Biomedical subjects

A Portner

Publications and source records attributed to A Portner.

At least 19 recordsLinked to original sources

PubMed2005

Neuraminidase inhibitors as antiviral agents.

The enzyme neuraminidase (NA) is an attractive target for antiviral strategy because of its essential role in the pathogenicity of many respiratory viruses. NA removes sialic acid from the surface of infected cells and virus particles, thereby preventing viral self-aggregation and promoting efficient viral spread; NA also plays a role in the initial penetration of the mucosal lining of the respiratory tract. Random screening for inhibitors has identified only low-affinity and nonselective viral NA inhibitors. Selective, high-affinity inhibitors of influenza virus neuraminidase, zanamivir and oseltamivir, were developed using computer-aided design techniques on the basis of the three-dimensional structure of the influenza virus NA. These drugs were highly efficient in inhibiting replication of both influenza A and B viruses in vitro and in vivo and were approved for human use in 1999. Subsequently, the same structure-based design approach was used for the rational design of inhibitors of the parainfluenza virus hemagglutinin-neuraminidase (HN). One of these compounds, BCX 2798, effectively inhibited NA activity, cell binding, and growth of parainfluenza viruses in tissue culture and in the lungs of infected mice. Clinical reports indicate high efficiency of NA inhibitors for prophylaxis and treatment of influenza virus infection, good tolerance, and a low rate of emergence of drug-resistant mutants. Future experimental and clinical studies should establish the viability of NA inhibitors for the treatment of other respiratory virus infections.

Acetamides↗

PubMed2001

Two regions of the P protein are required to be active with the L protein for human parainfluenza virus type 1 RNA polymerase activity.

The paramyxovirus P protein is an essential component of the viral RNA polymerase composed of P and L proteins. In this study, we characterized the physical and functional interactions between P and L proteins using human parainfluenza virus type 1 (hPIV1) and its counterpart Sendai virus (SV). The hPIV1 P and SV L proteins or the SV P and hPIV1 L proteins formed complexes detected by anti-P antibodies. Functional analysis using the minigenome SV RNA containing CAT gene indicated that the hPIV1 P--SV L complex, but not the SV P--hPIV1 L complex, was biologically active. Mutant SV P or hPIV1 P cDNAs, which do not express C proteins, showed the same phenotype with wild-type P cDNAs, indicating that C proteins are not responsible for the dysfunction of SV P--hPIV1 L polymerase complex. Using the chimeric hPIV1/SV P cDNAs, we identified two regions (residues 387--423 and 511--568) on P protein, which are required for the functional interaction with hPIV1 L. These regions overlap with a previously identified domain for oligomer formation and binding to nucleocapsids. Our results indicate that in addition to a P--L binding domain, hPIV1 L requires a specific region on P protein to be biologically functional as a polymerase.

Amino Acid Sequence↗

PubMed2001

Receptor specificities of human respiroviruses.

Through their hemagglutinin-neuraminidase glycoprotein, parainfluenza viruses bind to sialic acid-containing glycoconjugates to initiate infection. Although the virus-receptor interaction is a key factor of infection, the exact nature of the receptors that human parainfluenza viruses recognize has not been determined. We evaluated the abilities of human parainfluenza virus types 1 (hPIV-1) and 3 (hPIV-3) to bind to different types of gangliosides. Both hPIV-1 and hPIV-3 preferentially bound to neolacto-series gangliosides containing a terminal N-acetylneuraminic acid (NeuAc) linked to N-acetyllactosamine (Galbeta1-4GlcNAc) by the alpha2-3 linkage (NeuAcalpha2-3Galbeta1-4GlcNAc). Unlike hPIV-1, hPIV-3 bound to gangliosides with a terminal NeuAc linked to Galbeta1-4GlcNAc through an alpha2-6 linkage (NeuAcalpha2-6Galbeta1-4GlcNAc) or to gangliosides with a different sialic acid, N-glycolylneuraminic acid (NeuGc), linked to Galbeta1-4GlcNAc (NeuGcalpha2-3Galbeta1-4GlcNAc). These results indicate that the molecular species of glycoconjugate that hPIV-1 recognizes are more limited than those recognized by hPIV-3. Further analysis using purified gangliosides revealed that the oligosaccharide core structure is also an important element for binding. Gangliosides that contain branched N-acetyllactosaminoglycans in their core structure showed higher avidity than those without them. Agglutination of human, cow, and guinea pig erythrocytes but not equine erythrocytes by hPIV-1 and hPIV-3 correlated well with the presence or the absence of sialic acid-linked branched N-acetyllactosaminoglycans on the cell surface. Finally, NeuAcalpha2-3I, which bound to both viruses, inhibited virus infection of Lewis lung carcinoma-monkey kidney cells in a dose-dependent manner. We conclude that hPIV-1 and hPIV-3 preferentially recognize oligosaccharides containing branched N-acetyllactosaminoglycans with terminal NeuAcalpha2-3Gal as receptors and that hPIV-3 also recognizes NeuAcalpha2-6Gal- or NeuGcalpha2-3Gal-containing receptors. These findings provide important information that can be used to develop inhibitors that prevent human parainfluenza virus infection.

Animals↗

PubMed2001

Role of matrix and fusion proteins in budding of Sendai virus.

Paramyxoviruses are assembled at the surface of infected cells, where virions are formed by the process of budding. We investigated the roles of three Sendai virus (SV) membrane proteins in the production of virus-like particles. Expression of matrix (M) proteins from cDNA induced the budding and release of virus-like particles that contained M, as was previously observed with human parainfluenza virus type 1 (hPIV1). Expression of SV fusion (F) glycoprotein from cDNA caused the release of virus-like particles bearing surface F, although their release was less efficient than that of particles bearing M protein. Cells that expressed only hemagglutinin-neuraminidase (HN) released no HN-containing vesicles. Coexpression of M and F proteins enhanced the release of F protein by a factor greater than 4. The virus-like particles containing F and M were found in different density gradient fractions of the media of cells that coexpressed M and F, a finding that suggests that the two proteins formed separate vesicles and did not interact directly. Vesicles released by M or F proteins also contained cellular actin; therefore, actin may be involved in the budding process induced by viral M or F proteins. Deletion of C-terminal residues of M protein, which has a sequence similar to that of an actin-binding domain, significantly reduced release of the particles into medium. Site-directed mutagenesis of the cytoplasmic tail of F revealed two regions that affect the efficiency of budding: one domain comprising five consecutive amino acids conserved in SV and hPIV1 and one domain that is similar to the actin-binding domain required for budding induced by M protein. Our results indicate that both M and F proteins are able to drive the budding of SV and propose the possible role of actin in the budding process.

Actins↗

PubMed2001

Nucleocapsid incorporation into parainfluenza virus is regulated by specific interaction with matrix protein.

The paramyxovirus nucleoproteins (NPs) encapsidate the genomic RNA into nucleocapsids, which are then incorporated into virus particles. We determined the protein-protein interaction between NP molecules and the molecular mechanism required for incorporating nucleocapsids into virions in two closely related viruses, human parainfluenza virus type 1 (hPIV1) and Sendai virus (SV). Expression of NP from cDNA resulted in in vivo nucleocapsid formation. Electron micrographs showed no significant difference in the morphological appearance of viral nucleocapsids obtained from lysates of transfected cells expressing SV or hPIVI NP cDNA. Coexpression of NP cDNAs from both viruses resulted in the formation of nucleocapsid composed of a mixture of NP molecules; thus, the NPs of both viruses contained regions that allowed the formation of mixed nucleocapsid. Mixed nucleocapsids were also detected in cells infected with SV and transfected with hPIV1 NP cDNA. However, when NP of SV was donated by infected virus and hPIV1 NP was from transfected cDNA, nucleocapsids composed of NPs solely from SV or solely from hPIVI were also detected. Although almost equal amounts of NP of the two viruses were found in the cytoplasm of cells infected with SV and transfected with hPIV1 NP cDNA, 90% of the NPs in the nucleocapsids of the progeny SV virions were from SV. Thus, nucleocapsids containing heterologous hPIV1 NPs were excluded during the assembly of progeny SV virions. Coexpression of hPIV1 NP and hPIV1 matrix protein (M) in SV-infected cells increased the uptake of nucleocapsids containing hPIV1 NP; thus, M appears to be responsible for the specific incorporation of the nucleocapsid into virions. Using SV-hPIV1 chimera NP cDNAs, we found that the C-terminal domain of the NP protein (amino acids 420 to 466) is responsible for the interaction with M.

Animals↗

PubMed2000

Crystallization of Newcastle disease virus hemagglutinin-neuraminidase glycoprotein.

The hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was isolated by cleaving HN (cHN) from reconstituted virosome with chymotrypsin. N-terminal sequence analysis of the purified cHN showed that chymotrypsin cleavage had occurred at amino acid 123, freeing the C-terminal 454 amino acids. The purified cHN retained its neuraminidase and receptor binding activities and reacted with specific monoclonal antibodies, showing that the isolated cHN was biologically and antigenically functional. The crystals of the cHN were obtained in acetate buffer (pH 4.6) containing polyethylene glycol 3350 and ammonium sulfate and belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimension of approximately a = 72 A, b = 78 A, and c = 198 A. Crystals of cHN grown in the presence of sialic acid (Neu5Ac) were grown in HEPES buffer (pH 6.2) containing polyethylene glycol 3350 and belong to the hexagonal space groups P6(1) or P6(5) with unit cell dimensions of a = b = 137.5 A and c = 116.6A. The orthorhombic crystals produced in this study diffract X rays to at least 2.0-A resolution, thereby setting the stage for the solution of the three-dimensional structure of the HN glycoprotein of a paramyxovirus.

Amino Acid Sequence↗

PubMed2000

Molecular cloning and expression of human parainfluenza virus type 1 L gene.

The large (L) protein, a subunit of paramyxovirus RNA polymerase complex is responsible for the majority of enzymic activities involved in viral replication and transcription. To gain insight of the functions of the L protein, we cloned the L gene of human parainfluenza virus type 1 (hPIV1) and sequenced the entire gene. The L gene, which was 6800 nucleotides, encoded a protein of 2223 residues with a calculated molecular weight of 253657. The predicted amino acid sequence was highly homologous with that of Sendai virus (SV) L (86% identity). The hPIV1 L protein expressed from the cloned L gene bound hPIV1 P expressed in the same cells. When cells were transfected with hPIV1 L, P and NP genes together with SV minigenome RNA containing a chloramphenicol acetyltransferase (CAT) gene (Send-CAT), RNA was transcribed, and CAT proteins were detected. These results indicate that the protein encoded by the cloned hPIV1 L gene was biologically functional and that the hPIV1 polymerase complex recognized SV transcription initiation and termination sequences to produce viral transcripts.

Amino Acid Sequence↗

PubMed2000

Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase.

Paramyxoviruses are the main cause of respiratory disease in children. One of two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN), has several functions in addition to being the major surface antigen that induces neutralizing antibodies. Here we present the crystal structures of Newcastle disease virus HN alone and in complex with either an inhibitor or with the beta-anomer of sialic acid. The inhibitor complex reveals a typical neuraminidase active site within a beta-propeller fold. Comparison of the structures of the two complexes reveal differences in the active site, suggesting that the catalytic site is activated by a conformational switch. This site may provide both sialic acid binding and hydrolysis functions since there is no evidence for a second sialic acid binding site in HN. Evidence for a single site with dual functions is examined and supported by mutagenesis studies. The structure provides the basis for the structure-based design of inhibitors for a range of paramyxovirus-induced diseases.

Amino Acid Sequence↗

PubMed1999

Human parainfluenza virus type 1 matrix and nucleoprotein genes transiently expressed in mammalian cells induce the release of virus-like particles containing nucleocapsid-like structures.

The matrix (M) protein plays an essential role in the assembly and budding of some enveloped RNA viruses. We expressed the human parainfluenza virus type 1 (hPIV-1) M and/or NP genes into 293T cells using the mammalian expression vector pCAGGS. Biochemical and electron microscopic analyses of transfected cells showed that the M protein alone can induce the budding of virus-like particles (vesicles) from the plasma membrane and that the NP protein can assemble into intracellular nucleocapsid-like (NC-like) structures. Furthermore, the coexpression of both the M and NP genes resulted in the production of vesicles enclosing NC-like structures, suggesting that the hPIV-1 M protein has the intrinsic ability to induce membrane vesiculation and to incorporate NC-like structures into these budding vesicles.

Animals↗

PubMed1998

Cytoplasmic domain of Sendai virus HN protein contains a specific sequence required for its incorporation into virions.

In the assembly of paramyxoviruses, interactions between viral proteins are presumed to be specific. The focus of this study is to elucidate the protein-protein interactions during the final stage of viral assembly that result in the incorporation of the viral envelope proteins into virions. To this end, we examined the specificity of HN incorporation into progeny virions by transiently transfecting HN cDNA genes into Sendai virus (SV)-infected cells. SV HN expressed from cDNA was efficiently incorporated into progeny Sendai virions, whereas Newcastle disease virus (NDV) HN was not. This observation supports the theory of a selective mechanism for HN incorporation. To identify the region on HN responsible for the selective incorporation, we constructed chimeric SV and NDV HN cDNAs and evaluated the incorporation of expressed proteins into progeny virions. Chimera HN that contained the SV cytoplasmic domain fused to the transmembrane and external domains of the NDV HN was incorporated to SV particles, indicating that amino acids in the cytoplasmic domain are responsible for the observed specificity. Additional experiments using the chimeric HNs showed that 14 N-terminal amino acids are sufficient for the specificity. Further analysis identified five consecutive amino acids (residues 10 to 14) that were required for the specific incorporation of HN into SV. These residues are conserved among all strains of SV as well as those of its counterpart, human parainfluenza virus type 1. These results suggest that this region near the N terminus of HN interacts with another viral protein(s) to lead to the specific incorporation of HN into progeny virions.

Amino Acid Sequence↗

PubMed1998

The M2 ectodomain is important for its incorporation into influenza A virions.

M2 is an integral protein of influenza A virus that functions as an ion channel. The ratio of M2 to HA in influenza A virions differs from that found on the cell surface, suggesting selective incorporation of M2 and HA into influenza virions. To examine the sequences that are important for M2 incorporation into virions, we used an incorporation assay that involves expressing M2 from a plasmid, transfecting the plasmid into recipient cells, and then infecting those cells with influenza virus. To test the importance of the different regions of the protein (extracellular, transmembrane, and cytoplasmic) in determining M2 incorporation, we created chimeric mutants of M2 and Sendai virus F proteins, exchanging corresponding extracellular, transmembrane, and cytoplasmic domains. Of the six possible chimeric mutants, only three were expressed on the cell surface. Of these three chimeric proteins, only one mutant (with the extracellular domain from M2 and the rest from F) was incorporated into influenza virions. These results suggest that the extracellular domain of M2 is important for its incorporation into virions.

Amino Acid Sequence↗

PubMed1997

Elevated expression of the human parainfluenza virus type 1 F gene downregulates HN expression.

Interactions involved in the expression of parainfluenza glycoproteins were examined by expressing cDNA clones of the HN and F genes from human parainfluenza virus type-1 (hPIV1) or Sendai virus (SV) in recombinant Semliki Forest virus (recSFV) or vaccinia-T7 expression vectors. We found that expression of a cloned F protein gene of hPIV1 resulted in downregulation of the HN proteins of hPIV1 or SV. Compared to the amount of HN expressed in the absence of F, coexpression of HN and F led to about 70% reduction in HN. This reduction of HN was observed in both total cell lysates and in protein localized on the cell surface. In contrast to hPIV1 F, SV F did not suppress the expression of HN. Northern blot analysis indicated that similar levels of HN mRNA accumulated in the absence or presence of hPIV1 F. The reduction of HN protein expression by hPIV1 F was detectable after as little as a 10-min labeling period, suggesting that downregulation occurred at the level of translation or at an early stage of protein folding. In hPIV1-infected cells, the amount of F protein synthesized was only about 15% of that of HN, whereas SV F is expressed at high levels. When the level of F in hPIV1-infected cells was artificially increased by recSFV, HN expression was suppressed. The reduction of F protein production in hPIV1-infected cells was regulated at the level of transcription. Characterization of mRNAs produced in hPIV1-infected cells showed that only 20% of the hPIV1 F mRNAs were monocistronic transcripts; 80% were bicistronic M-F readthrough mRNAs. Because proteins are suggested to be synthesized from only the first cistron of bicistronic mRNA in paramyxovirus (T. C. Wong and A. Hirano (1987) J. Virol. 61, 584-589), production of F protein is likely suppressed by transcriptional regulation in hPIV1-infected cells. These results suggest that F is capable of downregulating the synthesis of HN, but that this is normally prevented in hPIV1-infected cells by suppression of F protein synthesis by transcriptional regulation.

Animals↗

PubMed1997

Intranasal Sendai virus vaccine protects African green monkeys from infection with human parainfluenza virus-type one.

Human parainfluenza virus-type I (hPIV-1) infections are a common cause of "group" and hospitalizations among young children. Here we address the possibility of using the xenotropic Sendai virus [a mouse parainfluenza virus (PIV)] as a vaccine for hPIV-1. Sendai virus was administered to six African green monkeys (Cercopithecus aethiops) by the intranasal (i.n.) route. A long lasting virus-specific antibody response was elicited, both in the serum and nasal cavity. Sendai virus caused no apparent clinical symptoms in the primates, but live virus was detected in the nasal cavity for several days after inoculation. No virus was detected after a second dose of Sendai virus was administered on day 126 after the initial priming. Animals were challenged with hPIV-1 i.n. on day 154. All six vaccinated animals were fully protected from infection while six of six control animals were infected with hPIV-1. The antibody responses induced by Sendai virus immunizations proved to be greater than those induced by hPIV-1. These results demonstrate that unmanipulated Sendai virus is an effective vaccine against hPIV-1 in a primate model and may constitute a practical vaccine for human use.

Administration, Intranasal↗

PubMed1997

Inter- and intra-patient sequence diversity among parainfluenza virus-type 1 nucleoprotein genes.

Parainfluenza viruses (PIV) have been categorized into four discrete types (types 1-4), based on antigenic similarities. Here is described an evaluation of nucleoprotein (NP) sequence variability among nine patients infected with the type 1 virus. The examination of short segments of the NP sequence was sufficient to define significant variability both within and between patient samples. These data, in conjunction with previous studies of hemagglutinin-neuraminidase and fusion protein sequences from PIV-infected patient populations suggest a lack of absolute stability among isolates within each virus type. Potentially, antigenic variability exists to the extent that an immune response elicited toward one isolate may not be fully protective against another of the same type. Thus, sequence variability could contribute to natural re-infections with PIV, as well as to previous vaccine failures. Results highlight the importance of analyzing viruses that break through vaccine-induced immunity, in order to measure the influence of virus diversity on PIV vaccine outcome.

Amino Acid Sequence↗

PubMed1995

A single amino acid changes enhances the fusion promotion activity of human parainfluenza virus type 1 hemagglutinin-neuraminidase glycoprotein.

Clinical isolates of human parainfluenza virus type 1 in our laboratory were found to induce significantly different degrees of syncytium formation in CV-1 cells. Sequence analysis of high- and low-fusion strains suggested that the hemagglutinin-neuraminidase (HN) protein was responsible for the differences in fusion activity. We exploited the strain differences to define the specific amino acid residues of the HN protein which were responsible for the low and high fusion activities. The HN proteins of the two low-fusogenic strains 8389 and 45785, and the highly fusogenic strain C35, were expressed in HeLa T4+ cells and their fusion promotion activities were compared. When coexpressed with C35 F, HNs from the low-fusogenic viruses were associated with much lower fusion activity than was C35 HN, suggesting that the HN proteins modified the fusogenicity of the viruses. To identify the region of the HN protein responsible for this difference, we constructed a series of chimeric HN cDNAs combining 8389 and C35 sequences. All chimeric HNs that contained C35 sequence in the central 36% of the protein exhibited high fusion promotion activity. Further analysis by site-directed mutagenesis showed that a single Asn-to-Lys substitution at position 242 converted 8389 HN to a highly fusion-promoting molecule. Thus, the globular head of the HN molecule is involved in fusion promotion activity.

Amino Acid Sequence↗

PubMed1995

Molecular evolution of the F glycoprotein of human parainfluenza virus type 1.

Human parainfluenza virus type 1 (hPIV1) is a major cause of upper and lower respiratory tract infections among children. Immunity is mediated at least in part by antibody to the fusion (F) surface glycoprotein. Thus, genetic variation in the F gene could influence host range, virulence, and immunity. To examine the genetic diversity among hPIV1 isolates, the F genes of hPIV1 isolates from a single geographic location were sequenced and compared with the F gene of a strain isolated in 1957. Genetic variation was 2.2%-3.4%, averaging 0.8 amino acid changes per year. Changes were progressive over time, and virus evolution was dominated by a single lineage. Three of 7 isolates tested did not induce syncytium formation in tissue culture. This phenotype could not be ascribed to a single unique mutation in the F gene, but these 3 isolates had mutations in the transmembrane region of the HN gene. It is unlikely that the limited genetic evolution of the F gene will be an obstacle to vaccine development.

Amino Acid Sequence↗

PubMed1994

Regions on the hemagglutinin-neuraminidase proteins of human parainfluenza virus type-1 and Sendai virus important for membrane fusion.

To study the contributions of the hemagglutinin-neuraminidase (HN) and the fusion (F) glycoproteins in virus-induced membrane fusion, the HN and F proteins of human parainfluenza virus type-1 (hPIV-1) and Sendai virus (SV) were expressed in HeLa T4+ cells using the vaccinia virus-T7 RNA polymerase transient expression system. Expression of F protein alone did not induce cell fusion. However, coexpression of homologous F and HN proteins resulted in extensive syncytium formation by hPIV-1 or SV glycoproteins, which supports the proposal that both the F and HN glycoproteins are necessary for membrane fusion. To investigate the function of HN in membrane fusion, we coexpressed heterologous combinations of the HN and F proteins of hPIV-1 and SV. No fusion was observed when SV HN and hPIV-1 F proteins were coexpressed. In contrast, the coexpression of hPIV-1 HN and SV F induced extensive cell fusion. These results suggest that specific interaction between HN and F is required to induce membrane fusion. To locate regions that are essential to the fusion promoting activity, chimeric HN proteins of SV and hPIV-1 were constructed. The chimeric proteins coexpressed with the SV or hPIV-1 F proteins indicated that some regions in the middle 62% of HN contribute to the fusion-promoting activity. To determine the role of the transmembrane region of HN on fusion-promoting activity, mutant HN proteins were expressed and their biological activities examined. Mutation of hPIV-1 HN at residue 55 from cysteine to tryptophan did not affect cell binding, neuraminidase activities, or homooligomer formation, but did result in the loss of cell fusion activity. The mutation of the same cysteine residue to glycine retained the fusion-promoting activity, suggesting that a sulfhydryl moiety is not specifically required at position 55, but the structure of the residue that occupies the position is important in fusion-promoting activity.

HN Protein↗

PubMed1994

Virus-specific CD8+ T-cell memory determined by clonal burst size.

Although some viruses, particularly the herpes viruses, may never be eliminated from the body, others like influenza A, regularly reinfect humans and boost waning crossreactive CD8+ T-cell immunity. Prolonged T-cell memory is found for viruses that are unlikely to be re-encountered and which do not persist in the host genome, indicating that CD8+ T-cell memory might be independent of continued (or sporadic) antigenic exposure. A feature of virus-specific CD8+ T-cell memory is that antigen-specific cytotoxic T-lymphocyte precursors (CTLp) are greatly increased and remain high throughout life. The idea that persistence of the inducing antigen is essential is based on experiments in which adoptively transferred CD8+ memory T cells could not be detected for more than a few weeks in naive recipient mice without secondary challenge. Here we show that restimulation of such chimaeric mice with an inducing Sendai virus antigen increases the clonal burst size more than 7-fold within 8 days, making memory CTLp easier to detect in the longer term. We find that Sendai-virus-specific CTLp are maintained for > 250 days in irradiated uninfected recipients, including reconstituted beta 2-microglobulin-/- mice. To determine whether a source of viral peptide can persist after primary infection, we gave Sendai-virus-specific Thy1.1+ memory spleen cells to naive mice that had been minimally depleted of Thy1.2+ T cells, or to comparable recipients that had recovered from infection with Sendai virus or influenza virus. Although antibody against Sendai virus was never found in the naive recipients, Sendai-virus-specific CD8+ memory T cells were maintained equally well in each case for > 100 days after cell transfer. We find no evidence for persisting depots of viral protein that might feed into the endogenous processing pathway and maintain virus-specific CD8+ T-cell memory.

Animals↗