This leaves the insertion of polybasic. Virus Evol. Nat. The proximal origin of SARS-CoV-2 | Nature Medicine 53), this is inferred to have occurred before the divergence of RaTG13 and SARS-CoV-2 and thus should not influence our inferences. All three approaches to removal of recombinant genomic segments point to a single ancestral lineage for SARS-CoV-2 and RaTG13. and P.L.) Sequencing from Malayan pangolins collected during anti-smuggling operations in southern China detected coronavirus lineages related to SARS-CoV-2. However, inconsistency in the nomenclature limits uniformity in its epidemiological understanding. Of the nine breakpoints defining these ten BFRs, four showed phylogenetic incongruence (PI) signals with bootstrap support >80%, adopting previously published criteria on using a combination of mosaic and PI signals to show evidence of past recombination events19. Katoh, K., Asimenos, G. & Toh, H. in Bioinformatics for DNA Sequence Analysis (ed. Visual exploration using TempEst39 indicates that there is no evidence for temporal signal in these datasets (Extended Data Fig. Researchers in the UK had just set the scientific world . The ongoing pandemic spread of a new human coronavirus, SARS-CoV-2, which is associated with severe pneumonia/disease (COVID-19), has resulted in the generation of tens of thousands of virus . Lam, H. M., Ratmann, O. It is RaTG13 that is more divergent in the variable-loop region (Extended Data Fig. Specifically, we used a combination of six methods implemented in v.5.5 of RDP5 (ref. A single 3SEQ run on the genome alignment resulted in 67 out of 68sequences supporting some recombination in the past, with multiple candidate breakpoint ranges listed for each putative recombinant. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Ge, X. et al. In the variable-loop region, RaTG13 diverges considerably with the TMRCA, now outside that of SARS-CoV-2 and the Pangolin Guangdong 2019 ancestor, suggesting that RaTG13 has acquired this region from a more divergent and undetected bat lineage. Using these breakpoints, the longest putative non-recombining segment (nt1,88521,753) is 9.9kb long, and we call this region NRR2. For the current pandemic, the novel pathogen identification component of outbreak response delivered on its promise, with viral identification and rapid genomic analysis providing a genome sequence and confirmation, within weeks, that the December 2019 outbreak first detected in Wuhan, China was caused by a coronavirus3. A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist Su, S. et al. We considered (1) the possibility that BFRs could be combined into larger non-recombinant regions and (2) the possibility of further recombination within each BFR. Cell 181, 223227 (2020). Biol. We thank T. Bedford for providing M.F.B. Virological.org http://virological.org/t/ncovs-relationship-to-bat-coronaviruses-recombination-signals-no-snakes-no-evidence-the-2019-ncov-lineage-is-recombinant/331 (2020). It is available as a command line tool and a web application. Maclean, O. We focused on these three non-recombining regions/alignments for divergence time estimation; this avoids inappropriate modelling of evolutionary processes with recombination on strictly bifurcating trees, which can result in different artefacts such as homoplasies that inflate branch lengths and lead to apparently longer evolutionary divergence times. Open reading frames are shown above the breakpoint plot, with the variable-loop region indicated in the Sprotein. PureBasic 53 13 constellations Public Python 42 17 The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. The 2009 influenza pandemic and subsequent outbreaks of MERS-CoV (2012), H7N9 avian influenza (2013), Ebola virus (2014) and Zika virus (2015) were met with rapid sequencing and genomic characterization. However, for several reasons, nucleotide sequences may be generated that cover only the spike gene of SARS-CoV-2. 04:20. ISSN 2058-5276 (online). COVID-19: A Catastrophe or Opportunity for Pangolin Conservation? - Nature wrote the first draft of the manuscript, and all authors contributed to manuscript editing. Published. The coverage threshold and consensus sequence generation threshold were set to 20 and 90 respectively. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. =0.00025. Li, X. et al. 16, e1008421 (2020). M.F.B. As informative rate priors for the analysis of the sarbecovirus datasets, we used two different normal prior distributions: one with a mean of 0.00078 and s.d. 82, 48074811 (2008). Host ecology determines the dispersal patterns of a plant virus. COVID-19: Time to exonerate the pangolin from the transmission of SARS Humans' selfish, speciesist treatment of these animals could be the very reason why the novel coronavirus exists. Why Can't We Just Call BA.2 Omicron? - The Atlantic In case of DRAGEN COVID Lineage tool, the minimum accepted alignment score was set to 22 and results with scores <22 were discarded. Evol. Rambaut, A., Lam, T. T., Carvalho, L. M. & Pybus, O. G. Exploring the temporal structure of heterochronous sequences using TempEst (formerly Path-O-Gen). Emerg. By mid-January 2020, the virus was spreading widely within Hubei province and by early March SARS-CoV-2 was declared a pandemic8. N. China corresponds to Jilin, Shanxi, Hebei and Henan provinces, and the N. China clade also includes one sequence sampled in Hubei Province in 2004. But some theories suggest that pangolins may be the source of the novel coronavirus. Except for specifying that sequences are linear, all settings were kept to their defaults. performed Srecombination analysis. Biol. We call this approach breakpoint-conservative, but note that this has the opposite effect to the construction of NRR1 in that this approach is the most likely to allow breakpoints to remain inside putative non-recombining regions. Nat Microbiol 5, 14081417 (2020). Genet. Don't blame pangolins, coronavirus family tree tracing could prove key Due to the absence of temporal signal in the sarbecovirus datasets, we used informative prior distributions on the evolutionary rate to estimate divergence dates. This commit does not belong to any branch on this repository, and may belong to a fork outside of the repository. To employ phylogenetic dating methods, recombinant regions of a 68-genome sarbecovirus alignment were removed with three independent methods. Lancet 383, 541548 (2013). A pneumonia outbreak associated with a new coronavirus of probable bat origin. Scientists trying to trace the ancestry of SARS-CoV-2, the virus responsible for COVID-19, have found the pangolin is unlikely to be the source of the virus responsible for the current pandemic. These are in general agreement with estimates using NRR2 and NRA3, which result in divergence times of 1982 (19482009) and 1948 (18791999), respectively, for SARS-CoV-2, and estimates of 1952 (19061989) and 1970 (19321996), respectively, for the divergence time of SARS-CoV from its closest known bat relative. 35, 247251 (2018). July 26, 2021. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. Prolonged SARS-CoV-2 Infection and Intra-Patient Viral Evolu : The A second breakpoint-conservative approach was conservative with respect to breakpoint identification, but this means that it is accepting of false-negative outcomes in breakpoint inference, resulting in less certainty that a putative NRR truly contains no breakpoints. There is a 90% DNA match between SARS CoV 2 and a coronavirus in pangolins. 2 Lack of root-to-tip temporal signal in SARS-CoV-2. Sci. A tag already exists with the provided branch name. Trends Microbiol. Forni, D., Cagliani, R., Clerici, M. & Sironi, M. Molecular evolution of human coronavirus genomes. Provided by the Springer Nature SharedIt content-sharing initiative, Molecular and Cellular Biochemistry (2023), Nature Microbiology (Nat Microbiol) Sci. Biol. PubMed & Holmes, E. C. A genomic perspective on the origin and emergence of SARS-CoV-2. Add entries for pangolin-data/-assignment 1.18.1.1 (, Really add a document on testing strategy. c, Maximum likelihood phylogenetic trees rooted on a 2007 virus sampled in Kenya (BtKy72; root truncated from images), shown for five BFRs of the sarbecovirus alignment. Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia. The Artic Network receives funding from the Wellcome Trust through project no. Evol. CoV-lineages GitHub On first examination this would suggest that that SARS-CoV-2 is a recombinant of an ancestor of Pangolin-2019 and RaTG13, as proposed by others11,22. Pink, green and orange bars show BFRs, with regionA (nt 13,29119,628) showing two trimmed segments yielding regionA (nt13,29114,932, 15,40517,162, 18,00919,628). Nevertheless, the viral population is largely spatially structured according to provinces in the south and southeast on one lineage, and provinces in the centre, east and northeast on another (Fig. Nguyen, L.-T., Schmidt, H. A., Von Haeseler, A. P.L. Specifically, progenitors of the RaTG13/SARS-CoV-2 lineage appear to have recombined with the Hong Kong clade (with inferred breakpoints at 11.9 and 20.8kb) to form the CoVZXC21/CoVZC45-lineage. 92, 433440 (2020). Because there is no single accepted method of inferring breakpoints and identifying clean subregions with high certainty, we implemented several approaches to identifying three classic statistical signals of recombination: mosaicism, phylogenetic incongruence and excessive homoplasy51. The variable-loop region in SARS-CoV-2 shows closer identity to the 2019 pangolin coronavirus sequence than to the RaTG13 bat virus, supported by phylogenetic inference (Fig. Google Scholar. with an alignment on which an initial recombination analysis was done. With horseshoe bats currently the most plausible origin of SARS-CoV-2, it is important to consider that sarbecoviruses circulate in a variety of horseshoe bat species with widely overlapping species ranges57. Sequences were aligned by MAFTT58 v.7.310, with a final alignment length of 30,927, and used in the analyses below. Of the countries that have contributed SARS-CoV-2 data, 30% had genomes of this lineage. We compiled a dataset including 27human coronavirus OC43 virus genomes and ten related animal virus genomes (six bovine, three white-tailed deer and one canine virus). We say that this approach is conservative because sequences and subregions generating recombination signals have been removed, and BFRs were concatenated only when no PI signals could be detected between them. Mol. This new approach classifies the newly sequenced genome against all the diverse lineages present instead of a representative select sequences. Pangolins may have incubated the novel coronavirus, gene study shows Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus. Zhou et al.2 concluded from the genetic proximity of SARS-CoV-2 to RaTG13 that a bat origin for the current COVID-19 outbreak is probable. 5). Based on the identified breakpoints in each genome, only the major non-recombinant region is kept in each genome while other regions are masked. This dataset comprises an updated version of that used in Hon et al.15 and includes a cluster of genomes sampled in late 2003 and early 2004, but the evolutionary rate estimate without this cluster (0.00175 substitutions per siteyr1 (0.00117,0.00229)) is consistent with the complete dataset (0.00169 substitutions per siteyr1, (0.00131,0.00205)). To evaluate the performance procedure, we confirmed that the recombination masking resulted in (1) a markedly different outcome of the PHI test64, (2) removal of well-supported (bootstrap value >95%) incompatible splits in Neighbor-Net65 and (3) a near-complete reduction of mosaic signal as identified by 3SEQ. 4), but also by markedly different evolutionary rates. Nature 583, 286289 (2020). PubMed Li, Q. et al. For the HCoV-OC43, MERS-CoV and SARS datasets we specified flexible skygrid coalescent tree priors. Individual sequences such as RpShaanxi2011, Guangxi GX2013 and two sequences from Zhejiang Province (CoVZXC21/CoVZC45), as previously shown22,25, have strong phylogenetic recombination signals because they fall on different evolutionary lineages (with bootstrap support >80%) depending on what region of the genome is being examined. Five example sequences with incongruent phylogenetic positions in the two trees are indicated by dashed lines. In such cases, even moderate rate variation among long, deep phylogenetic branches will substantially impact expected root-to-tip divergences over a sampling time range that represents only a small fraction of the evolutionary history40. Sarbecovirus, HCoV-OC43 and SARS-CoV data were assembled from GenBank to be as complete as possible, with sampling year as an inclusion criterion. All authors contributed to analyses and interpretations. A new coronavirus associated with human respiratory disease in China. Boni, M.F., Lemey, P., Jiang, X. et al. Nat. PLoS Pathog. Gray inset shows majority rule consensus trees with mean posterior branch lengths for the two regions, with posterior probabilities on the key nodes showing the relationships among SARS-CoV-2, RaTG13, and Pangolin 2019. Bryant, D. & Moulton, V. Neighbor-Net: an agglomerative method for the construction of phylogenetic networks. b, Similarity plot between SARS-CoV-2 and several selected sequences including RaTG13 (black), SARS-CoV (pink) and two pangolin sequences (orange). These residues are also in the Pangolin Guangdong 2019 sequence. 26 March 2020. It allows a user to assign a SARS-CoV-2 genome sequence the most likely lineage (Pango lineage) to SARS-CoV-2 query sequences. Decimal years are shown on the x axis for the 1.2 years of SARS sampling in c. d, Mean evolutionary rate estimates plotted against sampling time range for the same three datasets (represented by the same colour as the data points in their respective RtT divergence plots), as well as for the comparable NRA3 using the two different priors for the rate in the Bayesian inference (red points). In the meantime, to ensure continued support, we are displaying the site without styles The presence in pangolins of an RBD very similar to that of SARS-CoV-2 means that we can infer this was also probably in the virus that jumped to humans. covid19_mostefai2021_paper/01_CreateObjects.r at master HussinLab and X.J. Our third approach involved identifying breakpoints and masking minor recombinant regions (with gaps, which are treated as unobserved characters in probabilistic phylogenetic approaches). 82, 18191826 (2008). Possible Bat Origin of Severe Acute Respiratory Syndrome Coronavirus 2 Its genome is closest to that of severe acute respiratory syndrome-related coronaviruses from horseshoe bats, and its receptor-binding domain is closest to that of pangolin viruses. Share . Thank you for visiting nature.com. You signed in with another tab or window. & Li, X. Crossspecies transmission of the newly identified coronavirus 2019nCoV. Lam, T. T. et al. BEAST inferences made use of the BEAGLE v.3 library68 for efficient likelihood computations. In regionA, we removed subregion A1 (ntpositions 3,8724,716 within regionA) and subregion A4 (nt1,6422,113) because both showed PI signals with other subregions of regionA. Concatenated region ABC is NRR1. 206298/Z/17/Z. performed codon usage analysis. Evol. Webster, R. G., Bean, W. J., Gorman, O. T., Chambers, T. M. & Kawaoka, Y. Evolution and ecology of influenza A viruses. Its origin and direct ancestral viruses have not been . The consistency of the posterior rates for the different prior means also implies that the data do contribute to the evolutionary rate estimate, despite the fact that a temporal signal was visually not apparent (Extended Data Fig. Eden, J.-S., Tanaka, M. M., Boni, M. F., Rawlinson, W. D. & White, P. A. Recombination within the pandemic norovirus GII.4 lineage. 1) and thus likely to be the product of recombination, acquiring a divergent variable loop from a hitherto unsampled bat sarbecovirus28. Biazzo et al. Adv. Scientists defined the pangolin lineage of this variant to be B.1.1.523 and it was originally recognized as a variant under monitoring on July 14, 2021. This produced non-recombining alignment NRA3, which included 63 of the 68genomes. Phylogenetic Assignment of Named Global Outbreak LINeages, The pangolin web app is maintained by the Centre for Genomic Pathogen Surveillance. While it is possible that pangolins, or another hitherto undiscovered species, may have acted as an intermediate host facilitating transmission to humans, current evidence is consistent with the virus having evolved in bats resulting in bat sarbecoviruses that can replicate in the upper respiratory tract of both humans and pangolins25,32. pango-designation Public Repository for suggesting new lineages that should be added to the current scheme Python 968 73 pangolin Public Software package for assigning SARS-CoV-2 genome sequences to global lineages. Influenza viruses reassort17 but they do not undergo homologous recombination within RNA segments18,19, meaning that origins questions for influenza outbreaks can always be reduced to origins questions for each of influenzas eight RNA segments. COVID-19 lineage names can be confusing to navigate; there are many aliases and if you want to catch them all to examine further in data analyses it helps to Allen O'Brien on LinkedIn: #r #rstudio #rstats #pangolin #covid19 #datascience #epidemiology Are pangolins the intermediate host of the 2019 novel coronavirus (SARS-CoV-2)? As illustrated by the dashed arrows, these two posteriors motivate our specification of prior distributions with standard deviations inflated 10-fold (light color). the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Use of Genomics to Track Coronavirus Disease Outbreaks, New Zealand & Andersen, K. G. Pandemics: spend on surveillance, not prediction. Genetics 176, 10351047 (2007). Here, we analyse the evolutionary history of SARS-CoV-2 using available genomic data on sarbecoviruses. Global epidemiology of bat coronaviruses. Collectively our analyses point to bats being the primary reservoir for the SARS-CoV-2 lineage. Divergence time estimates based on the HCoV-OC43-centred rate prior for the separate BFRs (Supplementary Table 3) show consistency in TMRCA estimates across the genome. PubMed At present, we analyzed the diversity of SARS-CoV-2 viral genomes in India to know the evolutionary patterns of viruses in the country through their pangolin lineage and GISAID-Clade. D.L.R. Anderson, K. G., Rambaut, A., Lipkin, W. I., Holmes, E. C. & Garry, R. F. The proximal origin of SARS-CoV-2. Menachery, V. D. et al. Google Scholar. Evol. Did Pangolin Trafficking Cause the Coronavirus Pandemic? 5 (NRR1) are conservative in the sense that NRR1 is more likely to be non-recombinant than NRR2 or NRA3. Virus Evol. Biol. 2). Google Scholar. The construction of NRR1 is the most conservative as it is least likely to contain any remaining recombination signals. Phylogenetic supertree reveals detailed evolution of SARS-CoV-2, Origin and cross-species transmission of bat coronaviruses in China, Emerging SARS-CoV-2 variants follow a historical pattern recorded in outgroups infecting non-human hosts, Inferring the ecological niche of bat viruses closely related to SARS-CoV-2 using phylogeographic analyses of Rhinolophus species, Genomic recombination events may reveal the evolution of coronavirus and the origin of SARS-CoV-2, A Bayesian approach to infer recombination patterns in coronaviruses, Metagenomic identification of a new sarbecovirus from horseshoe bats in Europe, A comparative recombination analysis of human coronaviruses and implications for the SARS-CoV-2 pandemic, Pandemic-scale phylogenomics reveals the SARS-CoV-2 recombination landscape, https://github.com/plemey/SARSCoV2origins, https://doi.org/10.1101/2020.04.20.052019, https://doi.org/10.1101/2020.02.10.942748, https://doi.org/10.1101/2020.05.28.122366, http://virological.org/t/ncov-2019-codon-usage-and-reservoir-not-snakes-v2/339, http://virological.org/t/ncovs-relationship-to-bat-coronaviruses-recombination-signals-no-snakes-no-evidence-the-2019-ncov-lineage-is-recombinant/331. 110. J. Med Virol. In addition, sequences NC_014470 (Bulgaria 2008), CoVZXC21, CoVZC45 and DQ412042 (Hubei-Yichang) needed to be removed to maintain a clean non-recombinant signal in A. Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins. a, Breakpoints identified by 3SEQ illustrated by percentage of sequences (out of 68) that support a particular breakpoint position. In March, when covid cases began spiking around India, Bani Jolly went hunting for answers in the virus's genetic code. The lineage B.1 has been the major basal and widespread lineage from the initial SARS-CoV-2 spread and it became the more prevalent lineage in Colombia ( 13 ), while the B.1.111 lineage, first detected in the USA from a sample collected on March 7, 2020 and subsequently in Colombia on March 13, 2020 is currently circulating and mainly represented Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. This statement informs us of the possibility that a virus has spilled over from a very rare and shy reptile-looking mammal . Virological.org http://virological.org/t/ncov-2019-codon-usage-and-reservoir-not-snakes-v2/339 (2020). 68, 10521061 (2019). Below, we report divergence time estimates based on the HCoV-OC43-centred rate prior for NRR1, NRR2 and NRA3 and summarize corresponding estimates for the MERS-CoV-centred rate priors in Extended Data Fig. However, formal testing using marginal likelihood estimation41 does provide some evidence of a temporal signal, albeit with limited log Bayes factor support of 3 (NRR1), 10 (NRR2) and 3 (NRA3); see Supplementary Table 1. Mol. A counting renaissance: combining stochastic mapping and empirical Bayes to quickly detect amino acid sites under positive selection. The canine viral genome was excluded from the Bayesian phylogenetic analyses because temporal signal analyses (see below) indicated that it was an outlier. A deep dive into the genetics of the novel coronavirus shows it seems to have spent some time infecting both bats and pangolins before it jumped into humans, researchers said . Biol. Article Viral metagenomics revealed Sendai virus and coronavirus infection of Malayan pangolins (Manis javanica). Yuan, J. et al. Because the estimated rates and divergence dates were highly similar in the three datasets analysed, we conclude that our estimates are robust to the method of identifying a genomes NRRs. Since experts have suggested that pangolins may be the reservoir species for COVID-19, the scaly anteater has been catapulted into headlines, news reports, and conversationsand some are calling COVID-19 "the revenge of the . performed recombination analysis for non-recombining alignment3, calibration of rate of evolution and phylogenetic reconstruction and dating. You are using a browser version with limited support for CSS. 95% credible interval bars are shown for all internal node ages. The idea is that pangolins carrying the virus, SARS-CoV-2, came into contact with humans. PubMed PubMed Central This is evidence for numerous recombination events occurring in the evolutionary history of the sarbecoviruses22,33; specifying all past events in their correct temporal order34 is challenging and not shown here. These datasets were subjected to the same recombination masking approach as NRA3 and were characterized by a strong temporal signal (Fig. It is available as a command line tool and a web application. This underscores the need for a global network of real-time human disease surveillance systems, such as that which identified the unusual cluster of pneumonia in Wuhan in December 2019, with the capacity to rapidly deploy genomic tools and functional studies for pathogen identification and characterization. The Sichuan (SC2018) virus appears to be a recombinant of northern/central and southern viruses, while the two Zhejiang viruses (CoVZXC21 and CoVZC45) appear to carry a recombinant region from southern or central China. Eight other BFRs <500nt were identified, and the regions were named BFRAJ in order of length. 2). J. Virol. Early detection via genomics was not possible during Southeast Asias initial outbreaks of avian influenza H5N1 (1997 and 20032004) or the first SARS outbreak (20022003). The existing diversity and dynamic process of recombination amongst lineages in the bat reservoir demonstrate how difficult it will be to identify viruses with potential to cause major human outbreaks before they emerge. Intragenomic rearrangements involving 5-untranslated region segments in SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses, Crystal structure of the CoV-Y domain of SARS-CoV-2 nonstructural protein 3, Association of underlying comorbidities and progression of COVID-19 infection amongst 2586 patients hospitalised in the National Capital Region of India: a retrospective cohort study, Molecular characterization of horse nettle virus A, a new member of subgroup B of the genus Nepovirus, Molecular phylogeny of coronaviruses and host receptors among domestic and close-contact animals reveals subgenome-level conservation, crossover, and divergence. 5. Our results indicate the presence of a single lineage circulating in bats with properties that allowed it to infect human cells, as previously described for bat sarbecoviruses related to the first SARS-CoV lineage29,30,31. Biol. 4 we compare these divergence time estimates to those obtained using the MERS-CoV-centred rate priors for NRR1, NRR2 and NRA3. J. Virol. 1. Membrebe, J. V., Suchard, M. A., Rambaut, A., Baele, G. & Lemey, P. Bayesian inference of evolutionary histories under time-dependent substitution rates. 1a-c ), has the third-highest number of confirmed COVID-19 cases in the state of So. 21, 255265 (2004). Chernomor, O. et al. volume5,pages 14081417 (2020)Cite this article. ac, Root-to-tip (RtT) divergence as a function of sampling time for the three coronavirus evolutionary histories unfolding over different timescales (HCoV-OC43 (n=37; a) MERS (n=35; b) and SARS (n=69; c)). 3 Priors and posteriors for evolutionary rate of SARS-CoV-2. Nature 579, 265269 (2020). Using the most conservative approach to identification of a non-recombinant genomic region (NRR1), SARS-CoV-2 forms a sister lineage with RaTG13, with genetically related cousin lineages of coronavirus sampled in pangolins in Guangdong and Guangxi provinces (Fig. By 2009, however, rapid genomic analysis had become a routine component of outbreak response. Identifying the origins of an emerging pathogen can be critical during the early stages of an outbreak, because it may allow for containment measures to be precisely targeted at a stage when the number of daily new infections is still low. Rev. Posterior distributions were approximated through Markov chain Monte Carlo sampling, which were run sufficiently long to ensure effective sampling sizes >100.
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