Potyviridae. This slippage results in either an insertion or a substitution, depending on whether the RNA duplex realigns following the insertion. In this study we investigated whether this phenomenon is a conserved feature of superfamily I viral RdRps, by inserting a range of potyvirus-derived slip-prone sequences into a picornavirus, Theiler’s murine encephalomyelitis virus (TMEV). Deep-sequencing analysis of viral transcripts indicates that the TMEV polymerase ‘slips’ at the sequences U6–7 and A6–7 to insert additional nucleotides. Such sequences are under-represented within picornaviral genomes, suggesting that slip-prone sequences create a fitness cost. Nonetheless, the TMEV insertional and substitutional spectrum differed from that previously determined for the potyvirus polymerase.

The genusTorovirus(subfamilyTorovirinae, familyCoronaviridae, orderNidovirales) encompasses a range of species that infect domestic ungulates including cattle, sheep, goats, pigs and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV) we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyse the relative expression levels of the known torovirus proteins and transcripts, chimaeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle) and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilises a unique combination of discontinuous and non-discontinuous RNA synthesis to produce its subgenomic RNAs; indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated ORFs, located within the so-called 5’ UTR. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens.ImportanceToroviruses infect cattle, goats, pigs and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens including severe acute respiratory syndrome (SARS) coronavirus and they share some common features, however the mechanism that they use to produce subgenomic RNA molecules differs. Here we performed deep sequencing to determine how equine torovirus produces subgenomic RNAs. In doing so, we also identified two previously unknown open reading frames “hidden” within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus recently associated with congenital diseases and neurological complications. As for all flaviviruses, the ZIKV RNA genome is expected to encode a single polyprotein with all the enzymatic activities required for viral replication. Here, we report the discovery of multiple non-canonical open reading frames (ORFs) identified by ribosome profiling. In both mammalian and insect cells infected with Asian/American and African ZIKV strains, we observed translation of previously unrecognised upstream ORFs (uORFs) in the 5′ region. In the Asian/American ZIKV lineage, ribosomes translated uORF1 and uORF2 that initiated from non-AUG start codons, whereas in the African ZIKV lineage, these two uORFs were fused into a single uORF (African uORF). Using a reverse genetics system, we examined the impact on ZIKV fitness of the expression of single or dual uORFs by analysing a panel of mutant viruses. We found that expression of the African uORF, and more significantly, the Asian/American uORF1, modulated virus growth and tropism in human cortical neurons and 3D organoid tissue, indicating that these novel uORFs contribute to ZIKV neurotropism. Although ZIKV uORFs are expressed in mosquito cells, they did not have a detectable effect on transmission by the mosquito vectorin vivo. Our discovery of ZIKV uORFs sheds new light on ZIKV-induced neuropathogenesis and raises the question of their existence in other neurotropic flaviviruses.

Background Deep vein thrombosis is a major global health issue, responsible for thousands of deaths each year. While thrombi can form under a variety of circumstances, lack of mobility significantly increases risk and therefore non-ambulant patients are frequently fitted with anti-embolism stockings on admission to hospital, to aid blood flow, prevent pooling and thus clotting. Anti-embolism stockings are the most widely used non-invasive medical device on the market and are believed to reduce the risk of deep vein thrombosis by 40%. Despite their widespread use in hospitals world-wide, there is remarkably little research addressing their use or reconditioning and a wide variety of different reconditioning protocols are used in hospitals. Objective The objective of this study was to establish the impact of different wear and reconditioning protocols on the pressure delivering ability of anti-embolism stockings. Design/methods A laboratory investigation was undertaken to evaluate the pressure delivering ability of 2 major global brands of anti-embolism stockings over 5–8days of simulated wear (extension on static cylinders) and 4 different reconditioning protocols. 1 set of samples was continuously ‘worn' for 8days without reconditioning, 1 set of samples was ‘worn' for 5days with a day of relaxation between each day of ‘wear', 1 set was ‘hand washed' and 1 set was machine washed and then allowed to relax between each day of ‘wear'. The pressure was measured at the beginning and end of each period of ‘wear'. Setting This study was undertaken in a conditioned textile testing laboratory that complies with BS EN ISO 139:2005+A1:2011. Results The pressure exerted by anti-embolism stockings reduced by between 15 and 24% after 24h of wear, it reduced by between 21 and 32% when worn continuously for 8days. Allowing stockings to rest for a day between days of wear allowed them to recover slightly but this recovery was only temporary. Washing stockings regenerated their pressure delivering potential significantly and machine washing allowed some to recover to exert more pressure than they had when new. Conclusions Different brands of anti-embolism stockings exert different pressures on the same size of leg, when correctly fitted. The pressure exerted by anti-embolism stockings decreases with use but the correct pressure gradient is maintained if correctly fitted. Washing stockings after 24h of wear is effective in restoring their pressure delivering abilities and in some cases can surpass their ‘as new' pressure delivering ability.

Full length endogenous feline leukemia virus (FeLV) proviruses exist within the genomes of many breeds of domestic cat raising the possibility that they may also exist in a transmissible exogenous form. Such viruses would share receptor usage with the recombinant FeLV-B subgroup, a viral subgroup that arises in vivo by recombination between exogenous subgroup A virus (FeLV-A) and endogenous FeLV. Accordingly, all isolates of FeLV-B made to date have contained a "helper" FeLV-A, consistent with their recombinatorial origin. In order to assess whether endogenous viruses are transmitted between cats, we examined primary isolates of FeLV for which the viral subgroup had been determined for the presence of a subgroup B virus that lacked an FeLV-A. Here we describe the identification of two primary field isolates of FeLV (2518 and 4314) that appeared to contain subgroup B virus only by classical interference assays, raising the possibility of between-host transmission of endogenous FeLV. Sequencing of the env gene and U3 region of the 3' long terminal repeat (LTR) confirmed that both viral genomes contained endogenous viral env genes. However the viral 3' LTRs appeared exogenous in origin with a putative 3' recombination breakpoint residing at the 3' end of the env gene. Further, the FeLV-2518 virions also co-packaged a truncated FeLV-A genome containing a defective env gene, termed FeLV-2518(A) whilst no helper subgroup A viral genome was detected in virions of FeLV-4314. The acquisition of an exogenous LTR by the endogenous FeLV in 4314 may have allowed a recombinant FeLV variant to outgrow an exogenous FeLV-A virus that was presumably present during first infection. Given time, a similar evolution may also occur within the 2518 isolate. The data suggest that endogenous FeLVs may be mobilised by acquisition of exogenous LTRs yielding novel viruses that type biologically as FeLV-B.

The genomes of all animal species are colonized by endogenous retroviruses (ERVs). Although most ERVs have accumulated defects that render them incapable of replication, fully infectious ERVs have been identified in various mammals. In this study, we isolated a feline infectious ERV (RD-114) in a proportion of live attenuated vaccines for pets. Isolation of RD-114 was made in two independent laboratories using different detection strategies and using vaccines for both cats and dogs commercially available in Japan or the United Kingdom. This study shows that the methods currently employed to screen veterinary vaccines for retroviruses should be reevaluated.

Objectives: Currently, there is a deficit of information on policies regarding oral hygiene practices in Toronto daycares. It is unknown if any tooth-brushing programs are in existence and if children are permitted to follow professional advice on oral hygiene. The main objectives of this investigation were to a) determine the prevalence of oral care policies in daycares and b) examine the availability of resources. Methods: Telephone interviews were conducted with daycare supervisors using a pretested questionnaire. Summary statistics and the chi-square test were used to analyze the results. Results: Two hundred forty-nine questionnaires were completed (response rate of 99.6 percent), representing 38 percent of the total daycare population (650) in Toronto. Eighty-three percent did not have a policy on oral care and 11 percent would not cede to requests from parents or medical professionals to brush teeth. However, 50 daycares indicated that their centers used to have a tooth-brushing program, and most (79 percent) were open to establishing an oral care policy. Fifteen percent reported not having proper sinks for tooth brushing. Conclusions: Many daycares do not have a policy regarding oral hygiene. A policy that encourages and provides guidance on safe tooth-brushing procedures is needed and may improve the oral health of preschool children.

Highlights • Hepatitis C virus (HCV) is a significant human pathogen, causing severe liver disease. • Accurate quantification of viral titres is essential for the majority of assays. • The current methods of HCV titration and quantification are laborious and imprecise. • We report a novel method for calculating infectious HCV titres using the IncuCyte ZOOM. • This method has applications for screening of novel antiviral compounds., Summary Hepatitis C virus (HCV) is a significant human pathogen infecting 3% of the world population. An infectious molecular clone capable of replicating and releasing infectious virions in cell culture has only been available since 2005, leaving a significant knowledge gap concerning post-RNA replication events such as particle assembly, trafficking and release. Thus, a fast, efficient and accurate method of measuring infectious viral titres is highly desirable. Current methods rely upon manual counting of infected cell foci and so are both labour-intensive and susceptible to human error. Here, we report a novel protocol, which utilises the IncuCyte ZOOM instrument and related software to accurately count infected cells and extrapolation of this data to produce an infectious titre, reported as infectious units per millilitre (IU/mL). This method reduces cost, time and error in experiments. We also demonstrate that this approach is amenable to high-throughput compound screening, thereby expediting the identification of novel antivirals.

The 5′ untranslated region (5′UTR) of the recently described non-primate hepacivirus (NPHV) contains a region with sequence homology to the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) and GB virus B (GBV-B). Here, we demonstrated internal translation initiation by the NPHV 5′UTR in a bicistronic vector. An RNA stem–loop upstream of the NPHV IRES was structurally distinct from corresponding regions in HCV and GBV-B, and was not required for IRES function. Insertion of the NPHV stem–loop into the corresponding region of the HCV 5′UTR within the HCV subgenomic replicon significantly impaired RNA replication, indicating that long-range interactions between the 5′UTR and cis-acting downstream elements within the NPHV genome are not interchangeable with those of HCV. Despite similarities in IRES structure and function between hepaciviruses, replication elements in the NPHV 5′UTR appear functionally distinct from those of HCV.

It is generally accepted that all primary isolates of feline leukemia virus (FeLV) contain a subgroup A virus (FeLV-A) that is essential for transmission. In contrast, FeLV-B is thought to arise de novo in the infected animal through RNA recombination events with endogenous FeLV transcripts, presumably through copackaging of RNA from endogenous FeLV and exogenous FeLV-A. Here, we report the complete genome sequences of two novel strains of FeLV-B (FeLV-2518 and FeLV-4314) that were isolated in the absence of FeLV-A. The env genes of these isolates have been characterized previously, and the 3′ recombination sites have been identified. We describe herein the 5′ recombination breakpoints of each virus. These breakpoints were found to be within the signal peptide of the env gene and the reverse transcriptase-coding region, respectively. This is the first report of a recombination site within the pol gene of an FeLV-B genome and the first genetic characterization of multiple independently arising FeLV-B isolates that have been identified without a functional FeLV-A ancestral virus.

Binding of the Rac1-specific guanine-nucleotide-exchange factor, Tiam1, to the plasma membrane requires the N-terminal pleckstrin homology domain. In the present study, we show that membrane-association is mediated by binding of PtdIns(4,5)P2 to the pleckstrin homology domain. Moreover, in 1321N1 astrocytoma cells, translocation of Tiam1 to the cytosol, following receptor-mediated stimulation of PtdIns(4,5)P2 breakdown, correlates with decreased Rac1-GTP levels, indicating that membrane-association is required for GDP/GTP exchange on Rac1. In addition, we show that platelet-derived growth factor activates Rac1 in vivo by increasing PtdIns(3,4,5)P3 concentrations, rather than the closely related lipid, PtdIns(3,4)P2. Finally, the data demonstrate that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 bind to the same pleckstrin homology domain in Tiam1 and that soluble inositol phosphates appear to compete with lipids for this binding. Together, these novel observations provide strong evidence that distinct phosphoinositides regulate different functions of this enzyme, indicating that local concentrations of signalling lipids and the levels of cytosolic inositol phosphates will play crucial roles in determining its activity in vivo.

The tumour suppressor PTEN is a PtdIns(3,4,5)P(3) phosphatase that regulates many cellular processes through direct antagonism of PI 3-kinase signalling. Here we show that oxidative stress activates PI 3-kinase-dependent signalling via the inactivation of PTEN. We use two assay systems to show that cellular PTEN phosphatase activity is inhibited by oxidative stress induced by 1 mM hydrogen peroxide. PTEN inactivation by oxidative stress also causes an increase in cellular PtdIns(3,4,5)P(3) levels and activation of the downstream PtdIns(3,4,5)P(3) target, PKB/Akt, that does not occur in cells lacking PTEN. We then show that endogenous oxidant production in RAW264.7 macrophages inactivates a fraction of the cellular PTEN, and that this is associated with an oxidant-dependent activation of downstream signalling. These results show that oxidants, including those produced by cells, can activate downstream signalling via the inactivation of PTEN. This demonstrates a novel mechanism of regulation of the activity of this important tumour suppressor and the signalling pathways it regulates. These results may have significant implications for the many cellular processes in which PtdIns(3,4,5)P(3) and oxidants are produced concurrently.

We conducted a survey during the 1999 2000 school year to obtain valid estimates of the oral health status of a probability sample of children in the 4 regions of the newly amalgamated city of Toronto. The results will be used in developing recommendations for programs to address the oral health problems identified. The Dental Indices System is the Ontario protocol whereby information on the oral health status and treatment needs of children can be obtained by direct assessment of the children. One of 2 specially trained dental hygienists examined each child's teeth and periodontal tissues using sterilized mouth mirrors and blunt probes with a standard light source. Overall, there were 3657 participants in the survey, of whom 2435 were aged 7 or 13 years; these 2 age groups formed the basis for the analysis. Forty percent of those aged 7 or 13 had had one or more decayed teeth. Approximately 7% of children in the younger age group had at least one condition requiring urgent care. Dental fluorosis of moderate severity (Tooth Surface Index of Fluorosis 2) was found among 14.0% of 7-year-olds, 12.3% of 13-year-olds and 13.2% of the 2 age groups combined. The prevalence of fluorosis was of the same order as all but one of the more recent studies performed in Toronto. The prevalence may fall as the recently imposed reduction in concentration of fluorides in city water takes effect. On the basis of these findings of fluorosis, Toronto Public Health should continue to monitor levels of dental fluorosis and caries and should continue its efforts to inform parents of very young children about the safe use of fluoridated dentifrice.

BACKGROUND:\ud \ud The development of anaemia in feline leukaemia virus (FeLV)-infected cats is associated with the emergence of a novel viral subgroup, FeLV-C. FeLV-C arises from the subgroup that is transmitted, FeLV-A, through alterations in the amino acid sequence of the receptor binding domain (RBD) of the envelope glycoprotein that result in a shift in the receptor usage and the cell tropism of the virus. The factors that influence the transition from subgroup A to subgroup C remain unclear, one possibility is that a selective pressure in the host drives the acquisition of mutations in the RBD, creating A/C intermediates with enhanced abilities to interact with the FeLV-C receptor, FLVCR. In order to understand further the emergence of FeLV-C in the infected cat, we examined primary isolates of FeLV-C for evidence of FeLV-A variants that bore mutations consistent with a gradual evolution from FeLV-A to FeLV-C.\ud \ud RESULTS:\ud \ud Within each isolate of FeLV-C, we identified variants that were ostensibly subgroup A by nucleic acid sequence comparisons, but which bore mutations in the RBD. One such mutation, N91D, was present in multiple isolates and when engineered into a molecular clone of the prototypic FeLV-A (Glasgow-1), enhanced replication was noted in feline cells. Expression of the N91D Env on murine leukaemia virus (MLV) pseudotypes enhanced viral entry mediated by the FeLV-A receptor THTR1 while soluble FeLV-A Env bearing the N91D mutation bound more efficiently to mouse or guinea pig cells bearing the FeLV-A and -C receptors. Long-term in vitro culture of variants bearing the N91D substitution in the presence of anti-FeLV gp70 antibodies did not result in the emergence of FeLV-C variants, suggesting that additional selective pressures in the infected cat may drive the subsequent evolution from subgroup A to subgroup C.\ud \ud CONCLUSIONS:\ud \ud Our data support a model in which variants of FeLV-A, bearing subtle differences in the RBD of Env, may be predisposed towards enhanced replication in vivo and subsequent conversion to FeLV-C. The selection pressures in vivo that drive the emergence of FeLV-C in a proportion of infected cats remain to be established.