Considering that vaccination against smallpox with live vaccinia virus led to serious adverse effects in some cases, the WHO, after declaration of the global eradication of smallpox in 1980, strongly recommended to discontinue the vaccination in all countries. This led to the loss of immunity against not only smallpox but also other zoonotic orthopoxvirus infections in humans over the past years. An increasing number of human infections with zoonotic orthopoxviruses and, first of all, monkeypox, force us to reconsider a possible re-emergence of smallpox or a similar disease as a result of natural evolution of these viruses. The review contains a brief analysis of the results of studies on genomic organization and evolution of human pathogenic orthopoxviruses, development of modern methods for diagnosis, vaccination, and chemotherapy of smallpox, monkeypox, and other zoonotic human orthopoxvirus infections. Based on the report of the Global Commission for the Certification of Smallpox Eradication of the World Health Organization (WHO) , it was solemnly proclaimed at the 33rd World Health Assembly (WHA) on 8 May 1980 that all the nations of the Earth have defeated this especially dangerous infection. In order to prevent accidental spread of the causative agent of this infection from laboratories storing samples of variola  virus (VARV), measures have been applied to reduce the number of these laboratories. In 1975 there were 75 laboratories storing VARV in different countries. By 1984, however, there remained only two laboratories that received the status of WHO Collaborating Centers for Smallpox and Other Poxvirus Infections—the Institute for Viral Preparations (IVP), Moscow, USSR and the Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA .Although tightly controlled by WHO, these stocks of viable VARV strains were considered a potential biohazard. Therefore, at the fourth meeting of the Ad Hoc Committee on Orthopoxvirus Infections in Geneva in March 1986, the following solution was proposed: “The Committee reviewed the need for retaining the remaining stocks of variola virus. It was noted that the variola gene pool could be cloned into non-expressing sites in bacterial plasmids, for future studies of variola virus. … Because implementation of a decision to destroy all variola virus stocks could be irrevocable, some 60 virologists working in 21 countries had been consulted by a Committee member before the meeting; only 5 thought that variola virus should be maintained indefinitely. Taking these facts into consideration, the Committee concluded that the cloned DNA provided sufficient reference material to resolve any future diagnostic problem involving suspected smallpox and that research studies of variola requiring culture of the virus were no longer justified. Thus, in the Committee’s opinion, there was no need to retain stocks of viable variola virus any longer.” .In December 1990, the 5th meeting of the Ad Hoc Committee on Orthopoxvirus Infections reported the following: “All stocks of variola virus and materials containing variola virus must be destroyed by December 1993.” Given the planned destruction of collections of VARV strains, it was important to reliably conserve the genetic material of different VARV isolates in a biologically safe form. In order to preserve information about this unique virus, the Committee considered it necessary to carry out preliminary studies on genome sequencing. National VARV genome sequencing programs proposed by the Russian Federation  VECTOR, Koltsovo, Novosibirsk region) and the United States (Center for Disease Control and Prevention (CDC), Atlanta, Georgia and the Institute for Genomic Research, Gaithersburg, Maryland) were approved; and a Technical Committee was established to oversee these DNA sequencing efforts .By the middle of 1992, Russian researchers were the first ones to complete the genome sequencing and the bioinformatic analysis of a highly virulent VARV major strain isolated in India in 1967 during an outbreak of smallpox with a case-fatality rate of 31% ,6,7. TheAfter publication of the report of the WHO Committee on planned destruction of VARV stocks, there has been a debate about the necessity of these measures among the scientific community. In particular, during the IXth International Congress of Virology held in Glasgow in August 1993, a special plenary session was held; however, no consensus was reached on this issue ,12.At the 6th meeting of the Ad Hoc Committee on Orthopoxvirus Infections , it was decided to postpone the destruction of VARV stocks for some time and store the collections of DNA and plasmid-cloned fragments of VARV genome in two international repositories: SRC VB VECTOR (Russia) and CDC (USA) . In addiTaking into account the potential hazard of working with viable VARV in Moscow, VARV stocks were transferred from IVP (Moscow) to the SRC VB VECTOR  on September 27, 1994 based on a joint order of a number of ministries of the Russian Federation and the Russian Academy of Medical Sciences. Following the inspection of the highest biosafety level laboratory planed for VARV research, by the WHO panel in 1995, the WHO officially established the WHO Collaborating Center for Orthopoxvirus Diagnosis and Repository for Variola Virus Strains and DNA at the SRC VB VECTOR in 1997. The right to store VARV stocks and their genomic DNA at the SRC VB VECTOR was approved by the WHA resolution 49.10 and then confirmed by WHA resolutions 52.10, 55.15, and 60.1.On 25 May 1996, the 49th WHA adopted a recommendation to postpone the date for possible eradication of VARV stocks to 30 June 1999 . As a wiIn 1999, the WHO Advisory Committee on Variola Virus Research was established. The Committee began to supervise VARV studies and hold annual meetings of the scientists involved in the study of this virus and development of the methods for diagnosis, prevention, and treatment of smallpox and other human orthopoxvirus infections.de novo. All this makes the proposed destruction of VARV stocks pointless and indicates the need to intensify research to develop new methods for diagnosing, preventing, and treating not only smallpox but also other human orthopoxvirus infections.Proponents of the concept of compulsory eradication of the existing two official VARV repositories proceeded from the fact that destruction of these stocks would solve the issue of potential danger of virus release into the environment. The drawback of this viewpoint is that there is no confidence in unintentional or intentional informal conservation of VARV strains in any country. For example, forgotten glass ampoules with live VARV, which had been stored for more than 60 years, were found in a low-temperature refrigerator at the National Institutes of Health (USA) in 2014 . PhenomeOrthopoxvirus of the Poxviridae family includes human pathogenic species Variola virus (VARV) and related zoonotic Monkeypox virus (MPXV), Cowpox virus (CPXV), Camelpox virus (CMLV), and Vaccinia virus (VACV) [The genus s (VACV) ,2.Zoonotic orthopoxviruses are usually isolated from animals that are in close proximity to humans  and serve as an intermediate host of the virus, whose natural reservoir is often other wild animals. Therefore, the name of orthopoxvirus species usually does not indicate the real animal that is the natural virus reservoir.VARV, the causative agent of smallpox, can only infect humans; there is no natural reservoir  for this virus. Until its eradication, smallpox was one of the most dangerous human infectious diseases. At least 400 million people died from this infection in less than 80 years in the 20th century, when mass vaccination and intensive anti-epidemic measures against smallpox were carried out . Two subGeographical variations with different mortality rates were found among variola major epidemics. The Indian subcontinent and adjacent areas had the highest mortality rate (up to 40%) in the second half of the 20th century, while significantly lower mortality rates 5–15%) were observed in Indonesia and Western, Central, and Eastern Africa [5% were oThe severity of smallpox infection depends on the age of the infected person. Analysis of the outbreaks of variola major in the 20th century showed that the mortality can exceed 40–50% in unvaccinated children < 5 years of age and adults > 40 years of age, while a lethal outcome is much less common (5–15%) in unvaccinated people aged 10–30 years .Smallpox is a highly contagious infection: it is only slightly inferior to measles and chicken pox in this parameter. The pharynx/nasopharynx and the respiratory tract are the entry gate for infection. VARV can also enter the body through damaged skin. The secondary attack rate of variola major infection in close contact averaged > 58% for susceptible individuals. The data of the smallpox eradication program demonstrated that one patient infected an average of five contacts; although the real number was often much higher (up to 20–35 people) ,2.MPXV causes a human infection that clinically resembles discrete smallpox and sometimes (up to 10% of cases) causes death . Unlike An important difference between human mpox and smallpox was considered to be the low efficiency of human-to-human transmission of MPXV, which until recently prevented the transition of local outbreaks of the disease into widespread epidemics. However, recent studies have indicated an increasing efficiency of MPXV spread in the human population. According to the WHO, the number of people infected due to human-to-human transmission was 29.6% of the total number of registered cases by the end of the first Monkeypox Project (1986). The ratio changed dramatically by 1997 (73.0%). The number of identified generations in the chain of human-to-human transmission of mpox has also increased to eight. Analysis of the available data made it possible to conclude that mpox incidence in humans in the DRC  had increased 20 times by 2010  [An expansion of the distribution of human mpox has been recently noted in Africa. According to the WHO, confirmed cases of mpox in humans and animals have been found in the Central African Republic, Cameroon, DRC, Liberia, Nigeria, the Republic of the Congo, and Sierra Leone in 2010–2018 .The first mpox outbreak in humans outside the African continent was registered in 2003. The causative agent of this infection was introduced into the USA by infected animals imported from Ghana for sale as pets . The MPXA human mpox outbreak in Nigeria has recently attracted attention. A total of 414 suspected cases of mpox in humans, including 152 laboratory-confirmed cases, were identified in 2017–2019. The largest number of patients were aged 21–40 years. Mpox deaths were reported in 7 cases, with 4 patients being co-infected with HIV ,34. ViraThe important characteristics of this mpox outbreak in Nigeria were the following: the infection has repeatedly spread outside Africa, and mpox has been reported in travelers from Nigeria to Israel in September 2018, to the United Kingdom in September 2018, December 2019, and May 2021, to Singapore in May 2019, and to the USA in July and November 2021. Moreover, one of these patients infected a healthcare worker with mpox in the UK ,36.After the low virulent MPXV clade IIb spread in the gay, bisexual, and other men who have sex with men (GBMSM) community, a new stage of virus evolution began. After the mass gay pride parade in Gran Canaria on 5–15 May 2022, human mpox cases were registered in many countries on different continents, with the vast majority of them among the GBMSM population . The sudSuch a widespread mpox epidemic in humans may result in accelerated evolution of MPXV and emergence of virus variants with greater efficiency of human-to-human transmission and/or increased pathogenicity . TherefoCowpox in humans is a sporadic disease that occurs when CPXV is transmitted from an infected animal  to humans. CPXV is relatively low pathogenic to humans; however, it has a very wide range of susceptible animals. CPXV infection could be fatal in immunocompromised individuals ,43 and fRecently, the incidence of orthopoxvirus infection caused by zoonotic VACV  has also significantly increased in farm animals  and humans in India, Brazil and Colombia ,58,59,60Vaccinia virus [Determination of VACV origin is one of the most complicated issues. This virus has been used for many decades to vaccinate people against smallpox; this “variolae vaccinae” virus was consider to derive from CPXV introduced into the practice of smallpox vaccination by Edward Jenner in 1796 . Therefoia virus . At the ia virus .Horsepox virus (HSPV) genome had been sequenced. Phylogenetic analysis of the conserved region indicated that HSPV is closely related to sequenced isolates of VACV and rabbitpox virus, clearly grouping together these VACV-like viruses [Vaccinia virus decades later, were widely used for smallpox vaccination in the 19th century [The question of VACV origin was somewhat clarified after the complete  viruses . After t viruses ,65. In t viruses . Apparen century . A recen century .In terms of its biological properties and phylogenetic analysis of the complete viral genome sequence, CMLV is closest to VARV compared to other orthopoxviruses . It had The most direct and effective method to study the evolution of closely related viruses is whole genome sequencing and computer analysis of the obtained data. In order to establish evolutionary relationships between different human pathogenic orthopoxviruses after having sequenced the genomes of the first viruses CPXV  and MPXVThe accumulated data made it possible for the first time to conduct a comparative analysis of the genomes of all species of human pathogenic orthopoxviruses, perform the first phylogenetic study of this group of viruses, and reveal the evolutionary relationships between them. We have found for the first time that West African and South American VARV strains form a separate subtype (clade) with significant differences in their genome organization from all other studied geographic VARV variants . At the Based on the analysis of available archival data on smallpox epidemics, history of ancient civilizations, and the latest data on evolutionary relationships between orthopoxviruses, we hypothesised that smallpox could have emerged repeatedly in the past as a result of evolutionary changes in the zoonotic progenitor virus and disappeared due to the insufficient population of isolated ancient civilizations . The onlDespite the fact that orthopoxvirus infections have characteristic visual manifestations (skin lesions), experience shows that clinical diagnosis is often erroneous .The development of the polymerase chain reaction (PCR) method has led to creation of modern approaches for detection and identification of trace amounts of microorganisms in analyzed samples with high specificity and in a short period of time. At the same time, it eliminates the need to carry out manipulations with highly dangerous live infectious agents, including VARV and MPXV.Numerous methods have been proposed for PCR detection of different orthopoxvirus species ,92,93,94Oligonucleotide microarrays can also detect trace amounts of a test material in a sample. One of the important advantages of oligonucleotide microarrays over other methods of PCR-based diagnosis is the possibility to analyze multiple genetic loci simultaneously, which significantly increases method reliability. Various diagnostic oligonucleotide microarrays have been developed for reliable species-specific detection of orthopoxviruses ,101,102.Rapid development of sequencing technologies currently makes it possible to quickly obtain information on complete nucleotide sequence of the genome of an organism of interest. Thus, data on detected cases of unusual orthopoxvirus infections are increasingly supplemented by genome-wide sequences of these viral isolates ,104. Thevariola) was the first historic example of such protection against an infectious disease.The most reliable way to prevent any viral disease is vaccination. Vaccination against smallpox (Latin variolation (stands for variola inoculation), caused a moderately severe disease and provided further reliable protection against smallpox. However, 0.5–2% of variolated patients would die, which prevented the widespread use of this procedure [Smallpox survivors were easily identified by the characteristic scars on their face skin , which were left on the sites of pustules after the loss of dry crusts; these people became resistant to smallpox whenever a new outbreak of the disease occurred. Apparently, these observations formed the basis for inoculation of infectious material obtained by collecting skin crusts from smallpox patients into skin incisions  or intranasally made on healthy people in India and China. This procedure, called rocedure .In 1798, English physician E. Jenner described a new, safer procedure for protecting against smallpox . Rural rvariolae vaccinae” ) instead of the term cowpox and called the procedure “vaccine inoculation.” In 1803, Richard Dunning proposed the shortened term “vaccination.” In 1881, at the 7th International Congress of Medicine in London, Louis Pasteur suggested using the term vaccination for all protective immunization procedures against any infectious disease [Given these findings, to emphasize the protective effect of the used infectious entity against smallpox, E. Jenner introduced the term “ disease .It should be noted that the kingdom of viruses was discovered only a century after introduction of smallpox vaccination into practice. Orthopoxviruses are antigenically and immunologically similar to each others, they participate in serological cross-reactions and provide immune protection. This provided a reliable way to protect against smallpox by inoculating a person with either CPXV or HSPV, which were later widely replaced by VACV resulting in less pronounced adverse reactions during vaccination. This vaccine turned out to be so effective that it allowed for smallpox eradication during mass vaccination of people and strict epidemiological surveillance under the auspices of the WHO. This is the first and so far the only example of eradication of a particularly dangerous human viral infection in the history of mankind ,2.The cessation of vaccination against smallpox after 1980 meant that a huge part of the world’s population is now not anymore immune to not only smallpox but also other zoonotic orthopoxvirus infections. This creates a new risk of possible circulation of zoonotic orthopoxviruses in the human population, which may lead to a change in the ecology and range of sensitive hosts for different orthopoxviruses.Human monkeypox is of particular concern. In conditions of a long absence of vaccination of the population and a much more frequent infection in people, MPXV can acquire not only high transmissibility but also high pathogenicity, which is characteristic of VARV, as a result of natural evolution. If this happens, humanity will face a problem much more difficult than smallpox eradication. This is primarily due to the fact that, unlike for VARV, the natural reservoir of MPXV is various African rodents.In order to prevent the development of outbreaks into widespread epidemics and thereby reduce the risk of emergence of highly pathogenic orthopoxvirus due to natural evolution, the researchers’ efforts should be focused on creation of new generations VACV-based safe live vaccines ,78. VACVThe first-generation live smallpox vaccine was a VACV preparation obtained by virus propagation on the skin of calves and other animals. Its use for mass vaccination is currently limited due to the relatively large number of possible severe complications , which iCurrently, VACV vaccine strains are produced in mammalian cell cultures, and these preparations are classified as second-generation smallpox vaccines . The ACAAttenuated third-generation smallpox vaccines are created by multiple passages of a specific VACV strain in a heterologous host cell culture. For example, the most studied third-generation smallpox vaccine strain MVA was obtained after a large number of passages of the Ankara VACV strain in chicken embryo fibroblasts. The MVA strain genome contains multiple mutations and extended deletions compared to the DNA of the original VACV strain. MVA is incapable of replicating in most mammalian cells, including human cells .To date, a third-generation smallpox vaccine based on the MVA-BN strain, manufactured by Bavarian Nordic (Denmark), has passed numerous clinical trials, including studies in patients with atopic dermatitis and HIV ,111,112.The third-generation smallpox vaccine LC16m8, which was licensed in Japan, was obtained based on the VACV strain Lister by multiple passages in primary rabbit kidney cells at low temperatures (30 °C). Clinical studies of VACV LC18m8 have shown a significant reduction in side effects compared to the conventional Lister strain vaccine. The attenuation of the vaccine strain is mainly due to a mutation in the B5R gene, which encodes an extracellular virion protein. The protective efficacy of LC16m8 was shown to be comparable to that of the parental strain Lister in experiments in various animal models ,115,116.A new approach to producing fourth-generation attenuated smallpox vaccines involves the use of genetic engineering techniques to introduce targeted deletions/insertions to disrupt genes regulating the body’s defense responses against viral infection. The first variant of such VACV studied in detail is the NYVAC strain; the NYVAC genome lacks a block of 12 genes and contains 6 impaired genes. NYVAC was shown to induce significantly lower immunity against smallpox in humans compared to the conventional vaccine .A highly attenuated VACdelta6 variant was obtained by sequential introduction of targeted deletions/insertions in six individual genes of the LIVP strain at the SRC VB VECTOR ,119,120.Marmota bobak) infected with MPXV, have also been actively used in recent years [Chemotherapeutic drugs, the search for which has gained some success in the last twenty years, may be important for the treatment of human orthopoxvirus infections. The initial screening for orthopoxvirus replication inhibitors is usually carried out in cell cultures. Since there is no adequate animal model of smallpox, testing of potential drugs against smallpox has to be performed in surrogate animal models of smallpox infection. Substances that have shown high antiviral activity in vitro are usually studied in animal models such as intranasal or aerosol infection of mice with ectromelia virus (ECTV) and CPXV and monkeys infected with MPXV . Rabbitsnt years ,126,127.Compound ST-246 presents the greatest interest as a therapeutic drug against smallpox, mpox, and other orthopoxvirus infections; it inhibits the last stage of enveloped virion assembly and prevents orthopoxvirus virion release from the infected cell ,130,131.An ST-246 analogue called NIOCH-14 also showed high activity against orthopoxvirus infections in various animal models ,134,135.A viral DNA polymerase inhibitor, the nucleotide analogue Cidofovir approved for clinical use in cytomegalovirus retinitis, proved to be an effective anti-orthopoxvirus drug . CidofovConsidering that the widespread use of antiviral drugs results in emergence of resistant virus strains, effective treatment of possible cases of smallpox, mpox, and similar orthopoxvirus infections requires further search for new chemotherapeutic drugs with different molecular targets in orthopoxviruses.The cessation of smallpox vaccination more than 40 years ago and the subsequent loss of herd immunity in humans against not only smallpox but also other orthopoxvirus infections promotes an increased spread of zoonotic orthopoxviruses among people. This, in turn, can contribute to the natural selection of highly pathogenic and epidemic virus strains. The human mpox epidemic, which spread in many countries in 2022, is the first big challenge for researchers and physicians.Considering that this might happen in the future, WHO efforts have been focused on development and testing of modern methods for orthopoxvirus identification, creation of safe new-generation smallpox vaccines, and chemotherapy drugs against VARV and other orthopoxviruses pathogenic to humans at the two WHO Collaborating Centers for Smallpox  since the beginning of the 21st century ,139. TheNevertheless, it is important to continue research in these fields in order to create new safe and highly effective means to prevent and treat orthopoxvirus infections. Diagnostic methods should focus on rapid identification of not only VARV and MPXV but also CPXV, VACV, and CMLV. Given the increased number of outbreaks of orthopoxvirus infections in animals and humans in recent years and their potential danger, constant monitoring of these infections should be embraced in all parts of the world. This will reduce the risk of small outbreaks developing into widespread epidemics and, thereby, decrease the chance of re-emergence of a highly contagious and pathogenic orthopoxvirus. It is also important that stocks of new vaccines and chemotherapeutics should be widely available and in sufficient quantities to treat the emerging biological threats effectively.


This cross-sectional study evaluates the association of generic competition and changes in product coverage and cost-sharing by using Medicare data and estimated out-of-pocket costs for the oral specialty drug abiraterone. Multiple recent generic market entrants across 2 commonly used dosage units (250 and 500 mg) make abiraterone ideal for evaluating these associations.Generic competition lowers drug pricesSTROBE reporting guideline. The University of Minnesota deemed this cross-sectional study exempt from review, and informed consent was waived because we used deidentified data. The study followed the 4 We calculated point-of-sale prices (excluding rebates and discounts) and out-of-pocket costs assuming monthly fills, standard plan benefit design, and no other medication use. We also calculated a best-case scenario of 2022 out-of-pocket costs  using the Medicare Part D Plan Finder,5 assuming the patient was diagnosed in January 2022. We analyzed data using Excel version 16.61 (Microsoft).We studied 4 abiraterone products, including 250- and 500-mg generic and brand-name tablets, for a common total dosage of 1000 mg/d. We used the FDA Orange Book to identify market entry of generic products approved through May 2022. We used Medicare Prescription Drug Plan Formulary and Pricing Information Files (2018-2022) to identify product coverage and expected annual out-of-pocket costs.The proportion of Medicare Part D plans covering generic abiraterone increased from 0% for the 250- and 500-mg formulations in 2018 to 100% and 79%, respectively, in 2022 . BetweenExpected annual out-of-pocket costs ranged from $7491 to $10 148 across products and years . In 2022Despite preferred coverage over brand-name products on Medicare Part D formularies, generic abiraterone was associated with modest savings for beneficiaries in this cross-sectional study. In fact, out-of-pocket costs were higher for the 500-mg generic vs brand-name products in 2022. A patient able to price shop could pay approximately $1100 for a 1-year supply of 250-mg generic abiraterone—one-seventh of the mean annual out-of-pocket costs.4 Second, out-of-pocket costs for Part D beneficiaries can be higher with generic products despite lower list prices (vs brand-name products) because of a program design flaw.4 Finally, we observed a broad range of out-of-pocket costs for the same drug and/or dose. Emphasizing this point, a patient without insurance can obtain an annual supply of abiraterone for $2202 from an online pharmacy platform—lower than through their health plan.6This study has several key findings. First, although plans rapidly covered generic products, reductions in out-of-pocket costs were delayed. The 250-mg generic product  is now less costly than its brand-name counterpart, but the 500-mg product is not. It took more than 3 years for costs to decrease, mirroring data reporting that it takes multiple years and/or entrants for robust price competition.A limitation of this study is that Medicare formulary files capture drug prices from the prior 3 months and may not reflect current prices. Regardless, Medicare Part D beneficiaries do not immediately benefit from price reductions associated with generic entry for specialty drugs. Proposals to correct the flaw that makes high-cost generic drugs more expensive to patients than brand-name drugs include eliminating manufacturer discounts from out-of-pocket spending calculations for brand-name drugs or extending the discount to generics. Alternatively, recent proposals to redesign Medicare Part D eliminate the coverage gap and cap out-of-pocket spending, which would avoid penalizing beneficiaries for generic drug use.


Active hypusine-modified initiation elongation factor 5A is critical for cell proliferation and differentiation, embryonic development, and innate immune response of macrophages to bacterial infection. Here, we demonstrate that both virus infection and double-stranded RNA viral mimic stimulation induce the hypusination of eIF5A. Furthermore, we show that activation of eIF5A is essential for the replication of several RNA viruses including influenza A virus, vesicular stomatitis virus, chikungunya virus, mayaro virus, una virus, zika virus, and punta toro virus. Finally, our data reveal that inhibition of eIF5A hypusination using the spermidine analog GC7 or siRNA-mediated downmodulation of eIF5A1 induce upregulation of endoplasmic reticulum stress marker proteins and trigger the transcriptional induction of interferon and interferon-stimulated genes, mechanisms that may explain the broad-spectrum antiviral activity of eIF5A inhibition. Polyamines have emerged as important molecules in the replication of different viruses such as Dengue virus, Chikungunya virus, Zika virus, Rift Valley fever virus, La Crosse virus, poliovirus, cytomegalovirus, coxsackievirus B3 virus, semliki forest virus or herpes simplex virus, and targeting polyamines has been suggested as a potential antiviral approach . In addiTogaviridae, Orthomyxoviridae, Flaviviridae, Phenuiviridae, and Rhabdoviridae. Finally, our data reveal that downmodulation of eIF5A1 or its inhibition trigger endoplasmic reticulum (ER) stress, as shown by increased phosphorylation of PERK and the activation of eIF2α which promotes the induction of type I interferon and type I-interferon stimulated genes, mechanisms that may contribute to the control of virus replication.In this report, we show that stimulation by double stranded RNA (dsRNA) or some RNA virus infection induce eIF5A hypusination. Activation of eIF5A is modulated by NF-κB and is essential for the efficient replication of viruses belonging to different families such as In vivogen) was used at a final concentration of 5 µg/ml. Smart-pool small interfering RNAs (siRNAs) against eIF5A1  and on-target plus non-targeting siRNAs  were purchased from Dharmacon. A retrovirus vector expressing short hairpin RNA (shRNA) targeting p65 subunit of NF-kB was kindly provided by Scott Lowe lab , 1% L-Glutamine , and 1% Penicillin-Streptomycin . Vero, Vero-E6, and HeLa cells were grown in Minimal Essential medium supplemented with 10% FBS, 1% L-Glutamine, and 1% Penicillin-Streptomycin. Treatment with the spermidine analogue and competitive Deoxyhypusine Synthase (DHS) inhibitor N1-guanyl-1,7-diamine-heptane (GC7)  was carried out at a concentration of 20 µM unless otherwise indicated. IKK 16 (Abcam) and BAY 11-7082 (Sigma-Aldrich) were used at a concentration of 100 nM and 1 μM, respectively. Poly I:C  according to the manufacturer’s instructions.Cells were collected in SDS sample loading buffer and, once separated in SDS-page gels, proteins were transferred to 0.45 μm nitrocellulose membranes. The primary antibodies used were anti-HA , anti-eIF5A , anti-phospho-eIF2α , anti-phospho-PERK , anti-p65 , anti-GAPDH , anti-VSV G , anti-actin , anti-influenza A virus PB2 protein , anti-influenza A virus NS1 protein , anti-Alphavirus E1 and NSP1 , anti-PT35S-methionine (100 µCi/ml) for the times indicated in the figure. Total lysates were analyzed by SDS-polyacrylamide gel electrophoresis, fixed and dried. The radioactively labeled proteins were visualized by autoradiography.Cells were infected at a multiplicity of infection (MOI) of 5, washed twice with methionine-free DMEM and labelled by incubation in methionine-free DMEM supplemented with Infections were carried out with vesicular stomatitis virus (VSV) of Indiana strain or recombinant VSV expressing GFP (rVSV-GFP), the mouse-adapted influenza A/PR78/34 (PR8) virus, mayaro virus , una virus , chikungunya virus , zika virus , and punta toro virus . All arboviruses were a kindly gift of Dr. Scott Weaver from WRCEVA, UTMB, USA. MAYV, UNAV, CHIKV, ZIKV, PTV, and VSV were resuspended in serum-free medium, and PR8 was resuspended in 0.3% BSA in PBS. One hour after inoculation with the virus, the unbound virus was discarded and fresh medium was added. In the case of influenza virus, TPCK-trypsin (2.0 μg/ml) was added to the medium. Virus yields were measured by plaque assay in BSC40 (VSV), MDCK (PR8), Vero-E6 (MAYV and UNAV), and Vero cells . VSV-GFP infectivity values were determined by flow cytometry (FACS) analysis using a FACSCalibur cytometer (BD Biosciences).Purification of total RNA was carried out using the RNeasy minikit (Qiagen), reverse transcription (RT-PCR) was performed using the reverse transcription system kit (Promega), and Q-RT-PCR was carried out using SYBR Green Power PCR Master Mix in a RealPlex 4 Thermocycler (Eppendorf). The oligonucleotides used are listed in Comparison between two groups of data was carried out by Student’s t test. Values were expressed as mean +/- SF of at least three independent experiments. A p value <0.05 was considered statistically significant.We first evaluated the eIF5A status in response to virus infection. A549 cells were infected with vesicular stomatitis virus (VSV) or influenza A virus (IAV) at a MOI of 5 and at different times after infection we analyzed the activation of eIF5A using Western blot analysis with anti-hypusine antibody. We observed a significant upregulation in the levels of hypusinated eIF5A after infection with VSV (Interferon had been reported to induce depletion of polyamines  and a deOrnithine decarboxylase (ODC) is a rate-limiting enzyme for polyamine synthesis that can be transactivated by NF-κB . Therefo35S-methionine was carried out. We observed a clear inhibition of host protein synthesis at 2 h after VSV infection , Alphaviruses mayaro (MAYV), una (UNAV), and Chikungunya (CHKV), or the Phlebovirus Punta Toro (PTV). We observed a clear decrease in the synthesis of viral proteins and/or a significant decrease in the viral titer of MAYV , (PID2021-126510NB-I00), and Xunta de Galicia (ED431G 2019/02). RS and SV are predoctoral fellows funded by Xunta de Galicia-Consellería de Cultura, Educación y Ordenación Universitaria . JG-S was supported by grants from Secretaría Nacional de Ciencia, Tecnología e Innovación de Panamá (SENACYT), grant number FID18-100, Ministerio de Economía y Finanzas, grant number 19911.012 and Sistema Nacional de Investigación (SNI from SENACYT), grant number 23-2021. This work was also partly supported by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), an NIAID funded Center of Excellence for Influenza Research and Response  to AG-S.We thank Richard Cadagan for excellent technical support.The AG-S laboratory has received research support from Pfizer, Senhwa Biosciences, Kenall Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines, Atea Pharma and Merck, outside of the reported work. AG-S has consulting agreements for the following companies involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, Pharmamar, Paratus, CureLab Oncology, CureLab Veterinary and Pfizer, outside of the reported work. AG-S is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Roseburia intestinalis has received attention as a potential probiotic bacterium. Recent studies have demonstrated that changes in its intestinal abundance can cause various diseases, such as obesity, enteritis and atherosclerosis. Probiotic administration or fecal transplantation alter the structure of the intestinal flora, offering possibilities for the prevention and treatment of these diseases. However, current monitoring methods, such as 16S rRNA sequencing, are complex and costly and require specialized personnel to perform the tests, making it difficult to continuously monitor patients during treatment. Hence, the rapid and cost-effective quantification of intestinal bacteria has become an urgent problem to be solved. Aptamers are of emerging interest because their stability, low immunogenicity and ease of modification are attractive properties for a variety of applications. We report a FluCell-SELEX polyclonal aptamer library specific for R. intestinalis isolated after seven evolution rounds, that can bind and label this organism for fluorescence microscopy and binding assays. Moreover, R. intestinalis can be distinguished from other major intestinal bacteria in complex defined mixtures and in human stool samples. We believe that this preliminary evidence opens new avenues towards aptamer-based electronic biosensors as new powerful and inexpensive diagnostic tools for the relative quantitative monitoring of R. intestinalis in gut microbiomes. R. intestinalis was first isolated from human feces in 2002 [R. intestinalis is thus promising as a new probiotic for maintaining intestinal health and the treatment of diseases [R. intestinalis in the intestine of untreated UC and CD patients [R. intestinalis significantly ameliorated DSS- and TNBS-induced colitis [R. intestinalis was found in the intestine of patients with the constipated irritable bowel syndrome (C-IBS) than in the controls [Roseburia was closely associated with faster colonic transit [Roseburia supplementation was found to significantly improve the ALD symptoms by inhibiting hepatic inflammation and steatosis [R. intestinalis plays an essential role in maintaining intestinal health for the prevention and treatment of digestive diseases [Cyclocarya paliurus (CCPP) could alleviate the symptoms of type 2 diabetes (T2DM) by increasing the production of short-chain fatty acids (SCFAs), represented by R. intestinalis, and promoting SCFA production through the construction of T2DM [R. intestinalis in the gut than in healthy individuals [R. intestinalis and body mass index (BMI) [R. intestinalis abundance in the gut and obesity. The study of cancer-associated microorganisms is also crucial in the cancer research process [R. intestinalis abundance is much lower in the gut of colorectal cancer (CRC) patients than in normal subjects, making it a reliable novel biomarker in CRC [ in 2002 . It is a in 2002 . Previou in 2002 ,5,6. Inf in 2002 ,8. In on in 2002 . The use in 2002 ,11. As adiseases ,13,14. Epatients ,16,17. I colitis ,19. A si transit . An earl transit . By consteatosis . Subsequteatosis . This fu of T2DM . An asseividuals . In anotex (BMI) , suggestr in CRC ,29.12–1016 single-stranded deoxyribonucleic acid (ssDNA) molecules and each sequence contains fixed primer binding sites and a random region in the center of the molecule. After incubation of the random library with the target cells during the screening process, the DNA sequences bound to the target cells are amplified by PCR, and the next round of screening is performed [R. intestinalis cells with high affinity, allowing for the specific labeling of cells for fluorescence microscopy, binding assays and to distinguish R. intestinalis from other gut bacteria. The initial experiments demonstrate that measurements will be possible in complex mixtures and stool samples, which suggests the R. intestinalis polyclonal aptamer library presented here is a promising binding entity for the construction of electronic biosensors. To our knowledge, we are the first to develop this specific DNA aptamer library for the labeling of this highly promising auspicious probiotic bacterium, to pave the way for the future development of biosensors for the specific detection of R. intestinalis.Electronic biosensors are easy to handle, often portable, and, in principle, can analyze in real-time, making them suited for measuring biomarkers . In receerformed ,36. Baseerformed ,36. The erformed . To omitR. intestinalis. The labeling efficiency developed slowly with the respective libraries from round one to round five, being clearly below the detection limit, in the later stages of the SELEX it increased from round five to seven, while introducing counter-selection with mixtures of bacteria in round six. Interestingly, the labeling was impaired for the round seven library , when the number of bacteria in the 500 µL reaction system was varied from 101 to 103 CFU. The experimental detection limit of R. intestinalis for the aptamer library Ri 7_2 was 101 CFU. Thus, the high sensitivity of the aptamer library Ri 7_2 for identifying R. intestinalis also provides the possibility of detecting trace amounts of R. intestinalis in the fecal bacteria. Finally, to further validate the potential of the aptamer library Ri 7_2 for practical applications, we first obtained the exact amount of Roseburia in the feces of two healthy volunteers  or fecal bacteria (108 CFU) for the binding assay. The relative fluorescence intensity of R. intestinalis as a control group was 100%. In contrast, the relative fluorescence intensity of the experimental group represented the relative abundance of Roseburia in the fecal bacteria. We obtained 4.75% and 6.18% for proband 1 and proband 2, respectively, of Roseburia in the fecal bacteria of the probands by NGS . Interestingly, the tendency that proband 2 had more Roseburia species was also visible in the aptamer analysis, while the relative difference between both of the analysis techniques was 55.8% lower in the aptamer experiment for proband 1 compared to the NGS value, and 20.7% for proband 2, respectively. Without yet having determined the binding affinity of library Ri 7_2 against other non-intestinalis Roseburia species, these results suggest that Ri 7_2 meets the expectation that binding occurs mainly below the genus level. This may not only open the opportunity to develop novel sub-genus detection assays for R. intestinalis, but also in turn, with additional dedicated libraries, for other Roseburia species. We have recently developed a graphene field-effect transistor-based sensor chip for the quantification and differentiation of a biological marker involving polyclonal aptamer libraries [R. intestinalis polyclonal aptamer library as a binding entity on such a chip, may allow in the near future the development of sensitive and cost-efficient monitoring devices for the rapid and easy supervision of e.g., probiotic supplementation studies with R. intestinalis, and also in a generalized form for all other health-relevant gut bacteria.Microbiome NGS analysis involves the sequencing of 16S rRNA genes, that are highly conserved in prokaryotes and can thus be amplified even from yet unknown organisms . Howeverprojects ,35,36,40R. intestinalis (DSM-14610), P. distasonis (DSM-29491), A. muciniphila mucT (DSM-22959), A. stercoricanis (DSM-13633), B. producta (DSM-29491) and R. microfusus (DSM-15922) were cultivated in Schaedler Broth Medium at 37 °C under anaerobic conditions.The bacteria strains (40)-TTGACTAGTACATGACCACTTGA-3′. The initial library consisted of three parts. The random part contained a randomly varying central region composed of 40 nucleotides and two fixed sequences of 23 nucleotides each, which could be precisely and complementarily bound to the primers. The other parts were comprised of Cyanine 5-labeled forward primer (Cy5-P1) 5′-Cy5-TAGGGAAGAGAAGGACATATGAT-3′, and phosphate-labeled reverse primer (Phosphate-P2) 5′-Phosphate-TCAAGTGGTCATGTACTAGTCAA-3′ .The random sequence library was synthesized and purified . The sequence was as follows: 5′-TAGGGAAGAGAAGGACATATGAT-NA. muciniphila mucT, A. steroricanis, P. distasonis, B. producta and R. microfusus. The aptamers that did not bind to the control bacteria were obtained, which could be further screened by target SELEX to gain an aptamer library specifically targeting R. intestinalis. The Cell-SELEX could be divided into the following steps.The Cell-SELEX included counter SELEX as well as target SELEX. In this chapter, the control bacteria used in counter SELEX were 600 of the bacterial solution was adjusted to 1.The cells were incubated under anaerobic conditions for 21–24 h and subsequently centrifuged at 6000 rpm for 1 min. They were then washed three times with 1× DPBS buffer. Finally, the ODA total of 0.5 nmol of the original library or 10/5 pmol ssDNA library were added to 500 µL 1× DPBS and incubated at 95 °C for 5 min, ice bath for 5 min, and then left at room temperature for 20 min to ensure that the aptamers had the same 3D structure.R. intestinalis at 37 °C. The supernatant was then removed by centrifugation at 3000 rpm for 2 min, and finally, the pellet was washed with 1× DPBS to discard unbound nucleic acids  and tRNA (10 mg/mL) with increasing amounts were added to increase the stringency and then incubated with cids see .R. intestinalis was collected by subsequent centrifugation at 11,000 rpm for 1 min.The cells from the previous step were resuspended in 100 µL 1× DPBS and incubated at 95 °C for 5 min to disrupt the aptamer 3D structure and separate the cells from the aptamer. The aptamer bound to The collected aptamers were amplified by PCR. The amplification conditions were as follows: 2 min at 80 °C; 2 min at 85 °C; 2 min at 90 °C; 3 min at 94 °C; followed by 25 cycles of 30 s at 94 °C; 30 s at 56 °C; 10 s at 72 °C; then 2 min at 72 °C. Subsequently, the PCR product was purified  and then decomposed into single-stranded DNA by lambda-exonuclease catalysis . The treated sample was finally purified by an optimized PCR clean-up kit  to obtain the new DNA pool for the following selection rounds, in which the required binding buffer was added with 1.5 volumes of isopropanol and 10 µL of NaAc solution (pH 5).R. intestinalis was preprocessed by the above method  with 5 pmol of the activated Cy5-labeled aptamer library in 500 µL of 1× DPBS for 30 min at 37 °C. Subsequently, the supernatant was removed by centrifugation at 3000 rpm for 2 min, and the pellet was resuspended in 100 µL of 1× DPBS buffer after washing three times to obtain the cell-bound aptamers after elution. Finally, the fluorescence intensity to determine the screening status was measured using an Infinite M200 spectrophotometer  at an excitation wavelength of 637 nm and an emission of 670 nm.The thod see . The binR. intestinalis and the control strains, including other five intestinal bacterium A. muciniphila mucT, A. steroricanis, P. distasonis, B. producta and R. microfusus, were mixed in different ratios. The ratios of the control strains and R. intestinalis were reduced from 0:1  to 1:0 . The semi-quantitative analysis was determined by incubating the increasing amount of R. intestinalis with 5 pmol of activated Cy5-labeled aptamer library in 500 µL of 1× DPBS for 30 min at 37 °C. Each group of samples was postprocessed, as described above  with varying concentrations of aptamer candidates in 500 µL of 1× DPBS for 30 min at 37 °C. Finally, the dissociation constants (Kd) of the fluorescent aptamers were determined by fitting the dependence of the fluorescence intensity on the concentration of the aptamers to the equation Y = Bmax × X/(Kd + X) using GraphPad PRISM 8 , with Y = the measured fluorescence, Bmax = the maximal fluorescence and X = concentration of the aptamers.The bacterial solution of bove see . The binR. intestinalis, A. muciniphila mucT, A. steroricanis, P. distasonis, B. producta and R. microfusus were pre-treated by the above method (see 600 = 1) for 30 min at 37 °C. After centrifugation at 3000 rpm for 2 min, the supernatant was removed. The pellet was washed once with 500 µL of 1× DPBS and then resuspended in 500 µL of 1× DPBS. Afterwards, 100 µL of each bacterial mixture was transferred to a 96-well microplate. Finally, the fluorescence imaging of each group was obtained using fluorescence microscopy, which was performed using a Leica DMi8 coded  at ×40 magnitude under transmitted light with the Y5 filter (excitation: 590–650 nm and emission: 662–738 nm).The bacterial solution of thod see . A totalthod see . SubsequThe fecal samples from two lean healthy volunteers were used in this study. Volunteers were recruited from Ulm University and signed a written informed consent form. Permission was received from the local Ethics Committee of Ulm University (no. 30/20). In addition, the study was designed and conducted following the regulations governing the use of human study participants and in strict accordance with the standards set by the Declaration of Helsinki.600 of the bacterial solution of one.The stool samples were weighed, added to extraction buffer (1× DPBS), and vortexed for 1 min until no fecal pellets were visible. The fecal pellets were then removed by filtration. The filtrate was centrifuged at 6000 rpm for 1 min, followed by three washes with 1× DPBS buffer, and finally adjusted to the ODThe fecal samples were collected using INTEST.pro  and then measured and analyzed by BIOMES laboratory , using 16s rRNA NGS for fecal bacterial abundance, according to Lilja et al., 2021 .R. intestinalis  and fecal bacteria (108 CFU)  see , respectR. intestinalis. We have described the possibility of using these aptamers in various diagnostic methods and demonstrated that the specific library Ri 7_2 retains its binding function towards this important member of the gut microbiome, even in binding analyses using human stool samples. Without also having yet identified single aptamers as the target structures on the cell membrane, we believe that this opens new avenues for the rapid and reliable diagnosis of R. intestinalis in a new generation of biosensors in the future.In summary, we provide the first aptamer library as a high-affinity binding entity with specificity towards


Ensifer adhaerens HY-1 is an industrial strain that also produces cobalamin. However production outputs are poor and the specific synthesis pathways require characterization. In this study, the whole genome sequence of E. adhaerens HY-1 was generated and annotated, and genes associated with cobalamin biosynthesis were identified. Then, three genes, CobSV, CobQ, and CobW were identified as the most efficient ones for enhancing cobalamin synthesis. By transcriptome sequencing of E. adhaerens HY-1 cells at different growth stages, 65 endogenous promoters with different gradient strengths were identified. After combined expression of different strength promoters and key genes, a high cobalamin-producing recombinant strain, ‘hmm’ (genotype: PmetH-CobSV-PibpA-CobQ-Pmdh-CobW), was generated. Cobalamin production was 143.8 mg/L in shaking flasks, which was 41.0% higher than the original strain. Cobalamin production was further enhanced to 171.2 mg/L using fed-batch fermentation. Importantly, our data and novel approach provide important references for the analysis of cobalamin synthesis and other metabolites in complex metabolic pathways.Cobalamin is an essential human vitamin widely used in the pharmaceutical, food, and feed additive industries and currently produced by bacteria or archaea.  It participates as a coenzyme in various metabolic processes where it promotes the transfer of methyl groups, promotes cell maturation, and participates in the isomerization of specific compounds [Cobalamin  or Cbi (anaerobic pathway) series of genes [de novo synthesis pathway requires the participation of more than 30 genes [Escherichia coli, a de novo cobalamin synthesis pathway was constructed and cobalamin production was optimized to 307.0 μg/g DCW [The synthesis of cobalamin requires the formation of porphyrin ring, which is mainly divided into two forms : one is of genes  Fig. 1)Cob  increased the production cobalamin by 41% compared to the original strain. Cobalamin production was further enhanced by a fed-batch fermentation. Our study provides valuable insights on the enhancement of cobalamin synthesis via metabolic engineering and the comprehensive dissection of complex metabolic pathways for the production of other key metabolites.pathways . In this22.1E. adhaerens HY-1 supplied by Hunan Hongying Biological Technology Co., Ltd . Strain names and genotypes are shown  agar plates or in LB medium at 30 °C. Shaking flask studies were performed in complex media containing 100 g/L beet molasses, 0.7 g/L (NH4)2SO4, 0.02 g/L MnSO4, 0.02 g/L ZnSO4·7H2O, 1.5 g/L MgSO4·7H2O, 1.8 g/L (NH4)2HPO4, 0.02 g/L CoCl2, 0.01 g/L 5,6-dimethylbenzimidazole (DMBI), and 0.2 g/L emulsified silicone oil (pH = 7.3–7.4).2PO4, 0.1 g/L ZnSO4·7H2O, 0.084 g/L CoCl2, 0.07 g/L DMBI, 0.83 g/L FeCl3, 0.18 g/L glycerophosphate, 50 g/L corn syrup, 0.05 g/L MgCl2, 1.3 g/L MgSO4, 1.3 g/L (NH4)2SO4, 0.63 g/L urea, and 1.0 g/L CaCO3 (pH = 7.5–7.8).Fermentation medium contained 85 g/L maltose, 15 g/L betaine, 1.0 g/L KH2. Appropriate antibiotics were added to media: kanamycin (50 μg/mL) and gentamicin (50 μg/mL). Batch fermentations were performed in 250 mL shaking flasks containing 30 mL culture medium at 220 rpm at 30 °C on a reciprocal shaker . For fed-batch fermentations, the feeding medium was intermittently fed at different fermentation time .Feeding medium contained 500 g/L glucose, 240 g/L betaine, 0.4 g/L DMBI, and 0.4 g/L CoCl2.3E. adhaerens HY-1 genomic DNA was extracted using Sangon Biotech Bacterial DNA kits . Genome sequencing was performed by Genewiz . E. adhaerens HY-1 genome annotation was completed using the RAST (https://rast.nmpdr.org/) website. The genomic data of the E. adhaerens HY-1 has been submitted to a sharing platform National Microbiology Data Center  and the number was NMDCN00010QE. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/), genes related to cobalamin synthesis in E. adhaerens HY-1 were obtained and identified. Gene sequences are shown  and RNA-Seq performed on samples by Genewiz  using the HiSeq 2500 platform. Based on RNA-Seq analyses, the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) value of each gene was generated. According to the selection criterion, that is the FPKM values of genes were relative stable at the sampled points in the strain growth period and the average FPKM values of strain growth period were relative high. For our analyses, we selected 65 genes with the FPKM values spanning from 12.86 to 976159.7 and the average FPKM values of growth period in the 2225.6–296,023.2 range. , 1 mL of LB medium was added to recover for 4 h, and then centrifuged and spread on the plate for culture.2.7E. adhaerens HY-1 strains expressing fluorescence (mCherry) plasmids were inoculated into 30 mL fermentation medium in 250 mL shaking flasks (approximately 10% volume). Cultures were grown at 30 °C at 220 rpm, with samples removed every 12 h and washed three times in PBS. mCherry fluorescence intensity  and optical density (absorbance at 600 nm) were measured using a microplate Multi-Mode Reader  at different growth stages. Fluorescence intensity levels were defined as relative fluorescence units (RFU) divided by cell density (RFU/OD600) [E. adhaerens HY-1 strain harboring Pdnak was used as a control for promoter screening.Recombinant U/OD600) . Fluores2.8600). Cobalamin was measured as follows: 400 μL fermentation broth was collected and 40 μL NaNO2 12.5% (w/v) and 40 μL glacial acetic acid added. The mixture was boiled for 30 min and centrifuged at 14,000 rpm for 5 min. Then, 500 μL supernatant was removed and 300 μL ddH2O and 200 μL KH2PO4 added. The mixture was resolved on a reverse phase C-18 column  using high performance liquid chromatography (HPLC)  operating at 38 °C monitored at 361 nm. The mobile phase consisted of 28% methanol run at 0.8 mL/min for 18 min.Cell densities were monitored by measuring the optical density at 600 nm .All the fermentation processes were performed in triplicate and the results were presented as mean values. The experimental data was analyzed by Origin 2019b. 33.1E. adhaerens HY-1 genome was sequenced and annotated using RAST software. All cobalamin synthesis-related genes from E. adhaerens HY-1 were compared with the E. adhaerens Casida A genome in the National Center for Biotechnology Information database. Using this approach, the following key information was ascertained. Based on the metabolic de novo synthesis of cobalamin by E. adhaerens in the KEGG database and E. adhaerens HY-1 genome sequencing results, several cobalamin synthesis genes were identified  were used for comparative analysis. Three genes, CobSV, CobQ, and CobW were identified as the most efficient ones for enhancing cobalamin synthesis. Overexpression of CobSV, CobQ, and CobW with Pdnak enhanced the production of cobalamin by 14.4%, 12.2% and 19.9%, reaching 109.4 mg/L, 107.3 mg/L and 114.6 mg/L, respectively , middle log (96 h), post log (144 h), and stationary phases (180 h)  was 5.8-fold higher than Pdnak , and 12.8-fold higher than the weak promoter, PgroES  (mCherry by weak promoter (PglyA), medium-strong promoter (Pdnak) and a strong promoter (PpurU), the result was consistent with the measurement of fluorescence intensity  . At earle weaker A. In the 28,005) D. By anantensity .Fig. 3Fl3.4CobSV, CobQ, and CobW were combined for overexpression analyses. This generated 27 different combinations that were expressed in E. adhaerens HY-1 using the pBBR1MCS-5 plasmid (hmm strain (genotype: PmetH-CobSV-PibpA-CobQ-Pmdh-CobW) was the most effective in accumulating cobalamin at 134.8 mg/L; this was 41.0% higher than the control strain (95.6 mg/L). It was showed that the combination of strong promoters with CobSV could generally yield better results. However, despites the CobSV was expressed by strong promoter, the strains hhl, hlh and hml just accumulated a small amount of cobalamin. Additionally, the cobalamin produced by strain hhh, in which the three key genes were all expressed by strong-promoters, was lower than those of the strain hmm and hlm, in which some genes were expressed by relatively weak promoters. These data indicated that CobSV was essential for cobalamin synthesis, and the performance of its function needed to well balancing expression of CobQ and CobW.From our promoter strength and rate-limiting studies, different strength promoters and the three key genes,  plasmid . The hmm3.5pbbr strain carrying pBBR1MCS-5 served as controls. Expression data are shown (hmm strain was 130.1 mg/L, 30.1% higher than the original HY-1 strain. When fermentation continued for 9.5 days, cobalamin production was further enhanced to 171.2 mg/L, which was 45.6% higher than the original HY-1 strain.The eight engineered strains obtained above with positive effects on cobalamin production were further investigated using the fed-batch fermentation. Medium was fed on days 3–5 and cobalamin production detected in samples at 7.5 and 9.5 days. The original HY-1 strain and the re shown . When co4E. adhaerens HY-1 strain de novo synthesizes cobalamin and has been used for cobalamin production in industry. To further understand strain characteristics, genome sequencing and annotation studies were performed. Genes related to cobalamin synthesis in E. adhaerens HY-1 were analyzed. We showed that cobalamin synthesis required the DMBI precursor for production [CobSV, CobQ, and CobW were identified by gene overexpression studies with the promoter Pdnak, and displayed considerable influences on cobalamin synthesis. Then, 65 promoters with different gradient strengths were identified by the transcriptome sequencing of cells at different growth stages. Using combinatorial overexpression of the obtained key three genes with different strength promoters, cobalamin levels reached 134.8 mg/L in shaking flasks, which were 41.0% higher than the original HY-1 strain. Finally, cobalamin production was further enhanced to 171.2 mg/L using a fed-batch fermentation strategy.The oduction . Three kE. adhaerens, Sinorhizobium meliloti and P. freudenreichii [S. meliloti belongs to the same species as E. adhaerens and aerobically synthesizes cobalamin [P. freudenreichii is reported to anaerobically synthesize cobalamin [E. adhaerens, few reports are available. In this study, based on rate-limiting step analyses and the optimization of key gene expression, cobalamin production was enhanced to 171.2 mg/L using a fed-batch fermentation strategy. Currently, this is the highest cobalamin titer produced by E. adhaerens.It was previously reported the cobalamin metabolic pathway involved more than 30 genes . Howevernreichii . S. meliobalamin . Howeverobalamin . P. freuobalamin , but celobalamin . For cobE. adhaerens synthetic biology tools were available for study. In previous work, the genome sequence of E. adhaerens OV14 was analyzed and the strain was reported for usage as a vector for plant plasmid transformations [E. adhaerens by quick freezing with liquid nitrogen [E. adhaerens were lacking which meant metabolic regulation strategies were impossible to implement. The pBBR1MCS-5 plasmid could be successfully transformed into E. adhaerens HY-1 cells and laid the foundations for the genetic manipulations of the industrial producer. Additionally, the gradient-strength promoters identified from our transcriptome analyses now form a valuable reference database for future E. adhaerens research.Unlike most typical model microorganisms, limited rmations . In addinitrogen . NonetheCobW remains unclear, but it was a significant player in cobalamin synthesis. The last step of cobalamin synthesis involves the conversion of adenosylcobinamide-GDP to adenosylcobalamin, which was encoded by CobSV and identified as the rate-limiting step. This has not been previously reported. CobQ, which encodes adenosylcobyric acid synthase, was identified as promoting adenosylcobyric acid production. Besides, overexpression of upstream genes in the cobalamin synthesis pathway, such as HemA, HemB, HemC and HemD led to dramatically decrease of cobalamin production. It was speculated that the potential accumulation of intermedium metabolites caused by overexpressing these upstream genes could disrupt the metabolism of cobalamin. Since the lack of standards for these complicated intermediates, the specific mechanism should be further investigated.The cobalamin synthesis pathway is subjected to feedback inhibition by various regulatory mechanisms in strains that accumulate cobalamin. It was previously noted that cobalamin synthesis was regulated by highly conserved RNA structures, riboswitches , which rE. adhaerens HY-1 strain. In the future, other strategies could be implemented to further enhance cobalamin synthesis. For example, the DMBI precursor is required for cobalamin synthesis but it exerts inhibitory effects on bacterial growth [In conclusion, a high titer cobalamin-producing strain was identified via promoter screening, rate-limiting step analyses, and combined gene expression. Cobalamin production levels reached 171.2 mg/L, which were increased by 45.6% when compared to the original l growth . Thus, al growth . Becausel growth . In addil growth  as well S. X., and Z. X. designed the study and wrote the manuscript. J. Z., and W. Z critically revised the manuscript. Z. X., S. Y., and Y. Z. performed the experiments and analyzed the results. J. Z., S. X., and W. Z. designed and supervised the project. All authors discussed the results and commented on the manuscript.The authors declare no competing interests.


Four divergent groups of duck astroviruses (DAstVs) have been identified that infect domestic ducks. In March 2021, a fatal disease characterized by visceral urate deposition broke out in 5-day-old Beijing ducks on a commercial farm in Guangdong province, China. We identified a novel duck astrovirus from the ducklings suffering from gout disease. The complete genome sequence of this DAstV was obtained by virome sequencing and amplification. Phylogenetic analyses and pairwise comparisons demonstrated that this DAstV represented a novel group of avastrovirus. Thus, we designated this duck astrovirus as DAstV-5 JM strain. DAstV-5 JM shared genome sequence identities of 15–45% with other avastroviruses. Amino acid identities with proteins from other avastroviruses did not exceed 59% for ORF1a, 79% for ORF1b, and 60% for ORF2. The capsid region of JM shared genetic distances of 0.596 to 0.695 with the three official avastrovirus species. suggesting that JM could be classified as a novel genotype species in the Avastrovirus genus. Meanwhile, JM shares genetic distances of 0.402–0.662 with all the other known unassigned avastroviruses, revealing that it represents an additional unassigned avastrovirus. In summary, we determined that the DAstV-5 JM strain is a novel genotype species of avastrovirus. Astroviridae family and are infectious, non-enveloped, positive sense, single-stranded RNA viruses. Astroviruses infect a wide variety of hosts. According to their host range, the Astroviridae family is divided into two genera: Mamastrovirus and Avastrovirus. The two genera comprises 22 species according to the International Committee on Taxonomy of Viruses (ICTV), namely Mamastroviruses 1–19 and Avastroviruses 1–3 (Avastroviruses genus include Avastrovirus 1 (turkey astrovirus 1), Avastrovirus 2 (avian nephritis virus 1 and avian nephritis virus 2) and Avastrovirus 3 (turkey astrovirus 2 and duck astrovirus 1). These Avastroviruses are tightly linked to avian diseases, such as hepatitis  belong to the epatitis , white cepatitis , and gouepatitis . BesidesTo date, chicken astrovirus and goose astrovirus have been found to cause avian gout diseases. Chicken astrovirus was reported as the causative agent of chicken gout in India and Malaysia –9. GooseIn the present study, we discovered a new duck astrovirus DAstV-5 in ducklings suffering from gout disease. We conducted virome sequencing and obtained the complete genome sequence of the virus. Phylogenetic analyses and pairwise comparisons demonstrated that this virus was different from all the other previously known astroviruses, and thus we proposed it as a novel genotype species of duck astrovirus.2 for 48 h. For virological detection, kidney and liver tissue samples were homogenized in phosphate-buffered saline (PBS) and pooled together for three cycles of freezing and thawing. The suspensions were centrifuged at 12,000 × g for 10 min and then the supernatant was collected for virus RNA/DNA extraction using a Tianlong Nucleic Acid Extraction & Purification Kit T180H . The extracted RNA/DNA was subjected to polymerase chain reaction (PCR) amplification to detect goose astrovirus (GAstV), avian orthoreovirus virus (ARV), duck Tembusu virus (DTMUV), novel duck parvovirus (NDPV), duck circovirus (DCV), goose parvovirus (GPV), duck enteritis virus (DEV), duck adenovirus (DAdV), avian influenza virus (AIV), and Newcastle disease virus (NDV). The primers used to amplify the genome sequences were detailed to our previous research  immediately. The plates were incubated at 37°C with 5% COresearch , 18 and Five grams liver tissue was ground. After three rounds of freeze-thawing, the tissue was centrifuged at 12,000 g for 5 min to remove the precipitate. The supernatant was centrifuged at 160,000 g for 2 h at 4°C with HIMAC CP 100wx ultracentrifuge . The supernatant was used to extract DNA/RNA with an R6662-02 MagPure Viral DNA/RNA Mini LQ Kit . The whole genome was amplified using REPLI-g Cell WGA & WTA Kit . Sequencing libraries were generated using an NEB Next® Ultra™ DNA Library Prep Kit for Illumina®  following the manufacturer's recommendations. The library was sequenced on an Illumina Novaseq 6,000 and 150 bp paired-end reads were generated. The raw data were processed using SOAPnuke (v1.5.6) to acquire the clean data for subsequent analysis .https://blast.ncbi.nlm.nih.gov/Blast.cgi). The authenticity and similarity of the sequences were validated. The most homologous virus strain was used as a reference strain. The sequence was assembled by using reference mapping.Firstly, we used BWA software (v0.7.17) to compare the clean reads with ribosome database (Silva.132) and host database respectively. If the comparison length was <80% of the total length of reads, the reads were filtered. Then the host sequences were removed. The BWA software (v0.7.17) was used to compare the clean reads to virus reference database (Virus-NT), And we filtered the comparison results whose comparison length was <80% of the total length of reads for preliminary virus classification. Secondly, we used Megahit software (v1.1.2) to assemble the clean data into contigs. The contigs that compared to the host sequence database by BLAST software (v2.9.0+). And the host sequences were removed. Finally, we used BLAST software  to compare the unique contigs with virus database (separated from the NT database). If the alignment similarity was ≥80%, the alignment length was ≥500 bp, and e ≤1e-5, the contig was defined as virus sequence. If the alignment length was ≥100 bp, and e ≤1e-5, the contig was defined as suspected virus sequence. After comparing contigs with the virus database obtained from the NT database and the virus database obtained from the NR and HMM (vpfs and vfam) databases, we obtained the consensus sequences as the candidate virus sequence. The virome sequencing and data analysis were performed by Magigene Biotechnology Corporation . The candidate virus sequence alignments were performed in NCBI Nucleotide BLAST site (Four pairs of PCR primers were designed to amplify the gaps  using 2 https://smart.embl-heidelberg.de/. The stem-loop II-like motif (s2m) was evaluated using the online Rfam analysis tool (http:/rfam.xfam.org). The open reading frames (ORFs) were analyzed by DNAMAN version 8.0 (Lynnon Biosoft). Phylogenetic trees for the complete genome and the deduced amino acid sequences of the three open reading frames (ORFs) were constructed in the MEGA 7.0 software using the neighbor-joining method, with 1,000 bootstrap replications  employing the Clustal W method.Transmembrane (TM) domains and functional proteins were predicted using the Sample Modular Architecture Research Tool (SMART) analysis tool ications . Amino aMamastroviruses and Avastrovirus) was evaluated using Recombination Detection Program (RDP) software Version 4.101 with the RDP, GENECONV, Bootscan, MaxChi, Chimaera, SiScan, and 3Seq methods . Finally, we identified that 6 contigs are virus sequences. The other 148 contigs are identified as bacterial sequences and host sequences. The six virus sequences include five duck astrovirus sequences and one chicken stool-associated gemycircularvirus sequence. The chicken stool-associated gemycircularvirus sequence was 741 bp in length. And the astrovirus sequences were 253 bp to 1,006 bp in length. The astrovirus segments were homologous to previously published nucleotide sequences. And the sequence similarities were 52–77% . Among these 307 contigs, 153 contigs were phage sequences and the rest 154 contigs were other virus sequences. Then we put these 154 contigs separately into the online NCBI Nucleotide BLAST site  should range between 0.576 to 0.742, and 0.204 to 0.284 between and within groups, respectively  can be found below: Ethical review and approval were not required for the study because the samples used in the present study were taken from field animals by veterinarians. The samples were taken and submitted to Institute of Animal Health, Guangdong Academy of Agricultural Sciences.ML and MS supervised this project and designed the experiments. JZ, YH, LL, JD, and RK collected samples and performed experiments. JZ analyzed data and prepared manuscript. All authors read and approved the final manuscript.This work was supported by the Modern Agricultural Industrial Technology System Innovation Team of Guangdong Province (2020KJ137), the Special fund for scientific innovation strategy construction of high level Academy of Agriculture Science , the National Natural Science Foundation of China (No. 32102691), Guangdong Basic and Applied Basic Research Foundation (2021A1515012071), Guangzhou Basic and Applied Basic Research Foundation (202102021162), the Innovation Fund of Guangdong Academy of Agricultural Sciences-Industrial Special Project (202144), Guangzhou Science and Technology Plan project (201906040005), and Science and Technology Plan Projects of Guangdong Province (Grant No. 2021B1212050021), and the Collaborative Innovative Center of Guangdong Academy of Agricultural Sciences (XT202207).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


An in-vivo study using Danio rerio as a model organism was done to examine the toxicity and the removal efficiency of the samples. It was observed that chromium water treated with alginate–SPIONs beads, which were removed after water treatment showed less damage to the fishes when compared to SPIONs and alginate beads treated with chromium water where the SPIONs and alginate beads were not removed after the treatment period.Nanoparticles are potential candidates for wastewater treatment especially for the removal of heavy metals due to their strong affinity. Many biopolymers are used as adsorbents and encapsulation of nanoparticle onto them can increase their efficiency. In this study, SPIONs, alginate, and SPIONs incorporated on alginate beads have been synthesized and characterized both microscopically and spectroscopically. These were then used for the removal of chromium metal and the percentage of removal was evaluated using a batch adsorption study. The percent removal of chromium using SPIONs, alginate and alginate–SPIONs beads were recorded to be 93%, 91% and 94%, respectively. The adsorption of chromium using SPIONs and alginate–SPIONs beads followed the Tempkin isotherm, whereas adsorption of chromium metal by alginate beads was found to be homogeneous in nature and followed the Langmuir isotherm with an R Water is one of the world’s natural resources, and its availability in its purest form is critical for all living things . EffluenThese nano—adsorbent beads are good nanocomposite materials for removing heavy metals from wastewater in a continuous treatment process and are proposed to be nontoxic in nature, less expensive, to have high hydrophilicity and high efficiency ,17. In c3) and ferrous chloride (FeCl2) as precursor agents: 5.4 g of ferric chloride (FeCl3) and 1.9 g of ferrous chloride (FeCl2) were mixed in 1 L of nitrogen—purged water. Then the solution was reduced by adding 40 mL of 2 M sodium hydroxide (NaOH) to it. The mixture was stirred vigorously until a black precipitate appeared. It was then centrifuged at 5000 rpm, and the pellets were collected to obtain SPIONs, which were later lyophilized [SPIONs were synthesized using a chemical co—precipitation method prepared using ferric chloride (FeClphilized .6H7O6) solution was prepared and added dropwise to a mixture containing 10% of calcium chloride (CaCl2) and 1 mL of Tween 20 to form beads. Sodium alginate was transformed into beads after interacting with calcium ions. These beads were then filtered from the solution using muslin cloth [25 mL of 1.2% sodium alginate  was added to it as a stabilizing agent. 125 mg of SPIONs was then added to the mixture and stirred for 40 min until the SPIONs were completely dispersed in the solution. Then the solution was added dropwise to the solution containing 10% of calcium chloride and 1 mL of Tween 20 to form beads. The beads were then filtered from the solution using muslin cloth [The UV–Visible absorption spectra of SPIONs were taken at room temperature using UV 3600 PLUS  with a variable wavelength between 200 and 800 nm. Fourier Transform Infrared Spectroscopic experiments were carried out to check the functional groups using a IRTRACER 100 . Scanning Electron Microscopic micrographs and elemental composition were recorded using a JSM—IT 200 . The crystalline structure of the sample was determined by X-ray crystallography . The determination of the hysteresis loop was performed by Vibrating Sample Magnetometer  and its stability was checked using Zeta Potential analysis .The alginate beads and alginate–SPIONs beads were characterized using UV–Visible spectroscopy , Fourier Transform Infrared Spectroscopy , and crystallinity was observed using X-ray crystallography .2SO4, which produces Cr6+ dissociation, and further treated with 0.5% of diphenyl carbazide, which gives a reddish—violet color by binding to Cr6+. It was then spectroscopically measured at 540 nm [The effective removal of chromium metal using SPIONs, alginate beads, alginate–SPIONs beads was done using batch adsorption studies by calculating triplicate OD values. The DPC method  was used to investigate the chromium ion adsorption ,26. The 2 and ln qe in order to determine the adsorption pattern, where Ce is the equilibrium concentration of adsorbate in mg/L, qe is the amount of adsorbate adsorbed upon the adsorbent, and ε2 is the adsorption potential based on the Polanyi potential.The adsorption pattern and the mechanism of adsorbate removal by adsorbent was examined using four types of isotherm models—Langmuir, Freundlich, Tempkin and Dubinin—Radushkevich (D—R) isotherm. A graph between 1/qe and 1/Ce was plotted for the Langmuir isotherm, ln qe vs. ln Ce for the Freundlich, the Tempkin isotherm was investigated by plotting qe vs. ln Ce, and D—R was determined by plotting between εDanio rerio were purchased from Tarun fish farm, Chennai and kept in a glass aquarium filled with water with sufficient and continuous aeration. The fishes were introduced into the test environment after three to four days of acclimating to the new circumstances. The water was changed once a week and dry food fed twice a day. The initial pH of the water was at 7.2 ± 0.3 [Male .2 ± 0.3 .in-vivo study for the effective removal of chromium using SPIONs, alginate beads, and alginate–SPIONs beads was done against Danio rerio. Test 1 with 1 ppm of chromium water was used to test the survivability of zebrafishes. In Test 2, 7 g of synthesized SPIONs were added to 5 L of water. In Test 3, 7 g of SPIONs were mixed with 1 ppm chromium water and was not removed. In Test 4, SPIONs—treated water was used, whereby 7 g of SPIONs were added to 1 ppm of chromium water and the SPIONs were withdrawn after 1 h of interaction before introducing the fishes. In Test 5, 7 g of synthesized SPIONs were put into the water, and after 1 h of interaction they were removed. In Test 6, SPIONs—encapsulated alginate beads were added to 1 ppm of chromium water and not removed. In Test 7, SPIONs—encapsulated alginate beads were put into chromium water and removed after 1 h of interaction. In Test 8, 7 g of alginate beads were introduced to 1 ppm of chromium water and were not removed. Ten fishes were allowed inside the aquarium which contained the aforementioned conditions. All the fishes were fed regularly, as mentioned.An The damage that occurred in the fishes on exposure to the chromium water and SPIONs was analyzed using histology studies. The fishes were taken from each test on the 5th and 10th days and were put into 10% of formaldehyde solution for analysis. The organs of the fish—eyes and gills—were then subjected to Hematoxylin and Eosin (H&E) staining and examined using a fully inverted microscope .The uptake of chromium and iron metals by the fish was examined on the 5th and 10th days using ICP—MS (Inductively Coupled Plasma Mass Spectrometry) through the microwave digestion method. The whole test fish was taken from the aquarium and immersed in 5 mL of concentrated nitric acid for 15 min. A few drops of concentrated hydrofluoric acid were then added and microwaved until fully digested. A clear solution was obtained by centrifugation which was then taken and kept at 4 °C and then analyzed using Agilent 7700x—ICP—MS .The peak at the range between 250 and 300 nm shows the presence of iron oxide nanoparticles, and the peak between 200 to 400 nm shows the light absorption of the alginate beads a,b. A br−1. A peak around 3400 to 3300 cm−1 was due to hydroxyl groups and is seen in all the samples. A large peak seen in −1 depicts the presence of Fe–O vibrations of SPIONs [−1, 453 cm−1, and 586 cm−1 showed the presence of an OH group on the surface of SPIONs and the presence of the Fe–O bond of SPIONs, respectively. In another study, the presence of Fe3O4 particles in the Alg—Ce magnetic bead was depicted by the presence of a novel absorption band at 561 cm−1 [The functional groups present in the SPIONs, alginate beads and SPIONs—coated alginate beads were analyzed using Fourier Transform Infrared Spectroscopy (FTIR) . The shif SPIONs  reported561 cm−1 .3O4 face—centered cubic structure.The crystalline structure of the samples was examined through X-ray diffraction (XRD) . The cryThe surface morphology and the elementary composition of the SPIONs were examined using Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Analysis (EDX) . The sizThe charges of the SPIONs were observed using Zeta Potential analysis . The peaThe magnetism of the SPIONs was examined using a vibrating sample magnetometer (VSM) . The hysBatch adsorption experiments on the removal of chromium utilizing SPIONs, alginate beads, and alginate–SPIONs beads were studied using the DPC method. The conditions such as adsorbate concentration, adsorbent concentration, pH, and contact time were all optimized.The optimized condition for the effective removal of chromium from water was studied . The maxThe adsorbate concentration of 7 ppm was removed efficiently by alginate beads with a removal percentage of 81.61% . Under tThe removal of chromium using alginate–SPIONs beads was found to be higher than that of SPIONs and alginate beads . 7 ppm o2) values of each isotherm (Highlighted in yellow). The adsorption of chromium using SPIONs and alginate–SPIONs beads was found to be ionic in nature, as it followed the Tempkin isotherm with an R2 of 0.6003 and 0.7215. Homogeneous adsorption of chromium on alginate beads was recorded, as it followed the Langmuir isotherm with the highest R2 value of 0.9784. The removal of chromium using SPIONs was found to follow the Freundlich isotherm, with the highest R2 of 0.9228 [The pattern by which the adsorbent adsorbs the adsorbent is studied using four kinds of isotherm model: the Langmuir isotherm, the Freundlich isotherm, the Tempkin isotherm, and the D—R isotherm . The besf 0.9228 . Bilici f 0.9228  reportedf 0.9228 .The damage caused by the exposure to the chromium and the adsorbents was evaluated by histology study. Histology images taken on the 5th day are shown in On the 10th day of exposure, Test 1 showed increased damage in the eyes and gill regions a,b. HighThe accumulation of chromium and the iron in fish was evaluated using ICP—MS analysis . From thDanio rerio), and therefore it may be one of the better and lower—cost biosorbents for removing heavy metals. The presence of absorbents was found to cause lethal impacts to the animals; therefore, it is suggested not to allow the adsorbents into the treatment system following the treatment process.SPIONs, alginate beads and alginate–SPIONs beads were successfully synthesized, characterized and evaluated for the removal of chromium from contaminated water. It was concluded that the removal of chromium by alginate–SPIONs beads is higher than that of SPIONs and alginate beads with the optimized adsorbate and adsorbent concentration of 7 ppm and 0.08 g/10 mL, respectively, at pH 8 with a contact time of 1 h. The toxicity study also revealed that chromium water treated with the alginate–SPIONs beads (where the adsorbents were removed) imposed less damage on zebrafish (