Pestivirus A  and B species , members of the Pestivirus H are mainly cattle pathogens. Viruses belonging to the Pestivirus H group are known as HoBi-like pestiviruses (HoBiPev). Genetic and antigenic characterization suggest that HoBiPev are the most divergent pestiviruses identified in cattle to date. The phylogenetic analysis of HoBiPev results in at least five subgroups (a–e). Under natural or experimental conditions, calves infected with HoBiPev strains typically display mild upper respiratory signs, including nasal discharge and cough. Although BVDV1 and BVDV2 are widely distributed and reported in many South American countries, reports of HoBiPev in South America are mostly restricted to Brazil. Despite the endemicity and high prevalence of HoBiPev in Brazil, only HoBiPev-a was identified to date in Brazil. Unquestionably, HoBiPev strains in BVDV vaccine formulations are required to help curb HoBiPev spread in endemic regions. The current situation in Brazil, where at this point only HoBiPev-a seems present, provides a more significant opportunity to control these viruses with the use of a vaccine with a single HoBiPev subtype. Despite the lack of differentiation among bovine pestiviruses by current BVDV tests, the reduced genetic variability of HoBiPev in Brazil may allow reliable identification of cases within the region. On the other hand, introducing foreign ruminants, biologicals, and genetic material to South America, especially if it originated from other HoBiPev-endemic countries, should consider the risk of introducing divergent HoBiPev subtypes.Along with viruses in the  Flaviviridae underwent extensive taxonomic revisions in the past few years, leading to many of its members' classification or reclassification. For decades, only four viral species were officially recognized and historically known as Bovine Viral Diarrhea Virus 1 and 2 (BVDV1 and BVDV2), Classical Swine Fever Virus, and Border Disease Virus. These species are now classified as Pestivirus A, B, C, and D. Several pestiviruses described as “atypical” are currently officially recognized at the species level  and BVDV2 (Pestivirus B) demonstrated that HoBiPev was the most divergent ruminant pestivirus identified to date  upper respiratory signs , 35–37. More prominent disease cases involving HoBiPev were reported in Italy and China, including severe respiratory signs and diarrhea , 8. The A key ruminant pestivirus characteristic is its tropism for fetal tissues and the establishment of persistent infection . Such peSpecific control measures for HoBiPev, including commercial vaccines, are unavailable. Due to the antigenic similarities that HoBiPev shares with BVDV1 and BVDV2, a study evaluating the potential cross-protection was conducted. Virus neutralization assays with HoBiPev-a, BVDV1-b, BVDV2-a, and border disease virus were performed using the serum of cattle immunized by either a MLV or killed vaccine containing both BVDV species . It was in vitro and in vivo, clearly demonstrated that robust immunity to either BVDV1 or 2 would have limited effect controlling the spread of HoBiPev in the event of introduction into a naïve population.Subsequently, we demonstrated limited fetal protection to HoBiPev in cows that generated either a BVDV1 or BVDV2 PI in a previous gestation . HoBiPevThe antigenic variability among isolates belonging to the same pestivirus subtypes is described . Not sur0.6 TCID50/ml and 100.3 TCID50/ml, while for VetMAX-Gold were 100.6 TCID50/ml and 102.3 TCID50/ml. However, virotype sensitivity to HoBiPev-c and -d was decreased compared to HoBiPev-a detection level  and VetMAX-Gold bovine virus diarrhea RNA test kit , designed for BVDV, demonstrated suitability in detecting positive samples from experimentally generated HoBiPev PI calves . The calon level .4.6 TCID50/ml and 106.3 TCID50/ml , which is based on detecting the Erns protein, mostly excels in detecting samples with HoBiPev-a . AlthougCID50/ml . Other ECID50/ml , 51.Currently, there is no nationwide eradication program for BVDV in South America, and the BVDV control is entirely voluntary at the farm or regional level. In addition, vaccination use may be restricted due to its costs, and testing may be limited to commercial ELISA kits that do not identify the specific pestivirus strain. All these factors hamper a comprehensive understanding of the true epidemiology of ruminant pestiviruses in the region. Despite these challenges, many countries in South America have reported BVDV types 1 and 2 and multiple subtypes within those viruses , 52–59. Considering that Brazil borders 10 countries or territories in South America, it is likely that either HoBiPev has disseminated to additional regions or there is an imminent risk of its spread. Despite the high prevalence of HoBiPev in cattle herds in Brazil, with strong evidence for it being the most prevalent ruminant pestivirus in at least the north-eastern region, the description of infection in other species in South America is restricted to water buffalos . DespiteIndependently of the HoBiPev subtype, most acute infections are clinically indistinguishable from typical, uncomplicated BVDV1 and BVDV2 infections. The descriptions of MD-like disease associated with HoBiPev infections in different parts of the world suggest that highly fatal forms of HoBiPev infection do exist. Typically, most of the PI animals die at an early age, usually within the first 6 months of life . The desin vivo and in vitro studies suggested low to no cross-protection. In the long-term, countries in South America or Asia using BVDV vaccines may provide a favorable scenario for HoBiPev becoming the most common ruminant pestiviruses in additional regions.The genetic and antigenic diversity of pestiviruses is also a well-known nemesis of vaccine design. Whereas, the benefits of using vaccines with partial protection are debatable from the standpoint of accelerating virus divergence, it may help decrease the emergence of PI animals . Based oIt is untested whether HoBiPev strains in BVDV vaccine formulations are required to help curb HoBiPev spread. The situation in Brazil and Argentina, where only HoBiPev-a was identified, provides a better opportunity to control these viruses with the addition of a single subtype in the vaccine compared to the other regions in the world.Despite the critical need for specific HoBiPev diagnostic, no commercial test is available. Pestivirus-free regions and BVDV endemic regions will certainly benefit from commercially available kits with the capacity to differentiate BVDV from HoBiPev. The discovery of divergent HoBiPev subtypes also questions the efficacy of in-house tests previously designed for HoBiPev-a detection , 51, 61.FB and JR conceived and wrote the article. All authors contributed to the article and approved the submitted version.JR was employed by the company Ridpath Consulting, LLC. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Viral infections are the leading cause of childhood acute febrile illnesses motivating consultation in sub-Saharan Africa. The majority of causal viruses are never identified in low-resource clinical settings as such testing is either not part of routine screening or available diagnostic tools have limited ability to detect new/unexpected viral variants. An in-depth exploration of the blood virome is therefore necessary to clarify the potential viral origin of fever in children. Metagenomic next-generation sequencing is a powerful tool for such broad investigations, allowing the detection of RNA and DNA viral genomes. Here, we describe the blood virome of 816 febrile children (<5 years) presenting at outpatient departments in Dar es Salaam over one-year. We show that half of the patients (394/816) had at least one detected virus recognized as causes of human infection/disease , 12% rotaviruses, 11% human herpesvirus type 6). Additionally, we report the detection of a large number of viruses  not yet known to cause human infection/disease, highlighting those who should be on the radar, deserve specific attention in the febrile paediatric population and, more broadly, for surveillance of emerging pathogens.Trial registration:ClinicalTrials.gov identifier: NCT02225769. Humans are infected by a large number of eukaryotic DNA and RNA viruses that, together with prokaryotic phages and endogenous viral elements, comprise what we define as the human virome. Eukaryotic viruses are a diverse group of DNA and RNA viral agents replicating in a variety of human cells and tissues and engaging in a vast permutation of interactions with the human immune system. Consequently, the presentation of human viral disease depends on both viral and host factors, where a virus may only be pathological in a certain situation (e.g. following immunosuppression), whilst being considered  as commensal or merely an incidental finding in a “healthy” situation.The composition of the human virome does not only vary between these populations but it also changes over time within individuals according to their personal history of exposure and immunity and thus, their geographic location, access to vaccination and their genetic make-up. Metagenomic next-generation sequencing (mNGS) has the ability to detect any viral RNA or DNA sequence in a given sample and thus, it represents the most appropriate tool with which to explore the human virome .It is recognized that we have only uncovered a fraction of the so-called virosphere , 3. WhilThe human virome has been described in stool, skin, nasopharyngeal swabs or breast milk . Investide novo assembly. We report the full diversity of their blood virome, including novel viruses and those unexpected or not previously reported in humans that deserve specific attention.Infections and fever in sub-Saharan Africa represent the most frequent motivation for paediatric outpatient consultation . The majThe serum samples tested by mNGS come from a larger cohort of 3192 consecutively paediatric patients (2–59 months of age), recruited between December 2014 and February 2016, at nine outpatient clinics in Dar es Salaam, Tanzania, with the inclusion criteria of “acute febrile illness” (axillary temperature ≥ 37.5°C). The detailed characteristics of this cohort are described in Keitel et al [A sub-cohort of interest comprising 1233 patients was then selected  based onA total of 823 patients (resulting in 823 sera samples) were then randomized from this sub-cohort of interest and submitted retrospectively to the entire process of mNGS (detailed below). Only samples with a minimum of one million reads for both RNA and DNA libraries were considered for further bioinformatic analysis, resulting in the exclusion of 4 samples. Three samples were further excluded; two consecutive to a technical problem encountered during the library procedures, and one due to an overrepresentation of insect sequences after analysis of raw sequencing data . TherefoThis study was approved by the local IRB in Tanzania by the Ifakara Health Institute and the National Institute for Medical research (paediatric cohort: IHI/IRB/EXT/16-2015 and NIMR/HQ/R.8a/Vol. IX/1789). IRB approval was also granted for analyses undertaken in Switzerland by the Ethikkommission Nordwest- und Zentralschweiz (EKNZ UBE-15/03). Written informed consent was obtained from the parent or guardian of the patients before enrolment. The trial was registered in ClinicalTrials.gov, identifier NCT02225769.g for 10 min. Then, 200 ul are transferred in a new collection tube and treated with 40 U of Turbo DNAse (2U/ul) + 24 ul of 10X DNAse Buffer . Of the total resulting 244, 240 μl were recovered to perform the nucleic acid extraction. Two nucleic acid extraction procedures were then carried out separately for RNA and DNA virus genome extraction  as previously described [Two hundred twenty microliters (ul) of each serum sample was centrifuged 10,000× escribed . BrieflyFor RNA virus detection (RNA protocol), ribosomal RNA was removed using the Ribo-Zero Gold depletion kit  prior to library preparation. RNA libraries were prepared with the TruSeq total RNA preparation protocol (Illumina) before being multiplexed by four on the HiSeq 2500 platform (Illumina) using the 2 × 100 bp paired-end protocol. The mean of total number of read pairs obtained was 4.45E7 (range from 2E6 to 1.09E8).For DNA virus detection (DNA protocol), libraries were prepared with the Illumina Nextera XT protocol (12 PCR cycles) before being multiplexed by six on the HiSeq 4000 platform (Illumina) using the 2 × 100 bp paired-end protocol. The mean of total number of read pairs obtained was 5.46E7 (range from 2E6 to 2.52E8).Both RNA and DNA library concentrations were measured using Qubit (Life Technologies). The size distribution of fragments was controlled using a 2200 TapeStation .To assess the presence of potential mNGS contaminants , a “no-template” control (NTC) submitted to the whole procedure was included to each sequencing run. In order to assess the entire process efficiency  each sequencing run included a virus-spiked positive control (PC). This latter is also useful in addition to the NTC samples to evaluate the presence of potential sporadic mNGS contaminants. For the RNA virus procedure, the PC consists of a canine distemper virus (CDV)-spiked positive control (RNA-PC), whereas for the DNA virus procedure, a baculovirus  harboring 793 nucleotides of the CDV fusion gene (corresponding to positions 5505 to 6297 from the GenBank KY971529 reference) was used as spiked-positive controls (DNA-PC).FeVir) designed to detect all vertebrate viruses and a de novo assembly sequencing approach.For each sample, the reads generated by the Illumina platforms (RNA and DNA libraries) were processed for virus detection using a bioinformatics pipeline  , a curatIndex hopping/lane cross-contamination was removed using a 1% threshold. An important challenge in the interpretation of mNGS data is to distinguish clinically significant viruses from those that are known to be medically irrelevant and also to differentiate novel viruses (not previously described in humans) from those that are more likely to be environmental or mNGS reagent contaminants . TherefoHerpesviridae, each gene’s results were summed using a non-redundant and non-overlapping list of genes.For segmented viruses, results for each segment were summed. For Those of “recognized clinical significance” including viruses known to infect humans and well recognized in clinical practice as disease-causing agents. As for most pathogens the spectrum of associated illness associated with this group of viruses ranges from mild symptoms (or even asymptomatic infection) to life-threatening disease.Anelloviridae and human pegivirus-1.Commensal viruses of “undetermined clinical significance” that comprise viruses that infect human but for which the association with a specific disease has not yet been established. Most of these viruses however have shown to interact with the human immune response and have been referred to by some as commensal agents. The two main representatives are Those of “unknown significance” that represent viral agents not usually recognized as infecting humans and/or have never fulfilled Koch’s postulates that would attribute a theorical pathogenic role . Thus, iViral hits were classified into 3 groups:de novo analysis was performed as previously published [de novo assembly was performed using IDBA-UD (v.1.1.3) [Anelloviridae contigs were filtered out and processed separately. The newly obtained sequences were then cross-checked by BLAST  against GenBank (Nucleotid collection (nr/nt), Non-redundant protein sequences (nr)) and NCBI conserved domain tool. Novel virus contigs with a presumed bacterial or fungi host were not considered for further analyses.The ublished . First, ublished . Then, dv.1.1.3)  and the v.1.1.3)  against v.1.1.3) . AnellovFeVir pipeline, using SNAP. For each new hit, mapped reads and coverage metrics were then computed. Only results with ≥300 nucleotides of coverage were considered. Results for viruses detected in processes controls were not reported.Sample and control FASTQ files were additionally filtered for low-complexity sequences, using tagdust (v2.31). Remaining reads were mapped against assembled genomes that were not already detected by the previously described Anelloviridae contigs with a complete ORF1 larger than 600 amino acids were separated into Alpha-, Beta- and Gamma-torquevirus genera using multiple alignment (muscle v3.8.31) with the reference sequences of ORF1. Alternative “ACG” initiation codon was also used to detect Alphatorquevirus ORF1.To estimate the proportion of assembled contigs that was “new” compared to reported sequences in GenBank , we first excluded non-primate sequences, then filtered as above and added to the new assembled sequences. For the three genera, complete ORF-1 sequences were separated in clusters of 80% nucleotide identity, using cdhit-est (CD-HIT v4.6). For each cluster, sequences of 90% identity were selected using cdhit-est. Then representative sequences were aligned two by two using muscle (v3.8.1551), when two clusters contain reference sequences that shared more than 80% nucleotide identity, clusters were merged.Anelloviridae of the same genus present in each sample, the filtered FASTQ files were mapped to the ORF1 representative sequence using SNAP, for each cluster, positive detection was considered if 50% or more of the ORF1 was covered.To determine the number of distinct Anelloviridae contigs that contained a complete ORF1, circular genomes were linearized starting after the GC rich region and incomplete genomes were trimmed the same way. Finally, representative genomes were selected at 90% nucleotide identity with cdhit-est. Then filtered FASTQ files were mapped against the representative genomes with SNAP. Metrics were computed and results were processed as described above . Results with the best total genome coverage value for each genus were reported if previously found negative with the FeVir pipeline.Additionally, for each of the assembled https://www.unige.ch/virology/en/krona/); those known to cause disease in humans and of “recognized clinical significance” ), those known to be present in humans and of “undetermined clinical significance” ) and a third group of viruses not usually described in humans and of “unknown significance” ).In this large population of febrile paediatric outpatients recruited in East Africa, we report the detection of a large diversity of RNA and DNA virus sequences that we classify into 3 groups   and rotaviruses  ).Of the 816 children, 394 (48.3%) harboured RNA and/or DNA sequences specific for a virus recognized as causing human diseases (Supplementary Table 1). This group was dominated by RNA viruses, predominantly from members of the Picornaviridae family were detected, including EV-A, B and C , rhinovirus (RV)-A and C , hepatovirus A (n = 10) and parechovirus A (n = 9)  whereas the novel HAstV (i.e. HAstV MLB/VA) were observed in 17 cases . We also identified Norwalk virus (n = 12), human immunodeficiency virus 1 (n = 7), Sapporo virus (n = 4), human coronavirus OC43 (n = 2), human parainfluenza virus type 4a (n = 1) and the Wistar RA 27/3 rubella vaccine strain in one case. Finally, only one patient had detectable sequences of dengue virus (serotype 2), which corroborates clinical diagnoses and on-site rapid testing, that the study did not occur during a dengue virus outbreak.A total of 98/816 (12%) samples had detectable rotavirus sequences, and only one corresponded to the vaccine G1P [Betaherpesvirinae subfamily followed by mastadenovirus species. Notably, a significant proportion of these agents were also recovered at the RNA level (i.e. RNA protocol).Concerning the DNA viruses, by far the most frequent ones were members of the Betaherpesvirinae ), human herpesvirus type 6 (HHV-6) was the most frequent , followed by human herpesvirus type 7 (HHV-7) (n = 36) and cytomegalovirus (CMV) (n = 15) (n = 4), herpes simplex type 1 virus (n = 1), and human herpesvirus 8 (n = 1). Among all HHV-6, HHV-7 and CMV positive samples listed above, a large proportion  were detected in both DNA and RNA libraries suggesting the presence of ongoing genome replication. Of note, 7 HHV-6, 2 HHV-7 and 5 CMV positive cases were detected in the RNA libraries only, which means that, overall, 81.1% of HHV-6 (n = 73/90), 83.3% of HHV-7 (n = 30/36) and 66.6% of CMV (n = 10/15) positive cases were detected in the RNA libraries.Among Specific sequences for human mastadenovirus (HAdV) were recovered in 28 samples and encompassed species A, C, E and F. Human bocavirus, parvovirus B19, trichodysplasia spinulosa-associated polyomavirus and BK virus were recovered in 10 or less samples each  and n = 803) was the most frequent, followed by Torque teno virus , and Torque teno midi virus  , human pegivirus-1 (HPgV-1) (n = 42), cardiovirus B (n = 2), and human polyomavirus 10 (n = 1)  and Figure 3(a–d)).In additionFeVir pipeline and/or de novo analysis  and Dicistroviridae genus (a family of viruses known to infect arthropods) identified in 84 samples and reported separately in Cordey et al [n = 50) were also detected, including two nearly complete sequences (n = 44) with a significant number of reads detected in the DNA libraries  that are not recognized as a cause of infection in humans were detected using either the ey et al . Porcinen = 27), providence-like viruses 1–2 (n = 24), dicistro-like viruses 1–2 (n = 12), bunya-like virus 1–3 (n = 3), and phlebovirus-like virus (n = 1) (n = 2), avian coronavirus (n = 1), iflavirus (n = 1), and mammalian orthoreovirus (n = 1) (n = 29), geminivirus (n = 11), human associated cycloviruses (n = 5), chicken stool-associated gemycircularvirus (n = 2), CRESS virus (n = 2), cyclovirus ZM36a (n = 2), avian gyrovirus 2 , bat associated cyclovirus 16 (n = 1), human associated gemyvongvirus 1 , human CSF-associated densovirus (n = 1), and taupapillomavirus 3 (n = 1)  in a population of young children with fever of unknown origin and/or severe outcomes who are living in Tanzania to pave the way of future clinical and diagnostic investigations. Beyond the well-recognized human viruses known to cause diseases and for some of them screened in routine clinical practice, or widely considered as “commensal,” we identify numerous novel, divergent and unexpected viruses that show mNGS to be a powerful tool for the surveillance of unrecognized or emerging viral infections. Among these unexpected viruses, were a large proportion never before described in humans, of mammalian, arthropod or other invertebrate origin. As the detection of a viral sequence does not necessarily reflect the cause of the disease, further matched studies on a broader geographic, demographic and clinical scope would be required to better differentiate between background noise, abortive infections or novel causes of clinical disease.Many previous investigations have surveyed viruses in the animal kingdom, including non-vertebrates, in order to extrapolate a probabilistic list of viruses that could emerge in humans . Howeverde novo analysis, the raw data were analysed with the FeVir pipeline . The mNGS analysis reported the detection of a large diversity of RNA and DNA virus sequences. Of the 816 children, 394 (48.3%) were found positive for at least one virus of “recognized clinical significance.” Anelloviridae were detected in all except one sample and up to 92.2% of samples were found co-infected by all three anellovirus genera . In addition, a large number of sequences were detected that related to 33 arthropod, vertebrate or mammalian virus species that are so far unknown to cause human infections; the most frequent being dicistroviruses, porcine parvoviruses 4–6 and ambidensoviruses.We conducted two bioinformatics analyses in parallel for each sample: In addition to traditional To assess the presence of potential environmental or laboratory reagent contaminants, NTC and PC submitted to the whole procedure were included to each series. Despite this, mNGS data must be interpreted with caution  as sporadic contaminations cannot be ruled out e.g. the mNGS analysis reported in the blood for so-called respiratory viruses only one HPIV-4a positive sample with 4 reads detected as well as two human coronavirus OC43 positive samples with ≤10 reads each  (Supplementary Table 1).Herpesviridae, Picornaviridae and the Reoviridae family members were the most frequently detected among the 4 DNA (Herpesviridae > Adenoviridae > Parvoviridae > Polyomaviridae) and 9 RNA  virus families observed ). Indeed, among the 35 viral species detected and considered of clinical significance, we observed a predominance of human enteroviruses (13.8%), rotaviruses (12%), HHV- 6 (11%) and HHV-7 (4.4%). Overall, rotavirus A sequences were the most frequently detected and confirm that rotavirus infections frequently result in viremia in paediatric patients [n = 36) in our cohort. Although commercial or in-house molecular assays are available, HHV-7 is rarely (if ever) tested even in high-resource settings. Although great care was taken during the RNA virus genome extraction (RNA protocol), we cannot rule out rare DNA contaminations from inter-/lower phases after phase separation. However, given their significant proportions, the detection of RNA for HHV-6 and HHV-7 in the RNA libraries suggest ongoing viral replication in the majority of positive cases. Some viruses are also notable for their absence, for instance, Epstein–Barr was not detected by mNGS analysis in any children. We also show the wide circulation of EV in East Africa, and report a potential new EV-A71 genogroup H. Additional investigations on the prevalence and global distribution of this genogroup as well as its associated disease will give potentially important information about its neurotropic potential. Continuous genomic studies on the evolution of EV-A71 are important to detect new mutants or recombinants with epidemic potential [Concerning the viruses considered as being of “recognized clinical significance,” sequences related to patients , 39. Simotential . Such suPicornaviridae family members we observed two samples harbouring Cardiovirus B. This virus was isolated in cell culture from stool samples collected from a child with fever of unknown origin and named the Saffold virus [de novo analysis only. This demonstrates that, although more time-consuming, a de novo approach is complementary to database-oriented pipelines and recommended in such surveillance studies to detect highly divergent viruses. To support this statement, 13 RV-C and 13 RV-A genomes were de novo assembled by Ramesh A and colleagues in their mNGS study [Interestingly, although its relationship with clinical pathology is currently difficult to establish, among different ld virus . Since tld virus  and has ld virus . Among tld virus  and our GS study .Our results further highlight that HAstV has a significant circulation in the global population, with a higher prevalence of novel HAstV (2.1%) than classical HAstV (0.9%). This observation is in agreement with previous reports and underscores the clinical pertinence of novel HAstV in the paediatric population .Anelloviridae is still debated [Anelloviridae are considered major members of the human blood virome and possibly one of the most abundant virus in blood samples [The potential clinical implication of  debated . Among s debated  with inc debated . Anellov samples . Reflect samples , anellov samples , 53. In A wide range of RNA and DNA viruses previously unknown to infect humans were observed. These deserve specific attention in further studies in the febrile paediatric population in Africa. Indeed, the aim of this study was to characterize the diversity of the viral landscape in the blood of febrile Tanzanian children. However, while these viruses were discovered in febrile children, the absence of a non-febrile comparison group means that we cannot definitively associate the febrile episode to the presence of the viral sequences. While this specific question is beyond the scope of our study, the associations between the considerable diversity of metagenomics data and clinical features is yet to be explored. Therefore, although extremely challenging, it would be important to perform a similar mNGS survey analysis on blood samples from an asymptomatic paediatric cohort with comparable demographic and epidemiological characteristics to better clarify any potential clinical causality.In conclusion, this blood virome study on a population of young children with fever of unknown origin and/or severe outcomes living in Tanzania highlighted several DNA and RNA viral sequences, and provides a large list of novel viruses that should be considered as a potential cause of human infections, some certainly being candidates for future fever investigations.Click here for additional data file.


Phaseolus biodiversity, we determined the identity and distribution of viruses infecting wild and domesticated Phaseolus spp. in the Mesoamerican Center of Domestication (MCD) and the western state of Nayarit, Mexico. We used small RNA sequencing and assembly to identify complete or near-complete sequences of forty-seven genomes belonging to nine viral species of five genera, as well as partial sequences of two putative new endornaviruses and five badnavirus- and pararetrovirus-like sequences. The prevalence of viruses in domesticated beans was significantly higher than in wild beans , and all samples from domesticated beans were positive for at least one virus species. In contrast, no viruses were detected in 80–83% of the samples from wild beans. The Bean common mosaic virus and Bean common mosaic necrosis virus were the most prevalent viruses in wild and domesticated beans. Nevertheless, Cowpea mild mottle virus, transmitted by the whitefly Bemisia tabaci, has the potential to emerge as an important pathogen because it is both seed-borne and a non-persistently transmitted virus. Our results provide insights into the distribution of viruses in cultivated and wild Phaseolus spp. and will be useful for the identification of emerging viruses and the development of strategies for bean viral disease management in a center of diversity.Viruses are an important disease source for beans. In order to evaluate the impact of virus disease on  Phaseolus spp. is needed to quantify the impact of viruses on bean cultivation and the effect of human management on population heterogeneity, biodiversity loss and disease risk. Common bean (Phaseolus vulgaris L.) is likely the most susceptible plant species in the Leguminosae to virus infection [Virus diseases are a significant threat to agriculture production. The emergence of indigenous and newly introduced viruses is a potential threat to biodiversity in centers of origin and plant domestication, reviewed in ; therefonfection  and one nfection .P. vulgaris L.) [There are two major genetic pools of common bean, the Mesoamerican and Andean, reviewed in . Genomicaris L.) ,6, with aris L.) . In Mexiaris L.) . The analysis of RNA sequences from plant samples through next-generation sequencing (NGS) is an effective method for virus detection, reviewed in . Among NPhaseolus spp. Our results provide insights into the distribution of viruses in domesticated and wild Phaseolus spp., as well as information that could be useful for the identification of emerging viruses and the development of strategies for common bean viral disease management.In aims to expand the knowledge on the diversity, distribution, and dissemination of viruses infecting domesticated and wild beans in the MCD and surrounding areas, symptomatic and asymptomatic samples from field-grown common bean and wild bean populations were collected in Guanajuato, Jalisco, and Nayarit, Mexico, and were analyzed through the sRSA approach using the Illumina platform. The data obtained revealed the presence of nine known viral species, two putative novel alphaendornaviruses, and five virus-like sequences of putative badnaviruses and pararetroviruses, and significant differences in the prevalence of viruses between domesticated and wild Phaseolus spp.) and domesticated bean (P. vulgaris L.) with and without symptoms of viral infection were collected in the MCD and the western State of Nayarit. The foliar tissues were collected and preserved in silica gel with a moisture indicator, as reported in Chiquito-Almanza et al. [Foliar tissues from 627 plants of wild  and sequenced on an Illumina HiSeq 2000 system in 50-bp single-end mode. Seventy-two samples were used to construct 53 sRNA libraries, 26 of which were from domesticated bean species (25 from P. vulgaris and one from P. acutifolius), while 27 were from the wild bean species P. acutifolius, P. coccineus, P. leptostachyus, P. lunatus, P. microcarpus, and P. vulgaris. All libraries were constructed from a single sample except for the composite libraries CJ16-25Lc and CJ25-56Lc from domesticated beans and SG5-136Lc, SG7-245Lc, SJ9-34Lc, SJ19-24678Lc, and SN41-2345Lc from wild beans, which were made of the equimolar mixtures of total RNA from three to six plants collected from the same spot as the single sample in the population ; RNA quantity was determined using a NanoDrop 8000™ analyzer , and RNA quality was checked by agarose gel electrophoresis. Total RNA (3 μg) was shipped to Macrogen Inc. , where sRNA libraries were constructed with the TruSeqpulation .https://github.com/kentnf/VirusDetect/tree/master/tools/sRNA_clean, accessed on 16 October 2020) to trim the 3’ adaptor sequences and remove the sRNA reads without adaptor sequences and those of low quality or shorter than 15 nt after trimming. In order to determinate the size distribution of sRNA, trimmed sRNA sequences between 15–40 nt were aligned to the common bean genome (NCBI: GCA_000499845.1) using the Bowtie v1.1.1 program [http://virusdetect.feilab.net/cgi-bin/virusdetect/index.cgi, accessed on 18 October 2020); the common bean genome and plant virus reference database in the VirusDetect software was used as a reference to subtract host sRNA and for reference-guided assemblies through aligning sRNA sequences, and other parameters applied were coverage = 0.1 and depth = 5. SnapGene V4.1.9.  was used for the annotation and reconstruction of complete and near-complete genomes of viruses from the pooled sRNA reads using the complete genomes of the viruses with the highest bit score as a reference  method, according to Chiquito-Almanza et al. . The absVirus prevalence in each region was calculated considering individual plants as the percentage of the number of individual samples with at least one virus divided by the number of total individual samples. The Mann–Whitney U-test was used to determine the significance of the differences between wild and domesticated beans concerning viral prevalence in the MCD and Nayarit using the number of samples of individual plants that contained at least one virus from the total of individual samples tested as input data.Bean common mosaic virus (BCMV), Bean common mosaic necrosis virus (BCMNV), Peanut mottle virus (PeMoV), Phaseolus vulgaris alphaendornavirus 1 (PvEV-1), Phaseolus vulgaris alphaendornavirus 2 (PvEV-2), Cowpea mild mottle virus (CPMMV), the DNA A components of Bean golden yellow mosaic virus (BGYMV), and Bean latent virus (BLV); the amino acid sequences of the RNA-dependent RNA polymerase (RdRp) of the Bean-associated cytorhabdovirus (BaCV) isolate CN3; the putative novel alphaendornaviruses Phaseolus leptostachyus alphaendornavirus (PlepEV) and Phaseolus lunatus alphaendornavirus (PluEV). The RdRp domains of PlepEV and PluEV were predicted using the NCBI Conserved Domain-Search tool . The MEGA X package [The nucleotide sequences of the complete and near-complete reconstructed genomes were used to establish phylogenetic relationships with other members of their respective viral families that displayed best bit scores in BLAST searches. The phylogenetic analysis was performed using the complete genome nucleotide sequences of the isolates of  package  was used package .The sequencing of the sRNA libraries generated between 3.4 and 52.3 million raw reads per library. Trimming adapters and low-quality reads resulted in 2 to 49.2 million clean reads. In order to determine the size distribution of sRNA, trimmed reads between 15–40 nt were aligned to the common bean genome. Between 34% and 89% of the sRNA in each library were from the host; the unmapped sRNA reads with highly enriched vsiRNAs were between 11% and 66% of the total sRNA counts in each library . The mosForty-seven complete or nearly complete genomes of nine known viral species, two sequences of 3581 bp and 2878 bp of two putative novel alphaendornaviruses, and five badnavirus- and pararetrovirus-like sequences were assembled . The virPapaya cytorhabdovirus (PpVE) from Ecuador  and P. leptostachyus (sample SJ8-5), and the latter sample was also infected with PeMoV; BCMNV was found infecting P. leptostachyus (sample SJ12), while PvEV-1 and PlepEV infected two samples, one of P. vulgaris (sample SG1-1) and one of P. leptostachyus (sample SG29)  and eight with double infections (five with PvEV1 + PvEV-2 and three with BCMV + BCMNV). Conversely, among the wild bean samples from this region, 24 samples harbored no viruses, and five showed single infection  C. In thele SG29) .P. vulgaris (sample SN44-5), and a putative PluEV, infecting P. lunatus (SN35) , double infections  and triple infections (one each with BCMNV + BCMV + BGYMV and BCMNV + BCMV + BLV). In the wild beans, no virus was detected in 10 samples C, and sis (SN35) .These results show that BCMV and BCMNV were the most frequent viruses identified in domesticated and wild beans , and sinp < 0.001). In the MCD, 20 of the 21 samples of domesticate bean (95.2%) were infected by at least one viral species, while in wild beans, only six of 30 samples (20%) were infected; in Nayarit, these percentages were 100% (nine of the nine samples) and 16.7% (two of the 12 samples), respectively  and Jalisco (library CJ14-4Ls); meanwhile, PvEV-1 was also identified in one sample of wild bean from Guanajuato (library SG1Ls).Cluster 2 encompasses the higher diversity of viral species. Among the six viruses in this cluster, BCMNV is present in all the libraries and includes mainly samples of domesticated beans from Nayarit, and two samples of wild beans, one from Nayarit (SN44-5Ls) and one from Jalisco (SJ12Ls). This result confirms the high prevalence of BCMNV in common beans grown in Nayarit and the presence of this virus in wild and domesticated bean from Nayarit and Jalisco (library CJ25-56Lc in cluster 3a).Cluster 3a includes all libraries infected with BCMV, except those from libraries CN61Ls and CN30Ls from cluster 2. In addition, PeMov was identified in mixed infection along with BCMV in library SJ8-5Ls. Although most of the libraries in this cluster are from samples of domesticated beans, it also includes samples of wild beans (SJ9-34Lc and SG27-3Ls). Samples in this cluster were collected from Nayarit, Guanajuato and Jalisco. This result indicates the wide distribution of BCMV in the common bean that is grown in this region and confirms the presence of this virus in wild beans in the MCD.Several assembled contigs without detectable nucleotide homology but with amino acid identity to reference virus protein sequences were identified in 11 of the libraries . In ordeP. leptostachyus collected in Guanajuato and SN35 from P. lunatus collected in Nayarit. For identification purposes, the names Phaseolus leptostachyus alphaendornavirus (PlepEV) and Phaseolus lunatus alphaendornavirus (PluEV) were assigned. The partial nucleotide sequence of PlepEV consisted of 3581 nt (GenBank accession MT792848) and included one partial ORF of 3540 nt that was translated into a fragment of a putative polyprotein of 1179 aa, sharing 45.9% and 43.9% aa identity with the carboxy-terminal polyprotein of the unclassified alphaendornavirus Geranium carolinianum endornavirus  and the Phaseolus vulgaris endornavirus 3 isolate LA , Phaseolus coccineus badnavirus-like sequence SJ10-3 (PcBV-SJ10-3), Phaseolus coccineus pararetrovirus-like sequence SJ10-3 (PcPRV-SJ10-3), Phaseolus lunatus badnavirus-like sequence SN35 (PluBV-SN35), and Phaseolus lunatus pararetrovirus-like sequence SN35 (PluPRV-SN35).Five virus-like sequences with amino acid identity to the viral reference sequences were reconstructed . For ideP. coccineus sample with symptoms of local chlorosis lesions, while the sequence PluBV-SN35, consisting of 1378 bp, was reconstructed from a symptomless wild P. lunatus sample; in both samples, SJ10-3 and SN35, the pararetrovirus-like sequences PcPRV-SJ10-3 (2588 bp) and PluPRV-SN35 (1440 bp) were also identified; these translated into 862 aa and 480 aa proteins, respectively, with 49% and 32% aa identity to Citrus endogenous pararetrovirus polyprotein (GenBank accession KF800044).The sequence of PvBV-CG17 consisted of 1629 bp and was reconstructed from a sample from common bean cv. Azufrado Higuera with symptoms of local chlorosis lesions; in this sample, PvEV-1 (GenBank accession MG640416) was also identified. The sequence of PcBV-SJ10-3, consisting of 2604 bp, was reconstructed from a wild Badnavirus-like pelargonium vein banding virus . On the other hand, the sequence of PcBV-SJ10-3 exhibited two partial ORFs, one was translated into a putative protein of 58 aa with 41.3% aa identity to the ORF2 protein of the badnavirus cacao swollen shoot virus , and the other was translated into a putative protein of 808 aa with 43.4% aa identity to the P3 putative polyprotein of PVBV. Although the reconstructed sequences of PvBV-CG17, PcBV-SJ10-3, and PluBV-SN35 shared low nucleotide similarity with the available sequences of badnavirus in the GenBank, suggesting that they could correspond to a novel badnavirus species, the reconstructed fragment did not include the RT/RNAse H domain required for the demarcation of the species. Additionally, since badnaviruses and pararetroviruses had been reported as integrated fragmented sequences, the circularity of the genome needs to be confirmed.Both the PvBV-CG17 and PluBV-SN35 sequences exhibited one partial ORF, which was translated into putative proteins of 543 and 456 aa, respectively, with 39.0% and 35.6% aa identity to the putative P3 polyprotein (ORF3) of the unclassified Phaseolus spp. were collected in the western state of Nayarit since the occurrence of virus damaging in the common bean crop is particularly high in this area [Phaseolus spp. with presumptive viral symptoms, no viruses were identified; the symptoms observed in those samples could be due to nutrient deficiencies or the effect of other biotic or abiotic stresses that may show similarities to mosaic or mottling [In addition to the MCD, samples of wild and domesticated his area ,30, and mottling . Nine spB. tabaci represents a constraint to vegetable production [The number of virus genera found in the MCD was lower than in the western state of Nayarit A; a possoduction . MoreoveCapsicum annuum var. glabriusculum), a wild pepper plant under different anthropogenic influences, including cultivation and incipient domestication, support the fact that ecological conditions are a valuable predictor of virus prevalence and that human management is associated with a reduction of habitat biodiversity and genetic diversity of host species; in addition, as host plant density is increased, the viral disease risk is enhanced due to plant-to-plant proximity. Furthermore, virus infection decreases the survival and fecundity of wild chiltepin populations, hence negatively affecting fitness [The evidence indicates that the incidence of viral diseases in the wild populations sampled is low in contrast to domesticated bean, in which almost all samples showed single, double, or triple infections B,C. The  fitness . It is a fitness ,38. None fitness , who fouPhaseolus spp. in Mexico before.The viruses BCMV, BCMNV, BGYMV, and CPMMV have been documented infecting common bean and wild bean in Guanajuato, Jalisco, and Nayarit, Mexico ,30,40. IB. tabaci. BGYMV is an important non-seed-transmitted virus in areas with a high incidence of B. tabaci; the disease incidence and the extent of losses vary depending on the populations of the whitefly, the susceptibility of the bean cultivar, and environmental conditions, particularly rainfall, which affects both B. tabaci populations and cultural practices [Predicting epidemics of plant virus diseases and the causes that facilitate pathogen emergence is complex and depends on multiple factors, reviewed in ,41,42. Bractices . The vecractices .Papaya cytorhabdovirus with strains PpVE infecting papayas and BaCV infecting beans [B. tabaci MEAM1 [Papaya ringspot virus (PRSV), and no differences in leaf symptoms were observed between PRSV single-infected plants and plants co-infected with both viruses [Based on the high nucleotide sequence identity between the cytorhabdovirus BaCV-BR-GO and PpVE, it was proposed that the virus species would be named ng beans ; thus, Bci MEAM1 , the traci MEAM1  found th viruses . TherefoP. vulgaris plants at rates close to 100% [The presence of endornaviruses PvEV-1 and PvEV-2 in almost all the domesticated and wild samples from Guanajuato  could be to 100%  and have to 100% .Arachis hypogea), but it also infects susceptible genotypes of P. vulgaris, causing various reactions that range from mosaic, banding, leaf deformation and blistering, to systemic necrosis in genotypes that have only the dominant I gene; these reactions are similar to those caused by necrotic strains of BCMV and BCMNV. Although PeMoV is mechanically and non-persistently transmitted by aphids, transmission to common bean seeds is low (1%) [The main host of PeMoV is peanuts (low (1%) .Phaseolus spp. from the MCD and the western state of Nayarit suggest that the transmission of these viruses through insect vectors is highly efficient. Further studies are needed to know how and from where these viruses came into the wild bean populations of the MCD and Nayarit and if transmission occurred from cultivated into wild bean populations. The other virus that has the potential to emerge as an important pathogen is CPMMV, which is emerging as a soybean pathogen in Brazil, and it is re-emerging in cultivated common beans [Carlavirus that is not transmitted by aphids, and it is nonpersistently transmitted by B. tabaci (MEAM1), with a period of access to acquisition of 10 min and an inoculation period of 5 min [P. vulgaris) and wild bean , and some RT-PCR-positive samples did not present symptoms [Among the viruses identified in this study, BCMV and BCMNV likely present the highest phytosanitary risk due to their high incidence in the region and their efficient mechanisms of seed and vector transmission . BCMV anon beans , particuon beans . CPMMV iof 5 min . Althougof 5 min ,58. Furtof 5 min . In soybof 5 min ,50. In Msymptoms .CPMMV is classified in Annex 1/A1 in the European Union and in the United States as a quarantine pest, where it is considered to represent a potential risk to soybeans, beans, cowpea, and some species of the Solanaceae family ,62. MexiP. vulgaris cv Black Turtle Soup [The assembled sequences of PlepEV and PluEV share amino acid identity with the RdRp domains of PvEV-3 and GcEV, suggesting that they could be novel alphaendornaviruses; however, their complete genome sequences will be required for their classification. The plant samples in which PlepEV and PluEV were identified were symptomless, and like other endornaviruses that have been reported in economically important crops, their effect on the host has not been fully determined. For instance, PvEV-1 and PvEV-2 were not associated with visible pathogenic effects on tle Soup ; also, tThe badnavirus- and pararetrovirus-like sequences reconstructed in this study were obtained by sequencing since multiple single-nucleotide variations (SNVs) in the assembled contigs made scaffolding difficult. Some badnaviruses occur as integrated, complete, fragmented, and/or rearranged genomic sequences of the original circular viral DNA in their host plant genomes, in which case they are referred to as endogenous pararetroviruses or endogenous viral elements . Their iHigher viral diversity was found in Nayarit in comparison with the MCD. BCMV and BCMNV were the most prevalent viruses in wild and domesticated beans; these viruses, along with CPMMV, have the potential to emerge as an important pathogen because they are seed-borne and non-persistently transmitted viruses. Integrated disease management approaches using virus-resistant common bean cultivars and efficient management of insect vectors and weeds can reduce the dispersion and the production losses associated with these viruses. More studies are needed to confirm the putative viruses identified in this study and determine the effect of human management on population heterogeneity, biodiversity loss, and disease risk that these viruses represent for the domesticated common bean and wild bean populations.


These filaments were found to be similar to those that form the endoskeleton of assembled virions. The structures provide a molecular basis to understand the functions of M1 during the virus life cycle. Here, we compare and discuss the two structures, and explore their implications for the mechanisms by which the multifunctional M1 protein can mediate virus assembly, interact with viral ribonucleoproteins and act during infection of a new cell.Influenza A virus matrix protein 1 (M1) is the most abundant protein within virions and functions at multiple steps of the virus life cycle, including nuclear RNA export, virus particle assembly, and virus disassembly. Two recent publications have presented the first structures of full‐length M1 and show that it assembles filaments  Influenza A virus matrix protein 1 (M1) forms an endoskeleton underneath the membrane envelope and functions at multiple steps of the virus life cycle, including nuclear export, virus assembly and entry. Two recent publications presented the first structures of full‐length M1 protein in vitro and within the virus, providing a molecular basis to understand the multiple functions of M1. In order to resolve high‐resolution structural details, purified M1 protein has been assembled into arrays in vitro that mimic the arrangement in the virion, but are more regular. We achieved this by recombinantly expressing M1 (from strain A/Puerto Rico/8/1934 (H1N1), containing substitution K134R, that was found to improve polymerization quality and is found naturally in certain virus strains) and assembling it into helical arrays in the presence of nucleic acid, which likely acts as an assembly scaffold  et al. 7JM3). The structures of both helical arrays were determined using cryo‐electron microscopy (cryoEM) and helical reconstruction methods to 3.8 Å and 3.4 Å resolutions, respectively. The nucleic acid scaffolded arrays are formed by symmetric M1 dimers that are arranged in a D1‐symmetric helical array around two strands of nucleic acid  connects to the CTD. The CTD forms an alpha helical domain that is bound in trans to the NTD of a neighbouring M1 monomer . This observation is consistent with our hypothesis based on the comparison of the two in vitro structures that the amount of H9 extension in M1 can vary to accommodate different curvatures observed across different virions.We compared the structures of the two NTD Fig. . HoweverCTD Fig. . When cotly Fig. . The relin vitro assembled structure, while the structure of the M1 CTD is best represented by the CTD from the high‐salt in vitro assembled structure. The relative domain orientation and H9 extension likely correspond to an intermediate between the two in vitro structures.We therefore conclude that the NTD arrangement within the linear M1 oligomer for the major population of M1 in filamentous virions is best represented by the nucleic acid bound in vitro assembled M1 oligomers represent different functional states of M1. If this is the case, which states might they represent?M1 is a multifunctional protein, with different roles at different stages in the viral lifecycle, and it is therefore possible that the conformations observed in the two different The different conformations reveal sites of flexibility; changes in the NTD/NTD interface, in H9 and in the relative orientation of the NTD and CTD, allow M1 to form oligomers with different curvatures. These may accommodate small difference in curvature depending on virus diameter or the number of M1 helices, but may also accommodate different curvatures present at the spherically curved tip or at the neck of the growing virus.in vitro assembled structures contain a similar, conserved, ordered histidine cluster, we think that both structures are likely to exist above the pKa of histidine within the histidine cluster and therefore that both represent neutral pH conformations. M1 has been observed to form coil‐like structures when dissociated from the virus membrane after low pH exposure [It has long been suggested that M1 undergoes a change in both its conformational and oligomerization state when the virus encounters low pH in the endosome during virus entry , 22, 26.exposure , 15, 27,via nucleoprotein (NP) but potentially also directly via viral RNA [in vitro assemblies) is required for RNP binding and nuclear export. After entry, fusion and subsequent release of M1 from the viral membrane, and once in the neutral pH of the cytoplasm, it is conceivable that M1 adopts a new conformation, for example coordinating with and protecting viral RNA, although M1 needs to be detached from the vRNPs to allow their debundling and nuclear import , 16. A firal RNA , 6, 29, iral RNA , 32 and Overall, we entertain two speculative hypotheses regarding the functional relevance of the two conformations of the final 30 residues of the CTD. First, that both may represent conformations found within the virion: one, the CTD confirmation of the high‐salt assembled structure, may correspond to the one found within the M1 endoskeleton on the sides of filaments, while the other – that observed when scaffolded with nucleic acid – may represent a subpopulation of M1 within the virion, which interacts with other virion components such as the RNPs at the front tip of the filament. Second, that the nucleic acid scaffolded conformation of the CTD reflects a conformation that interacts with RNPs during nuclear or cytosolic transport. Testing these hypotheses will require further experimental data about conformational adaptability of M1 and about the different roles and interaction partners of M1 during the virus life cycle.in vitro and within the virion have provided important new insights into assembly of influenza A. They reveal NTD‐NTD, NTD‐CTD, and CTD‐CTD interactions between M1 proteins and show that the CTD can fold to interact with the NTD from another M1 monomer, leading to linear oligomerization of M1. This suggests a molecular mechanism for virus assembly: an M1 monomer binds into a growing strand, leading to folding of the CTD and creation of a site to which the next monomer can bind. Burying of a large surface area during this processive assembly may provide the energy for protrusion of the viral filament from the cell surface. The in situ structure reveals the membrane‐interacting surface of M1, while the higher‐resolution in vitro structures reveal a conserved histidine cluster, which may serve as the pH switch mediating M1 disassembly upon virus entry.The new structures of full‐length M1 determined in vitro structures and the in situ structure are all very similar, differences in the NTD–NTD interfaces, in H9 and at the end of the CTD hint at the conformational flexibility and multifunctional nature of the M1 protein. It is clear that there is still much to be learned about M1.Although the two


In Europe, squirrelpox virus is carried by non-native grey squirrels and spread into native red squirrel populations. The virus causes a large proportion of infected red squirrels to die and contributes to local declines and the replacement by grey squirrels. There are relatively few published studies quantifying the impact of disease amongst red squirrels. We present findings from a short-term study in north Wales, United Kingdom.Sciurus vulgaris) persisted in the coastal mainland woodlands of northern Gwynedd whilst sympatric with an invasive grey squirrel (Sciurus carolinensis) population suppressed by culling. Squirrelpox disease in the red squirrel population was recorded in 2017 and 2020/21. An autumn 2020 outbreak was associated with only 17.4% of animals caught and marked in the preceding June known to be present in March 2021. Despite an opportunistic data collection lacking the rigour of empirical experimental design, we observed low local survival rates similar to previously published accounts reported during major squirrelpox outbreaks. The use of a conservation dog to detect red squirrel carcasses resulted in positive detection and confirmation of a temporal and spatial expansion of one disease outbreak. The study is the first in Wales to use conservation dogs and the findings reinforce the vital strategic importance of geographical isolation reducing sympatry of red with grey squirrels in European regions where the introduced congener is a source of the squirrelpox infection.Native red squirrels ( Sciurus carolinensis) causes decline in native Eurasian red squirrel (Sciurus vulgaris) populations through resource competition [Strongyloides robustus) from the introduced to native sciurid, and infected red squirrels have lower survival rates than those that are not infected [The introduced Eastern grey squirrel  screening has revealed that 69% of healthy grey squirrels tested were positive for SQPV antibody presence .Anglesey contains the largest red squirrel population in Wales following the successful eradication of grey squirrels . Island Grey squirrels found on Anglesey were legally trapped and culled to prevent the spread of squirrelpox infection into the sympatric red squirrel population. In contrast, the risk of intra-specific infection occurring between the mainland and island red squirrel populations is harder to manage because individual movement across the Menai Strait cannot realistically be prevented and, as animals are fully protected by law, they cannot be trapped without a license. The risk of an infected mainland red squirrel crossing the sea channel and bringing infection onto Anglesey from Gwynedd is ever present and additional to the risk of infection being spread via grey squirrel dispersal.Regional red squirrel conservation requires the rapid detection and removal of grey squirrels from Anglesey, the suppression of mainland grey squirrel populations and the monitoring of red squirrels for signs of disease . On the Annual trapping could only be conducted in a proportion of the mainland woodland habitat between March and July because of game-rearing operations taking place . The resRed squirrel carcasses showing signs suggesting a pathological skin infection were submitted to the Animal and Plant Health Agency (APHA), Shrewsbury Veterinary Investigation Centre for gross post-mortem and associated laboratory testing. Examinations were undertaken within the APHA Diseases of Wildlife Scheme, a part of the GB Wildlife Disease Surveillance Partnership.Where appropriate, a sample of skin lesion was then submitted to the APHA Weybridge laboratory for Transmission Electron Microscopy (TEM) screening as described elsewhere , amplifiThe location of each recovered carcass was recorded. The cumulative data allowed the construction of the timeline of squirrelpox infection confirmation in red squirrels. All animals had a submission reference code which provides a unique catalogue entry for all pathological examinations and histology.Resource limitations precluded regular and systematic proactive ground searches to be undertaken to detect and recover dead squirrels. Instead, the regional conservation programme relied upon initial opportunistic discovery of carcasses by members of the public or contractors undertaking grey squirrel control. Where subsequent laboratory tests revealed squirrelpox infection, then limited searches were undertaken focusing on areas known to have high red squirrel activity. In one instance, a search was undertaken using a conservation dog trained to find dead red squirrels . This siVolunteers managed trail-cameras that monitored animal activity at point locations baited with sunflower seed. This encompassed five discrete and permanent woodland locations selected because (1) landowner permission allowed round the year access and (2) because the locations were away from areas with high public recreational activity . Volunteers would share recorded images suggestive of squirrelpox infection in a red squirrel. Camera trap models varied and both video and still image settings were used interchangeably.Live trapping involving the handling of captured red squirrels could only be undertaken by a licensed professional ecologist. This individual had limited time resource. Unmodified commercially available Albi™ single-catch Mink-traps baited with hazel nuts and sunflower seed were deployed. Traps were cleaned with Virkon S™ disinfectant between all captures. All traps were opened in the morning and closed after inspection three to four hours later. From June 2020 to September 2021, captured red squirrels were handled by transferring them into a plastic bag and then into a Virkon S™ sterilised wire mesh handling cone. Individuals were sexed, weighed to the nearest 5 g with a 1000 g Pesola™ spring-balance, had their hind shin measured to the nearest 0.1 mm and reproductive state categorised in earlier studies ,35. ThesMeasurements collected helped to gauge body condition using weight/skeletal size ratio. However, in the light of subsequent squirrelpox outbreaks, the information provided a useful insight into the relative conditions of groups of detected and undetected animals. Given that squirrelpox is likely spread by direct and indirect  we predicted that because larger sized animals may be more dominant they are therefore most likely to encounter infection.A unique Passive Integrated Transponder (PiT tag) (Peddymark™ 1.4 × 10 mm) was inserted under the skin at the nape of the neck or dorsal area of the back where the skin was relatively loose. This facilitated individual identification.Captured red squirrels were examined for signs of external disease. Red squirrels showing signs of infection were taken to a local veterinary practice for examination and professional assessment. When squirrelpox was confirmed in the population, handling of captured red squirrels was limited to reduce the risk of cross infection. Each squirrel was able to move into a bag placed to cover and enclose the front of the trap and the trap door, and when inside the bag, it was scanned for a PiT tag using a Peddymark™ ISO PM450 Scanner. Chipped animals were released unless earlier casual observation of the squirrel in the trap had suggested a potential lesion presence. Unmarked animals and those marked individuals that were to be more closely examined for signs of infection were placed in the handling cone and measured. The approach sacrificed collection of biometric data in order to reduce exposing animals to stress and limiting opportunities for accidental viral cross infection.Trapping interventions aimed to reduce grey squirrel abundance but, were not sufficient to eradicate grey squirrels. Any captured grey squirrel was euthanized using the cranial dispatch method . Grey sqAdult red squirrels were defined as individuals with a weight greater than 240 g. Adult grey squirrels were 450 g or greater in weight.Finally, data from a third-party seasonal contractor undertaking grey squirrel control were also available. The contractor was not licensed to handle captured red squirrels, but records of incidental trapping were recorded which provided a measure of return per unit trap effort.On 7 September 2017, a dead male (P1) red squirrel (APHA Ref 26-M0286-09-17) weighing 282 g was discovered in a live-capture trap being operated in Treborth Botanical gardens as part of ongoing grey squirrel control (UK grid reference SH 5505 7097). There was a small minor lesion on the lip. Hair loss was observed around the right eye, right whiskers and chin surrounding the bottom lip and at the margin between the foot pads and hairy skin on both front feet. The skin surrounding the right eye was noticeably pale and thickened, covering a circular area of approximately 1 cm diameter. Histological examination documented typical poxviral inclusions in keratinocytes within hair follicles, supporting a diagnosis of SQPV. TEM  revealedStaphylococcus aureus and non-haemolytic Escherichia coli were found to be present. SQPV particles were observed within lesions via TEM.Three weeks later, 27 September 2017, the carcass of a female (P2) (APHA Ref 26-M0280-10-17) weighing 244 g was recovered. A member of the public discovered the remains on the woodland floor within the Faenol estate (SH 539 702). Gross post-mortem recorded reddening of the skin present on the head in well circumscribed areas on the lower chin and lip extending to the left and right sides and a circular area on the right side of the face below the right nostril, approximately 5 mm diameter and the right upper lip. Affected areas were reddened and thickened with a crusty surface and some hair loss. There was also reddening around both eyes and of the skin of the digits of both front feet. Histology observed marked, diffuse orthokeratotic hyperkeratosis and scaling. The epidermal surface was colonised by large numbers of cocci, which were also found multifocally within follicles and the dermis, but without any inflammation (post-mortem growth). Haemolytic coagulase positive On 22 November 2017 the remains of a 410 g female (P3) red squirrel (APHA Ref 26-M0440-11-17) were found following a search near a camera location (UK grid reference SH5351 7048) from which images of a red squirrel with swollen eyes were recorded. Gross post-mortem noted the lower lip was swollen and there was an area of ulceration with a flap of necrotic skin extending from the bottom of the lower lip under the jaw approximately 1 cm diameter. Lesion material taken from around the eye and from the lip were both confirmed TEM positive for SQPV particles.A grey squirrel control contractor trapped in the Faenol woodland in February/March 2017, June/July 2017 and January/February 2018 and reported 1.79, 4.73 and 5.92 grey squirrel captures per 100 days trap effort and 1.14, 0.81 and 2.96 red squirrel captures per 100 days trap effort. He reported red squirrel captures throughout the area denoted by black in On 5 April 2019 a member of the public sent in images from a wildlife camera within Treborth gardens which showed a red squirrel with lesions around the eyes and on the digits . A visuan = 7) were lactating and were caught alongside a subadult female. Of 13 adult males , 10 had large scrotal testes and the mature animals were removed along with three subadults (<450 g). A total of 19 female and 15 male sympatric red squirrels were caught and marked (n = 14) whilst mean female weight  was 308.6 g ± 10.0 s.e. (n = 15).In the summer of 2020, live trapping across Faenol and Treborth  over eigd marked . This inLater that year, on 29 September 2020, a member of the public found a dead adult female (P4) red squirrel (APHA Ref 26-M0037-10-20) weighing 240 g within the Eithinog Nature Reserve (adjacent Treborth gardens). The carcass showed skin lesions characteristic of squirrelpox infection which was confirmed via TEM. Given the difficulties in searching for red squirrel bodies in woodland experienced in 2019, it was decided to use a conservation dog trained to recover dead red squirrels. On 18 October 2020 a systematic search was undertaken by ‘Max’ and his handler, both fully trained by Kryus Ltd . Red squirrel remains (P5) in the form of part of tail and clumps of body hair were found in a localised area within Faenol estate. PCR testing confirmed the presence of amplified SQPV DNA. This was the second confirmed outbreak in Gwynedd. Unfortunately, in the autumn of 2020 two cameras were stolen and because of COVID-19 restrictions, volunteers were unable to monitor the remaining cameras and they were removed to prevent further theft.Temporal land-access restrictions meant that significant areas were also inaccessible and therefore only a proportion of the woodland area trapped in June could be surveyed using traps from the autumn . Within n = 6) and male weight 320 g ± 14.0 s.e. (n = 5). A Mann–Whitney U-test did not reveal any significant difference , between female red squirrel weights in June and October, when pregnant animals were excluded from the analysis. Male body weight was also not significantly different .In October (30 September 2020–21 October 2020) the same survey effort (120 trap days across the same 15 traps) yielded 18 red squirrels (0.15 captures per trap day) and a single grey squirrel (0.01 captures per trap day). Nine (39%) of the red squirrels caught there in June were present, along with two animals caught and marked previously in adjacent habitats which were now inaccessible. There were seven unchipped squirrels caught: three males and four females. None of the handled animals were reproductively active. Female mean weight was 319 g ± 11.9 s.e. (On 31 January 2021 another TEM confirmed SQPV case was recorded (APHA Ref 26-M0024-02-21). The 320 g adult male (P6) was caught alive but died whilst being transferred to veterinary quarantine. Erosive skin lesions were present on the chin, lips and around both eyes. There were areas of hair loss on the bridge of the nose, overlying the thoracic inlet and on three digits of the left fore foot and two digits of the right hind foot. It is unclear whether this case was a continuation of autumn 2020 outbreak or was part of a third temporal cluster of infections. In March 2021, only two red squirrels were caught (14 days × 15 traps). These were both females chipped in June 2020 and both had also been caught in October 2020. A single grey squirrel was removed. Later in 2021, an additional male and female marked in June 2020 were recorded along with the recapture of a female red squirrel, first chipped in October 2020. These occurrences meant that at least one of seven (14.2%) October chipped red squirrels and four of the 23 (17.4%) June 2020 marked animals trapped within the limited woodland area accessible (15 traps) in autumn and winter months were known to have been alive there in March 2021.Live trapping allowed the partitioning of individual biometrics (weight and shin length) recorded between groups of animals subsequently detected (open triangles) or not found again (open circles) . The smaAlthough data collection was opportunistic rather than rigorously experimental, the information collected helped provide valuable insight into the local impact of disease outbreaks on red squirrel population size and individual survival. Despite the limitations of land-access restrictions, mark and recapture data collected before, during and after squirrelpox was first confirmed in 2020 was invaluable. These data provided snapshots of the presence, body weight, size and reproductive state of animals during the outbreak.The available mark-recapture data strongly suggests a decline in red squirrels associated with squirrelpox infection. It is unclear whether this was associated with a single chronic disease outbreak or successive outbreaks merging temporally. Only 17.4% and 14.2% of animals first marked in June and October 2020, respectively were known to be still alive in March 2021. It should be noted that we were unable to quantify dispersal or completely exclude that individual animals may have remained alive locally but undetected. It is therefore possible that the scale of squirrelpox-associated decline includes animals that remained alive and hence figures represent a maximum rather than absolute mortality. The 17.4% and 14.2% figures equate to 82.6%–85.8% ‘loss’ which is lower than the 93% decrease reported from Tollymore Forest, Northern Ireland between 2008 and 2011 and the estimated reduction to 6% of original density in a Lancashire population . High moDespite confirmation that squirrelpox mortality occurred in the autumn of 2017, autumn of 2020, and the late winter 2020/21, red squirrels persisted on the mainland side of the Menai Strait whilst remaining sympatric with a grey squirrel population suppressed by culling. It is unfortunate that 2017/2018 squirrel trapping data are limited and cannot provide a measure of the scale of red squirrel mortality. In addition, the contribution of ongoing island to mainland dispersal to mainland presence is unclear. Nevertheless, we confirmed that in 2021, four mainland resident females and a male were known to have survived through the previous autumn/late winter outbreak, indicating that mainland population persistence is not solely due to immigration.Sainsbury et al.  report sRecognising resource limitations in undertaking systematic human observer visual searches in woodland terrain often containing dense fern and ground cover, fallen trees and patches of laurel, the use of a conservation dog was invaluable in carcass detection and recovery. The subsequent successful amplification of SQPV DNA from hair  led to Whether the third confirmed 2020/21 squirrelpox case (found 31 January 2021) derived from a discrete inter-specific infection from grey squirrel to red squirrel or was part of an ongoing intra-specific viral spread (from case 1 on 29 September 2020) is impossible to determine. Carroll et al.  documentThe continual detection of successful grey squirrel dispersal onto the island remains a concern because although modelling suggests a resulting squirrelpox outbreak would not cause population extinction  the poteOur findings demonstrate the value in opportunistic study to further understanding of the impacts of pathogenic infections upon native wildlife. They reaffirm the ongoing threat posed by introduced grey squirrels to red squirrel populations and highlight the need for stringent efforts to be made to exclude grey squirrels from habitats occupied by native red squirrels.


Sepsis and meningitis are among the leading causes of neonatal deaths in sub-Saharan Africa (SSA). Neonatal sepsis caused ~400 000 deaths globally in 2015, half occurring in Africa. Despite this, there are few published data on the acute costs of neonatal sepsis or meningitis, with none in SSA.We enrolled neonates admitted to 2 hospitals in South Africa and Mozambique between 16 April 2020 and 1 April 2021. In South Africa all cases were microbiologically confirmed, but in Mozambique both clinically suspected and microbiologically confirmed cases were included. Data were collected on healthcare resource use and length of stay, along with information on household expenditure and caregiving. We used unit costs of healthcare resources in local currencies to estimate healthcare provider costs per patient and costs per household. Results were converted to 2019 international dollars (I$).We enrolled 11 neonates in Mozambique and 18 neonates in South Africa. Mean length of stay was 10 days  and 16 days , respectively. In Mozambique we estimated mean household costs of I$49.62  and hospitalization costs of I$307.58 . In South Africa these costs were I$52.31  and I$684.06 , respectively. We found substantial costs associated with acute neonatal bacterial  sepsis and meningitis in SSA. Our estimates will inform economic evaluations of interventions to prevent neonatal invasive bacterial infections. WHAT WAS KNOWN AND WHAT IS NEW?Globally, the few published studies on acute healthcare costs of neonatal sepsis and/or meningitis are from high-income countries, and mainly from the United States. There are no studies from sub-Saharan Africa despite the high disease burden in this region. Our study provides estimates of health care and household costs associated with acute neonatal sepsis and meningitis hospitalisations in Mozambique and South Africa.2. WHAT DID WE DO AND WHAT DID WE FIND?Using hospital records and discharge questionnaires of 29 neonates hospitalized in Mozambique and South Africa, we estimated mean costs to households and healthcare providers to be I$49.62  and I$307.58 , and I$52.31  and I$684.06 , respectively in 2019 I$.3. WHAT TO DO NOW IN PROGRAMMES?Neonatal complications including infections are an important cost to health systems and families, and are an important consideration for inclusion in financial protection schemes. Interventions to prevent neonatal sepsis and meningitis could reduce the costs to healthcare providers and households, as well as improving health outcomes.4. WHAT NEXT IN RESEARCH?Our results could be used in future research on the cost-effectiveness of interventions to prevent neonatal bacterial infections. Further costing studies with larger study populations, across a range of hospital settings and countries, and using similar tools to ours would make findings more generalisable and give insights into cost drivers and contextual differences.Streptococcus (GBS), led to about 400 000 deaths globally in 2015, half of which occurred in the African region [Although child mortality has halved in the last 2 decades, deaths in the neonatal period accounted for a larger proportion, approximately 47% (2.4 million) of all under-5 deaths in 2019 [n region .Despite this, there are few published studies on the cost of acute healthcare for sepsis and/or meningitis in neonates from low- and middle-income countries (LMICs), with data concentrated mainly in the United States (US) . FurtherThe study was conducted both in Mozambique (categorized as a low-income country) and South Africa (categorized as an upper-middle-income country). In 2019, the gross domestic product per capita based on the purchasing power parity was I$1336 in Mozambique and I$13010 in South Africa [This article is part of a series on GBS worldwide. This paper addresses the lack of empirical data in LMICs, and SSA specifically, by estimating the costs to the healthcare system and households during acute episodes of bacterial sepsis and/or meningitis among neonates admitted to hospital in Mozambique and South Africa.This costing study was a linked component of a larger study on the outcomes after invasive GBS disease in young infants, for which the protocol has been published previously . Data weWe enrolled all participants who met the inclusion criteria and who were admitted between 16 April 2020 and 1 April 2021 at the Chris Hani Baragwanath Academic Hospital, a tertiary-academic hospital situated in Soweto, South Africa; and the Manhiça District Hospital, the referral hospital for the district of Manhiça in Mozambique. The hospitals included in this study are public ones; healthcare is normally provided free of charge to all patients in Mozambique and to patients <6 years old in South Africa. Data collection took place during the middle of the severe acute respiratory syndrome coronavirus 2 pandemic, which prevented us from reaching the initial set sample size of 20 neonates per site.Most participants were identified prospectively on admission. About 14% (4/29) of the neonates were admitted from birth. These were already in hospital and kept in due to developing sepsis and/or meningitis shortly after birth. Neonates who died shortly after admission were not excluded from the study. Data on hospital resource use were collected from medical records, including information on length of stay, drug use, diagnostic tests, and surgical procedures . Unit coWe estimated household costs associated with the acute episode including direct and indirect costs, namely medical, transportation, and accommodation costs, as well as costs of informal caregiving. Healthcare provider costs were calculated based on the average length of stay, use of drugs, diagnostics, and supportive care, among others. Most unit costs were provided by the sites in local currencies for the year 2019. Wherever estimates were in another currency  and/or for another fiscal year, these were converted into the local currency using the appropriate exchange rate and/or inflated using the local consumer price index . To faciEthical ConsiderationsThe overarching protocol for this multicountry observational study was granted ethical approval at the London School of Hygiene and Tropical Medicine . Institutional review boards in each of the operating countries granted ethics approval , as well as the institutional review board of the World Health Organization . Data sharing agreements were jointly developed and signed by all collaborating partners. Data collection instruments used in this study are available in the Escherichia coli).Overall, 29 neonates (18 from South Africa and 11 from Mozambique) were enrolled in the study population, which is summarized in Hospitalization and household costs for the 2 countries are described in The mean length of stay of 16 days  in South Africa was substantially longer than in Mozambique, which was 10 days , and 11 days  when including only microbiologically confirmed cases. The mean hospitalization cost per neonate of I$684.06  in South Africa was also higher than the cost of I$307.58  in Mozambique. However, for microbiologically confirmed cases this increased to I$413.41 , primarily due to higher laboratory costs.Most households incurred no direct healthcare expenses related to their child’s acute infection. The mean healthcare expenditure per patient was I$40.04 in Mozambique and I$10.03 in South Africa, but median costs were zero in both countries.In both countries, food and accommodation at the hospital are provided to caregivers without charge, so the main direct costs were transportation expenses. On average, households spent I$9.58 in Mozambique and I$41.64 in South Africa on transportation. Therefore, the overall direct household costs were I$49.62  and I$52.31  in Mozambique and South Africa, respectively.In 97% of cases the caregiver accompanying the child to the hospital was the mother, and otherwise the father. Caregivers’ age ranged from 15 to 36 years. In Mozambique, caregivers reported spending 52 hours  on extra care. In South Africa, hours of informal care varied significantly between caregivers with a mean of 76 hours . Among caregivers, 79% did not earn an income, but all reported that none of the extra time spent caring for their child would have been normally spent in paid work.The mean healthcare provider costs of treating neonatal sepsis and meningitis were substantially higher in South Africa than Mozambique: I$684.06  vs I$307.58 , although this difference was smaller when including only microbiologically confirmed cases in Mozambique. Higher costs in South Africa can be partly explained by intercountry differences in resource unit costs, and by the shorter length of stay and more limited standard of care provided in Mozambique. However, the direct costs from the household perspective were more similar between the 2 countries. These were estimated to be I$49.62  in Mozambique and I$52.31  in South Africa. Such expenses were not considered catastrophic except for 2 households, 1 in each country, for whom these costs represented >40% of their monthly income [The lack of existing literature on this topic prevents any comparison with other studies in SSA. However, based on a few other LMIC studies, the hospitalization costs found in our study appear broadly consistent with previously reported cost of treating sepsis 2017 I$642) and meningitis (2017 I$198–I$8078) [2 and menWhile our study is one of the first to report costs associated with bacterial sepsis and meningitis in SSA, it has some limitations. First, our analysis was only descriptive since the small sample size prevented us from investigating the determinants of costs and differences between countries. Second, we collected data from only 1 hospital in each country, which together with the sample size limits the generalizability of our findings. Third, in Mozambique our results include suspected, as well as microbiologically confirmed cases of sepsis, which may underestimate the true costs. Last, this study does not include the healthcare costs associated with the longer-term impact of the disease such as neurodevelopment deficit. Furthermore, this study does not include preterm infants because it would require a larger sample size and controlling for potential cofounders, which is beyond the scope of this study.Despite these limitations, our multicountry study addresses an important gap in the literature that can inform policymaking. We found substantial costs associated with acute neonatal bacterial sepsis and meningitis in 2 countries in SSA, and our estimates will be useful to inform cost-effectiveness analyses of interventions to prevent invasive bacterial infections in neonates.Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciab815_suppl_Supplementary_MaterialClick here for additional data file.


The newly developed SWAT method allows achieving highly sensitive and highly specific DNA hybridizations. The detection and discrimination of the MTB and NTM strain in the clinical samples with the SBR and SNP discrimination ratios higher than 160:1 indicate the high clinical applicability of the SWAT.Highly sensitive (high SBR) and highly specific (high SNP discrimination ratio) DNA hybridization is essential for a biosensor with clinical application. Herein, we propose a method that allows detecting multiple pathogens on a single platform with the SNP discrimination ratios over 160:1 in the dynamic range of 10 Biosensors based on concurrent DNA-DNA hybridization for the simultaneous detection of various bacterial strains of the same species have unparallel importance in selecting accurate drug therapy ,2. DNA bm, °C), and high concentrations of chaotropic agents in hybridization buffers, have been used to increase the specificity  °C [m of the probes calculated by the nearest neighbor method was identical (±1.5 °C) with the Tm calculated by the latter approach  do not participate in hybridization. Similarly, the Tm of Probe3 reduces from 57 °C to 43 °C because three nucleotides (CCC) do not participate in hybridization. The decrease in respective Tm values is indicated by the significant reduction in signal intensities of Probe1 and Probe3 upon washing at a higher temperature. Here, the decrease in Tm due to mismatches is called Tmdm . In similar experiments, the Cy5-ssDNA target complementary to Probe1 showed specific hybridization with Probe1 and non-specific hybridization with Probe2 at 25 °C, indicated by the signal intensities of 60,000, respectively (Probe1 and Probe3 have one mismatch in their sequences compared to the probe binding region of the Cy5-ssDNA complementary to Probe2. Based on the explanation of ectively b.m of Probe2 for hybridization with the complementary Cy5-ssDNA target of Probe1 decreases from 57 °C to 45 °C because three nucleotides (GGC) do not participate in hybridization. The sharp decline in signal intensity of Probe2 upon washing at a higher temperature indicates the decrease in Tm. Similarly, the Tm of Probe3 for hybridization with the complementary Cy5-ssDNA target of Probe1 decreases from 57 °C to 33 °C because six nucleotides (CCC and GGC) do not participate in the hybridization process. The reduction in Tm is indicated by a lack of hybridization signal for Probe3, even at a washing temperature of 25 °C. Therefore, from the result of the hybridization behavior of Probe3 (Tm of 57 °C and Tmdm of 33 °C), it was concluded that washing at a temperature higher than Tmdm-10 °C could eliminate the non-specific hybridization and allow for the SNP discrimination ratio to be higher than 160:1.The signal intensity for the non-specific interaction of Probe2 with the Cy5-ssDNA target of Probe1 sharply decreases with the increase in washing temperature . Thus, by washing at 55 °C, the SNP discrimination ratio of Probe1 to Probe2 was only 2.2:1. However, it is interesting that Probe3 did not show any non-specific hybridization with the Cy5-ssDNA target complementary to Probe1. Therefore, even when washing at 25 °C, the SNP discrimination ratio of Probe1 to Probe3 was more than 160:1. These results of the non-specificity of Probe2 and the specificity of Probe3 can be rationalized by the fact that Probe2 has only one mismatch and Probe3 has two mismatches separated by five nucleotides, compared to the probe binding region of the Cy5-ssDNA target complementary to Probe1. Therefore, based on the explanation of m = 53 °C)) by adding the artificial mutation at the 5th position from the 3′ end. Probe2 was changed to Probe5 (5′-TAC CGG CCT ACG CGG GC-3′ (Tm = 55 °C)) by adding the artificial mutation at the 9th position from the 3′ end. Probe3 was changed to Probe6 (5′-TAC CGG CCC ACC TGG GC-3′ (Tm = 55 °C)) by adding the artificial mutation at the 5th position from the 3′ end.To further understand the concept, new probes were designed containing one artificial mutation see the . Probe1 As demonstrated in Similar results were also observed for the hybridization of the Cy5-ssDNA target complementary to Probe1 with Probe4, Probe5, and Probe6 c. Probe4m of Probe6 for the hybridization with complementary Cy5-ssDNA target of Probe2 decreases from 55 °C to 41 °C because four nucleotides (CC TG) do not participate in hybridization. Therefore, according to the Tmdm-10, the SNP-discriminating washing temperature for this probe should be higher at 31 °C. It is essential to note that the non-specific hybridization was observed as anticipated upon washing at 25 °C. The non-specific hybridization was eventually disappeared by hybridization at 35 °C, leading to a SNP discrimination ratio higher than 160:1.The hybridization patterns of Cy5-ssDNA targets complementary to Probe2 and Probe1 with Probe4, Probe5, and Probe6 can be rationalized by comparing their sequences. Probe6 has two consecutive mismatches compared to the probe binding region of the Cy5-ssDNA target complementary to Probe2. Therefore, the Tm of Probe4 for the hybridization with Cy5-ssDNA target complementary to Probe2 decreases from 53 °C to 23 °C because six nucleotides (GAC and GTG) do not participate in hybridization. Therefore, according to the Tmdm-10, the SNP discriminating washing temperature for this probe should be higher than 23 °C. Thus, Probe4 does not show non-specific hybridization with the complementary Cy5-ssDNA target of Probe2 at a washing temperature of 25 °C, which is higher than Tmdm-10.Similarly, Probe4 has two mismatches separated by more than four nucleotides, compared to the probe binding region of the Cy5-ssDNA target complementary to Probe2. The Tmdm for any given sequence can be obtained by the following equations,The data presented in MTB), Probe8 (M. Chelonae), Probe9 (M. Avium), Probe10 (M. Marinum), Probe11 (M. Abscessus), Probe12 (M. Kansasii), Probe13 (PC), Probe14 (PCR), and Probe15 (HC) on the AMCA slides, following the earlier report. The immobilized probes were allowed to hybridize at 25 °C with the Cy5-ssDNA obtained by the asymmetric PCR amplification of the targeted genomic DNA of the MTB and four NMT strains. The DNA chips were washed at 35 °C after hybridization in order to keep the experimental protocol simple. The obtained results are depicted in Therefore, the non-specific hybridizations can be eliminated by implementing washing temperatures higher than the SWAT after hybridization at 25 °C. The TB-NTM 9G DNAChips were used to investigate the applicability of the SWAT for the detection and discrimination of the MTB and NTM strains in the clinical samples. The TB-NTM 9G DNAChips were obtained by the immobilization of Probe7  remains a significant disadvantage of microarray-based methods. Several studies report on improving the SNP discrimination ratio by increasing the hybridization and washing temperatures (45–95 °C) and lengthy processes (4–16 h) . Higher In this regard, we have developed a method for determining the SNP-discriminating washing temperature (SWAT). The SWAT allows for the design of probes that do not show non-specific interactions, even at room temperatures, unlike the reported methods where they need to use high temperatures. The SWAT allows determining the washing temperature and helps to design the probe sequence such that the SBR and SNP-discriminating ratio is >160:1, compared to the reported methods (5:1). The present method is applied to detecting and discriminating MTB and well-known NTM strains responsible for infectious diseases. The results shown in In conclusion, the highly sensitive and specific hybridization of the target DNA of diagnostic importance with probes is an essential criterion for the clinical applicability of diagnostic methods based on DNA hybridizations. In this communication, we have presented an exemplary framework for the highly specific DNA hybridizations. The newly developed SWAT method allows achieving highly sensitive and highly specific DNA hybridizations with the hybridization and washing steps conducted at 25 °C. The detection and discrimination of the MTB and NTM strain in the clinical samples with the SBR and SNP discrimination ratios higher than 160:1 indicate the high clinical applicability of the SWAT. In turn, SWAT can boost the ongoing efforts to identify the MTB and respective NTM infections for accurate drug therapy.