Arbovirus infections represent a global public health problem, and recent epidemics of yellow fever, dengue, and Zika have shown their critical importance in Brazil and worldwide. Whilst a major effort for vaccination programs has been in the spotlight, a number of aptamer approaches have been proposed in a complementary manner, offering the possibility of differential diagnosis between these arboviruses, which often present similar clinical symptoms, as well as the potential for a treatment option when no other alternative is available. In this review, we aim to provide a background on arbovirus, with a basic description of the main viral classes and the disease they cause, using the Brazilian context to build a comprehensive understanding of their role on a global scale. Subsequently, we offer an exhaustive revision of the diagnostic and therapeutic approaches offered by aptamers against arboviruses. We demonstrate how these promising reagents could help in the clinical diagnosis of this group of viruses, their use in a range of diagnostic formats, from biosensors to serological testing, and we give a short review on the potential approaches for novel aptamer-based antiviral treatment options against different arboviral diseases. This review aims to cover the applications of aptamers as one among other strategies to face the great challenge offered by arboviruses in terms of public health in Brazil and worldwide. The use of such, not so new, tools against targets of a wide variety of molecular and biological natures has been increasing, and aptamer technology is establishing itself as one of the most significant branches of biological nanoparticle science. We start by defining arboviruses and their significance to human health, focusing our discussion on the current problems related to differential diagnosis. We subsequently provide an overview of their epidemiological panel, with emphasis on the context they have in Brazil, some cellular and molecular aspects related to viral metabolism, followed by a brief consideration of aptamer theory and technology, before finally discussing their various applications in arboviruses.Arthropod-borne viruses, named arboviruses, are an important global public health threat. They cause disease in humans after circulating among wild animals and subsequently being transmitted to domestic animals that act as hosts . Dengue Togaviridae; the flaviviruses from the family Flaviviridae; the bunyaviruses, nairoviruses, and phleboviruses, all from the family Bunyaviridae; the orbiviruses, family Reoviridae; the vesiculoviruses, family Rhabdoviridae; the thogotoviruses from the family Orthomyxoviridae [Arbovirus is a term that does not denote a taxonomy classification, but includes a wide variety of RNA virus taxes, such as the alphaviruses from the family oviridae .Arboviruses may be transmitted in two ways, although, by definition, they must replicate in an arthropod vector prior to transmission. They are mainly transmitted by biological transmission; the other mode of transmission, mechanical transmission, may occur through contaminated biological fluids. Details could be widely discussed here about the important aspects of viral transmission in order to better understand the peculiarities of epidemic spread. However, as this is not the focus of the review, we would only like to present some important aspects of the theme. Biological transmission can either be vertical  or horizontal . It could also occur vertically from an infected male directly to a female vector. However, the main route is an oral transmission from a vector to a vertebrate host by injection of infectious saliva during blood feeding ,2.Asfaviridae; Asfavirus), which is the only known DNA arbovirus. This variety of RNA genomes and the different replication strategies adopted by these types of viruses demonstrate that the transmission through arthropods has been evolutionarily selected a number of times [Arboviruses are RNA viruses, with the exception of the African swine fever virus  transmission in blood transfusions as well as organ transplantation procedures, but this ability to trigger iatrogenic infections is a characteristic of any viremia-producing arbovirus . This is2) covered by forests, especially in the Amazon region [Aedes aegypti mosquito, the principal vector of a number of arboviruses. Under these favorable conditions, dengue has become an endemic disease and, on a number of occasions, caused large urban outbreaks. Similarly, yellow fever has caused a number of recent outbreaks, and its potential urbanization is a constant threat, potentialized by the movement of populations from sylvatic transmission areas to large urban centers [Brazil is a tropical country with a great terrestrial extension 8,512,000 km,000 km2 n region . The pre centers . ClinicaAmong the most studied arboviruses are the viruses of the families Flaviviridae , Togaviridae , and Peribunyaviridae (Oropouche virus—OROV).Flavivirus genus includes about 39 species that are classified as arboviruses, with some being the cause of encephalitis, and others provoking hemorrhagic fevers in humans and animals. The flaviviruses have a viral particle measuring 40 to 60 nm in diameter, with an icosahedral symmetry protein capsid enveloped by a lipid envelope where the membrane proteins and spikes of glycoprotein are inserted. The genome is a single RNA strand with the positive polarity of about 11 kb, and it encodes three structural proteins and five nonstructural (NS) proteins as follows: 5’-C-preM-E-NS1-ns2a-ns2b-NS3-ns4a-ns4b-NS5-3’, which play a role in regulating and expressing the virus, such as replication, virulence, and pathogenicity [In the Flaviviridae family, the Aedes. Its transmission is mainly through the mosquito A. aegypti, but A. albopictus and A. polynesiensis are secondary vectors. The main transmission cycle of DENV involves only humans and mosquitoes in large, tropical, urban centers. The disease presents with symptomatology ranging from fever to hemorrhagic fever or dengue shock syndrome. Other symptoms can be related to pain in the form of myalgia, headache or retro-orbital pain, or bleeding, which, apart from the hemorrhagic fever, can include petechia, epistaxis, and bleeding of the gingiva [DENV virus is the most important flavivirus causing human disease in Brazil, and it is represented by four serotypes (DENV-1 to DENV-4). Viruses are kept in wild cycles involving small primates and mosquitoes of the genus  gingiva ,7.Culex declarator and C. coronator as vectors. Its reservoirs include various animal species such as birds, monkeys, sloths, armadillos, and marsupials [Another arbovirus that is widely disseminated in the Americas is SLEV, which is found in a number of countries from Canada to Argentina. SLEV is a neurotropic virus that, at least in the Amazon region of Brazil, is found to use rsupials . The disrsupials .Aedes and Psorophora mosquitoes [Rocio virus is also neurotropic in nature and can cause anything from asymptomatic infection to acute encephalitis, with an incubation period of 7 to 14 days. The symptoms are acute fever, headache, anorexia, nausea, vomiting, myalgia, and malaise. Signs of encephalitis are late and include mental confusion, reflex disturbances, motor disability, meningeal irritation, and cerebellar syndrome. Patients may also present seizures, abdominal distension, urinary retention, visual, olfactory, and auditory disorders, lack of motor coordination, impaired balance and swallowing, and memory disorders . The Rocsquitoes ,7.WNV was first isolated in the West Nile province of Uganda in 1937. The WNV transmission cycle involves mosquitoes and birds, and humans are an accidental host. About 80% of infected people are asymptomatic. Of those with symptoms, about 20% develop the characteristic febrile illness, with an incubation period of 2 to 14 days, causing fever, headache, fatigue, rash, palpable lymph nodes, and ocular pain. The most serious manifestations are meningitis and encephalitis [Another flavivirus is the Japanese encephalitis virus (JEV) that is considered to have its origin in Indonesia and Malaysia from where it has spread widely in Asia and as far as Australia to become the major cause of arbovirus-related encephalitis. Children and adolescents are mostly affected by this virus and constitute 75% of the registered cases. JEV presents a high fatality rate, which can be up to 30% in severe cases, but it is also the cause of permanent neurologic and/or psychiatric damage to some 30–50% of the victims. Whilst humans and horses can be infected by the virus, they are not able to infect mosquitoes due to the low viremia developed during the infection .A. aegypti is the main transmission vector, and the more complex wild cycle, where a broad range of mosquitoes can cause virus transmission. These include, in Africa, mosquitoes Aedes , and in America, mosquitoes Haemagogus  and Sabethe schloropterus [Yellow fever is an infectious, noncontagious disease caused by YFV in two basic cycles: an urban human–mosquito cycle in which ropterus . Whilst ropterus . The cliA. aegypti mosquitoes [Aedes species was the origin of the first isolation of ZIKV in Asia, in Malaysia (1969) [Aedes spp arboreal mosquitoes and nonhuman primates, and an urban cycle that is distinct both ecologically and evolutionarily between humans and peridomestic or domestic mosquitoes of the Aedes spp [ZIKV gets its name from the Zika forest in Uganda, where the virus was first identified in a rhesus monkey in 1947. This was followed a few years later by the first reported infection in humans in the Macnamara region of Nigeria, in 1954, and the isolation of the virus in squitoes ,13. The a (1969) . As witha (1969) . ZIKV deedes spp . It takeedes spp .Proechimys spp. rodents or Culex mosquitoes. Serological investigations suggest that it is an infection of rare occurrence, with a single case of reported human febrile infection. (b) Cacipacore virus, first isolated in 1977 from a bird, also in the Amazon region , but, to date, not associated with any disease in humans and no vector has yet been identified as responsible for its transmission. (c) Iguape virus, isolated in 1979 from a sentinel mouse in the coastal area of the rainforest of southeastern Brazil. Human diseases caused by Iguape are still unknown. (d) Ilhéus virus, isolated for the first time in 1944 from a pool of Aedes and Psorophora mosquitoes; it causes infection in humans and is characterized by severe headaches, chills, myalgia, and weakness, with or without mild encephalitis syndrome [An overview of emerging arboviruses also brings us other, less distributed and more localized diseases: (a) Bussuquara virus, first isolated in the state of Pará in the Amazon region of Brazil in 1956, from sentinel and wild animals such as the syndrome ,7.Orthobunyavirus, we have OROV. This virus is transmitted between sloths, marsupials, primates, and birds by the mosquito A. serratus and Culex quinquefasciatus. Remarkably, this virus has adapted to an urban cycle involving humans, with Culicoides mosquitoes from Pará (Brazil) as the main vector. OROV has already caused large outbreaks of acute febrile disease in the Amazon and central region of Brazil, with an estimated 500,000 cases of infection by this virus in Brazil in the last 48 years [Callithrix sp. in the state of Minas Gerais [In the Peribunyaviridae family, genus 48 years ,18,19. Ts Gerais .Phlebovirus genus of the Peribunyaviridae family, is endemic to the sub-Saharan region of Africa and has been reported to have spread to Egypt and become endemic to the whole of the Arabian Peninsula. This virus presents a number of symptoms that, in the cases of severe disease, can include hepatitis, encephalitis, and hemorrhagic fever. RVFV has further economic significance as it is responsible for abortion storms in livestock [Rift Valley fever virus (RVFV), ivestock ,21. The ivestock ,23. As hivestock ,25. In fivestock .Alphavirus genus, representative arboviruses are the MAYV, CHIKV, and eastern equine encephalitis viruses. MAYV was first isolated in 1954 from febrile workers in Trinidad. Subsequently, several individuals with fever and headache from the area of the Guamá River, state of Pará, Brazil, were also identified as infected by the virus. Several fever and arthralgia outbreaks in the Amazon region and the central plateau of Brazil and in various other Latin American countries have been attributed to MAYV. The main vectors are forest-inhabiting mosquitoes of the genus Haemagogus, and the vertebrate hosts are mammals. Symptoms associated with MAYV include fever, headache, myalgia, arthralgia, and, at times, arthritis. As has been described above with a number of other arboviruses such as dengue and RVFV, the rainy season increases MAYV occurrence, although MAYV illness persists throughout the year. The estimated transmission of MAYV in the city of Manaus alone is more than 2 million people, making MAYV a relevant public health problem, especially as control of the vector is nonviable and there is no vaccine or treatment available [In the Togaviridae family, vailable .A. aegypti and A. albopictus are the described vectors of CHIKV in the urban cycles, where symptoms appear 2–4 days postinfection and viremia in a further 5 days after the first symptoms appear. Typical symptoms of CHIKV include fever, as with other arboviruses, with common manifestations of arthralgia, backache, and headache. A maculopapular rash can also manifest as a symptom some days after the initiation of the disease and can last up to 10 days, as well as neuroretinitis or sand uveitis. More rare neurological diseases can also be present in the form of febrile seizures, meningitis, or encephalitis [Another zoonotic arbovirus of the Togaviridae family is CHIKV, which was initially isolated in Africa in 1952 from a contaminated primate. phalitis .It thus becomes clear that arboviruses are of great relevance to public health due to a number of factors, from the diversity of the infectious agents involved and the plurality of clinical manifestations to the lack of efficient differential laboratory diagnosis, lack of immunoprophylactic measures for most of the current infections, and the difficulty in implementing and maintaining educational and sanitary measures. Consequently, new approaches for rapid laboratory diagnosis and effective control and prevention of these infections are urgently required. Appropriate diagnosis is the key factor for the treatment of viral diseases when these infections are difficult to distinguish clinically. Especially at the onset, when the infection appears, patients present nonspecific signs and symptoms, often common in many of these diseases. Finally, due to their antigenic similarity, it is difficult to distinguish among some of these infections, even with serological tests, due to several cross-reactions among them.Arboviral diagnosis is essentially a great challenge, whether in clinical examination or in laboratory assessment, due to the particular characteristics of each set of symptoms, which could be present or not, the similarities between one or more symptoms, and the phylogenetic cross-reaction in serological tests that often occur, especially in endemic regions .Specificity is probably a major question related to most arbovirus diagnostic tests and it plays an important role in the differential identification of these ever-growing diseases. Many methods and strategies have been proposed to better discriminate these viruses, ranging from tests using traditional equipment for molecular diagnostic assays, supported by the high reliability of RT–PCR, nested RT–PCR and real-time RT–PCR machines to the use of point of care (PoC) immunochromatographic tests and novel biosensors.As discussed above, the majority of the arboviruses, at least at the early stages of the infection and during the light and medium stages of symptoms, present a clinical profile that is very similar, namely, fever, rush, headaches, and fatigue, that in an endemic area like Brazil could indicate any number of viruses. In addition, whilst molecular diagnostics are accurate, they are expensive, require specialized equipment and trained personnel, and are available only in large medical centers and central laboratory facilities. In the case of Brazil, as well as in many other tropical countries, this would also be affected by the logistics of shipping samples, maintaining a cold chain, and delays in processing and analyses. Finally, molecular diagnosis is only possible during the acute phase of the disease, which is during the first few days postinfection. On the other hand, serological tests are often incapable of specifically identifying antibodies from viruses of the same genus, particularly in the case of immunoglobulin G (IgG). Thus, whilst serological testing for immunoglobulin M (IgM) is reasonably accurate, IgG between dengue and Zika or Chikungunya and Mayaro is practically indistinguishable due to the cross-reactivity of such antibodies, giving false results and masking epidemiological data.In the last 25 years, aptamers and SELEX  technology have received great attention. Aptamers are single-stranded nucleic acid molecules consisting of DNA or RNA of about 20 to 80 bases, with molecular weights of 6 to 30 kDa, that adopt various three-dimensional conformations that bind to organic or nonorganic molecules—from single atoms to a wide range of proteins, as well as more complex targets such as whole cells, viruses, or bacteria. Aptamers are characterized by high specificity to target molecules and high binding affinity, inherent to the way they are selected. Thus, the dissociation constant of the aptamer/target molecule complex is frequently in the nanomolar or picomolar range ,32. The Similar to the conformational recognition that mediates antibody–antigen recognition and the formation of complexes, aptamers bind to their cognate targets with high specificity and affinity through various types of interaction, such as Van der Waals forces, hydrogen bonds, electrostatic interactions, and bonds between the complementary bases. Thus, aptamers are also referred to as “chemical antibodies” and are functionally used as antagonists, agonists, or target ligands and are applied in a variety of bioanalytical and biomedical assays, as well as therapeutic applications ,35.Aptamers are excellent alternatives to monoclonal antibodies, which have high production costs and important limitations such as temperature instability or low secretion by the obtained clone. Therefore, it becomes apparent that the affinity and specificity of these molecules position them as an important technology, not only for diagnostic and therapeutic purposes but also for bioanalytical applications ,36,37,38In order to address the problem of nuclease stability, modifications are often employed in order to stabilize the aptamers, including the design of spiegelmers or the chemical modification of either the oligonucleotide backbone or the 2′-position of the pyrimidine moiety. Mairal et al.  cited seMycobacterium tuberculosis and Staphylococcus aureus, among others. Despite the fact that we will not describe their work here because the main subject of their study is not within the scope of our review, these authors also listed a series of potential antiviral aptamers for therapy. Similar to Pan et al. [Appropriate diagnosis is important for defining medical care options in viral disease patients. However, viral infections, especially during the acute phase, often cause common, nonspecific symptoms that confuse the medical diagnosis. Pan et al.  presenten et al. , other sn et al. ,40. In tRapid population growth and the increased resistance to or lack of effective treatment of many viruses creates an obvious need for effective diagnosis, the ability to differentiate with high sensitivity and specificity between different viruses or other pathogens. One such rapidly growing area in viral diagnosis is based on biosensor technology, where aptamers can play an important role as recognition elements. Their low production cost compared to antibodies, their equal or better sensitivity and specificity, and their superior thermal stability, which eliminates the need for cold chain, are some characteristics that could make aptamers an important part of biosensor development. Aptamers can be a financially viable diagnostic solution during the acute phase of the disease, for the recognition of viral antigens soon after infection, as well as to monitor disease progression during treatment. SELEX offers a versatile and rapid PoC platform for aptamer selection, and the ease of chemical modification to further increase selective pressure could result in aptamers characterized by a precise detection capability of viremia levels that are well below those of currently available diagnostic assays ,39.Many strategies have been adopted in the use of aptamers, either in diagnostic or therapeutic products against arboviruses, but until now, the literature has focused mainly on ZIKV and DENV diagnostics, with some studies on the use of aptamers for diagnosis of tick-borne encephalitis virus (TBEV), RVFV, and CHIKV, among others.With regards to the use of aptamers in the diagnosis of arboviruses, Bruno et al.  demonstrEscherichia coli and purified in a Ni-charged resin from the cell lysate. Subsequently, a SELEX-type selection was performed to isolate families of RNA aptamers with binding properties characterized by high affinity and specificity for the target protein. The group identified GAUU and pyrimidine/guanine motifs present in both their selected aptamer sequences and in the coding region of the RVFV viral genome. A selected and subsequently truncated aptamer, labeled with fluorescein and studied in a fluorescence polarization system during a titration experiment with the target protein, demonstrated a high-affinity binding with a Kd of 2.6 µM, offering data that support the possibility of constructing a sensitive fluorescence-based sensor for the viral N protein, thus permitting viral testing or drug screening application development.Ellenbecker et al.  describeE. coli and purified in a Ni-charged resin. The purified recombinant protein was immobilized on immuno plates, where 15 rounds of a SELEX-based selection of aptamers were performed. The antiviral activity of the pool of aptamers was assessed using an isolate of the Siberian TBEV in pig embryo kidney cell (PK cell) culture. The concentration of infectious virus (PFU/mL—plaque-forming unit/mL) was determined by titration of the PFU in PK cell culture, and the neutralization index was calculated as the ratio between the titers of viral suspensions in the control and after the treatment with the pool of aptamers. As a result of the treatment with the pool of aptamers, the TBEV neutralization index I averaged logI = 1.16 ± 0.05, i.e., the amount of infectious virus in the suspension decreased approximately 17.5 times. This value is comparable with the results of the neutralization of the commercial human immunoglobulin against tick-borne encephalitis at a dilution of 1:80 (logI = 1.2–1.88). Since the analyzed pool apparently contained groups of aptamers with different inhibitory activity, it can be expected that this parameter will increase after additional rounds of selection. Aptamers specific for TBEV surface protein can also be used in diagnostic platforms. Today, enzyme immunoassay systems based on the immobilization of viral particles on antibodies are most commonly used for TBEV diagnosis. However, after obtaining a positive result, it is impossible to isolate the virus in physiological conditions for further research. On the other hand, if the virus is immobilized on aptamers, the virion–aptamer complex can be easily destroyed with deoxyribonuclease, and the probability of isolating intact viral particles is very high. In this case, the aptamers can be used to isolate live virus from natural and clinical specimens.For the development of therapeutics against TBEV, Kondratov et al.  obtainedEcoRI from aptamer-mediated inhibition. Active EcoRI is able to rapidly cleave multiple signaling molecules to generate a detectable signal. Upon analysis, a highly specific binding of the linker to its nucleic acid target was shown, and this quickly resulted in a distinct fluorescent signal. Furthermore, such a signal could be clearly distinguished from the control signal produced by nontarget sequences within 20 min. Moreover, when serotype-specific linkers were used, the resulted signal by different dengue serotypes was equal to that of the negative control non-dengue sequences. Thus, it was verified that serotype-specific aptamers were developed, and the test did not present any cross-reactivity between different dengue serotypes. When a linker capable of identifying all dengue serotypes was used, the test was found capable of distinguishing between dengue and non-dengue sequences. Thus, the biosensor developed was capable of specifically detecting and distinguishing between dengue serotypes with high specificity, as well as recognizing a pan-dengue sequence. Finally, apart from the efficiency of the described biosensor, the author pointed out its robustness and the potential low-cost of its reagents as a means of its viability in product development for the detection and control of infectious diseases.In relation to DENV diagnostics, Fletcher et al.  demonstrGandham et al.  describeBalinsky et al.  have shoAlejo-Cancho et al.  describe2O3  modified with 3-mercaptopropionic acid–MPA (SAMN@MPA) and bound to gold nanoparticles (AuNPs) conjugated with aptamers, forming the SAMN@MPA@AuNPs@aptamer that is a colorimetric immunosensor. For a better understanding of how to obtain these particles, we recommend reading the original article. Specific aptamers for the four dengue serotypes were modified in the 5′-terminus with a -SH group for binding to AuNPs. A pool containing the four dengue serotypes  was added to the SAMNs@MPA@AuNPs@aptamer complex and a decrease in the intensity of the absorbance peak was observed. The aptamer–virus conjugation causes a reduction of the surface area of AuNPs, justifying the observed reduction of absorbance and the wavelength displacement of the localized surface plasmon resonance signal. In addition, it is possible to observe a color change at each step of the immunosensor production according to the surface modifications of the hybrid nanomaterial. Thus, SAMNs only present an earthy yellow coloration, and AuNPs a red coloration. The SAMNs@MPA@AuNPs@aptamer complex showed a dark purple coloration caused by the aggregation of the aptamers in the nanoparticles, and after the addition of the pool with the four dengue serotypes, the solution showed a change in its coloration to green, indicating the detection of the virus caused by the antigen–aptamer binding. Tests with ZIKV and YFV have shown no false-positive response. The method described represents an easy, fast, low-cost, and specific base to a test, which could be used in field measurements for the diagnosis of dengue.Basso et al.  describeThe development of a Zika diagnostic assay was proposed using an aptamer-based ELASA for the highly specific and sensitive detection of Zika NS1 protein . NS1 is Escherichia coli and used to make monoclonal antibodies that were subsequently tested experimentally. Peptides and antibodies were conjugated to Eu NPs (europium nanoparticle beads) and experimentally tested using a fluorescence-linked sandwich immunosorbent assay (FLISA). The performance of the peptide-linked sandwich FLISA was evaluated in virus-spiked human serum and urine. In the interaction assays, the authors found that the aptamer peptide was able to detect the Zika protein at a minimum concentration of 0.16 µg/mL and bind to the virus at 1 × 105 tissue culture infective dose (TCID)50/mL. Importantly, the specificity of the test was evaluated using the dengue and Chikungunya viruses, for which there was no binding of the peptides. In FLISA, the performance of the assay significantly increased by at least 5 × 104 TCID50/mL when the virus was diluted in Dulbecco’s modified Eagle’s culture media. When the virus was diluted in the serum, although the signal strength of the test decreased, the detected titer remained 5 × 104 TCID50/mL. Interesting data demonstrated by the authors revealed that these results were obtained when pairs of peptides for capture and development were used in the assay, but a significantly lower titer of viruses is detected when pairs of monoclonal antibodies were used (1 × 106 TCID50/mL titer). When evaluating the titer in urine, the authors found no differences when using the pair of aptamer peptides or the pair of monoclonal antibodies; both the titers detected were 1 × 104 TCID50/mL. Interestingly, the authors commit in their conclusions that an advantage of using these aptamer peptides, instead of DNA aptamers, is the lack of quality clinical samples; the DNA aptamers used in aptamer-based ELISA assays have been reported to detect 1 ng/mL in 10% serum and monoclonal antibodies are still needed in the tests.It is relevant to point out the work of Kim et al. , which iA. aegypti salivary gland extract were coupled with AuNPs and tested. The Apt–AuNPs were able to recognize up to 10 ng of protein from the A. aegypti salivary gland extract as well as 15 ng of recombinant A. albopictus D7 protein. Another approach was to select and test several aptamers against recombinant ZIKV protein E. The aptamers were conjugated to AuNPs, and the aggregation, measured by absorbance, was positive when concentrations of 5 to 50 nM of protein were tested. When Apt–AuNPs were tested against the intact virus, the amount of 105 PFU of active ZIKV was detected in the test. In this work, the tests were performed in a buffer solution, but the authors reported that the ultimate goal was to detect these targets via end-use card-based diagnostics or to observe a color change of the mosquito abdomen after imbibing the Apt–AuNP solution. These preliminary results indicate that the use of this technique, employing Apt–AuNP-based colorimetric measurements, can be a useful tool for diagnosing mosquitoes carrying arboviruses.In parallel to strategies for the development of new techniques for the human diagnosis of arboviruses, Bosak et al.  describeAptamers capable of blocking enzyme MTase action have also been studied as the cap methylation of flavivirus RNA by MTase is an essential process of viral replication. The flavivirus replicates in the cytoplasm, and subsequently, it uses its own enzyme for RNA capping. Flavivirus MTase transfers a methyl group from S-adenosyl–L-methionine to both the N-7 position on the cap and the 2′-OH position of ribose on the first transcribed nucleotide. The flavivirus MTase domain is located at the N-terminal region of the NS5 protein and specifically methylates viral genome RNA by recognition of a specific sequence and structure of its 5′ untranslated region. This enzyme also presents guanylyl transferase activity. Han and Lee  commenteIn this review, we have shown that aptamers can be selected against a variety of viral targets of arboviruses of clinical and public health relevance, from viral capsids to envelop to nucleoproteins and their fragments. A number of complicated selection strategies can be optimized for diagnostic or therapeutic applications, whereby aptamers with the lowest dissociation constants or functional aptamers with neutralizing or enzymatic inhibition potential can and have been successfully selected.Such aptamers have been applied in diagnostic approaches that range from electrochemical or fluorescence biosensors to serological type assays such as ELASA or sandwich ELISA, alone, as a pair of aptamers, or in combination with monoclonal antibodies, with promising results. Similarly, several aptamers have been developed for therapeutic approaches, aiming at directly neutralizing the virus in question, or inhibiting the action of proteins or enzymes relevant for viral replication, thereby offering a therapeutic strategy in moments when no vaccine or other treatment is available. The appeal of a Zika therapeutic or prevention agent for pregnant women, which could avoid the potential for microcephaly or neurological damage to the baby, is an obvious example, as is the treatment for severe cases of arbovirus infections that lead to encephalitis or even death.Whether for early diagnosis or treatment, aptamers have been raised against a number of arboviruses and have shown promising results, high affinities and specificities, and other beneficial features such as increased stability and reduced size that can be exploited clinically and bring new and highly demanded products to the market.


Polycipiviridae is a family of picorna-like viruses with non-segmented, linear, positive-sense RNA genomes of approximately 10–12 kb. Unusually for viruses within the order Picornavirales, their genomes are polycistronic, with four (or more) consecutive 5′-proximal open reading frames (ORFs) encoding structural (and possibly other) proteins and a long 3′ ORF encoding the replication polyprotein. Members of species within the family have all been detected in ants or via arthropod transcriptomic datasets. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Polycipiviridae, which is available at www.ictv.global/report/polycipiviridae. Icosahedral particles approximately 33 nm in diameter have been observed by electron microscopy in samples prepared from ants infected with Solenopsis invicta virus 2, but not in samples prepared from non-infected ants  Table 1Table 1 . GenomesPicornavirales . ORF. ORF2]. Picornavirales.Replication has not been studied but is assumed to be similar to that of other members of the order Solenopsis invicta) and high virus loads have been detected in larvae, pupae, workers and queens [Solenopsis invicta virus 2 appears to establish a chronic, asymptomatic infection in fire ants .The family Polycipiviridae: www.ictv.global/report/polycipiviridae.Full ICTV Report on the family


COVID-19 caused by SARS-CoV-2 has already infected more than 6. 3 million people worldwide as of 1st June 2020 and caused a global medical emergency. Healthcare professionals have been struggling to devise appropriate therapeutic strategies against the virus mainly due to the diverse range of symptoms and multiple-organ failure in infected patients. Several broad-spectrum antiviral drugs are being used for treatment; however, there is yet no specific drug or vaccine against the virus. Multiple-organ failure due to hyperactivity of the immune system resulting in cytokine storms is a major reason for death among the 5% critically ill patients. In this article, we have discussed the damage caused by COVID-19 on different organs of the human body. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causal agent of Coronavirus disease 2019 (COVID-19), is a single-stranded RNA virus with a non-segmented genome. Like other coronaviruses, four structural proteins are present in assembled viruses: membrane protein (M), capsid protein (C), envelope protein (E), and spike glycoprotein (S) . The preDue to the lack of vaccine, immunization against the virus is not yet possible . Drugs l+ and CD8+ T cells have been identified in 100 and 70% of the convalescent patients, respectively. CD4+ T cell response was robust with maximum IgA and IgG against spike and membrane protein of SARS-CoV-2  should be present  symptoms . Anorexillus sp. . It willA case study on 214 COVID-19 patients from three special care centers of Union Hospital, Wuhan, revealed that 36.4% of the infected people had neurologic symptoms . The symAbnormal alanine transaminase (ALT) and aspartate aminotransferase (AST) levels are an indication of liver damage. Liver damage has been observed in about 15–53% of the cases with elevated bilirubin, AST, and ALT . The advThe occurrence of conjunctivitis has also been reported as a clinical manifestation of the infection and tears, and conjunctival fluids have tested positive for the virus in such cases . A doctoSome clinical manifestations have also been observed in the skin, hands, and toes 3–4 weeks after COVID-19 infection. In a study, out of six Spanish patients that had similar manifestations, two were tested positive for the virus weeks before. Multiple lesions were observed in the toes, fingers, soles, and heel . In anotAccording to World Health Organization, the common symptoms of the disease include dry cough, fatigue, and fever. Sore throat, nasal congestion, body aches, and pains are also common. However, several other manifestations related to the heart, lungs, liver, kidney, central nervous system, and gastrointestinal system failure is making treatment difficult for critically ill patients . It is sMP conceived the idea. AP wrote the manuscript and provided revisions to the manuscript. All authors have read and approved the final version of the manuscript.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.


The epidemic strain of Venezuelan equine encephalitis virus (VEE) 1B invaded south Texas in 1971. The success of the eventual containment and control of the virus invasion was the early recognition and immediate detection, cooperation, coordination, and participation among multiple federal agencies. There were 4739 wild vertebrate animals trapped on a ranch in the area with only 1 VEE virus isolation from a Virgina opossum (Didelphis virginiana). A large number of mosquitoes were also collected on the ranch and tested, resulting in 240 VEE virus isolations. Virus isolations were obtained from 58% of the 33 equines tested. Wild vertebrates did not play a significant role in the outbreak. In the letter below, Dr. McLean provides a first-hand account of the invasion of Venezuelan equine encephalitis virus 1B into South Texas. This invasion was met with a massive, coordinated, inter-agency response, which ultimately ended the outbreak. We believe this event represents the sole example to date in which invasion of an exotic zoonotic arbovirus into the United States has been eradicated, and exemplifies the interagency cooperation necessary to achieve such a successful outcome. During a time where we can expect the continued emergence of viruses that threaten human and animal health, such lessons from the field can be both informative and encouraging. Dr. McLean has led a very distinguished career, serving in leadership positions in both the Center for Disease Control (CDC) and the United States Department Agriculture (USDA), and we value his insight and perspective on this historic outbreak.The epidemic strain of Venezuelan equine encephalitis (VEE) 1B was responsible for epidemics in humans and equines in Ecuador in January 1969, and was subsequently introduced into coastal Guatemala and El Salvador (possibly in infected equines) in the summer of 1969. From there, the virus spread south to Honduras and Nicaragua, and north to Southern Mexico by November 1969. In 1970, the virus spread throughout those countries and north along the Gulf coast of Mexico toward Tampico, where it resulted in an epidemic there in March–April 1971. Descriptions of this outbreak have been published ,3,4.This continued northward progression alerted USDA Animal Plant Health Inspection Service (APHIS) and CDC to the likelihood of an invasion into the U.S. This impending hazard to the U.S. initiated the two agencies to start working together to develop contingency plans to address the hazard. A number of meetings were held among their staff and invited experts from other federal and state agencies and universities to develop strategies for prevention and control as well as to determine what investigations were needed. The U.S. Medical Research Institute of Infectious Diseases already had a VEE vaccine in their stock but it was not FDA/APHIS approved. However, it was made available to the team of investigators if they signed a voluntary release to protect them in Texas from VEE-1B virus infections that are quite debilitating in humans and kills equines. We were all willing to receive this unapproved (by FDA) vaccine so we could perform our duties in Texas. APHIS mobilized a number of teams of veterinarians, technicians, and administrators; CDC mobilized physicians, epidemiologists, entomologists (Dr. Vern Newhouse as leader), virologists , wildlife biologists (Dr. Robert McLean as leader), and technicians for each. Counterpart staff from public health and other state agencies in Texas joined in the effort.APHIS and CDC administrators and senior staff met in south Texas to develop local plans and establish administrative facilities and local and state connections and coordination. They were waiting for the first confirmed identification of VEE-1B virus in Texas (USA), whether equine, humans, or possibly mosquitoes or vertebrate animals. They could not enter Mexico to sample or test animals or humans to confirm the virus presence in nearby Northeast Mexico (across the Rio Grande river), and had to wait for it to spread into Texas. Arrangements were made to secure enough hotel and working space for the team members. All of the space at a motel in south Texas that was convenient for the potential affected area was reserved, and all of us later stayed there. We also used rooms to contain refrigerators and freezers, store equipment and supplies, setup a laboratory, and have meetings. It was the local epidemic investigation headquarters. APHIS had a large number of veterinarians there to deal with the investigation and control of VEE-1B virus in the equine population.The team leaders selected staff to help conduct investigations, collected equipment and supplies and arranged for shipment to Southeast Texas, reserved vehicles and hotel reservations, and waited for the orders to proceed after the first VEE-1B virus detection in south Texas near the U.S.-Mexico border was confirmed. The first detection was a sick equine near the border. All of the teams were quickly mobilized to South Texas.APHIS brought multiple teams of veterinarians for field investigations of affected equines and to establish quarantine and equine movement restrictions. CDC quickly setup a laboratory in the hotel to process and test blood samples for rapid detection of virus and antibody for infected equines and later humans after CDC epidemiologists began investigating human cases. All field supplies for all investigations of equine, human, mosquitoes, and wildlife were stored and distributed from that hotel, which was the headquarters for all of the entire U.S. government effort in South Texas. Coordination with state and local officials involved in the outbreak investigations was conducted, as well as with the press, from there.Mosquito control efforts were developed, organized, and conducted locally, then broadened as the epidemic zone expanded. Finally, the U.S. Air Force fleet of large insecticide spray aircraft were deployed as the epidemic spread widely threatening larger areas of Texas to the north .Because equines were the primary host for amplification, a big achievement for the control of the epidemic and the protection of equines was the very rapid emergency approval of the VEE vaccine by USDA and the production of the military VEE vaccine for use in equines by a private vaccine company. The vaccine was made available for use by private veterinarians throughout the state of Texas. One industrious veterinarian acquired state approval and distributed the vaccine by a rented private plane to veterinarians throughout the state. Control of the epidemic and advancement northward was achieved, but there were about 1800 equine cases in the southern half of the state by the time the epidemic was stopped more than two months later.At this time, I was the only wildlife biologist/epidemiologist at CDC and was in the Rabies branch of the epidemiology program. I was requested and agreed to head the wildlife investigations and selected some team members to assist. Luckily, I had just hired a biologist from south Texas who had worked for CDC at the public health unit at the border there that was closing and who grew up in that area. He had many contacts there and helped arrange in selecting a sampling location near the border on a large cattle ranch with great habitats for wildlife. The rancher allowed us free access to all of the ranch and provided help and guidance from his ranch manager.We set a large number of traps for rodents; smaller numbers of traps for larger mammals such as raccoons, opossums, and rabbits; and leg-hold traps for carnivores and captured tortoises by hand throughout the ranch. At the same time, we set up mist nets for birds throughout the area and operated some mist nets at night for bats. Canon nets were set up on the beach on South Padre Island to capture sea gulls. Mosquito light traps were placed within the lines of mist nets and mammal traps to obtain a comprehensive sampling of vector and vertebrate populations for a more thorough effort to detect the presence of VEE 1B virus. Entomologist Dr. Newhouse from CDC was the leader of the mosquito sampling effort, although we all worked together to conduct prolonged and intense field investigations during a multi-week period in August—September 1971.Follow-up investigations were conducted multiple times at these sampling sites by the vertebrate and vector teams during the next year to ensure that VEE 1B virus did not become established in South Texas.Some of the testing of blood specimens from vertebrate animals, humans and processed mosquitoes were conducted locally to obtain rapid results for the epidemiologists and mosquito control teams who were trying to stop the epidemic expansion.The majority of the testing was conducted at CDC in Atlanta. Virus isolation was performed by inoculating 0.02 mL of each sample by the intracerebral route into Swiss albino suckling mice. Virus isolates were identified by microtiter complement fixation tests. Virus neutralizing antibody in the vertebrate and human sera were tested in duck embryo cell culture by the plaque reduction neutralization test using VEE-TC-83 vaccine virus.This was a massive, coordinated effort to prepare for the impending invasion of the epidemic 1B strain of VEE virus into the USA at the Texas border. The success of the eventual containment and control of the virus invasion was the early recognition and immediate detection, cooperation, coordination, and participation among multiple federal agencies of USDA, CDC, U.S. Military, National Park Service, Department of Interior), multiple Texas state departments, and local agencies and their staff with valuable input from individual scientific experts. The leaders of these agencies were on the ground in South Texas to initiate this massive, coordinated federal/state response. A well-planned response was developed, and multiple teams were formed to investigate specific health aspects in the human and equine populations, and the mosquito vector and wild vertebrate host involvement. The effective control of virus transmission was initiated quickly through broad equine vaccination and quarantine and intensive mosquito control that prevented the epidemic spread to Northern Texas and surrounding states. The close cooperation and joint leadership among the federal, state, and local officials and the dedicated and exhaustive efforts by the scientists and public health officials and their staff was key for the success of this dramatic disease control effort. It was a good example for dealing with foreign disease invasion and control. A big advantage for the control of this invasion and impending epidemic in Texas was the prior knowledge about the virus strain, history of its origin, and movement from South America to Central America and northward along the Gulf coast of Mexico, and the information on the potential domestic and wild vertebrate hosts and vectors that would be affected.


RdRp (PB1) gene segment of a divergent negative-sense RNA virus, denoted Lauta virus (LTAV), in a native Australian gecko (Gehyra lauta). The presence of this virus was confirmed by PCR and Sanger sequencing. Phylogenetic analysis revealed that Lauta virus likely represents a newly described genus within the family Amnoonviridae, order Articulavirales, that is most closely related to the fish virus Tilapia tilapinevirus (TiLV). These findings provide important insights into the evolution of negative-sense RNA viruses and structural conservation of the viral replicase among members of the order Articulavirales.The discovery of highly divergent RNA viruses is compromised by their limited sequence similarity to known viruses. Evolutionary information obtained from protein structural modelling offers a powerful approach to detect distantly related viruses based on the conservation of tertiary structures in key proteins such as the RNA-dependent RNA polymerase (RdRp). We utilised a template-based approach for protein structure prediction from amino acid sequences to identify distant evolutionary relationships among viruses detected in meta-transcriptomic sequencing data from Australian wildlife. The best predicted protein structural model was compared with the results of similarity searches against protein databases. Using this combination of meta-transcriptomics and protein structure prediction we identified the  The development of next-generation sequencing (NGS) technologies, including total RNA sequencing (meta-transcriptomics), has revolutionized studies of virome diversity and evolution ,2,3. DesThe conservation of protein structures in evolution and the limited number of proteins folds (fold space) in nature form the basis of template-based protein structure prediction , providiThere is a growing availability of three-dimensional structural data in curated databases such as the Protein Data Bank (PDB), with approximately 11,000 viral protein solved structures that can be used in comparative studies. Importantly, these include a limited number (approximately 115) of structures of the RNA-dependent RNA polymerase (RdRp) from a variety of viral groups. Viral RdRp proteins are catalytic proteins (~460 to ~1930 residues) implicated in the low-fidelity replication of the genetic material of RNA viruses . The terAmnoonviridae as a newly recognized family of negative-strand RNA viruses present in fish (ICTV Master Species List 2018b.v2). Together with the Orthomyxoviridae, the Amnoonviridae are classified in the order Articulavirales, describing a set of negative-sense RNA viruses with segmented genomes. While the Orthomyxoviridae includes seven genera, four of these comprise influenza viruses (FLUV), and, to date, the family Amnoonviridae comprises a single genus—Tilapinevirus—which in turn includes only a single species—Tilapia tilapinevirus or Tilapia Lake virus (TiLV). The International Committee on Taxonomy of Viruses (ICTV) recently introduced the Oreochromis niloticus) in Israel and Ecuador [Orthomyxoviridae and Amnoonviridae have negative-sense, segmented genomes, the genomic organization of the Amnoonviridae comprises 10 instead of 7–8 segments [Orthomyxoviridae (~12–15 kb). To date, however, only the RdRp (encoded by a 1641 bp PB1 sequence) has been reliably defined and most segments carry proteins of unknown function. Importantly, comparisons of TiLV RdRp with sequences from members of the Orthomyxoviridae revealed the presence of four conserved amino acid motifs (I–IV) of size 4–9 residues each [TiLV was originally identified in farmed tilapine populations  and hybrid tilapia (O. niloticus x O. aureus). Herein, we report the discovery of a divergent virus from an Australian gecko (Gehyra lauta) using a combination of meta-transcriptomic and structure-based approaches, and employ a phylogenetic analysis to reveal its relationship to TiLV. Our work suggests that this Gecko virus likely represents a novel genus within the Amnoonviridae.Unlike other members of the avirales , TiLV apCarlia amax, Carlia gracilis, Carlia munda, Gehyra lauta, Gehyra nana, Heteronotia binoei, and Heteronotia planiceps were collected alive in 2013 from Queensland, Australia. Specimens were identified by mtDNA typing and/or morphological data. Livers were harvested and stored in RNAlater at -80 °C before downstream processing. All sampling was conducted in accordance with animal ethics approval (#A2012/14) from the Australian National University and collection permits from the Parks and Wildlife Commission of the Northern Territory (#45090), the Australian Government (#AU-COM2013-192), and the Department of Environment and Conservation (#SF009270).A total of seven individuals corresponding to the reptile species RNA extraction was performed using the RNeasy Plus minikit  following the manufacturer’s instructions. Each of the seven livers was extracted individually and then pooled in equal amounts. For RNA sequencing, ribosomal RNA (rRNA) was depleted using the RiboZero (epidemiology) depletion kit and libraries were prepared with the TruSeq stranded RNA library prep kit before sequencing on an Illumina HiSeq 2500 platform (100 bp paired end reads). Library preparation and sequencing was performed by the Australian Genome Research Facility (AGRF), generating a total of 22,394,787 paired end reads for the pooled liver RNA library. Raw Illumina reads were trimmed of sequencing adapters and low-quality bases with Trimmomatic v0.38 . The tri−5) were assigned as “orphan” contigs . These were then analysed using a protein structure-informed approach. Specifically, orphan contigs were translated into all six open reading frames (ORFs) using the getorf program [n = 57). To detect distant sequence homologies and predict viral protein structures, this subset of translated ORFs was then analysed using a template-based modelling approach as implemented in Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) [To further screen the meta-transcriptomic data, all the assembled sequences below the assigned threshold  . In brie/phyre2) . In tota/phyre2)  with def/phyre2) .https://www.ncbi.nlm.nih.gov/) and the HMMER web server v2.41.1 (http://www.ebi.ac.uk/Tools/hmmer) against the following profile databases using default e-value cut-offs to assign significance: (i) reference proteomes v.2019_09, downloaded on 2019-10-03 (https://proteininformationresource.org/rps/); (ii) Uniprot v.2019_09, downloaded on 2019-10-03 (https://www.uniprot.org/); (iii) Swiss-Prot v.2019_09, downloaded on 2019-10-03. Protein families were identified using Pfam v.32.0 (https://pfam.xfam.org/). In addition, conserved domains were annotated using the Conserved Domain Database (CDD) v.3.17 and the CD search tool (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). To detect additional contigs and better characterize the genome of the novel virus, we aligned the cDNA contigs against custom databases using DIAMOND v.0.9.32 [Articulavirales . We focused on the de novo-assembled contigs that had no significant hits using initial searches against the NCBI nucleotide and non-redundant databases. Accordingly, of 293,586 orphan contigs, 57 contained translatable ORFs of more than 1000 nt in length, and because we hypothesized that some may correspond to undetected virus sequences, we interrogated them using a protein structure prediction approach with template-based modelling (TBM) in Phyre2 [Orthomyxoviridae)  a,b. Thisviridae) . Despiteviridae) a.Articulavirales, including influenza A virus (FLUAV), TiLV, and infectious salmon anaemia virus (ISAV). Comparisons of the assembled contigs against a custom database containing only members of the Articulavirales were then performed to improve sequence alignments. Accordingly, the best hit matches were obtained to TiLV   . To iden< 10−15) . Lauta virus (reflecting the species name of the gecko in which it was identified), abbreviated as LTAV. Multiple sequence alignment of the RdRp between Lauta virus and other members the order Articulavirales identified a number of well conserved amino acid motifs (I–IV) ranging in length from 5 to 11 amino acids in length , comprises a distinct monophyletic group. The close relationship between LTAV and TiLV was supported by high UFBoot/SH-aLRT values (99%/99%)  c. Likewiavirales .Lauta virus was initially identified in the pooled sequencing library comprising a mix of several Australian reptile species. To identify the exact host species, we screened each individual species sample separately using RT-PCR and Sanger sequencing. As a result, we detected the presence of the novel Lauta virus RdRp sequence in liver tissue of G. lauta (paratype QM J96622) ( J96622) , a geckoG. lauta).Advances in protein modelling and sequence analysis based on structural comparisons with well-characterized protein templates constitute an attractive approach for the identification of highly divergent RNA viruses . The RdRLauta virus based on its structural similarity with the RdRp subunit PB1 of influenza virus (family Orthomyxoviridae)  [Amnoonviridae (We obtained a confident predicted 3D model for the RdRp of viridae) . Althougvirales) , additionviridae c. In thiArticulavirales revealed that Lauta virus was most closely related to TiLV, in turn suggesting that it represents a novel and divergent (and as yet unnamed) genus within the Amnoonviridae. To date, members of the Amnoonviridae have only been detected in fish [Lauta virus expands the host range of this family. Indeed, given the huge genetic distance between TiLV and LTAV, we expect that further uncharacterized phylogenetic diversity exists in the Amnoonviridae especially in fish and reptiles, and that more studies using the form of genomic surveillance performed here will capture a far greater diversity of negative-sense RNA viruses [Although compromised by the large evolutionary distances involved, phylogenetic analysis among members of the order  in fish , such th viruses ,39.Lauta virus, broadly consistent with observations made for TiLV [Articulavirales may constitute effective molecular fingerprints for these viruses. Unfortunately, the marked lack of sequence similarity meant that we did not recover any conclusive evidence regarding the presence of other genome segments in Lauta virus. Further studies that include sequencing, microscopy, and cell culture techniques are, therefore, required to fully characterize the genome of this novel virus.Comparisons of the RdRp subunit PB1 from different articulaviruses revealed the presence of four well conserved motifs in for TiLV . As suggfor TiLV ,41. Despfor TiLV ,41. HencG. lauta) highlights the importance of virus surveillance in native species. Although Lauta virus was detected in liver tissue, we currently cannot draw any conclusions regarding its pathogenic potential and impact on the health of G. lauta, particularly since a limited number of individuals were collected and all were apparently healthy. Additional research is therefore needed to establish the type of biological interaction between Lauta virus and G. lauta. While a previous study reported the isolation of the arbovirus Charleville virus (family Rhabdoviridae) in G. australis (possibly G. dubia based on its distribution) collected in Queensland [The identification of a novel virus in an Australian gecko (eensland ,37, this


The risk of contracting rabies is low for travellers. However, the number of Dutch travellers potentially exposed abroad following an animal-associated injury and needing post-exposure prophylaxis (PEP) has increased, resulting in increased costs.Here, we evaluated the costs and the cost-effectiveness of different pre- and post-exposure interventions in the Netherlands, taking into account the 2018 World Health Organization (WHO) recommendations for the prevention of rabies.A decision tree-based economic model was constructed. We calculated and compared the cost of different WHO pre-exposure prophylaxis (PrEP) recommendations, intramuscular vs intradermal vaccination and PEP subsequent to increased vaccination coverage in risk groups. We estimated cost-effectiveness, expressed as incremental costs per rabies immunoglobulin (RIG) administration averted, using a societal perspective. Statistical uncertainty regarding number of travellers and vaccination coverage was assessed.Total costs at the national level were highest using previous WHO recommendations from 2012, estimated at EUR 15.4 million annually. Intradermal vaccinations in combination with the current recommendations led to the lowest costs, estimated at EUR 10.3 million. Higher vaccination uptake resulted in higher overall costs. The incremental costs per RIG administration averted varied from EUR 21,300-46,800.The change in rabies PrEP and PEP recommendations in 2018 reduced total costs. Strategies with increased pre-travel vaccination uptake led to fewer RIG administrations and fewer vaccinations after exposure but also to higher total costs. Although larger scale intradermal administration of rabies vaccine can reduce total costs of PrEP and can positively influence vaccination uptake, it remains a costly intervention. Rabies is a preventable infectious zoonotic disease that is responsible for annually roughly 59,000 deaths worldwide . The majAn increasing number of Dutch travellers are potentially exposed to rabies virus abroad after an animal-associated incident (AAI) and consult a doctor for post-exposure prophylaxis (PEP) either abroad or after returning home. The National Institute for Public Health and the Environment (RIVM) receives on a daily basis requests for consultations by municipal health services, general practitioners, hospital specialists and medical repatriation organisations about potential rabies exposure accidents among Dutch travellers abroad or after returning home. The number of consultations increased from 184 in 2008 to 350 in 2014 and 450 in 2018 [According to the World Health Organization (WHO) classification, an AAI with potential exposure to rabies virus is defined as a category II or category III injury. A category II injury consists of nibbling of uncovered skin, minor scratches or abrasions without bleeding, while a category III consists of transdermal bites or scratches, contamination of mucous membrane or broken skin with saliva from animal licks. Depending on the category of exposure, PEP consists of extensive wound washing, administration of a series of rabies vaccines (RV) and RIG . The RV The current WHO recommendations on PEP treatment were presented in 2018 and differed from the previous recommendations mainly on two points: Firstly, the number of needed vaccination doses was lowered by one dose for both PrEP and PEP. Secondly, the dose of RIG was based on the anatomical localisation of the affected area instead of the body weight of the individual . MaximumIn the WHO recommendations, rabies vaccinations can be administered either IM or ID. One ID dose is 0.1 mL of vaccine, leading to an immune response comparable with IM administration but at a considerably lower dose, thus saving vaccine and costs ,13. A diThe current WHO recommendations for rabies PrEP and PEP, as implemented in the Netherlands, are expected to have a positive effect on the costs of rabies prevention for the individual traveller and at national level. The ID vaccination can also contribute to cost reduction. In the Netherlands, several studies have focused on clinical outcomes or on the effect of risk behaviour of Dutch travellers on the number of AAI ,15. HoweAssessment of risk groups), both at national level and per person vaccinated before travelling. Two time points were included in the model: before travelling at the travel clinic when a risk assessment is made, and after an AAI when PEP is considered.We investigated costs of several policy measures for rabies in the Netherlands using a decision tree-based economic model programmed in MS Excel 2010. With this model, costs of different prevention strategies, including their effect on subsequent post-exposure treatment can be calculated and compared per risk group . Thirdly, we analysed the effects of a 1.5-fold increase in the uptake of PrEP in risk groups, using IM administration (two applications). We assumed that the increased vaccination uptake would happen because of revised recommendations and only within the traveller population who obtained a medical consultation at Municipal Health Services (MHS), general practitioners (GPs) or another travel clinic before their journey. No additional campaigns to increase the number of travellers requesting a medical consultation before their journey were assumed, hence no additional campaign costs for the MHS and GPs were included.Assessment of risk groups), both at national level and per vaccinated person. The benefit of higher pre-travel vaccination coverage among risk groups is the reduced use of RIG and a less extensive vaccination scheme after possible exposure. We calculated the incremental costs per (extra) vaccinated person and incremental costs per RIG application averted as an indication of the cost-effectiveness of the prevention strategy. A time horizon of 1 year was considered. The economic model was employed from a societal perspective, including healthcare costs, costs of lost holidays and an impact on evacuation and repatriation of those travellers possibly exposed, costs that would have to be covered by the patients themselves, their travel insurance and/or their health insurance.PEP with vaccinations is indicated for category II exposures, according to WHO recommendations . PEP is In 2017, a case–control study was conducted among Dutch travellers to rabies-endemic countries in which the various determinants of possible exposure to rabies virus were investigated . The folSupplement).In the Netherlands, the decision whether rabies vaccinations are indicated before and/or after an AAI and the actual administration of the vaccinations are provided by MHS, GP services, some hospital’s outpatient clinics and some commercial vaccination centres. The decision-making is based on travel destinations, travel duration, accommodations and activities during travel and on personal conditions including vaccination history. For our model, we used data from travellers consulting the MHS Hart voor Brabant and MHS Amsterdam between 1 July 2016 and 1 July 2017 (for details see Supplement). We extrapolated the mean number of RIG administrations abroad (n = 64) in the ECA database between 2016 and 2018 to all travellers needing RIG abroad, taking into account the market share of ECA . For incidents where RIG was not indicated, registration data from all Dutch MHS were used. ECA also provided the number of incidents requiring only two vaccinations abroad (for travellers with PrEP). This figure was also extrapolated to the Netherlands. To prevent double counting, travellers from the ECA registry needing PEP who started their regimen abroad with a full series of four vaccinations but further completed their vaccinations at MHS in the Netherlands were not taken into account.The estimated annual number of people who presented to a healthcare provider because of an AAI while travelling is provided in www.cbs.nl). Detailed information about costs is provided in the Supplement.Estimated costs were determined for both PrEP and PEP courses, including costs for the vaccine and RIG, administration costs, management costs for handling PEP, costs of lost holidays and costs involved in moving the involved subjects from one facility to another in case the required treatment needed was not available . For theWe calculated the incremental cost-effectiveness ratio (ICER) per (extra) person vaccinated before travelling and the ICER per RIG administration averted as an indication of the cost-effectiveness of the prevention strategy. That means that we divided the difference in total costs by the difference in persons vaccinated before travelling and we divided the difference in total costs by the difference in RIG administrations.Statistical uncertainty with respect to the number of travellers to certain regions and to vaccination coverage estimates was considered using the Monte Carlo simulation technique and the add-in software Palisade @Risk 7.5 in which we sampled 10,000 random values from input distributions. All other variables were considered fixed by using average resource utilisation and fixed unit cost prices. Results are presented as means with 95% uncertainty intervals.Table S3 in the Supplement. The largest risk group were people younger than 35 years, followed by people travelling to south-eastern and western Asia. In all risk groups, a higher vaccination uptake led to higher overall costs, despite a decline in management and vaccination costs of possible rabies exposure incidents, and avoided annually between 4 and 60 cases requiring RIG. The costs per averted RIG administration varied from EUR 21,300 to EUR 46,800. The ICER per additional person vaccinated before travelling varied between EUR 98 and EUR 105 per additional vaccinated person. This is also illustrated in Supplementary Figures S1 and S2. Results of increasing the vaccination coverage with 150% in the identified risk groups are presented in This cost evaluation showed that the current WHO rabies PrEP and PEP recommendations, as implemented in 2018 in the Netherlands, were associated with a substantial reduction (16%) in societal costs. An additional cost reduction (33%) could be achieved by offering ID vaccination on a large(r) scale. Increased use of ID administration requires proper planning of the number of travellers who need a rabies vaccination in order to avoid wasting vaccine. Therefore, ID administration would not lead to vaccine savings if there are only a few cases. A hypothetical increased vaccination uptake in the defined risk groups would lead to higher overall costs, despite the smaller number of RIG administrations and a reduced number of vaccinations after an AAI. Even though the costs of RIG administration according to the new recommendations were lower , total costs were higher because of higher costs of PrEP when assuming a higher number of travellers vaccinated before travelling. As the incidence of AAI in travellers to endemic countries is low, increasing the vaccination uptake is a costly intervention with an ICER varying from EUR 21,300 to EUR 46,800 per avoided RIG administration. These results are not only relevant for the Netherlands but also for other European countries with a similar healthcare system. The risks of contracting rabies are comparable for travellers from other countries and the costs of preventing rabies will also be similar.Our results are in line with a previous modelling study by Hampson et al. . In thatThis study has several strengths and limitations. One of the strong points was the use of model input data from different MPH and a recently conducted case–control study. The first limitation concerns the period of data collection, during which the old vaccination recommendations still applied. It is plausible that the vaccination uptake will go up as the number of necessary doses and consequently the costs and efforts for the traveller decrease. Secondly, because of data restrictions, the model contained a time horizon of only 1 year while vaccination offers lifelong immunological memory, thereby underestimating the benefits of improved vaccination coverage, which accumulates with every next trip to a rabies-endemic region. At present, decisions to vaccinate before travelling are typically made on the basis of an individual risk–benefit assessment and individual willingness to pay for the vaccine . If futuPrEP is indicated for individuals who face occupational and/or travel-related exposures to rabies virus in specific settings or over an extended period of time . Many trOther preventive measures to be taken in endemic countries such as One Health approaches have proven (cost-)effective in controlling rabies in different areas of the world ,25. For There is some evidence that vaccination schemes can be further simplified, as Jonker and Visser found promising results that vaccinating with a single dose was already sufficient to induce an adequate anamnestic antibody response in all subjects, at least for 1 year . However


Species taxa are the units of taxonomy most suited to measure virus diversity, and they account for more than 70% of all virus taxa. Yet, as evidenced by the content of GenBank entries and illustrated by the recent literature on SARS-CoV-2, they are the most neglected taxa of virus research. To correct this disparity, we propose to make species taxa a first choice for communicating virus taxonomy in publications concerning viruses. We see it as a key step toward promoting research on diverse viruses, including pathogens, at this fundamental level of biology. Viruses were discovered as miniscule agents causing infectious diseases –3, and tSpecies are the units of evolution, and surveys of the corresponding taxa are used as a census of the abundance of natural biodiversity . Yet virThis can be illustrated by 2 examples. First, in GenBank, which is a popular entry point for everybody interested in biological information , the genSevere acute respiratory syndrome-related virus by the Coronaviridae Study Group (CSG) [Nature Microbiology were commented on by diverse media, including Nature and Science. Indeed, the name SARS-CoV-2 has been broadly accepted by the international community. Nevertheless, the readers of the SARS-CoV-2 literature will be hard-pressed to find a few mentions of the cognate species taxon of this virus (Severe acute respiratory syndrome-related virus) in these publications, regardless of whether or not the original CSG paper is acknowledged.The second example we present is the literature relating to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease 2019 (COVID-19) pandemic –23. Althup (CSG) . The papCoronaviridae, the genus Betacoronavirus or the subgenus Sarbecovirus. This disparity is especially notable since all of the aforementioned taxa were delineated and named at very different times. For example, the increasingly popular subgenus Sarbecovirus was first recognized only in 2018 [Severe acute respiratory syndrome-related virus [Fig 1), this example reveals a disconnect between what virologists consider to be the most informative ranks of virus taxonomy  and what taxonomists think of as the cornerstone of virus taxonomy (the species rank). Essentially, it means that the development of virology proceeds largely without the benefit of the species concept, which is at the foundation of modern biology.One might argue that everybody knows where SARS-CoV-2 belongs, making an explicit reference to its multirank hierarchical taxonomy excessive in papers produced at the time when practical matters of containing the pandemic are the absolute priority. Indeed, many researchers chose not to refer to the coronavirus taxonomy in their publications. However, if any mention of taxonomy is made, the reference is typically to the family  in 2018 , more thed virus . Given tHuman immunodeficiency virus 1 and Human immunodeficiency virus 2, respectively. This is incorrect. The acronyms refer to the viruses, human immunodeficiency virus type 1 and human immunodeficiency virus type 2, and not to the species [Hepacivirus C to address this problem [So why do virologists not mention virus species in their work? There are probably many reasons, including (1) a reluctance to accept viruses as biological entities, despite extensive evidence to the contrary; (2) a predominant focus of virus research and its applications on circulating (often pathogenic) viruses that are typically a small subset of the species taxon, although they may be sufficiently divergent to be mistaken for a “species,” tacitly or explicitly; (3) a tradition in virology for citing taxa at the family and genus ranks, mainly because these ranks were established first ; and (4) species . A simil problem . It is t problem , and the problem , may conCompared to the taxonomies of other biological entities, the development of virus taxonomy is uniquely supervised by a governing body, the International Committee on Taxonomy of Viruses (ICTV) . This syThis connection of taxonomy to virus research may not always be apparent, but it is easily seen if one looks at how the taxonomy of distinct virus families is developed in practice. This responsibility rests mainly with experts on the viruses belonging to different families, who join together to form specialized Study Groups (SG), such as the CSG mentioned above. There are currently more than 100 SGs, and the number is likely to increase dramatically in the next few years. Taxonomic proposals may be initiated by any researcher, but it is generally the SGs that delineate taxa using the methods and demarcation criteria believed, at the time, to be best for partitioning the natural diversity of the respective virus groups. This modular structure also allows for flexibility as the SGs are largely independent of each other and develop their remit as they see fit. These SGs are the main conduits to connect the ICTV to all who are concerned with viruses.Enterovirus C [We believe that to move the field forwards, the development of virus taxonomy has to be translated into research focusing on virus taxa, especially at the species level; a research area that is chronically underfunded, and how could it be otherwise? We are all aware of the chicken and egg mentality that bases research funding on prior research and creates a self-propagating circle of ignorance. What is needed is research that, for instance, includes the experimental verification of species by systematically probing the genetic compatibility of viruses at intra- and interspecies levels, as has been initiated for polioviruses and other viruses of the species ovirus C ,34. Anotovirus C . Only inhttps://ictv.global/).We call on all virologists, whether or not they are interested in taxonomy, to reflect on the importance of species taxa in their research and take the lead in referring to them in their publications and other communications. We hope that this will encourage others including, for example, GenBank to follow. Moreover, there should be more awareness among the editors of journals and science communications, as well as administrators of regulatory bodies, regarding the proper and consistent communication of virus species. In this regard, the ICTV has an important role to play in providing accurate and up-to-date information, which can be found in the current Master Species List and Virus Metadata Resource at the ICTV website   and biom


In finishing beef cattle farms, the limited space allowance and the fully slatted floor system could negatively affect animals’ performance, health and welfare. A larger individual space was provided as a strategy to mitigate lameness problems in a commercial farm. A larger space allowance may contain the spread of pathogens, leading to a reduction in the incidence of lameness and infectious diseases, and may decrease the risk of animals stepping into each other causing claw and leg injuries. Increasing the space allowance did not affect animal performance, whereas it had a positive implication through a significant reduction in the number of treatments of lame and sick animals.2, respectively. Bulls were fitted with collars that measured rumination time and activity. Three 8-h observational sessions were conducted to record behaviors. Data about health conditions were collected daily. No differences were found in the animals’ performance. However, performance results might have been impaired by the culling rate experienced during the trial, which prevented from keeping a consistent SKD. Behaviors did not differ between groups, except for rumination time, which was higher for LD bulls during the third observation (p < 0.05). However, rumination time, recorded by collars, did not vary among treatments. There were no differences in the percentage of sick or lame bulls, but the percentage of animals treated repeatedly due to relapse was higher for the HD group (p < 0.05). It was concluded that a larger space allowance could improve the health of bulls kept on fully slatted floors.This study aimed at assessing whether a reduction in stocking density (SKD) would mitigate lameness and positively affect the performance and health of Charolais bulls in an Italian commercial farm. Bulls were distributed in groups of 10 or 8 animals/pen for high (HD) or low density (LD) corresponding to an individual space of 3.5 or 4.7 m However, there are no current European regulations that contextualize standards regarding space allowance for fattening bulls. Beef cattle production is particularly relevant in Italy, which is the fourth main contributor for European production . The mai2 per bull weighing 250–500 kg was found to reduce ADG up to 300 g, owing to the drop in feed intake. A larger space allowance may lead to a higher average daily gain (ADG) and feed conversion ratio (FCR). In fact, stress affects gonads, resulting in lower hormonal secretion, which may affect performance . MoreoveGenetic selection over the years has focused on hypermuscularity traits which, at the same time, might exert a negative effect on immunological responsiveness, making bulls more vulnerable to stressors and pathogens ,13. FurtWe hypothesized that a larger space allowance could reduce the prevalence of lame bulls, improve health and positively affect animal performance in commercial farms characterized by a high incidence of lameness. This condition may be obtained by decreasing the number of animals raised per pen. Thus, the aim of the study was to test the effect of a reduction in stocking density on Charolais finishing beef cattle kept on fully slatted floor in a commercial farm with a high prevalence of lameness, in the perspective of reducing claw and leg problems and improving animal health and production efficiency.Treponema species. According to the farm biosecurity procedure set by the farm veterinarian, each pen was sanitized before the arrival of the successive occupants with the use of limestone and left idle for a short period of time. The study involved a group of Charolais bulls , which were purchased from the same cattle collection center in France and selected to be homogenous in weight (424 ± 30 kg), age (466 ± 55 days) and physical conformation. The minimum sample size for each group was reckoned taking into account a power of the test of 90%, a standard deviation of the ADG of 0.2 kg/d and a significant difference in ADG of 0.133 kg/d.The trial was carried out in a commercial beef cattle farm in the Rovigo province, in northeastern Italy, from July to October 2018, not applying any procedure that goes beyond the regular commercial production practice, as approved by OPBA . The farm happened to have a high prevalence (on average 15%) of lame bulls during the finishing phase in the previous years. Lameness was caused, among other factors, by digital dermatitis due to After spending 60 days for their backgrounding and transition period in a loose housing facility with straw bedding, the animals were moved into 12 contiguous, concrete, fully slatted pens (5 m wide and 7 m deep) in a roofed facility with full walls on three sides, for their growing (70 d) and finishing (61 d) phases.2/animal for the high-density group (HD) and 4.37 m2/animal for the low-density group (LD), respectively. The HD condition was the one routinely adopted by the farmer, whereas the LD condition was the one suggested by the research group as a compromise between animals’ and farmer’s needs. The stocking density within the pens changed over time because of severe culling. Therefore, to analyze the effect of density, we kept maintaining the LD category for pens where the average number of animals per day, within each period, was equal or lower than 8.0, whereas this value was higher in the HD category . The two levels of stocking density were 10 and 8 animals per pen corresponding to 3.50 mcategory . The aveBulls were fed the same total mixed ration (TMR). The ingredients and chemical composition of the diet are reported in To meet animals’ requirements, diet adjustments were made when bulls were moved from the growing phase (P1) into the finishing phase (P2). The day before the beginning of the trial, bulls were fitted with SCR collars  to measure their daily activity and rumination.Overall, the trial lasted 131 days. All bulls were weighed in the morning before feed distribution at the outset (day 1), at day 70, between the growing and finishing phases, and at the end of the trial, to calculate individual ADG. The DMI was reckoned for each pen by the difference between the amounts of TMR given in the morning (T0) and its residuals after 24 h. Pen feed conversion rate (FCR) was calculated by dividing DMI by ADG. For both diets, TMR and 24-h leftover samples were collected each week, whereas 2 fecal samples per pen were gathered once per period. The particle length distribution of TMR samples was obtained by using the updated version of the Penn State Particle Separator  that is Total tract digestibility was estimated using lignin as an internal marker as described by McDonald et al.  and SchäBehavioral observations were carried out three times during the experimental period, in 8-h sessions starting right after feed distribution at 09:30. Animals were observed by four trained assessors standing outside the pens in the feeding alley. Each assessor observed animals in 6 contiguous pens for 2 h, alternatively monitoring 3 HD and 3 LD pens . To reduce bias due to the observer effect, observers rotated every 2 h and changed position at each 2-h interval. Behavioral assessments of postures (standing/lying) and continuous different activities  of bulls in each pen were performed using the scan sampling technique  with a 5® assessment protocol for cattle [The individual health condition of the bulls was checked daily by the stock people as required by the legislation in force on farm animal protection and weekly by an experienced veterinarian throughout the trial. The animals were visually inspected from the feeding alley and the occurrence of any symptom was recorded. Health check recordings were performed according to the Welfare Qualityr cattle  and specr cattle  as an alTo measure the rumination time and the level of activity of individual animals, SCR collars were used. These collars are equipped with an internal accelerometer which records the movements of the head and sends the data to a receiver connected to a computer at 2-h intervals. The computer software  differentiates the type of movements and reckons the rumination time and the level of activity, ranging from 0 to 253 bits (binary digit), per interval . It giveA statistical analysis was carried out using the SAS software . All data were first tested for normal distribution using the Shapiro–Wilk test . Data on diet particle length distribution were submitted to an ANOVA model using period as the fixed effect. Data on DMI, ADG, FCR, digestibility, rumination and activity were analyzed through a mixed ANOVA model using the pen within stocking density as the random effect and period, stocking density and their interaction as fixed effects. Bonferroni correction was run for post-hoc pairwise comparisons between factor levels. Assumptions of the linear model on the residuals were graphically tested.Data on the number of treatments per animal were submitted to the non-parametric Kruskal–Wallis test to evaluate the effects of stocking density, whereas data on the number of animals treated or culled, expressed as percentages, were analyzed through a Z test and the culling rate was analyzed through an Fisher’s exact test.Behavioral data gathered using the scan sampling technique were transformed into relative frequencies of events per pen per hour and were processed using a mixed model that considered the main effects of observation session day, stocking density and their interaction using the Bonferroni adjustment. Data regarding events were transformed into number of events per bull in the 8-h observation session and submitted to non-parametric statistics using the Kruskal–Wallis test.As previously anticipated, SKD changed throughout the trial, as reported in Diet ingredients and chemical composition are listed in p = 0.011) and lame animal (p = 0.032) in LD pens.During the experiment, 35 animals needed to be treated for different kinds of problems. Specifically, 26 animals suffered from lameness, 4 from respiratory affections, 2 from leg lesions, 2 from urogenital problems and 1 from diarrhea. Once treated and recovered, 11 out of the 26 lame bulls and 1 out of the 4 bulls which had suffered from respiratory problems experienced a relapse, needing a second pharmacological treatment. Overall, 16 bulls, of which 4 belonged to LD and 12 to HD, were culled early due to severe lameness. The effects of SKD on health status are described in p < 0.001) content of medium (8–19 mm) particles and a lower (p < 0.001) content of short (4–8 mm) particles.The particle length distribution is reported in p < 0.001) and DMI (p < 0.001) and lower digestibility of both DM (p = 0.014) and NDF (p = 0.003). Since the interaction period × stocking density was not significant, it was not reported in the table.As reported in p = 0.015), whereas daily rumination, DA and DR did not differ between different diets and SKD. With regard to the effect of period, significance was approached for daily rumination, DA and DR, which were all higher in P1 than in P2 (p < 0.001). As for previously reported data, the interaction of period × SKD did not approach significance, thus it was omitted.Results obtained from SCR collar recordings of rumination and activity are shown in p = 0.024) was affected. No differences in social interactions like mounting, chasing and head/butt displacement were found between the two SKDs.Regarding the effect of the interaction between SKD and observation session, reported in Overall, in this trial, although the stocking density did not significantly affect the prevalence of sick or lame animals, it led to a significant change in the number of treatments necessary to an animal to fully recover from lameness or other diseases. Looking at the average number of treatments per sick or lame animal, it can be appreciated that, among the debilitated animals, bulls raised in HD conditions required a higher number of pharmacological interventions to recover or that they experienced more relapses. Indeed, a smaller space allowance favors contacts between animals and pathogen transmission and increases animal stress, thus weakening the immune system . Based oAs regards the TMR, the distribution of particle length was partitioned similarly to what was reported in previous studies ,28, wher2 in a study involving 237 steers of 450 kg of weight. The lack of association between average daily gain and space allowance found in our experiment could partially be explained by the culling rate we recorded throughout the trial. As a matter of fact, animals started with 3.5 and 4.37 m2 of space allowance for HD and LD, respectively. Nevertheless, they ended up having on average 3.68 m2 in HD and 4.42 m2 in LD during P1, and 4.07 m2 in HD and 4.62 m2 in LD during P2, and this might be seen as the main shortcoming of our study. It implied that the difference between the two levels of space allowance passed from 0.87 m2 per head at the beginning of the trial to 0.55 m2 in the second period, which likely was not great enough to affect ADG.Against our expectation, stocking density did not affect bulls’ performance. We expected that animals raised with a larger space allowance would have an increase in ADG. In fact, previous studies have shown that animals with a small space allowance have a shorter lying time which leads to a reduced daily gain . MarquisAs animals grow, DMI increases proportionally to their metabolic body weight ; thus, DWith respect to SCR collars recordings, activity recorded for LD bulls was higher compared with HD bulls, meaning that when bulls had more space, they were more likely to move. From P1 to P2, we observed a decrease in daily rumination (min) and DR, which meant that there was less variation in rumination between one day and another in the second part of the experiment . This wa2, 2.0 m2, 2.5 m2 and 3 m2, they recorded a reduced level of rumination for heifers housed in the 1.5 m2 space allowance. They attributed the result to a lower lying times of these animals. However, we did not record any changes for the latter. Thus, these different conclusions may be related to variations in the study designs other than space area like the different sex and breed. Moreover, if we look at rumination data recorded by SCR collars, no difference among groups can be appreciated. The reason for the difference between groups noted at the third observation session could be linked to a possible compensation that occurred during the rest of the day. In fact, collars recorded rumination time on a 24-h basis, and any fluctuation during the 8-h observational session might have been compensated in the nighttime.The only behavior affected by treatment was ruminating activity which, during the third observation, was higher for LD compared with HD. This was in line with what had been found by Fisher et al. ; in an eIncreasing space allowance improved animals’ health status, decreasing the need of further treatments due to relapses for sick and lame bulls. However, it was of no benefit to bulls’ performance, but it must be considered that the planned space allowance could not be maintained, due to the culling rate experienced throughout the trial. Despite the fact that we cannot draw robust inferences, both due to the changes in stocking density and the limited number of animals, results of this study could spur beef cattle farmers to increase space allowance to improve animal health status and welfare, in high lameness prevalence conditions in particular. Further research is needed to address the importance of increasing space allowance and investigate which is the adequate space allowance for Charolais bulls housed in indoor facilities.