SARS-CoV-2 Seroprevalence and Cross-Variant Antibody Neutralization in Cats, United Kingdom.

Anthropogenic transmission of SARS-CoV-2 to pet cats highlights the importance of monitoring felids for exposure to circulating variants. We tested cats in the United Kingdom for SARS-CoV-2 antibodies; seroprevalence peaked during September 2021-February 2022. The variant-specific response in cats trailed circulating variants in humans, indicating multiple human-to-cat transmissions over a prolonged period.

Distemper, extinction, and vaccination of the Amur tiger.

Canine distemper virus (CDV) has recently emerged as an extinction threat for the endangered Amur tiger (Panthera tigris altaica). CDV is vaccine-preventable, and control strategies could require vaccination of domestic dogs and/or wildlife populations. However, vaccination of endangered wildlife remains controversial, which has led to a focus on interventions in domestic dogs, often assumed to be the source of infection. Effective decision making requires an understanding of the true reservoir dynamics, which poses substantial challenges in remote areas with diverse host communities. We carried out serological, demographic, and phylogenetic studies of dog and wildlife populations in the Russian Far East to show that a number of wildlife species are more important than dogs, both in maintaining CDV and as sources of infection for tigers. Critically, therefore, because CDV circulates among multiple wildlife sources, dog vaccination alone would not be effective at protecting tigers. We show, however, that low-coverage vaccination of tigers themselves is feasible and would produce substantive reductions in extinction risks. Vaccination of endangered wildlife provides a valuable component of conservation strategies for endangered species.

Structure-Guided Identification of a Nonhuman Morbillivirus with Zoonotic Potential.

Morbilliviruses infect a broad range of mammalian hosts, including ruminants, carnivores, and humans. The recent eradication of rinderpest virus (RPV) and the active campaigns for eradication of the human-specific measles virus (MeV) have raised significant concerns that the remaining morbilliviruses may emerge in so-called vacated ecological niches. Seeking to assess the zoonotic potential of nonhuman morbilliviruses within human populations, we found that peste des petits ruminants virus (PPRV)-the small-ruminant morbillivirus-is restricted at the point of entry into human cells due to deficient interactions with human SLAMF1-the immune cell receptor for morbilliviruses. Using a structure-guided approach, we characterized a single amino acid change, mapping to the receptor-binding domain in the PPRV hemagglutinin (H) protein, which overcomes this restriction. The same mutation allowed escape from some cross-protective, human patient, anti-MeV antibodies, raising concerns that PPRV is a pathogen with zoonotic potential. Analysis of natural variation within human and ovine SLAMF1 also identified polymorphisms that could correlate with disease resistance. Finally, the mechanistic nature of the PPRV restriction was also investigated, identifying charge incompatibility and steric hindrance between PPRV H and human SLAMF1 proteins. Importantly, this research was performed entirely using surrogate virus entry assays, negating the requirement for in situ derivation of a human-tropic PPRV and illustrating alternative strategies for identifying gain-of-function mutations in viral pathogens.IMPORTANCE A significant proportion of viral pandemics occur following zoonotic transmission events, where animal-associated viruses jump species into human populations. In order to provide forewarnings of the emergence of these viruses, it is necessary to develop a better understanding of what determines virus host range, often at the genetic and structural levels. In this study, we demonstrated that the small-ruminant morbillivirus, a close relative of measles, is unable to use human receptors to enter cells; however, a change of a single amino acid in the virus is sufficient to overcome this restriction. This information will be important for monitoring this virus's evolution in the field. Of note, this study was undertaken in vitro, without generation of a fully infectious virus with this phenotype.

An investigation of the breadth of neutralizing antibody response in cats naturally infected with feline immunodeficiency virus.

Neutralizing antibodies (NAbs) are believed to comprise an essential component of the protective immune response induced by vaccines against feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) infections. However, relatively little is known about the role of NAbs in controlling FIV infection and subsequent disease progression. Here, we present studies where we examined the neutralization of HIV-luciferase pseudotypes bearing homologous and heterologous FIV envelope proteins (n = 278) by sequential plasma samples collected at 6 month intervals from naturally infected cats (n = 38) over a period of 18 months. We evaluated the breadth of the NAb response against non-recombinant homologous and heterologous clade A and clade B viral variants, as well as recombinants, and assessed the results, testing for evidence of an association between the potency of the NAb response and the duration of infection, CD4(+) T lymphocyte numbers, health status and survival times of the infected cats. Neutralization profiles varied significantly between FIV-infected cats and strong autologous neutralization, assessed using luciferase-based in vitro assays, did not correlate with the clinical outcome. No association was observed between strong NAb responses and either improved health status or increased survival time of infected animals, implying that other protective mechanisms were likely to be involved. Similarly, no correlation was observed between the development of autologous NAbs and the duration of infection. Furthermore, cross-neutralizing antibodies were evident in only a small proportion (13 %) of cats.

Rapid evolution of the env gene leader sequence in cats naturally infected with feline immunodeficiency virus.

Analysing the evolution of feline immunodeficiency virus (FIV) at the intra-host level is important in order to address whether the diversity and composition of viral quasispecies affect disease progression. We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at a rate of 1.16×10(-3) substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3-V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3-V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation. Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximum evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared with human immunodeficiency virus might explain why many naturally infected cats do not progress rapidly to AIDS.

Emergence of CD134 cysteine-rich domain 2 (CRD2)-independent strains of feline immunodeficiency virus (FIV) is associated with disease progression in naturally infected cats.

BACKGROUND: Feline immunodeficiency virus (FIV) infection is mediated by sequential interactions with CD134 and CXCR4. Field strains of virus vary in their dependence on cysteine-rich domain 2 (CRD2) of CD134 for infection. FINDINGS: Here, we analyse the receptor usage of viral variants in the blood of 39 naturally infected cats, revealing that CRD2-dependent viral variants dominate in early infection, evolving towards CRD2-independence with disease progression. CONCLUSIONS: These findings are consistent with a shift in CRD2 of CD134 usage with disease progression.

Feline immunodeficiency virus (FIV) env recombinants are common in natural infections.

BACKGROUND: Recombination is a common feature of retroviral biology and one of the most important factors responsible for generating viral diversity at both the intra-host and the population levels. However, relatively little is known about rates and molecular processes of recombination for retroviruses other than HIV, including important model viruses such as feline immunodeficiency virus (FIV). RESULTS: We investigated recombination in complete FIV env gene sequences (n = 355) isolated from 43 naturally infected cats. We demonstrated that recombination is abundant in natural FIV infection, with over 41% of the cats being infected with viruses containing recombinant env genes. In addition, we identified shared recombination breakpoints; the most significant hotspot occurred between the leader/signal fragment and the remainder of env. CONCLUSIONS: Our results have identified the leader/signal fragment of env as an important site for recombination and highlight potential limitations of the current phylogenetic classification of FIV based on partial env sequences. Furthermore, the presence of abundant recombinant FIV in the USA poses a significant challenge for commercial diagnostic tests and should inform the development of the next generation of FIV vaccines.

Assessing the feasibility of applying machine learning to diagnosing non-effusive feline infectious peritonitis.

Feline infectious peritonitis (FIP) is a severe feline coronavirus-associated syndrome in cats, which is invariably fatal without anti-viral treatment. In the majority of non-effusive FIP cases encountered in practice, confirmatory diagnostic testing is not undertaken and reliance is given to the interpretation of valuable, but essentially non-specific, clinical signs and laboratory markers. We hypothesised that it may be feasible to develop a machine learning (ML) approach which may be applied to the analysis of clinical data to aid in the diagnosis of disease. A dataset encompassing 1939 suspected FIP cases was scored for clinical suspicion of FIP on the basis of history, signalment, clinical signs and laboratory results, using published guidelines, comprising 683 FIP (35.2%), and 1256 non-FIP (64.8%) cases. This dataset was used to train, validate and evaluate two diagnostic machine learning ensemble models. These models, which analysed signalment and laboratory data alone, allowed the accurate discrimination of FIP and non-FIP cases in line with expert opinion. To evaluate whether these models may have value as a diagnostic tool, they were applied to a collection of 80 cases for which the FIP status had been confirmed (FIP: n = 58 (72.5%), non-FIP: n = 22 (27.5%)). Both ensemble models detected FIP with an accuracy of 97.5%, an area under the curve (AUC) of 0.969, sensitivity of 95.45% and specificity of 98.28%. This work demonstrates that, in principle, ML can be usefully applied to the diagnosis of non-effusive FIP. Further work is required before ML may be deployed in the laboratory as a diagnostic tool, such as training models on datasets of confirmed cases and accounting for inter-laboratory variation. Nevertheless, these results illustrate the potential benefit of applying ML to standardising and accelerating the interpretation of clinical pathology data, thereby improving the diagnostic utility of existing laboratory tests.

Serological and Molecular Investigation of SARS-CoV-2 in Horses and Cattle in Switzerland from 2020 to 2022.

Horses and cattle have shown low susceptibility to SARS-CoV-2, and there is no evidence of experimental intraspecies transmission. Nonetheless, seropositive horses in the US and seropositive cattle in Germany and Italy have been reported. The current study investigated the prevalence of antibodies against SARS-CoV-2 in horses and cattle in Switzerland. In total, 1940 serum and plasma samples from 1110 horses and 830 cattle were screened with a species-specific ELISA based on the SARS-CoV-2 receptor-binding domain (RBD) and, in the case of suspect positive results, a surrogate virus neutralization test (sVNT) was used to demonstrate the neutralizing activity of the antibodies. Further confirmation of suspect positive samples was performed using either a pseudotype-based virus neutralization assay (PVNA; horses) or an indirect immunofluorescence test (IFA; cattle). The animals were sampled between February 2020 and December 2022. Additionally, in total, 486 bronchoalveolar lavage (BAL), oropharyngeal, nasal and rectal swab samples from horses and cattle were analyzed for the presence of SARS-CoV-2 RNA via reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Six horses (0.5%; 95% CI: 0.2-1.2%) were suspect positive via RBD-ELISA, and neutralizing antibodies were detected in two of them via confirmatory sVNT and PVNA tests. In the PVNA, the highest titers were measured against the Alpha and Delta SARS-CoV-2 variants. Fifteen cattle (1.8%; 95% CI: 1.0-3.0%) were suspect positive in RBD-ELISA; 3 of them had SARS-CoV-2-specific neutralizing antibodies in sVNT and 4 of the 15 were confirmed to be positive via IFA. All tested samples were RT-qPCR-negative. The results support the hypotheses that the prevalence of SARS-CoV-2 infections in horses and cattle in Switzerland was low up to the end of 2022.

Recent developments in human immunodeficiency virus-1 latency research.

Almost 30 years after its initial discovery, infection with the human immunodeficiency virus-1 (HIV-1) remains incurable and the virus persists due to reservoirs of latently infected CD4(+) memory T-cells and sanctuary sites within the infected individual where drug penetration is poor. Reactivating latent viruses has been a key strategy to completely eliminate the virus from the host, but many difficulties and unanswered questions remain. In this review, the latest developments in HIV-persistence and latency research are presented.

Increase in SARS-CoV-2 Seroprevalence in UK Domestic Felids Despite Weak Immunogenicity of Post-Omicron Variants.

Throughout the COVID-19 pandemic, SARS-CoV-2 infections in domestic cats have caused concern for both animal health and the potential for inter-species transmission. Cats are known to be susceptible to the Omicron variant and its descendants, however, the feline immune response to these variants is not well defined. We aimed to estimate the current seroprevalence of SARS-CoV-2 in UK pet cats, as well as characterise the neutralising antibody response to the Omicron (BA.1) variant. A neutralising seroprevalence of 4.4% and an overall seroprevalence of 13.9% was observed. Both purebred and male cats were found to have the highest levels of seroprevalence, as well as cats aged between two and five years. The Omicron variant was found to have a lower immunogenicity in cats than the B.1, Alpha and Delta variants, which reflects previous reports of immune and vaccine evasion in humans. These results further underline the importance of surveillance of SARS-CoV-2 infections in UK cats as the virus continues to evolve.

Feline Injection-Site Sarcoma and Other Adverse Reactions to Vaccination in Cats.

Vaccine-associated adverse events (VAAEs), including feline injection-site sarcomas (FISSs), occur only rarely but can be severe. Understanding potential VAAEs is an important part of informed owner consent for vaccination. In this review, the European Advisory Board on Cat Diseases (ABCD), a scientifically independent board of feline medicine experts, presents the current knowledge on VAAEs in cats, summarizing the literature and filling the gaps where scientific studies are missing with expert opinion to assist veterinarians in adopting the best vaccination practice. VAAEs are caused by an aberrant innate or adaptive immune reaction, excessive local reactions at the inoculation site, an error in administration, or failure in the manufacturing process. FISS, the most severe VAAE, can develop after vaccinations or injection of other substances. Although the most widely accepted hypothesis is that chronic inflammation triggers malignant transformation, the pathogenesis of FISS is not yet fully understood. No injectable vaccine is risk-free, and therefore, vaccination should be performed as often as necessary, but as infrequently as possible. Vaccines should be brought to room temperature prior to administration and injected at sites in which FISS surgery would likely be curative; the interscapular region should be avoided. Post-vaccinal monitoring is essential.

Detection and Molecular Characterization of the SARS-CoV-2 Delta Variant and the Specific Immune Response in Companion Animals in Switzerland.

In human beings, there are five reported variants of concern of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). However, in contrast to human beings, descriptions of infections of animals with specific variants are still rare. The aim of this study is to systematically investigate SARS-CoV-2 infections in companion animals in close contact with SARS-CoV-2-positive owners ("COVID-19 households") with a focus on the Delta variant. Samples, obtained from companion animals and their owners were analyzed using a real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and next-generation sequencing (NGS). Animals were also tested for antibodies and neutralizing activity against SARS-CoV-2. Eleven cats and three dogs in nine COVID-19-positive households were RT-qPCR and/or serologically positive for the SARS-CoV-2 Delta variant. For seven animals, the genetic sequence could be determined. The animals were infected by one of the pangolin lineages B.1.617.2, AY.4, AY.43 and AY.129 and between zero and three single-nucleotide polymorphisms (SNPs) were detected between the viral genomes of animals and their owners, indicating within-household transmission between animal and owner and in multi-pet households also between the animals. NGS data identified SNPs that occur at a higher frequency in the viral sequences of companion animals than in viral sequences of humans, as well as SNPs, which were exclusively found in the animals investigated in the current study and not in their owners. In conclusion, our study is the first to describe the SARS-CoV-2 Delta variant transmission to animals in Switzerland and provides the first-ever description of Delta-variant pangolin lineages AY.129 and AY.4 in animals. Our results reinforce the need of a One Health approach in the monitoring of SARS-CoV-2 in animals.

SARS-CoV-2 in Domestic UK Cats from Alpha to Omicron: Swab Surveillance and Case Reports.

Although domestic cats are susceptible to infection with SARS-CoV-2, the role of the virus in causing feline disease is less well defined. We conducted a large-scale study to identify SARS-CoV-2 infections in UK pet cats, using active and passive surveillance. Remnant feline respiratory swab samples, submitted for other pathogen testing between May 2021 and February 2023, were screened using RT-qPCR. In addition, we appealed to veterinarians for swab samples from cats suspected of having clinical SARS-CoV-2 infections. Bespoke testing for SARS-CoV-2 neutralising antibodies was also performed, on request, in suspected cases. One RT-qPCR-positive cat was identified by active surveillance (1/549, 0.18%), during the Delta wave (1/175, 0.57%). Passive surveillance detected one cat infected with the Alpha variant, and two of ten cats tested RT-qPCR-positive during the Delta wave. No cats tested RT-qPCR-positive after the emergence of Omicron BA.1 and its descendants although 374 were tested by active and eleven by passive surveillance. We describe four cases of SARS-CoV-2 infection in pet cats, identified by RT-qPCR and/or serology, that presented with a range of clinical signs, as well as their SARS-CoV-2 genome sequences. These cases demonstrate that, although uncommon in cats, a variety of clinical signs can occur.

A Risk Factor Analysis of SARS-CoV-2 Infection in Animals in COVID-19-Affected Households.

A higher prevalence of SARS-CoV-2 infections in animals that have close contact with SARS-CoV-2-positive humans ("COVID-19 households") has been demonstrated in several countries. This prospective study aimed to determine the SARS-CoV-2 prevalence in animals from Swiss COVID-19 households and to assess the potential risk factors for infection. The study included 226 companion animals (172 cats, 76.1%; 49 dogs, 21.7%; and 5 other animals, 2.2%) from 122 COVID-19 households with 336 human household members (including 230 SARS-CoV-2-positive people). The animals were tested for viral RNA using an RT-qPCR and/or serologically for antibodies and neutralizing activity. Additionally, surface samples from animal fur and beds underwent an RT-qPCR. A questionnaire about hygiene, animal hygiene, and contact intensity was completed by the household members. A total of 49 of the 226 animals (21.7%) from 31 of the 122 households (25.4%) tested positive/questionably positive for SARS-CoV-2, including 37 of the 172 cats (21.5%) and 12 of the 49 dogs (24.5%). The surface samples tested positive significantly more often in households with SARS-CoV-2-positive animals than in households with SARS-CoV-2-negative animals (p = 0.011). Significantly more animals tested positive in the multivariable analysis for households with minors. For cats, a shorter length of outdoor access and a higher frequency of removing droppings from litterboxes were factors that were significantly associated with higher infection rates. The study emphasizes that the behavior of owners and the living conditions of animals can influence the likelihood of a SARS-CoV-2 infection in companion animals. Therefore, it is crucial to monitor the infection transmission and dynamics in animals, as well as to identify the possible risk factors for animals in infected households.

Feline Morbillivirus: Clinical Relevance of a Widespread Endemic Viral Infection of Cats.

Feline morbillivirus (FeMV) was first isolated in 2012 from stray cats in Hong Kong. It has been found in association with tubulointerstitial nephritis (TIN), the most common cause of feline chronic kidney disease (CKD). However, viral host spectrum and virus tropism go beyond the domestic cat and kidney tissues. The viral genetic diversity of FeMV is extensive, but it is not known if this is clinically relevant. Urine and kidney tissues have been widely tested in attempts to confirm associations between FeMV infection and renal disease, but samples from both healthy and sick cats can test positive and some cross-sectional studies have not found associations between FeMV infection and CKD. There is also evidence for acute kidney injury following infection with FeMV. The results of prevalence studies differ greatly depending on the population tested and methodologies used for detection, but worldwide distribution of FeMV has been shown. Experimental studies have confirmed previous field observations that higher viral loads are present in the urine compared to other tissues, and renal TIN lesions associated with FeMV antigen have been demonstrated, alongside virus lymphotropism and viraemia-associated lymphopenia. Longitudinal field studies have revealed persistent viral shedding in urine, although infection can be cleared spontaneously.

Feline Infectious Peritonitis: European Advisory Board on Cat Diseases Guidelines.

Feline coronavirus (FCoV) is a ubiquitous RNA virus of cats, which is transmitted faeco-orally. In these guidelines, the European Advisory Board on Cat Diseases (ABCD) presents a comprehensive review of feline infectious peritonitis (FIP). FCoV is primarily an enteric virus and most infections do not cause clinical signs, or result in only enteritis, but a small proportion of FCoV-infected cats develop FIP. The pathology in FIP comprises a perivascular phlebitis that can affect any organ. Cats under two years old are most frequently affected by FIP. Most cats present with fever, anorexia, and weight loss; many have effusions, and some have ocular and/or neurological signs. Making a diagnosis is complex and ABCD FIP Diagnostic Approach Tools are available to aid veterinarians. Sampling an effusion, when present, for cytology, biochemistry, and FCoV RNA or FCoV antigen detection is very useful diagnostically. In the absence of an effusion, fine-needle aspirates from affected organs for cytology and FCoV RNA or FCoV antigen detection are helpful. Definitive diagnosis usually requires histopathology with FCoV antigen detection. Antiviral treatments now enable recovery in many cases from this previously fatal disease; nucleoside analogues (e.g., oral GS-441524) are very effective, although they are not available in all countries.

Identification of novel subgroup A variants with enhanced receptor binding and replicative capacity in primary isolates of anaemogenic strains of feline leukaemia virus.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Feline tetherin efficiently restricts release of feline immunodeficiency virus but not spreading of infection.

Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and "OrfA" proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread.