Rattusrattus, Rattus norvegicus, and Musmusculus) within urban environments. Despite their potential impacton human health, relatively few studies have addressed the monitoring of theseviruses in rodents. Comprehensive control and preventive activities shouldinclude actions such as the elimination or reduction of rat and mousepopulations, sanitary education, reduction of shelters for the animals, andrestriction of the access of rodents to residences, water, and foodsupplies.This review focuses on reports of hepatitis E virus, hantavirus, rotavirus,coronavirus, and arenavirus in synanthropic rodents ( Rodents (Order: Rodentia) are distributed on all continents except forAntarcticaRattusrattus) were found in residential and industrial areas, while housemice (Mus musculus) and brown rats (Rattusnorvegicus) were captured in green areas and shantytownsIn a study conducted in Buenos Aires (Argentina), black rats Rattusrattus we,,,These animals are natural reservoirs of infectious diseases-Although little information is currently available on the size of the population ofsynanthropic rats, some indicators allow the subjective estimation of theirpresence, such as the presence of excrement, marks on walls, trails on the ground,food sources, or the visual observation of rodents and/or the damage caused bythem,To evaluate the size of rodent populations, the concept of the ‘minimum known number’is usedA survey carried out in 1529 dwellings in a low-income region of São Paulo (Brazil)showed an initial synanthropic rodent infestation rate of 40%, which was reduced to14.4% after the implementation of sanitary education and pest control,Lassa virus, among others-Besides synanthropic species, other rodents can cocirculate in rural environments andwild and urban interface areas, where they contact other animals and peopleOrthohantavirus, equally distributed betweenwomen and men (n=6). In contrast, similar studies showed higher frequency inmiddle-aged men due to their risk behaviors,Fernandes et al. (2019)Mastomysnatalensis infected by Lassa virus in these camps,which is facilitated by the dissemination routes of the virus Lassavirus.Refugee camps accommodate large number of people and consequently there isaccumulation of food and residues, attracting rodents (synanthropic and wild). InAfrica, studies have revealed the circulation of ,Aircrafts and ships may also contribute to the introduction of rodents and evendissemination of diseases in new areasM. musculus, R. rattus, andR. norvegicus), and thereby contribute to a betterunderstanding of disease epidemiology and prevention.Therefore, this article aims to review important viral agents disseminated bysynanthropic rodents collected in the field were selected, excluding the studies restricted to animalexperimentation.Among the resulting, studies mainly related to synanthropic rodents Orthohepevirus A and C havealready been described in rodentsThe hepatitis E virus (HEV) has a single-stranded RNA genome of approximately 7 kblength,Infection occurs via the fecal-oral route through the consumption of watercontaminated with excrement or the consumption of raw/undercooked meat and theviscera of infected animals,The virus shows tropism to digestive system and is eliminated in stool after 4 to 23days of infection-The symptoms are mainly nonspecific, and include fever, headaches, abdominal pain,but these infections may also be asymptomatic (depending on the HEV dose to whichthe patient was exposed), hindering the detection, and potentiating the agent’sspread,,Mortality rates are higher among infected people presenting previous liver disease,immunocompromised patients, and pregnant women, as they present higher chances ofrenal failure leading to deathR. rattus) and brown rats (R.norvegicus) as reservoirs and transmitters of this viral agent and itsprevalence rate remains unknownThe role of black rats (Orthohepevirus A has seven different genotypes (HEV1-7) defined bythe concatenated amino acid distance between the open reading frames of ORF1 and ORF2 (capsid proteins),,,R. norvegicus) strains with regard to the HEV-3 genotype,which is most closely related to the genotypes found in rabbitsThe presence of anti-HEV IgGrat HEV (genotype C1), belonging to theOrthohepevirus C group, has been reported in R.norvegicus and R. rattus, although its potential tocause disease in humans is still questioned,On the other hand, rat HEV,,In Vietnam, animals captured at bus stations and hospitals have tested positive forDetection methods for HEV include serological (specific IgG), histopathological, andmolecular (RT-PCR) techniquesHantaviridae family, are divided intofour genera: Loanvirus, Mobatvirus,Thottimvirus, and OrthohantavirusHantaviruses, belonging to the ProspectHill virusMore than 50 species of hantaviruses have been reported worldwideOrthohantavirus genus includes the greatest number of pathogenicspecies of public health importanceMurinae, Avicolinae, and Sigmodontinae families), which can harbordistinct forms of the disease.The Hantaan virus (HTNV) species was related to thisoutbreak. HTNV was isolated for the first time in 1978Apodemus agrarius; the viruswas detected in blood, urine, feces, and respiratory tract samples. In humans, thisspecies causes a severe form of HFRS, which has thus far been restricted to ruralareas of China, Korea, and Russia,The earliest reports of hemorrhagic fever with renal syndrome (HFRS), caused byviruses known as Old World hantaviruses (Europe and Asia), come from Chinesewritings dating from 960 BC. Later, in the Korean War (1951-1954), these diseasescaused the death of over 3,000 soldiersDobrava virus (DOBV) is also associated with HFRS syndrome, forwhich men are accidental hostsKurkino) to 12% (DOBV - Dobrava and DOBV -Sochi),Puumala virus (PUUV), is disseminated by Myodesglareolus, whose proliferation is favored by the underbrush vegetationof this region, and the spread of the virus is further affected by virus-hostcoevolutionThe most commonly detected viral agent of this group in Western Europe,Seoul virus usually causes mild infections and without medical carethe mortality rates reach 1%. The host of this virus (R.norvegicus) is found in urban areas, leading to a cosmopolitan distributionof the disease, in contrast to those caused by other Old World hantaviruses,El NiñoHantavirus cardiopulmonary syndrome (HCPS) is another pathology associated withagents of the genus New World hantaviruses (Americas), and appears to be related toclimatic phenomena such as Juquitiba virus, transmitted by Oligoryzomysnigripes, was detected by Silva et al. (1997). To date, the following viruses have been identified in thecountry: (a) Araraquara virus (transmitted by Necromyslasiurus rodents); (b) Castelo dosSonhos virus (Oligoryzomys utiaritensis); (c)Laguna Negra virus ; (d)Anajatuba virus (Oligoryzomys mattogrossae);and Rio Mamore virus (Oligoryzomys microtis),among othersThe first report of hantavirus in Brazil dates from 1993, whenThe incubation period of HCPS ranges between 16-24 days, with initial nonspecificHFRS-like symptomatology differentiated by pulmonary edema and lymphoid organimpairmentIn rodents, this virus causes a chronic infection with mild symptomatology such asdecreased growthSeoulvirus antibodies were found in R. norvegicus serum samplesAnjozorobe virus (Thailand Orthohantavirus)strain in R. rattus and M. musculus, suggestingviral spilloverThe occurrence of HFRS has not yet been reported in Brazil, despite serologicallypositive human and rodent samples-To our knowledge, there is no licensed vaccine available on the market that preventshantavirus infections. Several clinical trials at different stages are ongoing totest inactivated , DNA, and live attenuated vaccines forboth HFRS and HCPS, with effectiveness of approximately 93.77-100% beingreportedRotavirus genus within theSedoreovirinae subfamily of the Reoviridaefamily,Rotavirus (RV) belongs to the ,,,Myriad mechanisms of viral variability occur in RVs, such as point mutations,rearrangements, reassortments, and intragenic recombination, conferring greatgenetic diversity,-For the RVA group, it is necessary to adopt the notation ofGx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx, which considers all the variability presented bythe coding genes of the VP7-VP4-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 proteins,respectively,Although rotaviruses are considered species-specific, heterologous infections mayoccur,Synanthropic rodents are usually not associated with RVs, however, their efficiencyin disease transmission and their close contact with other animals and peoplehighlight their importance to the epidemiology of these virusesTransmission initially occurs through the fecal-oral route, via particles present inthe soil and water, causing diarrhea due to the loss of absorptive capacity ofinjured intestinal cells during viral replication,RVs are widely distributed in Brazil, and have been described in animals (both youngand adults) such as cattleR. norvegicus in Germany characterized asample of RVA, revealing close identity between the identified strain and otheranimal and human strains, namely, genotypes G3, P[3], and N2A metagenomic analysis of R. rattus collected on swine farmswere analyzed, and a sample of RVA was characterized(G3-P[3]-I1-R11-C11-M10-A22-N18-T14-E18-H13), demonstrating an atypical combinationof genotypesIn Italy, 40 fecal samples from ,,As the associated symptomatology is mainly nonspecific, the diagnosis can easily bemisleadingTo control this disease, animal vaccination (swine and cattle) should be carried out,mainly in females in the late gestation period. In production animals, prevalencerates may be higher than 90% in adults,Coronaviridae family;Letovirinae, which has one subgenus,Milecovirus, found only in frogs and a sea hare thus farOrthocoronavirinae, which is found in birds andmammals, and is divided into four genera due to the antigenic and geneticcharacteristics of the viruses, Coronaviruses are enveloped, nonsegmented, positive, single-stranded RNA virusesassociated with the structural N-phosphoprotein in a nucleocapsid with helicalsymmetryAlpha and Betacoronaviruses, while birds arethe gene source for Gama and DeltacoronavirusesAlpha and Betacoronaviruses arefound mainly in mammals, such as humans, dogs, cats, pigs, bats, mice, rats, horses,and cattle,-Gama and Deltacoronaviruses infectmainly birds, with exceptions such as the white whaleGamacoronavirus (Delphinapterus leucas)DeltacoronavirusPhylogenetic studies indicate that bats are the gene source forAlpha and Betacoronaviruses, six are ofpublic health importance, causing mild  to severerespiratory syndromes (SARS and MERS),,Among the Paguma larvata)Camelus dromedarius),,,Despite the many uncertainties about the epidemiology and reservoirs of severe acuterespiratory syndrome (SARS) and Middle Eastern respiratory syndrome (MERS), batshave been identified as the most likely reservoirs, while palm civets, membrane (M),and envelope (E) proteins. In addition, some coronaviruses include hemagglutininesterase (HE),The S protein, which shows great variability, is responsible for host specificitybecause its S1 and S2 subunits are used for binding the virus to host cellreceptors, and are associated with the antigenicity and pathogenicity of thevirus,,,Apodemus sp., Myodes glareolus,Arvicola terrestris, and Microtus sp.),collected between 2014 and 2016 in different regions of FranceAlphacoronavirus groups. This study also revealedAlpha and Betacoronavirus in bats, rabbits,and hedgehogs from the same areaMany similarities exist between the CoVs of rats and batsApodemus chevrieri, Apodemus ilex, andEothenomys fidelis), and found Alpha andBetacoronavirus in 13% (23 samples). An investigation conducted in China,Alpha andBetacoronaviruses  have beenidentified in rodents in China and Europe,,,Besides field investigations, rodent CoV also has an important role as Murinehepatitis virus (MHV) Some vaccine candidates are being developed only for MERS-CoV, includingwhole-virus, vectored virus, DNA, and protein-based vaccines, however, lack ofinvestment is delaying their developmentArenavirus, familyArenaviridae) are enveloped viruses with an RNA genome segmented intwo ambisense single stranded molecules: small (S) and large (L),-Arenaviruses  of complete genomesMammarenaviruses are correlated to the geographical location wheretheir hosts are foundLassa virus,Lujo virus, among others), and found mainly in Africa inrodents of the Murinae family as natural hostsLymphocytic choriomeningitis virus (LCMV) belongsto this category, it circulates globallyM. musculusJunin virus - ArgentinaMachupo virus - BolíviaSabiá virus - BrazilGuanarito virus - VenezuelaSigmodontinae rodents are the main hosts of this class,even though Tacaribe virus has already been described in batsAmblyomma americanum ticksMembers of both OW and NW arenaviruses can cause hemorrhagic fever and severe humandiseases affecting the central nervous systemM. musculus, collected fromresidential areas, detected 10% (8/80) of positives in serological analysis forLCMV. When brain samples of the same animals were submitted to RT-PCR, serologicallynegative individuals showed positive results in this second analysis, highlightingthe importance of parallel diagnosisIn Colombia, a study conducted with M.musculus) of which two were positive for LCMV by hemi-nested PCR (fromlung and kidney samples)A research conducted in French Guiana sampled 37 animals M.musculus Lassa virus (OW) is endemic in African countries, withseroprevalence reaching about 50% of the human population; this disease causes about5,000 deaths every yearMastomys natalensis is considered the main reservoir ofthe virusHylomyscus pamﬁ andMastomys erythroleucusLassavirus in M. natalensis and M.erythroleucus, R. rattusM. musculusR. rattus andR. norvegicus showed positive rates of 75% and 17%,respectively, and a new viral species, the Wenzhou virus, wasisolatedSurveys conducted in Nigeria reported positive animals for Junin virus is considered endemic in ArgentinaSabiá virus, reportedin São Paulo (Brazil) in 1994. Initial symptoms were described as flu-like , quickly leading to hemorrhage and death (within3 days),,In Brazil, the most remarkable arenavirus is Pinhal virus, has been characterized as aNew World arenavirus (line C), first isolated from vesper mice  in São Paulo (Brazil), but there is still no evidence that thisviral strain causes disease to humans,Pinhal virus, other arenaviruses have beenreported in Brazil: Xapuri virus was recently isolated from rodents(Neacomys musseri); Amaparí virus(Neacomys guianae); Cupixi virus; Flexal virus(unidentified Oryzomyini rodent); Oliveros virus(Necromys lasiurus); Latino virus andAporé virus (Oligoryzomys mattogrossae)A new arenavirus, namely Arenaviruses can be diagnosed using: (a) RT-PCR  followedby viral RNA sequencing for differentiation; (b) serology, through detection ofspecific IgG and IgM employing immunofluorescence and/or ELISA tests; (c) viralisolation in cell culture.-Recommended treatment is support therapy that can be combined with the antiviralribavirin, which should be administered during the first 7-10 days after infection.Despite its efficacy, there are significant side effects, such as hemolytic anemia,progressive weight loss, respiratory difficulty, insomnia, and dermatitis, amongothersR. rattus, R.norvegicus, and M. musculus, although with theadvancement of human populations, the interaction with wild rodents increases, anddifferent viruses can emerge. There are relatively few studies addressing themonitoring of viruses in these hosts, favoring the occurrence of outbreaks.In literature we find description of viral rodent-infections, usually from within thecontext of biological models or experimentation. In this review, we focus on rodentswithin an urban environment, especially Control and preventive activities should go beyond the elimination or reduction ofthe populations of these hosts and involve sanitary education to aid the humanpopulation in the reduction of shelters for the hosts, the restriction of rodentaccess to residences, and the reduction of their water and food supply. Basicsanitation actions are a generic but effective measure in the reduction of rodentsand, consequently, the propagation of diseases.


Eptesicus fuscus) is widely distributed and lives in small colonies that roost in cavities, often in human dwellings, leading to frequent human interaction. Viral metagenomic sequencing of samples from an E. fuscus bat submitted for rabies testing identified the first exogenous bat Deltaretrovirus. The E. fuscus deltaretrovirus (EfDRV) genome consists of the typical deltaretrovial 5′-gag-pro-pol-env-3′ genes along with genes encoding two putative transcriptional transactivator proteins distantly related to the Tax protein of human T-cell lymphotrophic virus and nuclear antigen 3B of Epstein-Barr virus. Searches of the E. fuscus genome sequence failed to identify endogenous EfDRV. RT-PCR targeting the EfDRV pol gene identified 4/60 (6.7%) bats with positive results. Together, these results suggest that EfDRV is exogenous. As all members of Deltaretrovirus are associated with T- and B-cell malignancies or neurologic disease, further studies on possible zoonosis are warranted.Bats host a large numbers of viruses, many of which are zoonotic. In the United States, the big brown bat ( Coronaviridae (severe acute respiratory syndrome coronavirus [SARS-CoV] and SARS-CoV-2), Paramyxoviridae (Hendra and Nipah viruses), Rhabdoviridae (rabies virus), and Filoviridae (Ebola virus) as exemplars. Many retroviruses, such as human immunodeficiency virus, are similarly zoonotic; however, only infectious exogenous gammaretroviruses have recently been identified in bats. Here, viral metagenomic sequencing of samples from bats submitted for rabies virus testing, largely due to human exposure, identified a novel, highly divergent exogenous Deltaretrovirus from a big brown bat (Eptesicus fuscus) in South Dakota. The virus sequence, corresponding to Eptesicus fuscus deltaretrovirus (EfDRV), comprised a nearly complete coding region comprised of canonical 5′-gag-pro-pol-env-3′ genes with 37% to 51% identity to human T-lymphotropic virus (HTLV), an infectious retrovirus that causes T-cell lymphoma. A putative tax gene with 27% identity to HTLV was located downstream of the pol gene along with a gene harbored in an alternative reading frame which possessed a conserved domain for an Epstein-Barr virus nuclear antigen involved in gene transactivation, suggesting a regulatory function similar to that of the deltaretrovirus rex gene. A TaqMan reverse transcriptase PCR (RT-PCR) targeting the pol gene identified 4/60 (6.7%) bats as positive for EfDRV, which, combined with a search of the E. fuscus genome that failed to identify sequences with homology to EfDRV, suggests that EfDRV is an infectious exogenous virus. As all known members of Deltaretrovirus can cause malignancies and E. fuscus is widely distributed in the Americas, often with a colonial roosting behavior in human dwellings, further studies are needed to investigate potential zoonosis.Bats are the reservoir for a large number of zoonotic viruses, including members of IMPORTANCE Bats host a large numbers of viruses, many of which are zoonotic. In the United States, the big brown bat (Eptesicus fuscus) is widely distributed and lives in small colonies that roost in cavities, often in human dwellings, leading to frequent human interaction. Viral metagenomic sequencing of samples from an E. fuscus bat submitted for rabies testing identified the first exogenous bat Deltaretrovirus. The E. fuscus deltaretrovirus (EfDRV) genome consists of the typical deltaretrovial 5′-gag-pro-pol-env-3′ genes along with genes encoding two putative transcriptional transactivator proteins distantly related to the Tax protein of human T-cell lymphotrophic virus and nuclear antigen 3B of Epstein-Barr virus. Searches of the E. fuscus genome sequence failed to identify endogenous EfDRV. RT-PCR targeting the EfDRV pol gene identified 4/60 (6.7%) bats with positive results. Together, these results suggest that EfDRV is exogenous. As all members of Deltaretrovirus are associated with T- and B-cell malignancies or neurologic disease, further studies on possible zoonosis are warranted. Bats are a large, ancient, and incredibly diverse order of mammals that inhabit all continents and ecosystems other than Antarctica . ImportaRetroviridae consists of a large, diverse array of single-stranded RNA (ssRNA) viruses that cause a number of clinically and economically significant diseases , causes losses to the U.S. dairy industry in excess of 200 million dollars annually due to decreased cow longevity, milk production, and sale price due to carcass condemnation resulting from lymphoma (The remaining lymphoma . Additiolymphoma . BLV haslymphoma .Eptesicus fuscus) submitted for rabies detection that tested negative for rabies virus to screen for potential novel and zoonotic viruses using viral metagenomic sequencing.Compared to domestic animals, very little viral surveillance is performed in wildlife. Bats are a reservoir of rabies virus and represent the most commonly detected rabies carriers in the United States. Additionally, bats are the reservoir of numerous zoonotic diseases worldwide and so are of particular concern when exposed to humans , 12. Hern = 60) were submitted to the South Dakota State University Animal Disease Research and Diagnostic Laboratory between March and July 2020. The lungs and heart were dissected from 20 bats and homogenized in a minimal volume of phosphate-buffered saline before centrifugation was performed at 20,000 × g for 5 min to pellet debris. Next, 80 μl of sample was transferred to a microcentrifuge tube and treated with a cocktail of nucleases to digest unprotected nucleic acids. Nucleic acid isolation, reverse transcription, second strand synthesis, and amplification were performed as previously described  most similar to HTLV. Metagenomic sequencing was performed on this sample multiple times. Approximately 1.1 million combined sequencing reads were used to assemble a contig 6,597 bp in length that was verified by Sanger sequencing. The sample was collected from a bat found dead in a private residence in South Dakota.gag in the pro overlap region that is very similar to the conserved slippage sequence 6(A)-8 nt-6(G)-11 nt-6(C). Similarly, a ribosomal slippage sequence matching HTLV-1 and HTLV-2 (TTTAAAC) was identified near the predicted 3′ end of pro in the region that overlaps pol. A putative Tax homolog was identified immediately downstream of env based on 25% identity with HTLV. An ORF-overlapping env encoded a predicted 238-amino-acid (a.a.) protein which failed to show significant homology to known proteins by BLASTP; however, it did possess a conserved domain for an Epstein-Barr virus nuclear antigen involved in gene transactivation, suggesting a regulatory role in viral replication similar to that identified for the deltaretrovirus rex gene  compared to adenine, guanine, and thymine (21.0% to 24.4%).Open reading frame (ORF) and BLASTP analysis identified viral proteins Gag-Pro-Pol-Env with 37% to 51% identity to proteins encoded by HTLV and STLV . Translarex gene . A finalRetroviridae genera. Also included in this analysis were Gag sequences from an endogenous Deltaretrovirus identified in Miniopteridae genomes  to the EfDRV genome. Next, a TaqMan PCR was designed targeting the EfDRV pol gene using the following primers and probe: forward primer, 5′-CTATTTCCTGGCTACTGACACC; reverse primer, 5′-GGTAAGGTAGTGATGGTGCG; probe, 5′-6-carboxyfluorescein (FAM)-CCTCGTGATTTGGCTCCTTGGGT. Quantitative reverse transcriptase PCR (QRT-PCR) was performed on the 20 bat samples subjected to metagenomic sequencing and additionally on 40 viscera homogenates prepared from bats submitted for rabies virus testing. Four of the 60 (6.7%) bats were positive for EfDRV , including the sample where EfDRV was identified by metagenomic sequencing. Amplification and sequencing of an 847-bp product spanning parts of the pro and pol genes using primers 621F (5′-CTCCTGCGAGCTTGTGCTAATG) and 1624R (5′-GGGAGAACTTCCCAAGCGTAAC) found 97% to 100% identity to the EfDRV genome. Treatment of sample nucleic acids with DNase did not affect Eptesicus fuscus deltaretrovirus (EfDRV) detection by QRT-PCR, while treatment with RNase rendered EfDRV undetectable. Together, these results suggest that EfDRV is an exogenous infectious virus.A BLASTN search of the Chlamydia infection (23–Miniopterus endogenous retrovirus.Recently, multiple exogenous gammaretroviruses related to koala retrovirus were identified in bats from Asia and Australia . Prior tnfection , 23–25. Deltaretrovirus and represents only the second report of an exogenous bat retrovirus. The identification of EfDRV from a bat with human exposure raises questions of possible zoonosis, considering that all members of this genus cause disease. Further research and surveillance are needed to determine if EfDRV poses a risk to human or animal health.EfDRV is the first described bat MT700539. Sequence reads were archived as BioProject PRJNA659740.The sequence described in this work was submitted to GenBank under accession no.


The COVID-19 pandemic has necessitated a multifaceted rapid response by the scientific community, bringing researchers, health officials, and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. The COVID-19 pandemic has necessitated a multifaceted rapid response by the scientific community, bringing researchers, health officials, and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction, and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals, including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens. Infection with the novel human betacoronavirus SARS-CoV-2 can cause a severe or fatal respiratory disease, termed COVID-19 13. As the4–Efficacy of virus inactivation depends on numerous factors, including the nature and concentration of pathogen, sample matrix, concentration of inactivation agent(s), and contact time. To date, there are limited data on the efficacy of SARS-CoV-2-specific inactivation approaches in the scientific literature, and risk assessments have largely been based upon inactivation information for genetically related coronaviruses. Previous studies have found that treatment with heat, chemical inactivants, UV light, gamma irradiation, and a variety of detergents is effective at inactivating the high-consequence human coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) –13. Howe–16–8 PFU/ml) to be able to demonstrate a significant titer reduction and reduces recovery of low-level residual virus from treated samples, making it difficult or impossible to distinguish complete from incomplete virus inactivation. In contrast, methods for purification of virus away from cytotoxic components in treated samples may also remove virus or affect virus viability. Accurate quantification of remaining infectious virus ideally requires complete removal of cytotoxicity without compromising assay sensitivity, which needs careful consideration of reagent and purification processes prior to the performance of inactivation tests.An important consideration in inactivation efficacy assay development is cytotoxicity, a typical effect of many chemical inactivants. To mitigate cytotoxic effects, the inactivation agent needs to be either diluted out or removed from treated samples prior to testing for infectious virus. Each of these methods for addressing cytotoxicity presents its own challenges. Sample dilution requires the use of high-titer stocks of virus . For plaque assays, 24-well plates were seeded the day before the assay (1.5 × 105 cells/well in MEM–10% FCS). Tenfold dilutions of virus stock were inoculated onto plates (100 μl per well) at room temperature for 1 h, overlaid with 1.5% medium-viscosity carboxymethylcellulose (Sigma-Aldrich), and incubated at 37°C in 5% CO2 for 3 days. For TCID50, 10-fold dilutions of virus stock (25 μl) were plated onto 96-well plates containing Vero E6 cell suspensions (2.5 × 104 cells/well in 100 μl of MEM–5% FCS) and incubated at 37°C in 5% CO2 for 5 to 7 days. Plates were fixed with 4% (vol/vol) formaldehyde–phosphate-buffered saline (PBS) and stained with 0.2% (vol/vol) crystal violet-water. TCID50 titers were determined by the Spearman-Kärber method  were cultured in modified Eagle's minimum essential medium (MEM) supplemented with 10% (vol/vol) fetal calf serum (FCS). Virus used was the SARS-CoV-2 strain hCOV-19/England/2/2020, isolated by PHE from the first patient cluster in the United Kingdom on 29 January 2020. This virus was obtained at passage 1 and used for inactivation studies at passage 2 or 3. All infectious work was carried out using a class III microbiology safety cabinet (MSCIII) in a CL3 laboratory. Working virus stocks were generated by infecting Vero E6 cells at a multiplicity of infection (MOI) of 0.001 in the presence of 5% FCS. Cell culture supernatants were collected at 72 h postinfection, clarified for 10 min at 3,000 × r method , 23.The commercial reagents evaluated in this study, along with their compositions (if known) and manufacturers’ instructions for use (if provided), are given in Table S1 in the supplemental material. Specimen transport reagents tested were the following: Sigma molecular transport medium , eNAT (Copan), Primestore molecular transport medium , Cobas PCR medium (Roche), Aptima specimen transport medium (Hologic), DNA/RNA Shield (Zymo Research), guanidine hydrochloride (GCHl) and guanidine thiocyanate (GITC) buffers containing Triton X-100 , and virus transport and preservation medium (inactivated) (BioComma). Molecular extraction reagents tested were the following: AVL, RLT, and AL ; MagNA Pure external lysis buffer and Cobas Omni LYS used for on-board lysis by Cobas extraction platforms (Roche); viral PCR sample solution (VPSS) and lysis buffer ; NeuMoDx lysis buffer (NeuMoDx Molecular); Samba II SCoV lysis buffer ; NucliSENS lysis buffer (bioMérieux); Panther Fusion specimen lysis tubes (Hologic); and an in-house extraction buffer containing guanidine thiocyanate and Triton X-100 (PHE Media Services). Detergents tested were the following: Tween 20, Triton X-100, and NP-40 Surfact-Amps detergent solution  and UltraPure SDS 10% solution (Invitrogen). Other reagents assessed included polyhexamethylene biguanide , formaldehyde and glutaraldehyde , and ethanol and methanol .g to remove PBS immediately before use. For all matrices aside from the Amicon Ultra columns, 100 μl of treated sample was added to each spin column, incubated for 2 min at room temperature, and then eluted by centrifugation at 1,000 × g for 2 min. For Amicon Ultra filters, 500 μl of sample was added and centrifuged at 14,000 × g for 10 min, which was followed by three washes with 500 μl of PBS. Sample was then collected by resuspending contents of the filtration device with 500 μl of PBS. To assess remaining cytotoxicity, a 2-fold dilution series of treated filtered sample was prepared in PBS, and 6.5 μl of each dilution was transferred in triplicate to 384-well plates containing Vero E6 cells (6.25 × 103 cells/well in 25 μl of MEM–5% FCS) and incubated overnight. Cell viability was determined by a CellTiter Aqueous One Solution cell proliferation assay (Promega) according to the manufacturer’s instructions. Normalized values of absorbance (relative to untreated cells) were used to fit a four-parameter equation to semilog plots of the concentration-response data and to interpolate the concentration that resulted in 80% cell viability (20% cytotoxic concentration [CC20]) in reagent-treated cells. All analyses were performed using GraphPad Prism .Specimen transport tube reagents were assessed undiluted unless otherwise indicated. For testing of molecular extraction reagents, mock samples were generated by diluting reagent in PBS at ratios given in the manufacturers’ instructions. Detergents, fixatives, and solvents were all assessed at the concentrations indicated in 6 to 1 × 108 PFU/ml) were treated in triplicate with reagents at concentrations and for contact times recommended in the manufacturers’ instructions for use, where available, or for concentrations and times specifically requested by testing laboratories. Where a range of concentrations was given by the manufacturer, the lowest ratio of product to sample was tested . Specimen transport tube reagents were tested using a ratio of 1 volume of tissue culture fluid to 10 volumes of reagent, unless a volume ratio of sample fluid to reagent was specified by the manufacturer. Detergents, fixatives, and solvents were tested at the concentrations and times indicated in the tables. All inactivation steps were performed at ambient room temperature (18 to 25°C). For testing of alternative sample types, virus was spiked into the indicated sample matrix at a ratio of 1:9 and then treated with test reagents as described above. All experiments included triplicate control mock-treated samples with equivalent volumes of PBS in place of test reagent. Immediately following the required contact time, 1 ml of treated sample was processed using the appropriately selected filtration matrix. Reagent removal for inactivation testing was carried out in a larger spin column format using Pierce 4-ml detergent removal spin columns (Thermo Fisher) or by filling empty Pierce 10-ml-capacity centrifuge columns (Thermo Fisher) with SM2 Bio-Beads, Sephacryl S400HR, or Sephadex LH-20 to give 4 ml of packed beads/resin. For purification using Amicon filters, two 500-μl samples were purified using two centrifugal filters by the method previously described and then pooled. For formaldehyde and formaldehyde with glutaraldehyde removal, one filter was used with one 500-μl sample volume, which was resuspended after processing in 500 μl of PBS and added to 400 μl of MEM–5% FBS. For inactivation of infected monolayers, 12.5-cm2 flasks of Vero E6 cells (2.5 × 106 cells/flask in 2.5 ml of MEM–5% FBS) were infected at an MOI of 0.001 and incubated at 37°C in 5% CO2 for 24 h. Supernatant was removed, and cells were fixed using 5 ml of formaldehyde or formaldehyde and glutaraldehyde at room temperature for 15 or 60 min. The fixative was removed, and monolayers were washed three times with PBS before cells were scraped into 1 ml of MEM–5% FBS and sonicated (three cycles of 10 s on and 10 s off at 100% power and amplitude) using a UP200St with VialTweeter attachment (Hielscher Ultrasound Technology). Supernatants were clarified by centrifugation at 3,000 × g for 10 min.For commercial products, virus preparations  sample was titrated to determine residual test buffer cytotoxicity. For TCID50 assays, neat to 10−7 10-fold dilutions were prepared, and for plaque assays, neat to 10−5 10-fold dilutions were prepared, both in MEM–5% FCS. TCID50 titers were determined by the Spearman-Kärber method  were inoculated with either 500 μl or 50 μl of treated filtered sample. Flasks were examined for cytopathic effect (CPE), and 500 μl of culture medium from each flask was used to inoculate new 12.5-cm2 flasks of Vero E6 cells after 7 days. If no CPE was observed, this process was continued for up to four serial passages. For the duration of the passage series, a flask of untreated cells was included as a control for cross-contamination between flasks, and a SARS-CoV-2-infected control was included to ensure suitable conditions for virus propagation. To distinguish CPE from any residual cytotoxicity associated with test reagents, samples of cell culture medium were taken from each flask at the beginning and end of each passage. Nucleic acid was extracted from cell culture medium manually using a QIAamp Viral RNA Minikit (Qiagen) or by using NucliSENS easyMAG or EMAG platforms . Viral RNA levels were quantified by quantitative reverse transcriptase PCR (qRT-PCR) specific for the SARS-CoV-2 E gene (TC) value across the course of a passage were observed.In parallel to virus quantification, 12.5-cm20 value for each combination of reagent and filtration method was interpolated . The CC20 was chosen as cells retain 80% viability at this concentration and enable distinction of active SARS-CoV-2 replication by visualization of CPE in the monolayer. 20 after filtration, with a value of <1 indicating complete removal of cytotoxicity. These data were used to determine the relative cytotoxicity removed by one filtration step for each combination of reagent and matrix , and Bio-Beads SM2 nonpolar polystyrene adsorbents. Reagents were diluted with PBS to the working concentrations recommended by the manufacturer  or to the concentrations indicated in the tables , followed by a single reagent removal step with each filtration matrix. Dilution series of filtered and unfiltered samples were generated to determine concentration-dependent cytotoxicity, from which the CCd matrix .20 although only the Bio-Bead SM2 filters were effective at removing all cytotoxicity. The chemical fixative combination of 2% formaldehyde and 1.5% glutaraldehyde was the most cytotoxic unfiltered, requiring a dilution of over 4,000 to reach the CC20, with only the Amicon Ultra columns able to remove 100% of the cytotoxicity. However, for the majority reagents (27/34) tested, filtration through at least one matrix type removed 100% of cytotoxicity, allowing neat eluate to be used directly in cell culture without further dilution. There were several exceptions to this: DNA/RNA Shield , 40% GHCl (99.1% using Pierce DRSC), 4 M GITC (99.7% using Pierce DRSC), MagNA Pure (99.7% using SM2), AL buffer (87.4% using S400HR), Cobas Omni LYS (97.0% using SM2), and NeuMoDx (93.4% using S400HR). For these reagents, filtered eluate was still cytotoxic when used undiluted in cell culture. However, CC20 values indicated that this remaining cytotoxicity would be removed by first or second (10−1 to 10−2) dilutions in the TCID50 assay, allowing evaluation of titer reduction using these reagents, with the caveat that the effective assay limit of detection (LOD) would be higher. Passing treated samples through more than one column or increasing the depth of the resin/bead bed within the spin column can also improve cytotoxicity removal for some reagents (unpublished data).All unfiltered reagents tested here were cytotoxic, but the degree of cytotoxicity varied considerably as did the optimal filtration matrix for each reagent. The detergent Tween 20 used at a 1% concentration was the least cytotoxic unfiltered, requiring a dilution factor of only 7.7 to reach the CC6 TCID50/ml, triplicate purifications of virus through Sephadex LH-20 or Pierce detergent removal spin columns resulted in recovery of 100% of input virus . The exceptions to this were PrimeStore MTM and 4 M GITC, from which no residual virus was detectable by either TCID50 assay or by the passaging of treated purified sample. While several contact times were evaluated for all of these reagents, length of contact time had no effect on either the level of virus titer reduction or whether virus remained detectable upon passage.Specimen transport tubes are designed to inactivate microorganisms present in clinical specimens prior to sample transport while preserving the integrity of nucleic acids for molecular testing. If effective, these products have the potential to streamline SARS-CoV-2 diagnostic processing in testing laboratories by eliminating the requirement for CL3 processing or, for activities carried out at CL2, permitting processing outside an MSC. The BS EN 14476 standard requires demonstration of a >4-logon tests , and we d matrix . However10 reduction in TCID50 titer for all but two molecular extraction reagents when they were evaluated using tissue culture fluid , shorter times (10 min) were as effective at reducing virus titers as longer contact times. Most reagents reduced viral titers to around the TCID50 assay LOD, indicating that any remaining virus posttreatment was present only at very low titers (<10 TCID50/ml), but higher levels of virus were recoverable from samples treated with some extraction buffers. For NeuMoDx lysis buffer, although titers were reduced by ≥4 log10 TCID50, an average of 91 (±38) TCID50/ml remained detectable. Similarly, buffer AVL reduced virus titers by 5.1 log10 TCID50, but after treatment virus was detectable in all treated sample replicates . However, after addition of four sample volumes of absolute ethanol following a 10-min contact time with AVL , a ≥5.9-log10 titer reduction was recorded, with no virus recoverable following passages in cell culture.We also sought to inform sample processing by examining inactivation by molecular extraction lysis buffers used in several manual and automated extraction protocols within SARS-CoV-2 diagnostic and research laboratories. We could demonstrate a ≥4-logre fluid . The exc10 titer reduction with no remaining infectious virus detectable. We additionally evaluated the tissue lysis buffer RLT using homogenized ferret lung as sample material, with treatment resulting in a ≥4.8-log10 titer reduction with no residual infectious virus detectable.Panther Fusion lysis buffer was further tested against a relevant clinical sample matrix, pooled fluid from oropharyngeal (OP) and nasopharyngeal (NP) swab specimens, resulting in a ≥5.1-log10 TCID50, respectively, while both concentrations of NP-40 reduced titers by ≥6.5 log10 TCID50 with no residual virus detectable following NP-40 treatment. In contrast, up to 0.5% Tween 20 had no effect on viral titers. Triton X-100 is commonly used in viral inactivation reagents, and here we show that at both 0.1% and 0.5% (vol/vol) concentrations, virus titers in tissue culture fluid were reduced by ≥4.9 log10 TCID50, even with less than 2 min of contact time  is a polymer used as a disinfectant and antiseptic, evaluated here as a potential lysis buffer, but it was able to reduce viral titers by only 1.6 log10 TCID50 at the highest concentration tested (2%).Fixation and inactivation of viruses by the addition of formaldehyde or of a combination of formaldehyde and glutaraldehyde is a well-established protocol, particularly for diagnostic electron microscopy , 31. A ttectable . When inSamples containing infectious SARS-CoV-2 require an initial inactivation step in primary containment  before further processing; given the rapid emergence of SARS-CoV-2, these inactivation protocols have been guided by existing data for other coronaviruses, and there is an urgent need to both confirm these historical data using the new virus and to validate new approaches for inactivating SARS-CoV-2. We therefore analyzed numerous commercially and commonly available reagents used by public health agencies and research laboratories around the world in their response to the pandemic. In addition, to address challenges of reagent cytotoxicity in inactivation evaluation, we provide data on the effectiveness of filtration methods for removing cytotoxicity from chemically treated samples.10 TCID50/ml  effectiveness of cytotoxicity removal, (ii) efficiency of virus recovery, and (iii) the ease of performing these methods within a containment space. Methods permitting complete removal of cytotoxic reagent components with no or little effect on virus recovery give assurance that low levels of residual virus, if present, could be detected in virus inactivation studies. During reagent testing, there were several instances where we noted residual cytotoxicity in the neat eluate, which is contrary to what was expected based on the initial reagent removal data and is likely due to the extended incubation period required for inactivation testing . In all cases, however, we were still able to enhance the levels of titer reduction detectable compared with what would have been achieved by sample dilution alone.In conclusion, we have evaluated methods for straightforward, rapid determination of purification options for reagents prior to inactivation testing, enabling establishment of effective methods for sample purification while minimizing virus loss. This is applicable to inactivation studies for all viruses (known and novel), not only SARS-CoV-2. We have applied these methods to obtain SARS-CoV-2 inactivation data for a wide range of reagents in use (or proposed for use) in SARS-CoV-2 diagnostic and research laboratories. In addition to guiding laboratory risk assessments, this information enables laboratories to assess alternative reagents that may be used for virus inactivation and nucleic acid extraction, particularly considering concerns about extraction reagent availability due to increased global demand caused by the COVID-19 pandemic. Furthermore, chemical treatments evaluated here are commonly used for inactivation of a wide range of different viruses and other pathogens, and the results presented may be used to directly inform and improve the design of future inactivation studies.


There is a great deal of variation in reported droplet production between different studies [e.g., Duguid , these fine droplets were detected 8 m downwind of the source and their maximum vertical position with distance downwind showed that some were still moving upwards, meaning that they were behaving as an aerosol affected by eddy diffusion  of a spherical object with the density of water in still air at 20°C is approximated to 0.00308 multiplied by the square of the particle diameter (in μm) . So, a rFactors affecting the survival and dispersal of the virus include the material the droplet is formed of . The loss of water by evaporation from the surface of droplets during their flight may affect the activity (activity is used here because the virus particle itself is not a viable organism but when active can infect a living cell) or loss of activity of the infectious agent being transported because the water may partially protect the enclosed virus from desiccation and variations of temperature, and exposure to oxygen, ozone, and other chemicals. Estimates made for SARS-CoV-2 persistence of activity while suspended in air range from up to 30 min to a half-life in air of several hours –19. Howe50 (the cut-off for collection of 50% of particles) values to be over 4 μm, meaning that droplets smaller than this are increasingly less likely to be collected (Following the study of Perryman et al. , it is collected .The persistence of activity of SARS-CoV-2 particles in airborne droplets appears to be sufficient to pose a threat. Droplets may not even require coughing because production of fine droplets containing influenza virus particles has been demonstrated simply from normal breathing . If subsThe article was written by JW and SP. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vector transmission of plant viruses is basically of two types that depend on the virus helper component proteins or the capsid proteins. A number of plant viruses belonging to disparate groups have developed unusual capsid proteins providing for interactions with the vector. Thus, cauliflower mosaic virus, a plant pararetrovirus, employs a virion associated p3 protein, the major capsid protein, and a helper component for the semi-persistent transmission by aphids. Benyviruses encode a capsid protein readthrough domain (CP-RTD) located at one end of the rod-like helical particle, which serves for the virus transmission by soil fungal zoospores. Likewise, the CP-RTD, being a minor component of the luteovirus icosahedral virions, provides for persistent, circulative aphid transmission. Closteroviruses encode several CPs and virion-associated proteins that form the filamentous helical particles and mediate transmission by aphid, whitefly, or mealybug vectors. The variable strategies of transmission and evolutionary ‘inventions’ of the unusual capsid proteins of plant RNA viruses are discussed. Potyviridae family [Bromoviridae), a small icosahedral virus composed of 180 identical CP subunits whose aphid transmission depends solely on the capsid protein [Transmission of plant viruses is a complex virus–vector–host interplay tuned in the course of evolution. This process is basically of two types, one specified by the requirement for a virus helper component protein (HC), which serves as an ‘adaptor’ between the virus capsid protein (CP) and a receptor in the vector, and the other by the direct interaction of the assembled CP with a vector receptor ,2,3. Thee family ,10. A cl protein ,12.In the process of evolution, a number of plant viruses have developed peculiar capsid proteins providing for interactions with the vector. Thus, CaMV, apart from the nonvirion helper component, employs the capsid protein p3 for semi-persistent aphid transmission. The capsid protein readthrough domain (CP-RTD), being a minor component of icosahedral virions, mediates persistent, circulative aphid transmission of luteoviruses. Likewise, benyviruses encode the CP-RTD exposed at one end of the rod-like helical particle, which serves for the virus transmission by soil fungal zoospores. Closteroviruses encode several CPs and virion-associated proteins that form a complex structure of the filamentous helical particles and provide for the transmission by aphid, whitefly, or mealybug vectors. This review focuses on variable transmission strategies and evolutionary ‘inventions’ of the unusual capsid proteins of plant viruses. An insight into the transmission mechanisms of these viruses is not only of fundamental but also of practical interest in view of significant economic losses from diseases such as beet rhizomania , cereal Cauliflower mosaic virus, a plant pararetrovirus, has a circular 8-kbp DNA genome and icosahedral particles (approximately 50 nm in diameter) built of 420 CP subunits . After tBrevicoryne brassicae and Myzus persicae are the major field vectors [CaMV is semi-persistently transmitted by several aphid species of which  vectors . The vir vectors . CaMV pa vectors ,21. The  vectors ,6,7 , BYDV MAV (Macrosiphum (Sitobion) avenae), BYDV RMV (Rhopasosiphum maidis) [Luteoviruses are transmitted by aphids in a persistent, circulative mode, with acquisition and inoculation access times of many hours and virus retention throughout life of the vector. The virus-vector interactions are highly specific, with each luteovirus transmitted by one or a few aphid species ,31. The Acyrthosiphon pisum, directly binds to the major CP of pea enation mosaic enamovirus and may be a receptor for the virus [Buchnera spp., binds luteovirus particles and influences their stability and transmission [Gray and Gildow  recognizhe virus . Severalhe virus ,35. An asmission , althougsmission .On the virus side, the CP-RTD appears to be the key transmission receptor. Luteovirus CP-RTD is a multifunctional protein containing the major CP domain, a proline-rich spacer between the CP and RTD sequences, the conserved N-terminal and variable C-terminal regions of the RTD ,37. The Benyviridae) is the causal agent of rhyzomania, a devastating and widely distributed disease of sugar beet [Beet necrotic yellow vein virus , which explains the benyvirus survival in the infested soil for years [P. betae, but the question as to whether the virus replicates in the vector remains unclear [BNYVV and several other viruses associated with rhyzomania  are transmitted by zoospores of soil fungus ot cells ,46. Thisot cells . Once thor years ,48. The P. betae zoospores [BNYVV particles are transmitted by the viruliferous zoospores upon fusion of the vector and the host cytoplasm . This isoospores ,53. It rClosteroviridae includes about 40 plant positive-sense RNA viruses characterized by several distinct features: (1) Semi-persistent mode of insect transmission (with the acquisition and inoculation access times of at least 30 min and the retention of a virus in its vector for up to 72 h); (2) phloem-limited or semi-phloem limited mode of plant infection ; (3) unique ‘rattlesnake-like’ structure of flexuous helical symmetry particles built of four proteins; (4) large RNA genomes (up to 20 kb); (5) the presence of the gene for a homolog of cell HSP70 chaperones and the duplicated genes or gene fragments  [Closterovirus, Ampelovirus, and Velarivirus) or divided among two or three genomic RNA segments (genus Crinivirus) (The family nor CPs) ,55,56. Tnivirus) .Crinivirus members that have the divided genomes , whiteflies (genus Crinivirus), or mealybugs (genus Ampelovirus). Transmission of lettuce infectious yellows virus (LIYV) by Bemisia tabaci New World has been studied in considerable depth with respect to vector retention sites and virus determinants of transmission [B. tabaci on artificial diets with LIYV particles or recombinant capsid proteins, followed by diets with fluorescently labeled antibodies to visualize the virus and the CPs in the vector by fluorescence microscopy and confocal laser scanning microscopy. It was found that LIYV associated with the anterior foregut (cibarium) of the vector biotype A, but not of the non-vector biotype B (MEAM1) [B. tabaci [Closteroviruses are semi-persistently transmitted by aphids (members of the genus smission . Chen etsmission  used seq (MEAM1) . Whitefl (MEAM1) . This is. tabaci . A frame. tabaci . It shou. tabaci ,75. Neve. tabaci , possiblClosterovirus genus are semi-persistently transmitted by aphids. The mode and parameters of BYV transmission by green peach aphid Myzus persicae were described in early studies [Myzus pesicae and Aphis fabae are the most efficient and are the principal vectors in the field [Toxoptera citricida is the most efficient vector [M. persicae detected by electrical penetration graph technique, where the vector’s stylet penetrates the membrane of phloem cells prior to the phloem salivation [Toxoptera citricida. The fluorescent CTV particles were associated with the lining of the aphid’s cibarium, and this specific binding was reduced by competition with extraneously added CPm, HSP70h, or p60, but not with any other CTV-encoded protein  [Members of the  studies ,77. The t vector . Transmilivation . Killinylivation  used the or p13) . Pre-tre or p13) . Ampelovirus are semi-persistently transmitted by mealybugs (Pseudococcidae) and soft scale insects (Coccidae) [Pseudococcus longispinus or Phenacoccus aceris, is capable of transmitting several individual viruses in the grapevine leafroll-associated virus complex [Planococcus citri, whereas Prator et al. [P. ficus. The virus binding was blocked in competition with the wheat germ agglutinin added to the mealybug artificial diet, thus providing further analogy with the CTV transmission pattern [Members of the genus occidae) ,82,83,84 complex , and fou complex . The dat complex  detectedr et al.  observed pattern . The virThe rapid evolution of viral RNA genomes, owing to the low fidelity of RNA-dependent RNA polymerases ,90 and RLuteoviridae and Benyviridae have adapted the unusual capsid protein CP-RTD clinging the virus particle to vector receptor(s) to ensure its retention and transmission. The TP-CP-RTD gene module of luteoviruses and the CP-RTD of benyviruses are likely to have arisen by gene overprinting, gene duplication, and divergence of coding sequences [Closteroviridae have followed a distinct evolutionary path that has led to acquisition of more complex  RNA replicase and super flexible capsids, which has allowed the expansion of their RNA genomes up to 19 kb [These evolutionary trends concern the capsid proteins influencing the plant RNA virus-vector interactions and vector transmission. Plant pararetroviruses have acquired a unique duet of proteins, the p3 CPm and p2 helper component, that mediate the virus attachment to a receptor in the aphid vector. Among RNA viruses, members of the families equences . The Cloto 19 kb ,97. The to 19 kb , the CPmto 19 kb ,55,56. Cto 19 kb ,100.Despite some progress in studying the transmission determinants of caulimoviruses, luteoviruses, benyviruses, and closteroviruses, many gaps remain in our knowledge, particularly concerning the spatial structure of virus proteins mediating vector transmission and the mechanisms of their interaction with the vector receptors. Further studies are expected to give insights into the co-evolution of the virus–vector relationships and their molecular biological nature, thereby providing a basis for the development of measures to control virus transmission and reduce crop losses from the diseases such as rhyzomania, citrus tristeza, and cereal yellow dwarf.


We report the discovery of a new orthobunyavirus, Cristoli virus, by means of shotgun metagenomics. The virus was identified in an immunodepressed patient with fatal encephalitis. Full-length genome sequencing revealed high-level expression of a virulence factor, possibly explaining the severity of the infection. The patient’s recent history suggests circulation in France. Orthobunyavirus genus of the Peribunyaviridae family contains numerous viruses, usually transmitted by mosquitoes .Broad-spectrum diagnostic tests, such as metagenomics methods, are useful to discover new viruses, especially in patients with encephalitis of unknown etiology . We tested a negative environmental control (isotonic sterile solution) in parallel. We prepared DNA libraries with Nextera XT DNA, and RNA libraries with RNA Human RiboZero TruSeq Stranded Total RNA Library Prep Kit . We pair-end sequenced libraries, 2 × 150 bp, with High Output Kit version 2 on a NextSeq500 device (Illumina). We analyzed the sequences with MetaMIC software  and contig alignment with the ViPR database (https://www.viprbrc.org). After manual curation of full-length viral sequences, we performed alignment and phylogenetic analyses with the closest known viral species using Muscle version 3.8.31 (http://www.drive5.com/muscle) and IQ-Tree version 1.3.11.1 (http://www.iqtree.org). We used the general time reversible plus gamma 4 plus invariate sites model of nucleotide substitution and 10,000 full maximum-likelihood bootstrap replicates.Identification of nonhuman sequences uses different databases, including cleaned National Center for Biotechnology Information nucleotide and nonredundant databases  that contain all known microorganisms. For new species, we conducted verification using an algorithm combining de novo assembly with Meta-SPAdes version 3.12.0 , medium (M), and small (S) segments were individualized and could be aligned with similar sequences from known orthobunyaviruses , coding for a protein of 2,261 aa in the L segment, which, by analogy with other orthobunyaviruses, corresponded to the RNA-dependent RNA polymerase  Figure 1In segment M, we identified a 4,336-bp ORF coding for a protein of 1,445 aa, corresponding to a polyprotein processed into the 2 envelope glycoproteins n and c (Gn and Gc) and nonstructural protein m (NSm) in orthobunyaviruses  Figure 1We identified 2 overlapping ORFs in the S segment, one corresponding to the nucleocapsid (N) protein , the other to nonstructural protein s . We found few differences with Umbre virus for these proteins .The amounts of viral RNAs we semiquantified by shotgun metagenomics were 5.4 log viral genomes/mg of biopsy for segment L, log viral genomes/mg of biopsy 5.0 for segment M, and 6.2 log viral genomes/mg of biopsy for segment S. For confirmation, we designed specific reverse transcription PCRs for Cristoli virus segments L, M, and S. Brain extracts were positive for all three, with amplicon sequences identical to those in the full-length genome  Figure 2Orthobunyavirus genus of the Peribunyaviridae family in an immunodepressed patient with slowly progressive fatal encephalitis. Cristoli virus is close to Umbre virus, a member of the Turlock serogroup not previously associated with human disease. Because the patient had not traveled during the year preceding the onset of her symptoms, our findings suggest that Cristoli virus is endemic to France. Like other members of its family, it could have been transmitted through a mosquito bite. Changes in the distribution of mosquito species related to climate change may make such transmissions more frequent in the future. In this context, the metagenomics method we developed is particularly useful to detect unusual or emerging infectious agents in patients with unassigned infectious syndromes.The etiologic diagnosis of encephalitis is difficult because >25% of cases remain of unknown cause (Peribunyaviridae genome organization with conserved motifs. The genome segment coding for the NSs protein was found in an amount 10 times higher than that of the other segments. Because NSs has been described as a virulence factor (Full-length sequence analysis of Cristoli virus revealed a classical Additional methods and figures for fatal encephalitis caused by Cristoli virus, France. Additional information about fatal encephalitis caused by Cristoli virus, France.


Nodaviridae. Nodaviruses have been identified in different hosts, including insects, fishes, shrimps, prawns, dogs, and bats. A novel porcine nodavirus was first identified in the United States by applying next-generation sequencing on brain tissues of pigs with neurological signs, including uncontrollable shaking. RNA1 of the porcine nodavirus had the highest nucleotide identity (51.1%) to the Flock House virus, whereas its RNA2 shared the highest nucleotide identity (48%) with the RNA2 segment of caninovirus (Canine nodavirus). Genetic characterization classified porcine nodavirus as a new species under the genus Alphanodavirus. Further studies are needed to understand the pathogenicity and clinical impacts of this virus.Nodaviruses are small bisegmented RNA viruses belonging to the family  Culex tritaeniorhynchus mosquitoes in the Japanese village of Nodamura , sug, sug9], 9.4–29%) . SimilarAlphanodavirus. Specifically, porcine nodavirus clustered together with three viruses  and two viruses (Shuangao insect virus 11 and Nodamura virus) in the RNA 1 nucleotide and amino acid trees, respectively  of RNA2 to the feral dog isolate at nucleotide and amino acid levels . TherefoBetanodavirus genus) mainly targets the central nervous system, including the brain, spinal cord, and retina. The infected fish suffer neurological disorders characterized by viral encephalopathy and retinopathy [Alphanodavirus, could experimentally infect suckling mice leading to hind-limb paralysis [The fish nodavirus known as nervous necrosis virus (inopathy ,24. In a


India reports the highest number of tuberculosis (TB) cases worldwide. Poverty has a dual impact as it increases the risk of TB and exposes the poor to economic hardship when they develop TB. Our objective was to estimate the costs incurred by patients with drug-susceptible TB in Bhavnagar (western India) using an adapted World Health Organization costing tool.We conducted a descriptive cross-sectional study of adults, notified in the public sector and being treated for drug-susceptible pulmonary TB during January–June 2019, in six urban and three rural blocks of Bhavnagar region, Gujarat state, India. The direct and indirect TB-related costs, as well as patients’ coping strategies, were assessed for the overall care of TB till treatment completion. Catastrophic costs were defined as total costs > 20% of annual household income (excluding any amount received from cash transfer programs or borrowed). Median and interquartile range (IQR) was used to summarize patient costs. The median costs between any two groups were compared using the median test. The association between any two categorical variables was tested by the Pearson chi-squared test. All costs were described in US dollars (USD). During the study period, on average, one USD equalled 70 Indian Rupees.Of 458 patients included, 70% were male, 62% had no formal education, 71% lived in urban areas, and 96% completed TB treatment. The median (IQR) total costs were USD 8 (5–28), direct medical costs were USD 0 (0–0), direct non-medical costs were USD 3 (2–4) and indirect costs were USD 6 (3–13). Among direct non-medical costs, travel cost  to attend health facilities were the most prominent, whereas the indirect costs were mainly contributed by the patient’s loss of wages . Four percent of patients faced catastrophic costs, 11% borrowed money to cover costs and 7% lost their employment; the median working days lost to TB was 30 (IQR: 15–45). A majority (88%) of patients received a median USD 43 (IQR: 41–43) as part of a cash transfer program for TB patients.Treatment completion was high and the costs incurred by TB patients were low in this setting. However, negative financial consequences occur even in low-cost settings. The role of universal cash transfer programs in such settings requires further study. India is the country with the largest share of the global burden of tuberculosis (TB) cases 27%), with the burden being highest among the poor . Poverty7%, with Studies in India reported the percentage of catastrophic costs among drug-susceptible TB patients to be between 7 and 32% –12. The The primary objective of the study was to estimate the costs incurred by drug-susceptible pulmonary TB patients in a semi-urban and rural setting in western India following the introduction of the DBT scheme. The secondary objective was to estimate the proportion of households facing catastrophic costs and to determine the association between catastrophic costs and failure to complete treatment.The study was conducted in all six semi-urban and three rural “blocks” (administrative zones) of the Bhavnagar region of Gujarat state in the western part of India. The semi-urban blocks were part of Bhavnagar city (population ~ 0.6 million), and the rural blocks were drawn from the surrounding, largely agricultural, countryside. Agriculture and daily wage labor are the primary occupations of residents in rural and urban areas of the district, respectively. The TB case notification rate in Bhavnagar district from the public and private sector was 1040 and 780 per million population, respectively, in the year 2019. The care for TB is offered free of cost in the public sector in a decentralized model through a network of public health facilities, the district hospital being the tertiary-level facility. Once initiated on treatment, the medicines and other necessary follow-up care is delivered to the patients from a nearby public health facility. Patients with drug-susceptible pulmonary tuberculosis are given a treatment of six months – two months of intensive phase (to rapidly eliminate the bacilli) followed by four months of continuation phase (to eradicate the dormant bacilli).We conducted a descriptive cross-sectional study of patients enrolled for the treatment of drug-susceptible TB. We included patients ≥ 18 years on treatment for drug-susceptible pulmonary tuberculosis, registered under the public sector in all six urban blocks of Bhavnagar city, and three of 11 randomly-selected rural blocks  of Bhavnagar district. We excluded patients who were previously treated or taking treatment under a private health facility or put on treatment for < 14 days .The list (treatment registers) of patients put on treatment is maintained in physical form by TB Health Visitors at the District TB Centre of Bhavnagar. TB health visitors are contracted by NTP for record-keeping of TB patients in geographical areas assigned to them. For each of the nine selected urban and rural blocks, a trained field investigator reviewed the list of patients put on treatment to identify those who appeared to be eligible for the study, and then visited their homes  to confirm eligibility and collect data. Enrollment occurred between January 2019 and June 2019. Once patients from all nine blocks were enrolled, the process was repeated in selected urban blocks till June 2019 (end of the study period).Following written informed consent, the field investigator administered a survey to collect basic demographic and clinical information (some of which was extracted from the TB treatment registers), and administered the adapted WHO costing tool . The WHOWe collected data on costs relative to the following time points based on patient recall: pre-diagnosis (the period from onset of symptoms until diagnosis), at the time of diagnosis, at the end of one-and-a-half month for adverse drug reaction or directly observed treatment (DOT) medicine collection visits, at the end of two months (first follow-up visit) and at the end of six months (treatment completion visit). The survey was administered at the homes of patients and covered all costs up until the time of enrollment in the study, that is, the patients were enrolled at variable time points of treatment. Depending on how far the patient was in the treatment phase, the field investigator later made up to three phone calls to collect data on the additional costs incurred until treatment completion.https://www.nikshay.in/) respectively.The questionnaire was translated into the local language (Gujarati) by a language translator. Compensation of USD 0.7 (INR 50) was given to each participant for their time after completion of the interview. Patients were then followed-up passively to obtain treatment outcomes. The field investigator extracted data on treatment outcomes and DBT from the TB treatment registers and NIKSHAY  to attend health facility, and the travel costs for DOT visits. Indirect costs included patient’s and accompanying member’s loss of wages, and a measure to assess economic impact calculated as monthly family income before TB minus monthly family income at the time of interview. The total costs incurred were a sum of direct medical, direct non-medical and indirect costs, and were said to be catastrophic if they exceeded 20% of their annual household income (excluding the DBT amount). Negative financial coping strategies  and social consequences  were also assessed. During the study period, on average, USD 1 equalled INR 70.To stratify patients based on socioeconomic status, data on ownership of assets including the type of house, number of rooms, television, and others were used to create a standard of living (SLI) index. Depending on the ownership of different assets, a summary score (range: 1–23) adapted from the National Family Health Survey (NFHS), was calculated , 20. SLIFailure to complete treatment was defined according to NTP standards, including loss to follow-up (stopped treatment for at least one consecutive month), sputum smear positivity at the end of treatment, or death while on treatment .We collected data on all eligible patients during the study period of January–June 2019. The data were entered into a computerized form and analyzed in the statistical software EpiInfo 7.2.4.0 . The total costs and its sub-components incurred by patients were described in median with inter-quartile range (IQR). The median costs between any two groups were compared using the median test. The association between catastrophic costs and failure to complete treatment was tested by the Pearson chi-squared test. We conducted sensitivity analyses using 15, 10, and 5% of one’s annual self-reported household income to define catastrophic costs.Out of the 2233 total patients, 462 patients were eligible for the study Fig. . Out of CI: 3–6%).The median age of the patients was 35 (IQR: 23–50) years and the median number of family members was 5 (IQR: 4–6). Out of the 458 patients, 70% were male, 62% had no formal education, 72% were married, 71% lived in urban areas and 17% lived in a nuclear family  (Table P <  0.001). Among direct non-medical costs, travel cost  to attend health facilities were the most prominent.The direct medical costs were minimal for the majority of the patients  Table . For theP < 0.001). The indirect costs were mainly contributed by the patient’s loss of wages .The median indirect costs among the patients were USD 6 (IQR: 3–13) and differed significantly among patients between private vs government providers . We found no significant difference in total costs between low and middle/high-income participants , but differed significantly among patients who first visited a private vs a government provider  and remained low in sensitivity analyses using lower thresholds of annual household income to define catastrophic costs . However, the odds of experiencing catastrophic costs were higher among patients visiting first a private vs a government provider . As a result of the lower overall costs in this population, the percentage who faced catastrophic costs was also low. Despite these low overall costs, 18% of families reported having to employ at least one negative financial coping strategy to manage their anticipated and actual costs.The median costs reported in the present study were lower than those reported in most other studies from India , 22, 23.When compared with other high-burden countries , 4–7, thAround one-fifth of the patients in the current study employed a negative financial coping strategy to cover the costs of TB care. Negative financial coping strategies, also called dissavings, are directly associated with costs incurred by TB patients and may be used as a proxy indicator of the financial protection mechanisms being employed by governments . FurtherSeveral aspects of the study context likely affected these results. Study setting being a small town, patients faced less direct non-medical costs  due to proximity to public health facilities. The setting where the present study was conducted has a wide network of government-run diagnostic and treatment facilities for tuberculosis. The public-private partnership model for TB control seems to be effective in Bhavnagar as the majority of patients had their first or second visit at a public health facility. The private practitioners seem to be aware of referring patients to public health facilities for reducing their economic hardships. Even though patients have to collect their medicines weekly, their medicine boxes are placed somewhere near to their homes . This decentralized model of DOT helps to minimize direct non-medical costs incurred for such visits.This was a single-center study in a semi-urban and rural setting with a well-functioning community-based DOT program that is achieving high levels of treatment success. Therefore the findings should only be generalized to similar settings in India. Even though this is one of the initial studies from India using the validated WHO tool  and adheWe conclude from the study that TB patients in our region incur minimal costs, perhaps due to the highly decentralized provision of diagnostic and treatment services through community-based DOT. These findings support the further expansion of community-based DOT models to reduce catastrophic costs in India and other countries. However, almost one in five participants undertook negative financial coping strategies to facilitate TB care, suggesting that negative financial consequences occur even in low-cost settings. Further research is needed to assess whether universal cash transfer programs achieve social protection targets in settings with well-functioning community-based DOT programs.Additional file 1. Median costs in USD by standard of living index. Table showing median (with inter-quartile range) costs incurred by 458 patients with drug-susceptible pulmonary tuberculosis on treatment during January–June 2019.Additional file 2. Association between type of provider at first visit and catastrophic costs. Table showing statistical association between type of provider at first visit and catastrophic costs incurred by 458 patients with drug-susceptible pulmonary tuberculosis on treatment during January–June 2019.


Phytophthora cactorum. The virus genome consists of a single dsRNA segment of 5699 bp with two open reading frames predicted to overlap with each other and encoding a putative capsid protein of 705 aa and an RNA-dependent RNA polymerase of 779 aa. Sequence comparisons indicated that this virus, designated as “Phytophthora cactorum RNA virus 1” (PcRV1), shares the highest sequence similarity with the unclassified Pythium splendens RNA virus 1 (58% RdRp aa sequence identity). Phylogenetic analysis revealed that these two oomycete viruses group together with Giardia lamblia virus  and several unclassified toti-like viruses from arthropods, fish and fungi. This is the first report of a toti-like virus in a member of the genus Phytophthora and the first virus characterized in P. cactorum.This report describes the complete genome sequence of a double-stranded RNA (dsRNA) virus infecting the oomycetous plant pathogen  Phytophthora cactorum (Leb. and Cohn) Schröeter is an oomycetous phytopathogen of the Kingdom Stramenopila that causes root, collar and crown rot as well as foliar and fruit infections on a broad range of hosts, including over 200 species of trees, ornamentals, and fruit crops [Betula spp., P. cactorum is mostly a problem in nurseries, causing stem lesions on seedlings [P. cactorum, whereas a few viruses are known in congeneric host species: alphaendornaviruses infect members of the Phytophthora taxon ‘douglasfir’ and P. ramorum [P. infestans [Astroviridae, whereas one is affiliated with a family-level group tentatively called ‘fusagraviruses’ and one is an unclassified member of the family Narnaviridae. In this study, we report the complete genome sequence of a new toti-like virus infecting P. cactorum, designated as "Phytophthora cactorum RNA virus 1" (PcRV1).it crops . On Betueedlings . Prior t ramorum , and fouPhytophthora cactorum isolate BirchT_KT09 was isolated in 2009 from a trunk lesion on silver birch (Betula pendula Roth.) in Denmark by Kirsten Thinggaard, cultivated on 2% malt extract agar plates, and identified based on its morphology and ribosomal internal transcribed spacer 2 (ITS 2) sequence. Total dsRNA was extracted from 2.5 g of cultivated mycelia using cellulose affinity chromatography [G + I with five gamma categories; 1.1 × 106 cycles for the MCMC algorithm, sampling one tree per 200 cycles; discarding 105 samples as burn-in.tography . A 5.7-ktography  except tThe complete genome sequence of PcRV1  consists of a single segment of 5,699 bp . The genome organization of PcRV1 corresponds to that found in members of the family Totiviridae [A shifty heptamer motif , which may facilitate − 1 programmed ribosomal frameshifting in virus transcripts, was observed at the end of ORF1 , have recently been detected in arthropods, fish and fungi [Pythium viruses PsRV1 and Pythium polare RNA virus 1 (PpRV1) form a highly supported clade and cluster among the new GLV-like viruses. Interestingly, Shiba et al. [Heterosiphonia pulchra, which also included transcripts related to Phytophthora cactorum. Thus, the natural sample of H. pulchra might have included an associated P. cactorum hosting a similar toti-like virus. The virus-like sequence from H. pulchra showed 37.0% aa sequence identity to the PcRV1 RdRp based on global alignment. Among the GLV-like viruses, the presence of a shifty heptamer motif together with the absence of a nearby RNA pseudoknot is also found in piscine myocarditis virus (PMCV) and Camponotus yamaokai virus (CYV) [Viruses resembling Giardia lamblia virus (GLV), the sole classified member of the genus nd fungi , 10, 14.a et al.  detectedTotivirus in the  family Totiviridae [Pythium virus PsRV1 exceeds this threshold but remains moderate (ca. 58%) and corresponds to the 60% maximum identity criterion set for different species of the genus Victorivirus in family Totiviridae. In addition, the hosts represent different oomycete genera. Therefore, we propose that these viruses should be considered members of different species. As more related species are described, the ICTV may need to adjust the species demarcation criteria for this virus group accordingly. It should be noted that strains of Pythium and Phytophthora often co-occur in soil and diseased plants, which could provide a potential route for interspecies virus transmission. However, no mechanisms for interspecies virus transmission between members of oomycete genera have been described thus far, and in the case of Phytophthora infestans, even intraspecies transmission of viruses between somatically incompatible isolates has been shown to be unsuccessful [According to the virus species demarcation criteria for the genus iviridae , virusesccessful .


Schmallenberg virus (SBV), a teratogenic orthobunyavirus that infects predominantly ruminants, emerged in 2011 in Central Europe, spread rapidly throughout the continent, and subsequently established an endemic status with re-circulations to a larger extent every 2 to 3 years. Hence, it represents a constant threat to the continent’s ruminant population when no effective countermeasures are implemented. Here, we discuss potential preventive measures to protect from Schmallenberg disease. Previous experiences with other arboviruses like bluetongue virus have already demonstrated that vaccination of livestock against a vector-transmitted disease can play a major role in reducing or even stopping virus circulation. For SBV, specific inactivated whole-virus vaccines have been developed and marketing authorizations were granted for such preparations. In addition, candidate marker vaccines either as live attenuated, DNA-mediated, subunit or live-vectored preparations have been developed, but none of these DIVA-capable candidate vaccines are currently commercially available. At the moment, the licensed inactivated vaccines are used only to a very limited extent. The high seroprevalence rates induced in years of virus re-occurrence to a larger extent, the wave-like and sometimes hard to predict circulation pattern of SBV, and the expenditures of time and costs for the vaccinations presumably impact on the willingness to vaccinate. However, one should bear in mind that the consequence of seronegative young animals and regular renewed virus circulation might be again more cases of fetal malformation caused by an infection of naïve dams during one of their first gestations. Therefore, an appropriate and cost-effective strategy might be to vaccinate naïve female animals of all affected species before the reproductive age. Peribunyaviridae, order Bunyavirales), was eventually identified in blood samples of acutely diseased cows by next-generation sequencing-based metagenomics [In summer and autumn 2011, an unidentified disease characterized by fever, decreased milk production, and diarrhea was noticed in cattle in Germany and the Netherlands. The causative agent of the observed clinical signs, a novel orthobunyavirus of the Simbu serogroup  as a basis ,99, SBV For the design of the SBV-specific vector vaccines, two organisms previously described for vaccine development against other cattle diseases have been selected, namely equine herpesvirus type 1 (EHV-1) and the poxvirus modified Vaccinia virus Ankara (MVA) . As an SNone of the aforementioned (DIVA-capable) candidate vaccines are currently commercially available. To our knowledge, the licensed inactivated vaccines are currently only used to a very limited extent. As they received their marketing authorization only after the first infection wave in Central Europe, which by itself resulted in a very high seroprevalence ,7,8,102,As mentioned above, it was not considered necessary to vaccinate the animals in regions with a high proportion of seropositive animals. This high seroprevalence rate acquired due to natural infections of the ruminant population presumably led to the only sporadic virus detections in the year 2013, predominantly in the young stock. However, in the following years the herd immunity declined since the livestock population dynamics strongly influence the duration of herd immunity. Due to the high rate of replacement of the seropositive animals by seronegative young animals under routine animal production conditions, the overall rate of seropositive animals declined, leading to a decrease in herd immunity . This deCulicoides biting midges and predominantly infects ruminants [European experiences during the outbreak of the reovirus bluetongue virus (BTV) in 2006/2007 have already demonstrated that vaccination of livestock against a vector-transmitted disease can play a major role in reducing the virus circulation or even in eradicating the disease from some regions ,105. BTVuminants . Based ouminants . Althouguminants . The masuminants . Regardiuminants , but do uminants ,110. Culicoides biting midges and the transmission pattern of arboviruses are highly dependent on vector ecology. Therefore, the most effective veterinary measure in response to the European BTV outbreak was vaccination. Overall, more than 100 million animals were vaccinated throughout Europe and, as a result, the incidence of BTV infections decreased rapidly , demonstIn terms of vaccination strategies against SBV-infections, one should consider the extent of the measures. Mass vaccination resulting in a high coverage proved to be highly effective against arboviruses, but a high coverage will be difficult, if not impossible, to achieve in the case of SBV when vaccination is voluntary, especially when the costs are not refunded, but have to be paid by the animal owners. As an example, the probability of seroconversion dropped below 10% when vaccination against BTV became optional in France after 2010, while about 80% of the animals were seropositive in December 2009 when vaccination was compulsory . HoweverTo investigate an optimal vaccine deployment for SBV, a stochastic mathematical model was developed and used to simulate the effect of different scenarios for Scottish ruminants . In ScotTherefore, the most appropriate strategy might be to vaccinate naïve female animals of all affected species before the reproductive age, especially in years of only low-level virus circulation and an associated low seroprevalence in the young stock. The unexpected emergence of SBV demonstrates the constant threat that yet unknown insect-transmitted viruses suddenly appear in previously unaffected regions, probably driven by a rapid change in global climate, trade and travel habits. Following its introduction into Europe, SBV established an endemic status with re-circulations to a larger extent every 2 to 3 years. Hence, it represents a constant threat to the ruminant population when no countermeasures, in particular vaccinations, are implemented. Previous experiences have already demonstrated that vaccination of livestock against a vector-transmitted disease can play a major role in reducing the virus circulation or even in eradicating the disease from some regions. However, the intermittent nature of SBV circulation and the expenditures of time and costs for the vaccinations, especially when the costs have to be paid by the animal owners, presumably impact on the willingness to vaccinate. Hence, a high coverage will be difficult to achieve. However, one should keep in mind that the consequence of regularly renewed virus circulation in combination with seronegative young stock may be again more cases of fetal malformation caused by an infection of naïve dams during one of their first gestations. Therefore, the most appropriate and cost-effective strategy might be to vaccinate naïve female animals of all affected species before the reproductive age.


Maize Chlorotic Mottle Virus (MCMV) detected the virus in the majority (97%) of the samples (n = 223). Analysis of a subset (n = 48) of the samples using NGS-Illumina Miseq detected MCMV and Sugarcane Mosaic Virus (SCMV) at a co-infection of 62%. The analysis further detected Maize streak virus with an 8% incidence in samples where MCMV and SCMV were also detected. In addition, signatures of Maize dwarf mosaic virus, Sorghum mosaic virus, Maize yellow dwarf virus-RMV and Barley yellow dwarf virus were detected with low coverage. Phylogenetic analysis of the viral coat protein showed that isolates of MCMV and SCMV were similar to those previously reported in East Africa and Hebei, China. Besides characterization, we used farmers’ interviews and direct field observations to give insights into MLN status in different agro-ecological zones (AEZs) in Kilimanjaro, Mayara, and Arusha. Through the survey, we showed that the prevalence of MLN differed across regions (P = 0.0012) and villages (P < 0.0001) but not across AEZs (P > 0.05). The study shows changing MLN dynamics in Tanzania and emphasizes the need for regional scientists to utilize farmers’ awareness in managing the disease.Sustainable control of plant diseases requires a good understanding of the epidemiological aspects such as the biology of the causal pathogens. In the current study, we used RT-PCR and Next Generation Sequencing (NGS) to contribute to the characterization of maize lethal necrotic (MLN) viruses and to identify other possible viruses that could represent a future threat in maize production in Tanzania. RT-PCR screening for  Maize is the most important cereal crop and a staple food in sub-Saharan Africa. With over 5 million hectares of its land planted with this crop and a per capita consumption of 128 kg, Tanzania is one of the largest producers and consumers of maize in eastern and southern Africa  and MwanMaize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV)  , 0.1 µL (10 µM) of the forward (5′-CGCGGCTGACAAGCAAAT-3′) and reverse primer (5′-ACTGGTTGTTCCGGTCTTG-3′) targeting MCMV, 2 µL cDNA and 7.8 µL of sterile water for a final volume of 20 µL. The thermocycler conditions were as follows: an initial denaturation at 95 °C for 3 min followed by 30 cycles of denaturation at 95 °C for 30 sec, annealing at 49.4 °C for 30 sec, elongation at 72 °C for 1 min and a final extension step at 72 °C for 15 min. The PCR products were visualized under UV light on a stained (gel red) 1.2% agarose gel following electrophoresis.All leaf samples collected were tested for the presence of MCMV using RT-PCR. Total RNA was extracted from the samples (n 1x TAE  and quann = 15), Arusha (n = 22), and Manyara (n = 11) were selected for NGS characterization of the MLN causal viruses. The samples (n = 48) were used for the construction of sequence libraries using the Illumina TruSeq RNA library prep kit following the manufacturer’s instructions . Briefly, 0.5µg of RNA/sample was fragmented using the fragmentation mix  followed by first strand and second strand cDNA synthesis. The double-stranded cDNA was purified using Agencourt AMPure XP magnetic beads  followed by end repair and adapters ligation. The ligated ds-cDNA was amplified in a PCR using universal and index primers. Resulting libraries were purified using Agencourt AMPure XP magnetic beads. Libraries’ quality and quantity were assessed with the Agilent Tape Station 2200 system  and a Qubit™ fluorometer , respectively. The libraries were then normalized to a concentration of 10nM, pooled, and diluted to a final concentration of 6.5 pM. Pooled libraries were sequenced on the Illumina MiSeq system at the BecA-ILRI Hub  generating 151 paired-end reads.Based on the results of the RT-PCR for MCMV, a subset of samples positive for MCMV from Kilimanjaro (n = 137) selected with the help of extension staff representing different villages  within the three regions of Northern Tanzania were interviewed about specific aspects relating to awareness and experiences of MLN in 2015. Farmers provided information about whether they had observed MLN symptoms (pictures of the symptomatic plants were shown to farmers) in their maize crop within the past four years (2012–2015), the approximate percentage of maize losses due to MLN in comparison with previous yields, seed types and origin used, the predominant insect pests identified by using pictures and maize insect pests identifier from CIMMYT [A random group of farmers . Means of symptomatic plants across villages within the regions and AEZs were computed and compared in a nested linear regression model. This study contributes to a better understanding of MLN in Northern Tanzania, by providing information on disease prevalence across different AEZs in Kilimanjaro, Arusha and Manyara regions in 2015, detection of the associated viruses and indicates possible measures to manage the disease. Furthermore, the study detected other potential viruses that could present a threat to maize production in Northern Tanzania. The complete sequences of the virus isolates reported in this study provide additional resources for the development of diagnostic tools and for enhancing understanding of genetic relatedness of isolates of MCMV, SCMV, and MSV across Africa and their management. Given that the observational and sampling component of the current study were limited in scope, we propose further studies to expand these aspects in order to assess the seasonal variations of the epidemics, the role of the interaction among multiple viruses on severity of MLN, how the genetic variability of SCMV affects MLN, and the role of vectors in respect to changes in the climatic factors.