Bat Coronavirus Research Articles

The Inclusive Review on SARS-CoV-2 Biology, Epidemiology, Diagnosis, and Potential Management Options.

A novel coronavirus member was reported in Wuhan City, Hubei Province, China, at the end of the year 2019. Initially, the infection spread locally, affecting the Wuhan people, and then expanded rapidly throughout the world. On 11 March 2020, the World Health Organization (WHO) proclaimed it a global pandemic. The virus is a new strain most closely related to a bat coronavirus (RaTG13) which was not previously discovered in humans and is now formally known as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavirus disease 2019 (COVID-19) is the disease syndrome that the SARS-CoV-2 virus triggers. It is suggested that SARS-CoV-2 can be transmitted through aerosols, direct/indirect contact, and also during medical procedures and specimen handling. The infection is characterized by isolated flu-like symptoms, but there may be specific signs of fever, fatigue, cough, and shortness of breath, as well as the loss of smell and breathing difficulty. Within this report, we tried to review the most current scientific literature published by January 2021 on various aspects of the outbreak, including virus structure, pathogenesis, clinical presentation, epidemiology, diagnostic approaches, potential therapeutics and vaccines, and prospects. We hope this article makes a beneficial impact on public education to better deal with the SARS-CoV-2 crisis and push a step forward in the near term towards its prevention and control.

Severe acute respiratory syndrome coronavirus 2 may exploit human transcription factors involved in retinoic acid and interferon-mediated response: a hypothesis supported by an in silico analysis

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19), resulting in acute respiratory disease, is a worldwide emergency. Because recently it has been found that SARS-CoV is dependent on host transcription factors (TF) to express the viral genes, efforts are required to understand the molecular interplay between virus and host response. By bioinformatic analysis, we investigated human TF that can bind the SARS-CoV-2 sequence and can be involved in viral transcription. In particular, we analysed the key role of TF involved in interferon (IFN) response. We found that several TF could be induced by the IFN antiviral response, specifically some induced by IFN-stimulated gene factor 3 (ISGF3) and by unphosphorylated ISGF3, which were found to promote the transcription of several viral open reading frame. Moreover, we found 22 TF binding sites present only in the sequence of virus infecting humans but not bat coronavirus RaTG13. The 22 TF are involved in IFN, retinoic acid signalling and regulation of transcription by RNA polymerase II, thus facilitating its own replication cycle. This mechanism, by competition, may steal the human TF involved in these processes, explaining SARS-CoV-2's disruption of IFN-I signalling in host cells and the mechanism of the SARS retinoic acid depletion syndrome leading to the cytokine storm. We identified three TF binding sites present exclusively in the Brazilian SARS-CoV-2 P.1 variant that may explain the higher severity of the respiratory syndrome. These data shed light on SARS-CoV-2 dependence from the host transcription machinery associated with IFN response and strengthen our knowledge of the virus's transcription and replicative activity, thus paving the way for new targets for drug design and therapeutic approaches.

A Half-Day Genome Sequencing Protocol for Middle East Respiratory Syndrome Coronavirus.

Recent coronavirus (CoV) outbreaks, including that of Middle East respiratory syndrome (MERS), have presented a threat to public health worldwide. A primary concern in these outbreaks is the extent of mutations in the CoV, and the content of viral variation that can be determined only by whole genome sequencing (WGS). We aimed to develop a time efficient WGS protocol, using universal primers spanning the entire MERS-CoV genome. MERS and synthetic Neoromicia capensis bat CoV genomes were successfully amplified using our developed PCR primer set and sequenced with MinION. All experimental and analytical processes took 6 h to complete and were also applied to synthetic animal serum samples, wherein the MERS-CoV genome sequence was completely recovered. Results showed that the complete genome of MERS-CoV and related variants could be directly obtained from clinical samples within half a day. Consequently, this method will contribute to rapid MERS diagnosis, particularly in future CoV epidemics.

A Review on COVID-19: The Epidemiology, Transmission, Impact on Pregnancy and Reproductive Health and Its Prevention

At the end of 2019, a novel coronavirus was identified as the cause of a cluster of pneumonia cases in Wuhan, a city in the Hubei Province of China. This results in an epidemic throughout China, followed by an increasing number of cases in other countries throughout the world. Currently, containment and mitigation measures have intensified and disease-modifying pharmacologic compounds and vaccines are being developed, COVID-19 continues to spread. The 2019 novel coronavirus (2019-nCoV/SARS-CoV-2) was shown to be a Beta-coronavirus (lineage B) closely related to SARS-CoV and SARS-related bat coronaviruses, and it has been reported to share a common receptor with SARS-CoV (ACE-2)At the time of this writing, the full magnitude of the public health impact poses a great threat to all humankind. More than 12 million patients have been infected globally and 564, 623 have died as of 10th July 2020. In this review, we highlight epidemiology, transmission, impact on pregnancy and reproductive health and future directions to control the spread of this fatal disease.

Identification of Cytotoxic T-Cell and B-Cell Epitopes in the Nucleocapsid Phosphoprotein of SARS-COV-2 Using Immunoinformatics

Last December, a novel coronavirus emerged in Wuhan city, China. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes a high intense acute respiratory syndrome with elevation mortality. Nucleocapsid phosphoprotein (NP) is one of the most structural proteins of the virus. NP possesses active immunogenicity for T-cell response. Because NP considered as a potential vaccine target, our study goal was to identify the cytotoxic T-cell (CTL) and B-cell epitopes inside NP peptides. Methods. We used a series of popular immunoinformatics and algorithm tools such as FASTA-NCBI, CLUSTAL-OMGA, T-COFFEE, SWISS-MODEL, CTLPred and its branches. Results. Homology modeling and alignment of SARS-CoV-2 NP showed high conserved residues compared with related sequences. Different types of the major histocompatibility complex (MHC) alleles were identified, specifically human leukocyte antigens (HLA-A) affinity for NP. We also demonstrate six B-cell epitopes with a high score above the threshold. Conclusions. We recorded high binder HLA-A*02:01 alleles matched between the novel coronavirus SARS-CoV-2 NP and the Bat coronavirus SARS-Bat-CoV NP. Identification of CTL response and B-cell predictions will be helpful in reverse immunogenetic approaches, hence in the strategy process of the plausible design of the vaccine.

SARS CoV-2 Spike Protein in silico Interaction With ACE2 Receptors From Wild and Domestic Species.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared a pandemic by the World Health Organization (WHO), and since its first report, it has become a major public health concern. SARS-CoV-2 is closely related to SARS-CoV and SARS-related bat coronaviruses, and it has been described to use angiotensin-converting enzyme 2 (ACE2) as a receptor. Natural SARS-CoV-2 infection in domestic and wildlife animals, measured by RT-qPCR, has been confirmed in different countries, especially from the Felidae family. In silico analysis of the interaction between the SARS-CoV-2 spike protein and the cellular receptor ACE2 in various animal species has suggested that wild felids and domestic cats could be susceptible to SARS-CoV-2 based on this interaction. Here, we performed a protein-protein molecular docking analysis of SARS-CoV-2 spike protein with the ACE2 receptor from different animals to elucidate the potential of those species as intermediate hosts or susceptible animals for SARS-CoV-2 infection. Compared to human ACE2, we found that ACE2 receptors from domestic cats and tigers could efficiently interact with RBD of SARS CoV-2 Spike protein. However, dog, ferret, and hamster ACE2 receptor interaction with SARS-CoV-2 S protein RBD was not predicted as favorable, demonstrating a potential differentiated susceptibility in the evaluated species.

SARS-CoV-2 infection is effectively treated and prevented by EIDD-2801.

Summary ParagraphAll known recently emerged human coronaviruses likely originated in bats1. Here, we used a single experimental platform based on human lung-only mice (LoM) to demonstrate efficient in vivo replication of all recently emerged human coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2) and two highly relevant endogenous pre-pandemic SARS-like bat coronaviruses. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats harbor endogenous coronaviruses capable of direct transmission into humans. Further detailed analysis of pandemic SARS-CoV-2 in vivo infection of LoM human lung tissue showed predominant infection of human lung epithelial cells, including type II pneumocytes present in alveoli and ciliated airway cells. Acute SARS-CoV-2 infection was highly cytopathic and induced a robust and sustained Type I interferon and inflammatory cytokine/chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for coronavirus infection. Our results show that therapeutic and prophylactic administration of EIDD-2801, an oral broad spectrum antiviral currently in phase II-III clinical trials, dramatically inhibited SARS-CoV-2 replication in vivo and thus has significant potential for the prevention and treatment of COVID-19.

Evidence for SARS-CoV-2 related coronaviruses circulating in bats and pangolins in Southeast Asia

Among the many questions unanswered for the COVID-19 pandemic are the origin of SARS-CoV-2 and the potential role of intermediate animal host(s) in the early animal-to-human transmission. The discovery of RaTG13 bat coronavirus in China suggested a high probability of a bat origin. Here we report molecular and serological evidence of SARS-CoV-2 related coronaviruses (SC2r-CoVs) actively circulating in bats in Southeast Asia. Whole genome sequences were obtained from five independent bats (Rhinolophus acuminatus) in a Thai cave yielding a single isolate (named RacCS203) which is most related to the RmYN02 isolate found in Rhinolophus malayanus in Yunnan, China. SARS-CoV-2 neutralizing antibodies were also detected in bats of the same colony and in a pangolin at a wildlife checkpoint in Southern Thailand. Antisera raised against the receptor binding domain (RBD) of RmYN02 was able to cross-neutralize SARS-CoV-2 despite the fact that the RBD of RacCS203 or RmYN02 failed to bind ACE2. Although the origin of the virus remains unresolved, our study extended the geographic distribution of genetically diverse SC2r-CoVs from Japan and China to Thailand over a 4800-km range. Cross-border surveillance is urgently needed to find the immediate progenitor virus of SARS-CoV-2.

Population Genomics Insights into the First Wave of COVID-19.

Full-genome-sequence computational analyses of the SARS-coronavirus (CoV)-2 genomes allow us to understand the evolutionary events and adaptability mechanisms. We used population genetics analyses on human SARS-CoV-2 genomes available on 2 April 2020 to infer the mutation rate and plausible recombination events between the Betacoronavirus genomes in nonhuman hosts that may have contributed to the evolution of SARS-CoV-2. Furthermore, we localized the targets of recent and strong, positive selection during the first pandemic wave. The genomic regions that appear to be under positive selection are largely co-localized with regions in which recombination from nonhuman hosts took place. Our results suggest that the pangolin coronavirus genome may have contributed to the SARS-CoV-2 genome by recombination with the bat coronavirus genome. However, we find evidence for additional recombination events that involve coronavirus genomes from other hosts, i.e., hedgehogs and sparrows. We further infer that recombination may have recently occurred within human hosts. Finally, we estimate the parameters of a demographic scenario involving an exponential growth of the size of the SARS-CoV-2 populations that have infected European, Asian, and Northern American cohorts, and we demonstrate that a rapid exponential growth in population size from the first wave can support the observed polymorphism patterns in SARS-CoV-2 genomes.

Structural genetics of circulating variants affecting the SARS-CoV-2 spike/human ACE2 complex

SARS-CoV-2 entry in human cells is mediated by the interaction between the viral Spike protein and the human ACE2 receptor. This mechanism evolved from the ancestor bat coronavirus and is currently one of the main targets for antiviral strategies. However, there currently exist several Spike protein variants in the SARS-CoV-2 population as the result of mutations, and it is unclear if these variants may exert a specific effect on the affinity with ACE2 which, in turn, is also characterized by multiple alleles in the human population. In the current study, the GBPM analysis, originally developed for highlighting host-guest interaction features, has been applied to define the key amino acids responsible for the Spike/ACE2 molecular recognition, using four different crystallographic structures. Then, we intersected these structural results with the current mutational status, based on more than 295,000 sequenced cases, in the SARS-CoV-2 population. We identified several Spike mutations interacting with ACE2 and mutated in at least 20 distinct patients: S477N, N439K, N501Y, Y453F, E484K, K417N, S477I and G476S. Among these, mutation N501Y in particular is one of the events characterizing SARS-CoV-2 lineage B.1.1.7, which has recently risen in frequency in Europe. We also identified five ACE2 rare variants that may affect interaction with Spike and susceptibility to infection: S19P, E37K, M82I, E329G and G352V. Communicated by Ramaswamy H. Sarma

Structure and binding properties of Pangolin-CoV spike glycoprotein inform the evolution of SARS-CoV-2

Coronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.

Dynamics of binding ability prediction between spike protein and human ACE2 reveals the adaptive strategy of SARS-CoV-2 in humans

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a novel coronavirus causing the COVID-19 pandemic in 2020. High adaptive plasticity on the spike protein of SASR-CoV-2 enables it to transmit across different host species. In the present study, we collected 2092 high-quality genome sequences of SARS-CoV-2 from 160 regions in over 50 countries and reconstructed their phylogeny. We also analyzed the polymorphic interaction between spike protein and human ACE2 (hACE2). Phylogenetic analysis of SARS-CoV-2 suggests that SARS-CoV-2 is probably originated from a recombination event on the spike protein between a bat coronavirus and a pangolin coronavirus that endows it humans infectivity. Compared with other regions in the S gene of SARS-CoV-2, the direct-binding sites of the receptor-binding domain (RBD) is more conserved. We focused on 3,860 amino acid mutations in spike protein RBD (T333-C525) of SARS-CoV-2 and simulated their differential stability and binding affinity to hACE2 (S19-D615). The results indicate no preference for SARS-CoV-2 infectivity on people of different ethnic groups. The variants in the spike protein of SARS-CoV-2 may also be a good indicator demonstrating the transmission route of SARS-CoV-2 from its natural reservoir to human hosts.

Scientific effort in combating COVID-19 in obstetrics and gynecology.

Scientific effort in combating COVID-19 in obstetrics and gynecology.

The SARS-CoV-2 Pandemic a Global Emergency: The Journey from Bats to Humans

Bats are considered as the reservoirs of coronaviruses (CoVs) which resulted in severe disease outbreaks in humans. Zoonotic coronaviruses responsible for Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), were originated from bats and remain highly pathogenic to humans during the last two decades. Recently, an acute respiratory disease outbreak which spread in Wuhan, China was caused by a novel coronavirus (SARS-CoV-2) also known as 2019-nCoV, received worldwide attention. The World Health Organization (WHO) officially declared the COVID-19 epidemic caused by SARS-CoV-2 as a public health emergency. The emergence of SARS-CoV-2 has been considered as the third highly pathogenic epidemic of the 21 st century. The SARS-CoV-2 belongs to β-coronavirus group with highly identical genome to bat coronavirus RaTG13, assuming bat as the natural host. According to WHO as of December 21, 2020, approximately 75,479,471 persons were infected with 16,86,267 deaths in 222 countries. Whereas the in Pakistan according to WHO as of December 21, 2020, around 4 57,288 persons were infected with 9, 330 death reported in the country. Keywords: SARS-CoV-2; 2019-nCoV; Bats; Human; Epidemic; Pandemic DOI: 10.7176/FSQM/105-07 Publication date: February 28 th 2021

Anti-inflammatory potential of Quercetin in COVID-19 treatment

SARS-CoV-2 is a betacoronavirus causing severe inflammatory pneumonia, so that excessive inflammation is considered a risk factor for the disease. According to reports, cytokine storm is strongly responsible for death in such patients. Some of the consequences of severe inflammation and cytokine storms include acute respiratory distress syndrome, acute lung injury, and multiple organ dysfunction syndromes. Phylogenetic findings show more similarity of the SARS-CoV-2 virus with bat coronaviruses, and less with SARS-CoV. Quercetin is a carbohydrate-free flavonoid that is the most abundant flavonoid in vegetables and fruits and has been the most studied to determine the biological effects of flavonoids. Inflammasomes are cytosolic multi-protein complexes assembling in response to cytosolic PAMP and DAMPs, whose function is to generate active forms of cytokines IL-1β and IL-18. Activation or inhibition of the NLRP3 inflammasome is affected by regulators such as TXNIP, SIRT1 and NRF2. Quercetin suppresses the NLRP3 inflammasome by affecting these regulators. Quercetin, as an anti-inflammatory, antioxidant, analgesic and inflammatory compound, is probably a potential treatment for severe inflammation and one of the main life-threatening conditions in patients with COVID-19.

Features of SARS-COV-2 infection and directions of drug and vaccine creation

Features of SARS-COV-2 infection and directions of drug and vaccine creation

Reviewing the Latest Findings on the Polypeptide Sequence of the SARS-CoV-2 S-Protein to Raise Questions about the Origins of RaTG13

The ongoing coronavirus disease outbreak started from a local seafood market of the Wuhan city (China). The first reported infections were six seafood street-sellers with severe pneumonia admitted to the intensive care unit of the Wuhan Jin-Yin-Tan Hospital. Upon metagenomics sequencing and phylogenetic analysis of the complete genomes of the virus isolates from these patients, RaTG13, a bat coronavirus previously detected in Rhinolophus affinis from the Chinese province of Yunnan, was identified as the most identical and closely-related coronavirus to the human pathogen, which was designated as SARS-CoV-2. The most updated evidences suggest adaptation of the human coronavirus SARS-CoV-2 from an animal reservoir, which supposedly underwent some sort of recombination. However, the processes to describe how adaptation of the virus from an animal to a human host may occur still cannot be explained. From a polypeptide sequence analysis of the S-proteins of SARS-coronaviruses, this contribution address questions about the origins of RaTG13, which still remains the most identical strain with SARS-CoV-2 described up to now.

Horizontal gene transfer and recombination analysis of SARS-CoV-2 genes helps discover its close relatives and shed light on its origin

BackgroundThe SARS-CoV-2 pandemic is one of the greatest global medical and social challenges that have emerged in recent history. Human coronavirus strains discovered during previous SARS outbreaks have been hypothesized to pass from bats to humans using intermediate hosts, e.g. civets for SARS-CoV and camels for MERS-CoV. The discovery of an intermediate host of SARS-CoV-2 and the identification of specific mechanism of its emergence in humans are topics of primary evolutionary importance. In this study we investigate the evolutionary patterns of 11 main genes of SARS-CoV-2. Previous studies suggested that the genome of SARS-CoV-2 is highly similar to the horseshoe bat coronavirus RaTG13 for most of the genes and to some Malayan pangolin coronavirus (CoV) strains for the receptor binding (RB) domain of the spike protein.ResultsWe provide a detailed list of statistically significant horizontal gene transfer and recombination events (both intergenic and intragenic) inferred for each of 11 main genes of the SARS-CoV-2 genome. Our analysis reveals that two continuous regions of genes S and N of SARS-CoV-2 may result from intragenic recombination between RaTG13 and Guangdong (GD) Pangolin CoVs. Statistically significant gene transfer-recombination events between RaTG13 and GD Pangolin CoV have been identified in region [1215–1425] of gene S and region [534–727] of gene N. Moreover, some statistically significant recombination events between the ancestors of SARS-CoV-2, RaTG13, GD Pangolin CoV and bat CoV ZC45-ZXC21 coronaviruses have been identified in genes ORF1ab, S, ORF3a, ORF7a, ORF8 and N. Furthermore, topology-based clustering of gene trees inferred for 25 CoV organisms revealed a three-way evolution of coronavirus genes, with gene phylogenies of ORF1ab, S and N forming the first cluster, gene phylogenies of ORF3a, E, M, ORF6, ORF7a, ORF7b and ORF8 forming the second cluster, and phylogeny of gene ORF10 forming the third cluster.ConclusionsThe results of our horizontal gene transfer and recombination analysis suggest that SARS-CoV-2 could not only be a chimera virus resulting from recombination of the bat RaTG13 and Guangdong pangolin coronaviruses but also a close relative of the bat CoV ZC45 and ZXC21 strains. They also indicate that a GD pangolin may be an intermediate host of this dangerous virus.

The Rhinolophus affinis bat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2

Bat coronavirus (CoV) RaTG13 shares the highest genome sequence identity with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among all known coronaviruses, and also uses human angiotensin converting enzyme 2 (hACE2) for virus entry. Thus, SARS-CoV-2 is thought to have originated from bat. However, whether SARS-CoV-2 emerged from bats directly or through an intermediate host remains elusive. Here, we found that Rhinolophus affinis bat ACE2 (RaACE2) is an entry receptor for both SARS-CoV-2 and RaTG13, although the binding of RaACE2 to the receptor-binding domain (RBD) of SARS-CoV-2 is markedly weaker than that of hACE2. We further evaluated the receptor activities of ACE2s from additional 16 diverse animal species for RaTG13, SARS-CoV, and SARS-CoV-2 in terms of S protein binding, membrane fusion, and pseudovirus entry. We found that the RaTG13 spike (S) protein is significantly less fusogenic than SARS-CoV and SARS-CoV-2, and seven out of sixteen different ACE2s function as entry receptors for all three viruses, indicating that all three viruses might have broad host rages. Of note, RaTG13 S pseudovirions can use mouse, but not pangolin ACE2, for virus entry, whereas SARS-CoV-2 S pseudovirions can use pangolin, but not mouse, ACE2 enter cells efficiently. Mutagenesis analysis revealed that residues 484 and 498 in RaTG13 and SARS-CoV-2 S proteins play critical roles in recognition of mouse and human ACE2s. Finally, two polymorphous Rhinolophous sinicus bat ACE2s showed different susceptibilities to virus entry by RaTG13 and SARS-CoV-2 S pseudovirions, suggesting possible coevolution. Our results offer better understanding of the mechanism of coronavirus entry, host range, and virus-host coevolution.

At Least Seven Distinct Rotavirus Genotype Constellations in Bats with Evidence of Reassortment and Zoonotic Transmissions.

Bats host many viruses pathogenic to humans, and increasing evidence suggests that rotavirus A (RVA) also belongs to this list. Rotaviruses cause diarrheal disease in many mammals and birds, and their segmented genomes allow them to reassort and increase their genetic diversity. Eighteen out of 2,142 bat fecal samples (0.8%) collected from Europe, Central America, and Africa were PCR-positive for RVA, and 11 of those were fully characterized using viral metagenomics. Upon contrasting their genomes with publicly available data, at least 7 distinct bat RVA genotype constellations (GCs) were identified, which included evidence of reassortments and 6 novel genotypes. Some of these constellations are spread across the world, whereas others appear to be geographically restricted. Our analyses also suggest that several unusual human and equine RVA strains might be of bat RVA origin, based on their phylogenetic clustering, despite various levels of nucleotide sequence identities between them. Although SA11 is one of the most widely used reference strains for RVA research and forms the backbone of a reverse genetics system, its origin remained enigmatic. Remarkably, the majority of the genotypes of SA11-like strains were shared with Gabonese bat RVAs, suggesting a potential common origin. Overall, our findings suggest an underexplored genetic diversity of RVAs in bats, which is likely only the tip of the iceberg. Increasing contact between humans and bat wildlife will further increase the zoonosis risk, which warrants closer attention to these viruses.IMPORTANCE The increased research on bat coronaviruses after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) allowed the very rapid identification of SARS-CoV-2. This is an excellent example of the importance of knowing viruses harbored by wildlife in general, and bats in particular, for global preparedness against emerging viral pathogens. The current effort to characterize bat rotavirus strains from 3 continents sheds light on the vast genetic diversity of rotaviruses and also hints at a bat origin for several atypical rotaviruses in humans and animals, implying that zoonoses of bat rotaviruses might occur more frequently than currently realized.