Chlamydia trachomatis, was described using different methodologies for their detection. Their presence was further used as biomarkers associated with anthropological and other relevant information to trace human migration into the Amazon region of Brazil. The approach also evidenced their microbiological origin, emergence, evolution, and spreading. The information obtained confirms much of the archeological information available tracing ancient and more recent human migration into this particular geographical region. In this article, the paleomicrobiological information on the subject was summarized and reviewed.Infectious agents are common companions of humans and since ancient times they follow human migration on their search for a better place to live. The study of paleomicrobiology was significantly improved in its accuracy of measurement with the constant development of better methods to detect and analyze nucleic acids. Human tissues are constantly used to trace ancient infections and the association of anthropological evidences are important to confirm the microbiological information. Infectious agents which establish human persistent infections are particularly useful to trace human migrations. In the present article, the evidence of infection by viral agents such as human T-lymphotropic virus 1, human T-lymphotropic virus 2, human herpes virus-8, JC virus, and a bacterium,  Viral agents are commonly associated to infections which are able to trace human ancient and recent migrations all over the world. In the early history of civilization small human groups started to move around the world to find safer environment and food. Their traveling into previously unknown areas brought also the interaction with new infectious agents. Recent human spread as a consequence of wars, famine, disasters, and many other modern dangers also carried old and new agents with greater or less chance to spread and cause severe prejudice.Mycobacterium tuberculosis in an ancient skeleton which progressed with the identification of infectious agents and their geographical spreading, as they were related to their respective paleo specimens  integrated DNA from fibroblasts of an AIDS patient , as wellenturies . Human penturies  and 16thenturies  centurieenturies . More reenturies .Persistence of viruses allows the application of molecular biology methods, but also of other methods; antibody detection using serological studies are indicative of the presence of the persistent virus infection. This approach coupled to molecular biology methods has allowed us to define the presence of past and old infections among specific population groups in the Amazon region of Brazil within epidemiologically closed groups of “quilombolas” (slave refugees) or native Indians and immigrants with little or no degree of miscegenation at all.Epidemiological studies, mostly descriptive ones, were important biological tools when associated to molecular biology approaches, particularly when dealing with the recent history of specific infectious agents. Besides describing the presence of the infectious agent, it also showed the valuable importance of surveillance when detecting the recent entry of new infectious agents among human population communities. In the recent history of the study of infectious agents in the Amazon region of Brazil, there are some examples of major relevance, including the involvement of native Indians with devastating agents such as hepatitis B virus (HBV) and HIV-1 .An extensive seroepidemiological study was performed with native Indians looking for the presence of infection markers of HBV. There was one community, namely the Parakana, which was found to be free of the agent. The main characteristic was that the samples were collected within 2 weeks after the initial contact with the community . Few yeaChlamydia trachomatis was similar, as the bacterium is thought to persist in the infected persons. The evaluation of the antibody response to the serotypes indicated their spreading. The specific methodological approach for each infectious agents is reported briefly below. Specific details are described in the original publications.The initial general approach for studying viruses and their relation to human migrations investigated the presence of antibodies. As we were dealing with agents which established persistent infections the positive reactions were also a confirmation of the presence of the virus in the human host. The following step was to generate information of the genotypes, or serotypes, circulating in the Amazon region of Brazil and to associate them with previously described geographical distribution of the virus and their variants. The approach for the study of Figure 1), belonging to communities from the States of Maranhao (Urubu-Kaapor), Amapa , Roraima (Yanomami), Amazonas (Yamamadi), Rondonia , and Para . Nucleic acid extraction was performed from positive samples in order to sequence regions of the gene env and LTR region to construct phylogenetic trees which allowed to describe the molecular subtype HTLV-2c. Detailed methodological steps were previously described  in serum samples of 26 native Indian tribes largely distributed in the Amazon region of Brazil . JC virus was detected in urine samples using the amplification of vp1 gene and IG region according to a previously described protocol  in the Brazilian Amazon involved 10 native Indian tribes from the States of Para , Maranhao (Urubu-Kaapor), and Rondonia (Surui), one Brazilian afro-descendant “quilombo” from the Rio Trombetas region in the West of the State of Para and from its capital, the city of Belem . An enzyme immune assay with four structural and non-structural antigens  were investigated among four native Indian groups  from the Amazon region (antigens  was usedescribed .Chlamydia were detected in the blood samples of: (i) patients from a university public hospital (respiratory and cardiac disease) in the city of Belem, Para; (ii) subjects from two major urban communities  of the State of Para; (iii) individuals from two isolated Afro-descendant communities located in Northern Brazil  who originated from runaway individuals during the slavery period; (iv) individuals from Mazagao, State of Amapa, a village originating from immigrants of North Africa; and (v) three native Indian groups from the State of Maranhao (Awa-Guaja), and the State of Para  . A commercial indirect immunofluorescence assay using serotype L2 of C. trachomatis as the antigen was used for the screening of antibodies to the genus Chlamydia. Positive samples were titrated and those with titers equal or greater than 512 were investigated to detect serological evidence of bacterium persistence . This subtype is endemically distributed in a large geographical setting (more than 5 million km2) and maintained under continuous transmission  among epidemiologically isolated human groups due to their physical, cultural, and linguistic barriers .The geographical distribution of HTLV-2 around the world shows that in the Americas, the virus is present from North to South America in the molecular subtypes HTLV-2a (predominantly in urban areas) and HTLV-2b, predominantly among native Indians groups from North, Central, and South America . The disOnce the virus reached its way into the community it was actively transmitted horizontally  and maintained within epidemiologically closed communities using the vertical transmission from mother to child, a characteristic of retroviral persistent infections with integration of the viral nucleic acid into the host genome . The widMore recently, the virus was detected in several major cities of Brazil, including Salvador , Belo HoFigure 4). Tome-Acu was a village settled by the Japanese immigrants for specific agricultural purposes . The virus kept a similar genetic sequence as imported from Japan.Brazil received large waves of migration around 100 years ago, in the beginning of the 20th century, from European nations  and at a smaller scale from Japan . Our gropurposes . The infFigure 6; The finding of polyomavirus infections in the Amazon region helped to show the close association of migration routes of viruses and humans during their migration pathways. JC virus was described in the following communities: types A (subtype EU), B , and C (subtype Af-1) among subjects living in Belem; type B, subtypes Af-2 and MY, among Afro-Brazilians; and type B, subtype MY, within the Surui Indians  during the slavery trade into Brazil. A second spread occurred in the “quilombos,” which occurred as early as in 1788 in the State of Para . This isHuman herpes virus-8 infections in the Amazon region showed a widespread agent both in urban and non-urban communities, including native Indians in Brazil, French Guiana, and Equador . The higk1) identified diversity of HHV-8 subtypes among urban and native Indians. Subtypes E and C were described in the Tiriyo and the Zo’e, respectively . Human herpes virus-8, subtype E, was also described among two other Brazilian Indian communities (Arawete and Assurini), in French Guiana (Wayampi), and in Equador , and the presence of a unique strain found solely within the Amazon region are strong evidence that the virus represents an ancient endemic infection of native Indians. Human herpes virus-8 is another example of how viruses which establish persistence can also help to understand the mobility of humans around the world in their search for new areas for a better living.C. trachomatis, a widespread infection all over the world, is a bacterium used by our group to describe a more recent massive migration that occurred in the 18th century.The political organization of Brazil dates from the 1500s, starting as a colony of Portugal, but with several invasions throughout the centuries from other European countries, including Spain, Netherlands, and France. Several other waves of immigrants were also reported and some remain unclear. C. trachomatis has been detected by our group, indirectly on several occasions among urban and non-urban communities including an extensive search among native Indians in the Amazon region of Brazil . Curiously, serorreactivity to serotypes E and J, strains usually sexually transmitted, were seen exclusively within the Mazagao village.Trachoma an eye infection and a severe inflammatory disease caused by The different methods by which infectious agents are detected in ancient specimens were used in a combined approach with archeology to detect past and ancient infections in order to improve the understanding of the origin, emergence, evolution, and spreading of viruses, bacteria, and other infectious agents. Similar methods were also used to describe recent emergence and introduction of infectious agents.C. trachomatis were described among Amerindian tribes, Afro-descendant communities, and in the trihybrid population living in the urban and non-urban areas of the Amazon region of Brazil. The presence of different and largely variable strains of those agents circulating in the region and infecting selected human host groups brought light to the association of the ethnic origin of the populations investigated. The molecular linkage of different strains to specific areas in the European, Asiatic, and African continents is excellent tools to highlight the role of infectious agents as markers for studying the early migration of human populations, reflecting their early and late history.In our previous studies, HTLV-2, JCV, HHV-8, and The present world situation of political instability in several countries is, again, causing a massive movement of humans from Haiti to Brazil and from the Middle East to Europe (and to a lesser extent to other continents). It is quite certain that we will soon observe the introduction and upsurge of viruses and other infectious agents into different geographical areas. The use of infectious agents capable of establishing persistence is of major importance as models to study their clinical interaction with humans and microbiological active surveillance will be necessary to follow up the present ongoing migratory wave.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Sodiomyces alkalinus. The first, Sodiomyces alkalinus partitivirus 1 (SaPV1), is placed within the genus Betapartitivirus and is related to Ustilaginoidea virens partitivirus 2. The taxonomic position of the second virus is less clear as it shares high (85%) amino acid sequence identity but significantly low (77%) nucleotide sequence identity of the capsid protein with Colletotrichum truncatum partitivirus 1. The third, the novel Sodiomyces alkalinus fusarivirus 1 (SaFV1), is related to Fusarium poae fusarivirus 1. All the viruses show efficient vertical transmission through asexual and sexual spores. These novel coexisting viruses do not evoke apparent phenotypic alteration to their fungal host. This is the first description of a viral infection in an alkalophilic fungus.Mixed infection by three dsRNA viruses, a novel betapartitivirus, a gammapartitivirus, and a novel fusarivirus, has been identified in four isolates of the obligate alkalophilic fungus  Partitiviridae, Reoviridae, Megabirnaviridae, Totiviridae, Chrysoviridae, Endornaviridae, Quadriviridae). Furthermore, a large number of ssRNA and dsRNA viruses have been described, but remain unclassified. It is believed that mycoviruses lack an extracellular phase in their lifecycles, and are not infectious as free particles. However, in case of Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV1), the purified particles were infectious and the virus could be transmitted extracellularly [Lycoriella ingenua (Diptera: Sciaridae) has been found to be infected with the mycovirus SsHADV1. Viruliferous adults of this insect could transmit the virus transovarially [Ascomycetes occurs by asexual spores and, more rarely, by sexual spores, while in Basidiomycetes mycoviruses are usually passed through sexual spores [Aspergillus niger resulted in reduced competitive fitness, and, in some cases, phenotypic alterations [Saccharomyces cerevisiae yeast governed by the cytoplasmic dsRNA virus [Chrysoviridae, Endornaviridae, Partitiviridae and Totiviridae that have co-evolved with their respective hosts for a long time, have been integrated into host genomes. Mycovirus-like sequences have been identified even in eukaryotic lineages, which were not expected to be hosts for these viruses, e.g. partitivirus-like sequences in monocots, flatworms, and sand fly; totivirus-like sequences in diatom, and animals; and chrysovirus-like sequences in plants [Mycoviruses are widespread in fungi, and can carry various types of nucleic acids that constitute their genomes. Most of the described mycoviruses possess dsRNA and positive-sense ssRNA genomes, however some ssDNA-, dsDNA-, and negative-sense ssRNA-viruses have also been reported –3. The sllularly . In addillularly –6. Recenvarially . Mycovirl spores , 9. A vil spores –11. Mosterations . In someerations –14. AnotNA virus . It has NA virus . Furthern plants –20. Bothn plants –21.Dothideomycetes, Leotiomycetes, and Sordariomycetes. Virulence varies considerably among fusariviruses. For instance, FgV1 is responsible for reducing mycotoxin production and virulence of F. graminearum strain DK21 [Fusariviridae is a recently proposed family of mycoviruses with a positive polyadenylated ssRNA genome of 6–7.7 kb long and two large open reading frames (ORFs)  and one  or two  small ORFs on the coding strand  Fig 1)..Fig 1). ain DK21 , while Rain DK21 –24.Partitiviridae), contrary to fusariviruses, are bi-segmented dsRNA viruses that infect plants, fungi, or protozoa [Cryphonectria parasitica characterized by reduced growth rate and enhanced pigmentation [Sclerotinia spp. and Botrytis cinerea [Alphapartitivirus from fungi and plants (14 species), Betapartitivirus from fungi and plants (17 species), Gammapartitivirus from filamentous fungi (8 species), Deltapartitivirus from plants (5 species), and Cryspovirus (1 species from protist in a recent taxonomy) [Partitiviruses  . Furtheraxonomy) . The keySodiomyces alkalinus, a soda-lake fungus with an unusual ability to grow optimally at very high ambient pH [In this study, we describe dsRNA viruses nested in bient pH . We showS. alkalinus isolated previously [2CO3−24 g, NaHCO3−6 g, NaCl– 5 g, KNO3−1 g, K2HPO4−1 g, malt extract– 17 g, yeast extract– 1 g, and agar– 20 g. The cultivation was at 27°C in the dark. To obtain colonies derived from single ascospores, cleistothecia were picked manually, rolled on the surface of water agar (2%) medium to remove residual mycelium, and then placed in 0.09% saline/0.005% Tween-80 solution and gently crushed. The released ascospores were plated onto the AA medium and young colonies were transferred to fresh AA plates. No inhibition or barrages were observed in the interaction zone between the strains grown next to each other although the mycelia did not fuse with each other  was prepared with Superscript IV (Invitrogen) following manufacturers’ instruction, and random hexamer primer. Relative concentrations of viruses were estimated by real-time reverse-transcription polymerase chain reaction analysis (RT-PCR). For SaFV1, SaPV1, and CtParV1-SAL, specific primers (5´-3´) SaFV1-1902f: GACATTTACCGAAAAGGAACT, SaFV1-2333r: CATAGACTTCACCTTGCTCA, SaPV1-405f: GCTCAACACATCATGAACAA, SaPV1-804r: CATCTATGCGTTCGTTAAGC, CtParV1-SAL-810f: CTTCTTGGTGTGGAGATCAA, and CtParV1-SAL-1181r: AGCAGCATTTCTATTGACCA were used.For relative quantification of viruses, total RNA was isolated using a NucleoSpin RNA Plant kit  from 5´-TGGACCTTAAAAATGCTTGG-3´ and 5´-GTGCCTTCAGTACATTAGCA-3´ and prepared with the PCR DIG labelling kit . Detection was performed using alkaline phosphatase-conjugated, anti-DIG antibody (Roche) and DuoLuX chemiluminescent/fluorescent substrate . Viral bands were detected by fluorescence.Northern blot analysis was conducted as described in . Total RSequencing libraries were prepared from the dsRNA template using TruSeq RNA Library Preparation kit (for the isolates F11 and F12) or MuSeek library preparation kit for Illumina (Thermo Scientific) (for the isolates F13 and F18) and then sequenced with a HiSeq 2500 ultra-high-throughput sequencing system in SE 100bp mode . The reads were trimmed and assembled in CLC Genomics Workbench 8.5.1 . Contigs were cured, gaps between them were filled by amplification with specific primers, and Sanger sequenced. Terminal sequences were obtained using the 5´ and 3´ RACE System (Invitrogen) and virus specific primers  Tables.http://rna.tbi.univie.ac.at.Sequence similarity searches were conducted using BLAST programs in the NCBI database. Multiple sequence alignments were carried out using the CLUSTALx  and MEGAQuantities of dsRNA amount were compared as following: dsRNA was separated on an agarose gel and stained with SYBR Green. The stained RNA was observed using Kodak EDAS 290 system and relative intensity of dsRNA bands, expressed as integrated area of the peak, was compared. Results were averaged from the three independent dsRNA isolations.S. alkalinus out of the known 18 contained detectable amounts of high-molecular weight dsRNA. These strains were collected in northeast Mongolia in 1999 (isolates F11 and F12) and in the Kulunda Steppe of Russia in 2002 (isolates F13 and F18), locations set about 2500 km apart [S. alkalinus isolates. There were no detected phenotypic manifestations or growth rate differences for the strains containing dsRNA bands, compared to the virus-free isolates. All strains readily produced fruiting bodies and showed intense sporulation on the AA agar medium. Horizontal transfer of the viruses by anastomoses contact was tested in triplicates on the isolates F13 and F18 with the dsRNA-free isolate F7, but no transferred dsRNA was detected in the recipient strain after visualizing total nucleic acids content on a gel. No cured isolate was obtained from the 124 subsequent cultures generated from hyphal tips of the isolate F13 treated with cycloheximide during 4 passages. Therefore, we cannot compare the phenotype of an infected isolate with its isogenic uninfected counterpart. All viruses could be transmitted vertically by asexual (conidia) and sexual (ascospores) spores. Virus-like particles of 35–40 nm in diameter were detected in S. alkalinus strain F13 extract using transmission electron microscopy after negative staining ..S. alkalstaining .S. alkalinus was 6240 nt long, excluding the poly(A) 3´-terminal tail, and had a GC content of 48.9%. Two ORFs were found on the coding strand of this RNA. The ORF1 encodes a putative polyprotein of 1526 aa (estimated mass of 172 kDa). An RdRp and a helicase motif were identified on this putative polyprotein (The longest dsRNA segment isolated from yprotein . PSI-BLAyprotein .−10 to 1e−40)  . No otheo 1e−40) . In addio 1e−40) . In ordeo 1e−40) . On thiso 1e−40) .The ORF1–ORF2 intergenic region of SaFV1 is 69 nt long and is able to fold several stem-loop structures of ΔG −15.7 kcal/mol at 20°C. The 3´ UTR of SaFV1 is 57 nt long and formed a highly stable structure with a calculated ΔG of −22.91 kcal/mol at 20°C .S. alkalinus isolates  were obtained and compared. The viral sequences from the isolates F11 and F12 were identical and genetically distinct from those of the isolates F13 and F18, which were closely related to one another than to the sequences in the isolates F11 and F12. The aa sequences of ORF1 shared 95.6–97.8% sequence identity, and the aa sequences of ORF2 shared 93.3–97.4% identity between the isolates  separates a cluster of the proposed genus Fusarivirus from two hypovirus clades (proposed Alphahypovirus and Betahypovirus genera) . All knoS. alkalinus  with 76% and 67% aa identity, respectively. The 5´ UTR is 67 nt long, while the 3´ UTR is 73 nt long. There are 38 As (76%) in the last 50 nt on the 3´ UTR, a feature commonly observed in betapartitiviruses. The smaller segment is 2122 nt long and contains one ORF encoding a CP protein 641 aa long of about 71 kDa. The noncoding regions are 50 nt and 146 nt long on the 5´and 3´ends, respectively. The last 50 nt on the 3´ UTR contains 14 As (28%). We conclude that these two dsRNA segments from S. alkalinus represent a novel virus in the genus Betapartitivirus, for which the name Sodiomyces alkalinus partitivirus 1 (SaPV1) is proposed. The 400 bp long amplified segment of the CP gene of SaPV1 from the isolates F11, F12, F13, and F18 was compared and analyzed for sequence polymorphisms. Five positions were variable among the isolates, and were nonsynonymous substitutions.The largest segment of 2402 nt contains one ORF and encodes a protein 753 aa long and with estimated mass of about 88 kDa. The RdRp domains typical for dsRNA viruses were found in this protein. The most similar sequences are the RdRps of Ustilaginoidea virens partitivirus 2 and Fusarium poae partitivirus 2 (both of genus Gammapartitivirus), hence, we regard this virus as a new strain  of CtParV1. The nucleotide sequences differed with the 80% and 77.1% respective identity with the corresponding RdRp and CP genes of CtParV1. Conversely, the 400 bp segments of the CP gene from the isolates F11, F12, F13, and F18 of S. alkalinus were 97.8–99.7% identical. This is a very rare example of presence of similar partitiviruses in such diverse hosts and environments. The nucleotide sequence variability between CtParV1 from C. truncatum from an unidentified crop in the U.S.A. [S. alkalinus obtained from a soda soil environment in Asia [The smallest dsRNA segments of 1.8 and 1.5 kbp contain one ORF each encoding the RdRp and CP proteins, respectively. Amino acid sequences of the encoded RdRp and CP share high identity (92% for RdRp and 85.9% for CP) with the corresponding genes of Colletotrichum truncatum partitivirus 1 (CtParV1)  viruses in the isolate F13 of een them .Newly generated viral sequences were deposited in GenBank with accession numbers KT983618, KY817860-61 for Sodiomyces alkalinus fusarivirus (SaFV1), KY296404-5, and KY484539-40 for Sodiomyces alkalinus partitivirus 1 (SaPV1), and Colletotrichum truncatum partitivirus 1 –isolate SAL (CtParV1-SAL), respectively.Sodiomyces alkalinus. Acremonium alcalophilum, the ex-type strain CBS 114.92, a closely related alkalophile, does not have detectable titers of dsRNA viral bands on agarose gel. The recently described alkalophilic Sodiomyces magadii and Sodiomyces tronii species [Emericellopsis alkalina . This makes S. alkalinus thus far the only filamentous alkalophilic fungus known to harbour dsRNA viruses.The virosphere of extremophiles remains largely understudied, with the exception of archaea and some extremophilic  bacteria, for which up to 80 complete viral sequences are known . The pre species  also seeDichanthelium lanuginosum, the fungus Curvularia protuberata, and Curvularia thermal tolerance virus (CThTV) that confers heat tolerance to the grass host [Fusarium culmorum confers salt tolerance to plants [S. alkalinus seems unlikely, since the fungus is not in symbiosis with other organisms and viruses were detected only in 4 out of 18 isolates. The fact that not all isolates of S. alkalinus have the viruses indicates that they are not involved in the adaptations to high salinity or high pH.There are several examples where the virus presence confers benefit to its host. For instance, a unique three-way plant–fungal–virus symbiosis has been described between a tropical panic grass from geothermal soils, ass host . In a coo plants . More tho plants . This fio plants . HoweverHelicobasidium mompa [Penicillium stoloniferum strain and their coincident expression has been demonstrated in the host [Ceratobasidium fungus associated with Pterostylis orchids, recently [It is noteworthy that the three viruses were present in all four isolates together. Viral co-infections by distantly related or congeneric viruses have been described relatively often in different hosts , 47–50 bum mompa . Penicilthe host –52. Extrrecently .S. alkalinus as seen from similar intensities of the dsRNA bands on the agarose gel and from qRT-PCR data. However, there are cases where the viruses are replication-competent and their frequencies are biased towards one virus over the other. For instance, the co-infection of radish (Raphanus sativus) by Raphanus sativus cryptic virus 1 and 2  showed a significantly larger amount of dsRNA from the former virus [Lentinula edodes) by Lentinula edodes mycovirus HKB (proposed genus Phlegivirus) and Lentinula edodes partitivirus 1  increased replication of the RdRp gene of LePV1 by almost 6-fold in comparison to the infection of LePV1 alone [Rosellinia necatrix by Rosellinia necatrix megabirnavirus 2 (RnMBV2) and Rosellinia necatrix partitivirus 1 (betapartitivirus). Specifically, double infection reduced mycelial growth down to 50–60% of what observed in single infections, and RnMBV2 was able to confer hypovirulence with the aid of a co-infecting partitivirus, while the individual viruses exhibited asymptomatic infections [Co-infection by two or more viruses may lead to complex interactions between viruses resembling synergism in plants and animals, enhancing fitness of the viruses, or antagonism, where the presence of one virus lowers the fitness of the others . In the er virus . The co-V1 alone . An unprfections .Heterobasidion parviporum infection with distinct partitiviruses suggested that co-infections by distantly related viral species are more stable than those caused by conspecific strains. Furthermore, mutual exclusion was observed in the same host in closely related HetRV6 strains [Ceratobasidium mentioned above [Observations of the  strains . Long-te strains . The difed above  it is suS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Aspergillus is arguably the most well studied fungal genus because of its medical, ecological and economical significance. Mycoviruses were initially detected in Aspergillus species almost 50 years ago and the field continues to be active today with ground-breaking discoveries. The aim of the present review is to cover the scientific progress in all aspects of mycovirology as exemplified by Aspergillus-focused research. Initially an overview of the population studies illustrating the presence of mycoviruses in numerous important Aspergillus species, such as A. niger, A. flavus, and A. fumigatus with be presented. Moreover the intricacies of mycovirus transmission, both inter- and intra-species, will be discussed together with the methodologies used to investigate viral dispersion in a laboratory setting. Subsequently, the genomic features of all molecularly characterized mycoviruses to date will be analyzed in depth. These include members of established viral families, such as Partitiviridae, Chrysoviridae and Totiviridae, but also more recent, novel discoveries that led to the proposal of new viral families, such as Polymycoviridae, Alternaviridae and, in the context of the present review, Exartaviridae. Finally, the major issue of phenotypic effects of mycoviral infection on the host is addressed, including aflatoxin production in A. flavus, together with growth and virulence in A. fumigatus. Although the molecular mechanisms behind these phenomena are yet to be elucidated, recent studies suggest that by implication, RNA silencing may be involved.Fungi, similar to all species, are susceptible to viral infection.  The diversity of known fungal viruses, or mycoviruses, has increased rapidly over the last few years and this trend is expected to continue, especially with the development and the widespread use of state-of-the-art RNA deep sequencing techniques. Currently, the International Committee for the Taxonomy of Viruses (ICTV) officially recognizes 17 taxa, 16 families and one genus that does not belong to a family; these accommodate (exclusively or not) mycoviruses with double-stranded (ds) RNA linear genomes, positive-sense, single-stranded (ss) RNA linear genomes including reverse transcribing RNA linear genomes, negative-sense ssRNA linear genomes or ssDNA circular genomes  have non-segmented genomes and icosahedral virions. Genera Totivirus and Victorivirus infect exclusively fungi, while other totiviruses infect protozoa. (2) Partitiviridae (partitus = “divided” in Latin) have two genomic segments, each one encapsidated separately in distinct icosahedral virions. Genus Gammapartitivirus infect exclusively fungi, genera Alphapartitivirus and Betapartitivirus, infect both plants and fungi, genus Deltapartitivirus exclusively infect plants and genus Cryspovirus infect protozoa. (3) Megabirnaviridae (megas = “large” in Greek + bi = “two” in Greek + RNA) have two genomic segments, encapsidated together in icosahedral virions. The sole genus in the family exclusively infects fungi. (4) Botybirnavirus  have two genomic segments encapsidated in spherical virions and exclusively infect fungi. (5) Chrysoviridae were first isolated from the ascomycete Penicilliumchrysogenum and have four genomic segments. Similarly to partitiviruses, each one of the four segments is encapsidated separately in distinct icosahedral virions. Chrysoviruses infect exclusively fungi. (6) Quadriviridae (quattuor = “four” in Latin) have four genomic segments encapsidated in spherical virions and the sole genus exclusively infect fungi. (7) Reoviridae (respiratory enteric orphan viruses) have 10–12 segments, which, unlike partitiviruses and chrysoviruses, are encapsidated together in a large icosahedral virion. Out of the fifteen genera comprising the family, only genus Mycoreovirus infect fungi while the rest infect plants, algae, invertebrates and vertebrates including humans. (8) Endornaviridae (endos = “within” in Greek + RNA) possess non-segmented genomes that are non-conventionally encapsidated and accommodated in host-derived lipid vesicles. Both genera Alphaendoviridae and Betaendoviridae infect fungi, while the former also infect plants and oomycetes.The dsRNA mycoviruses are the most commonly found and are classified in seven families and one genus: (1) Alphaflexiviridae genomes are encapsidated in filamentous virions and only genera Botrexvirus and Sclerodarnavirus infect fungi while the other five infect plants. (2) Gammaflexiviridae are very similar to Alphaflexiviridae, with genomes encapsidated in filamentous virions. The sole genus, Mycoflexivirus, exclusively infect fungi. Notably, Alphaflexiviridae and Gammaflexiviridae are the only mycoviral families that belong to an order, Tymovirales. (3) Barnaviridae (bacilliform RNA) have genomes encapsidated in bacilliform virions and exclusively infect fungi. (4) Hypoviridae (hypo = “reduced” in Greek) have non-conventionally encapsidated genomes and are accommodated in host-derived lipid pleomorphic vesicles. Hypoviruses exclusively infect fungi and often cause significant hypovirulence. (5) Narnaviridae (naked RNA) are the smallest, simplest known viruses with unencapsidated genomes. Genus Mitovirus are the most commonly found exclusively infect fungi and are localized in mitochondria. Genus Narnavirus infect fungi, oomycetes and protista and are localized in the cytoplasm. The positive-sense ssRNA mycoviruses that encode a reverse transcriptase enzyme and are incorporated into the hosts' genome are classified in two families, both forming round to ovoid virus like particles (VLPs): (1) In the Metaviridae genus Metavirus include species that infect fungi. (2) In Pseudoviridae genera Hemivirus and Pseudovirus include species that also infect fungi.The positive-sense ssRNA mycoviruses all accommodate non-segmented genomes and are classified into five families: (1) Mymonaviridae (myco + mona) and its sole genus Sclerotimonavirus  in the order Mononegavirales accommodate the only negative-sense ssRNA virus known to infect fungi, a linear molecule encapsidated in filamentous virions. Although the family exclusively infect fungi, the order Mononegavirales accommodate important human pathogenic viruses, including the family Pneumoviridae and the genera Ebolavirus, Morbillivirus (cause of measles) and Lyssavirus (cause of rabies).The family Genomoviridae, genus Gemycircularvirus (gemini-like + myco + circular) accommodate the only ssDNA virus known to infect fungi, a circular molecule encapsidated in isometric virions. Other members of the genus and the family were derived from environmental, plant-associated or animal-associated materialFinally, the family Aspergillus is a genus of filamentous fungi belonging to the class Eurotiomycetes, phylum Ascomycota and comprising >250 species. Aspergillus is one of the most well studied fungi with a worldwide distribution occupying numerous niches, while individual Aspergillus species are medically, ecologically and economically important . Since the 1980's screening large panels of Aspergillus isolates revealed the presence of dsRNA elements in various species  strains examined was shown to harbor two dsRNA elements 3.5 and 0.6 kbp in size where interestingly, the small element was localized in the mitochondrial fraction compatibility between fungal strains have been recognized, with distinct effects on mycovirus transmission  and A. oryzae and A. ficuum (recipients) by Liang and Chen (A. niger (donor) and A. tubingensis (recipient) by Varga et al. (A. niger (donor) and A. nidulans (recipient) by Coenen et al. (A. nidulans (donor) and A. niger (recipient) by van Diepeningen et al.  and slow (AfV-S) according to their electrophoretic mobility. Both AfV-F and AfV-S are isometric viruses, 33–37 nm in size  and its genomic organization is typical of members of the genus Victorivirus, family Totiviridae, with two overlapping ORFs expressed via a coupled termination/reinitiation mechanism. The upstream ORF encodes the CP, 78 kDa in size, while the downstream ORF encodes the viral RdRP, 92 kDa in size  in Greek), contains an ORF encoding an RdRP, 100 kDa in size, and an unusually long 5′-UTR  ca. 6 kbp in size isolated from A. niger strain Ind 1.7.8 , originally isolated from A. niger strain 341, has four dsRNA segments, ranging in size from 1.5 to 3.6 kbp, as its genome where the largest was sequenced and found encode an RdRP with 99% similarity to AfAltV RdRP.The proposed family Exartaviridae would include AfEx, Penicillium aurantiogriseum Gammapartitivirus, family Partitiviridae have been isolated from Aspergillus, Aspergillus ochraceus virus (AoV) from A. ochraceus strain FA0611  from A. fumigatus isolate 88; . The AfuCV genome consists of four dsRNAs, ca. 3.6, 3.2, 3.0, and 2.8 kbp in size, respectively, each one containing an ORF flanked by conserved 5′- and 3′-UTRs. The two largest dsRNAs encode the viral RdRP and CP, 128 and 107 kDa in size, respectively. AfuCV dsRNA3 and dsRNA4 encode two proteins 99 and 95 kDa homologous to those encoded by dsRNA4 and dsRNA3 of typical chrysoviruses, a phenomenon observed previously for Penicillium chrysogenum virus and Verticillium dahliae chrysovirus 1. The former protein contains a “phytoreo S7 domain,” shares significant sequence similarity with the N-terminus of the RdRP and is considered to interact with it and play a role in viral RNA binding and packaging  and consists of four dsRNAs, ranging in size from 2 to 3.7 kbp, and one 3.4 kbp fragment derived from the largest dsRNA has been sequenced and found to encode an RdRP  is the prototype member of the proposed family Polymycoviridae  rich protein that is hypothesized to coat the viral genome, which is non-conventionally encapsidated. The virus was also shown to be infectious to A. fumigatus protoplasts as dsRNA  and in liquid (biomass production) medium, while growth media that differ in their nutritional values often serve to illustrate more clearly the effect of the virus on its host. To date, virulence in A. flavus strain NRRL 5565, the production of carcinogenic aflatoxins was shown to be repressed by the presence of a mycovirus (Schmidt et al., A. flavus isolates. In A. clavatus strains, the presence of mycoviruses was not associated with the production of the mutagenic patulin either (Varga et al., In A. niger strain Ind 1.8.16 was observed to form aconidial sectors that demonstrated a reduced growth rate and increased dsRNA levels. A direct comparison of virus-infected and virus-free isogenic lines confirmed that the presence of dsRNA was associated with a statistically significant decrease in radial growth rate, spore production and competitive spore production that was more evident on minimal medium when compared to water agar, very minimal medium and complete medium (van Diepeningen et al., Virus-infected A. fumigatus isolate A-56 produces darker green pigmentation than the AfuCV-free isogenic strain and forms aconidial sectors. Additionally, its biomass in both minimal and Aspergillus complete liquid medium is significantly smaller (Bhatti et al., G. mellonella (Özkan and Coutts, A. fumigatus isolate 237y demonstrated aconidial sectoring and lighter pigmentation when compared to the AfuPV-1-free isogenic strain, together with a significantly slower radial growth and biomass production in both minimal and Aspergillus complete medium (Bhatti et al., G. mellonella (Özkan and Coutts, A. fumigatus isolate 293 with a AfuPmV-1-free isogenic strain revealed no statistically significant differences in radial growth rate or biomass production; however a statistically significant decrease in the survival rates of G. mellonella larvae infected with the AfuPmV-1-infected strain was observed. This was not accompanied with a concomitant increase in the fungal burden, suggesting that the hypervirulent effect is not due to fungal growth (Kanhayuwa et al., A. fumigatus isolate A78 harbors an uncharacterised virus with an electrophoretic profile identical to that of AfuPmV-1. Both virus-infected isolates show a minor increase in the intensity of pigmentation and form sectors (Kanhayuwa et al., Aspergillus complete medium. This was independently confirmed using three distinct cured isogenic virus-free colonies (Readman, G. mellonella larvae exposed to the virus-infected A78 isolate was noted, which was probably due to the faster growth of the strain as indicated by the fungal burden assay (Özkan and Coutts, A. fumigatus infections and the hypo- or hyper-virulent effects of the viruses on their host, it would be worthwhile to investigate further the presence of distinct mycoviruses in clinical samples in association with clinicopathological parameters and patient survival. Additionally, any potential links between mycoviruses and the worldwide problem of azole resistance (Perlin et al., The AfuCV-infected Readman, . AdditioAspergillus are still largely unexplored. However almost a decade ago, Hammond et al. (A. nidulans and some others, such as the non-conventionally encapsidated AniAltV, may be silenced themselves as part of the anti-viral defense of their host strains. RNA silencing as an anti-mycoviral defense was initially studied in C. parasitica, where deletion of important genes encoding dicer-like or Argonaut-like proteins could lead to severe growth defects in the virus-infected but not the virus-free strains (Segers et al., A. fumigatus. More specifically, 50% of total sRNAs where derived from AfuPmV-1, while the percentage was approximately half, 25 and 22% for AfuPV-1 and AfuCV, respectively. Additionally, sRNAs differentially expressed in virus-free and virus-infected samples were identified, suggesting a putative pathway for mediating observed mycovirus effects on fungal hosts (Özkan et al., The molecular mechanisms underpinning these phenotypes in d et al.  indicateAspergilli are more extensively studied than in any other fungal genus. The major established viral families, including Partitiviridae, Chrysoviridae, and Totiviridae, are well represented in Aspergillus species, while novel families such as Polymycoviridae, Alternaviridae and Exartaviridae have been proposed based on discoveries made in Aspergilli. Population studies and basic mycovirological research have provided valuable insights into the distribution and transmission of mycoviruses in medically, ecologically and economically important species. Finally, distinct phenotypes have been associated with the presence of mycoviruses although their molecular mechanisms are still unknown.Mycoviruses in Future studies should focus on elucidating the molecular mechanisms of host-mycovirus interactions, especially the effects of the mycovirus infection on the fungal host. Off-target RNA silencing is expected to play a role given the dsRNA nature of these viruses that act as both triggers and targets of the antiviral defense system. Additionally, host-virus protein-protein interactions via Eukaryotic Linear Motifs leading to disruption of host pathways may also be implicated in accordance with other host-pathogen systems. The mechanisms controlling mycovirus-induced phenotypic alterations, from reduced aflatoxin expression to hyper- or hypo-virulence are still unclear but such knowledge would facilitate genetic engineering of known mycoviruses in order to induce desired phenomena. The discovery of non-conventionally encapsidated viruses which are infectious as dsRNA, such as polymycoviruses, will facilitate the process by providing us with tractable systems to study and engineer at will.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


We report identification of an Oropouche virus strain in a febrile patient from Ecuador by using metagenomic sequencing and real-time reverse transcription PCR. Virus was isolated from patient serum by using Vero cells. Phylogenetic analysis of the whole-genome sequence showed the virus to be similar to a strain from Peru. Bunyaviridae, genus Orthobunyaviridae) with a tripartite genome consisting of large (L), medium (M), and small (S) segments. OROV causes a self-limiting acute febrile illness, Oropouche fever  is a negative-sense, single-stranded RNA virus , chikungunya virus (CHIKV), Zika virus, yellow fever virus, Mayaro virus, Plasmodium spp., Leptospira spp., and Rickettsia spp. by using real-time reverse transcription PCR (rRT-PCR) and conventional RT-PCR assays developed in-house or acquired commercially . The sample gave borderline results for DENV (quantitation cycle [Cq] 35.3) and CHIKV  and negative results for the other pathogens. We collected a blood sample from a consenting 41-year-old male patient in Esmeraldas, Ecuador, who sought treatment in April 2016 after 7 days of fever, headache, joint pain, muscle pain, and nausea. The patient reported that he had been in Esmeraldas for As an initial screen for other pathogens, we applied unbiased metagenomic sequencing. Analysis of sequencing reads by using Kraken, a system for assigning taxonomic labels to individual reads . We clasAfter confirmation of the presence of OROV by using a validated rRT-PCR . GeneticCulicoides paraensis midge, is absent in the Pacific Coast region, including Esmeraldas , which raises the question of alternative insect vectors in OROV transmission. Culex mosquitoes have previously been implicated as vectors in the OROV urban cycle, notably C. quinquefasciatus (Phylogenetic analysis  showed tDENV and CHIKV rRT-PCR results for this patient were inconclusive. The small proportion of DENV reads in the metagenomic data suggests DENV-1 infection is possible. Using ELISA to detect DENV and CHIKV-specific antibodies may help clarify the results.,,It is likely that cases of Oropouche fever go unreported or misdiagnosed. Clinical features of the disease are similar to those of other viral, protozoan, and bacterial diseases previously reported in Ecuador (This work highlights the need for increased surveillance of OROV in Ecuador and effective differential diagnostic assays to distinguish between emerging pathogens sharing common clinical descriptions to those already circulating. To clarify the true prevalence of this disease in Ecuador, the OROV rRT-PCR assay will be used to screen archived and newly collected samples from a cohort of patients seeking treatment for acute undifferentiated febrile illness during 2016–2017.Isolation process leading to identification of Oropouche virus.


Understanding how heat waves affect morbidity and mortality, as well as the associated economic costs, is essential for characterizing the human health impacts of extreme heat under a changing climate. Only a handful of studies have examined healthcare costs associated with exposures to high temperatures. This research explores costs associated with hospitalizations for heat-related illness (HRI) in the United States using the 2001 to 2010 Nationwide Inpatient Sample (NIS). Descriptive statistics of patient data for HRI hospitalizations were examined and costs of hospitalizations were reported using the all-payer inpatient cost-to-charge ratio. Costs were examined using a log-gamma model with patient and hospital characteristics included as fixed effects. Adjusted mean costs were then compared across racial groups. The mean costs of HRI hospitalizations were higher among racial/ethnic minorities compared to Whites, who accounted for almost 65% of all HRI hospitalizations. Observed differences in costs based on income, insurance, and gender were also significant. These results suggest that these populations are suffering disproportionately from health inequity, thus, they could shoulder greater disease and financial burdens due to climate change. These findings may have important implications in understanding the economic impact public health planning and interventions will have on preventing hospitalizations related to extreme heat. Understanding how heat waves affect morbidity and mortality, as well as the associated economic costs, is essential for characterizing the human health impacts of extreme heat under a changing climate. Current research finds that mortality and morbidity increase during heat waves ,2,3,4. FStudies on the economic impacts of climate change have mainly focused on emissions of greenhouse gases (GHGs), particularly carbon dioxide, and the direct effects GHGs have on global warming and the economy, compared to direct health costs ,12,13. TThis study aims to improve our understanding of the public health costs associated with high ambient temperatures by calculating the economic burden of hospitalizations due to heat-related illnesses in the United States from 2001 to 2010. This includes a targeted analysis of the differences across racial/ethnic and socioeconomic groups. The study findings are intended to inform future interventions aimed at reducing heat-related illness hospitalizations and the costs associated with climate impacts on the health and well-being of the most vulnerable populations including the poor and minorities.International Classification of Diseases, Ninth Revision , primary and secondary procedures, type and source of admission, discharge disposition, primary payer type, total hospital charges, and length of stay. Stratification and weighting variables allow calculation of national estimates and accounts for the complex sampling design [The primary source of data for this study was the 2001 to 2010 Healthcare Cost and Utilization Project (HCUP) Nationwide Inpatient Sample (NIS), which was developed by the Agency for Healthcare Research and Quality (AHRQ). The NIS is the largest all-payer care database, with data from approximately 8 million hospitalizations per year . The NIS includes data on patient demographics and hospital characteristics, primary and secondary diagnoses coded according to the g design .The study population for HRI consists of patients in the NIS with at least one diagnosis of a heat-related illness (ICD-9 codes 992.0–992.9), which includes a spectrum of environment-related heat conditions, such as heat stroke (ICD-9 code 992.0), heat syncope (992.1), cramps (992.2), and edema (992.7), heat exhaustion to fatigue (992.3–992.6), and other unspecified heat effects (992.8–992.9) from 2001 to 2010. Only hospitalizations occurring during the summer months (May–September) were included in the analysis. All other hospital admissions during this time period were also examined and used for comparison.The main outcome assessed in this study was total billed charges per hospitalization, which was assessed by mean adjusted costs. Total billed charges were converted to individual hospital all-payer inpatient costs by applying the AHRQ hospital-specific cost-to-charge ratios to total charges within the NIS dataset. Individual patient records were matched by hospital identification numbers and linked to AHRQ hospital-specific cost-to-charge ratios . For host-test was used the compare differences in adjusted cost means for gender. All statistical analyses were performed using SAS 9.4  and the study conforms to the HCUP data use agreement.Adjusted costs were calculated based on total-billed charges and applying the cost-to-charge ratios. Cost-to-charge ratios were supplied by the AHRQ and multiplied by the total-billed charges ). Adjusted hospitalization costs were then modeled using a log-gamma regression model to appropriately account for the non-normal distribution of costs ,24. IndeApart from the descriptive statistics, there were also notable differences in the comparison of costs associated with HRIs and all other hospitalizations . Using WFurther examination of racial groups shows that among HRI patients with no insurance, hospitalization costs were higher among all racial/ethnic groups compared to patients identified as White. Among patients from the lowest zip-code income quartile, Blacks, Hispanics and Asian/Pacific Islanders had higher costs compared to Whites . SimilarOur analyses indicated that, compared to Whites, higher costs for heat-related hospitalizations were associated with some racial/ethnic minorities, particularly those residing in lower zip-code income areas and among the uninsured. Additionally, although accounting for a smaller portion of HRI hospitalizations, being female was associated with adjusted mean costs significantly higher than males. Patients using Medicaid/Medicare were also associated with significantly higher costs than those with private or HMO insurance, which could correlate to the number of poor and elderly that are hospitalized for HRIs.While a prior study indicated that females were more likely than males to be hospitalized for heat-related respiratory illnesses , our stuOur findings also indicate that adults aged 40–64 years old have a larger percentage of HRI hospitalizations during this time period, though older adults aged 65+ years old had significantly higher costs for HRI hospitalizations. Older adults were also more likely to be covered under Medicare. Patterns of hospitalization costs in the United States have been following this trajectory, with mean costs of all hospitalizations greatest among those aged 45–85 years old and for patients using Medicaid/Medicare, with average costs increasing by 0.9%–2.5% annually between 2003 and 2013 . A studyResearch has shown that HRI hospitalizations have increased during our study period, with more recent climate data indicating that average global temperatures continued to rise during and after the 2001 to 2010 period ,32. StudWhen examining specific racial/ethnic hospitalizations, our results show that Blacks, Hispanics, and Asian/Pacific Islanders have higher hospital costs for HRIs than Whites. Racial/ethnic minorities are also at an increased risk for heat-related hospitalizations and mortality due to extreme heat events ,34,35,36Individuals with chronic medical conditions are also likely to incur greater costs associated with HRIs as certain conditions, such as respiratory or renal diseases, are known to be exacerbated by extreme heat. Preventative actions for reducing heat-related illness hospitalizations among the poor and minorities, such as using air conditioning, are limited, due to the impact of additional operating costs associated with having and using air conditioning or accessing cooling centers ,42. EffoThough the nationwide inpatient sample leverages a very large number of records over the study period, our study has several limitations. First, the NIS does not capture the specific date of admission, only day of the week and month, to protect patient confidentiality, so we could not link specific heat exposures to heat-related illnesses diagnoses. We limited the analyses to the summer months (May–September) as a proxy for exposure, which accounted for 95% of heat-related illness cases, though some could be unrelated to high ambient temperature exposure. Second, the NIS is an administrative database and the specificity and sensitivity of diagnosed cases are dependent on the coding. Administrative data is less accurate than clinical data and heat-related illnesses may often be under-reported. Although the NIS provides weights for national estimates, the 20% sample survey may be influenced by sampling error in which some populations may be over- or under-represented. Third, data from the NIS is also limited in its ability to track patients over time and after discharge. Patients diagnosed with severe heat-illnesses, like heat-stroke, may be transferred out of hospitals or discharged to rehabilitation facilities, which could incur additional costs not captured by this analysis. Additionally, patients that have less severe diagnoses of heat-illnesses may not be admitted to a hospital and only pass through emergency departments. This analysis only captures those patients that have been hospitalized and may not represent the total financial burden associated with heat-related morbidity and mortality.The results of this study suggest healthcare costs for HRI hospitalizations are disproportionally higher for poor people as well as for some racial/ethnic minorities. Though while minority groups may not necessarily be labeled as poor, they still may incur higher HRI healthcare costs. In addition, those with Medicare/Medicaid and other insurance types also had higher HRI hospitalizations costs. These groups have been defined in the literature as being vulnerable to extreme heat and have an increased burden of disease from heat-related morbidity and mortality. Many factors such as underlying medical conditions, healthcare access and behaviors, and socioeconomic status contribute to these disparities and in turn may contribute to a greater economic costs associated with hospitalizations for heat-related illnesses. As climate change and average global temperatures continue to pose a threat to our health and well-being, strategic and cost-effective interventions should be identified and implemented, especially among underserved populations. Our findings reinforce the need for additional assessments of the financial impact that climate change, specifically high ambient temperatures, will have on population health and the healthcare system within the United States.


There have been only limited studies assessing the economic burden of HIV/AIDS in terms of direct costs, and there has been no published study related to productivity costs in Nepal. Therefore, this study explores in detail the economic burden of HIV/AIDS, including direct costs and productivity costs. This paper focuses on the direct costs of seeking treatment, productivity costs, and related factors affecting direct costs, and productivity costs.This study was a cross-sectional, quantitative study. The primary data were collected through a structured face-to-face survey from 415 people living with HIV/AIDS (PLHIV). The study was conducted in six representative treatment centres of six districts of Nepal. The data analysis regarding the economic burden (direct costs and productivity costs) was performed from the household’s perspective. Descriptive statistics have been used, and regression analyses were applied to examine the extent, nature and determinants of the burden of the disease, and its correlations.Average total costs due to HIV/AIDS  were Nepalese Rupees (NRs) 2233 per month (US$ 30.2/month), which was 28.5% of the sample households’ average monthly income. The average total direct costs for seeking HIV/AIDS treatment were NRs 1512 (US$ 20.4), and average productivity costs (before adjustment for coping strategies) were NRs 721 (US$ 9.7). The average monthly productivity losses (before adjustment for coping strategies) were 5.05 days per person. The major determinants for the direct costs were household income, occupation, health status of respondents, respondents accompanied or not, and study district. Health status of respondents, ethnicity, sexual orientation and study district were important determinants for productivity costs.The study concluded that HIV/AIDS has caused a significant economic burden for PLHIV and their families in Nepal. The study has a number of policy implications for different stakeholders. Provision of social support and income generating programmes to HIV-affected individuals and their families, and decentralising treatment services in each district seem to be viable solutions to reduce the economic burden of HIV-affected individuals and households.The online version of this article (doi:10.1186/s12913-017-1976-y) contains supplementary material, which is available to authorized users. An estimated 36.9 million people were living with human immunodeficiency virus (HIV) or acquired immunodeficiency syndrome (AIDS) in the world. An estimated number of 1.2 million people died and 2 million people were newly infected by HIV/AIDS in a year [After the introduction of multiple antiretroviral therapy (ART), HIV/AIDS became a chronic disease, and there is a need to provide long-term care and support for the ill person. HIV/AIDS is concentrated among adults of working age, unlike other diseases , 3. LongIf a member of a farming household is affected by HIV/AIDS at a young and productive age, the household either reduces the size of their farm due to the reduction in the number of farm (family) workers  or hiresThe financial coping mechanism for ill health plays an important role in the economic impact experienced by households . HousehoPast studies about the economic impact of HIV/AIDS have reported that the disease-affected households generate relatively lower income than unaffected households , 15: peoSince the first case of HIV/AIDS was reported in 1988 in Nepal, the nature of the HIV epidemic has gradually moved from being a ‘low prevalence’ to a ‘concentrated epidemic’  among inLack of knowledge about health insurance or unavailability of such insurance for HIV/AIDS causes higher out-of-pocket payment for the disease-affected households in Nepal . This evPoverty is an important factor in the propagation of HIV/AIDS. Poor people are more vulnerable for many reasons, including exposure to high-risk behaviours and poor access to health services . HIV/AIDExamining the overall spending in Nepal, we note that US$ 24.5 million was spent on HIV/AIDS. Of this total spending, 90% came from external sources and 10% from domestic sources . AlthougThere were few studies conducted on the direct costs to the households, but there were no studies which reported the productivity costs due to HIV/AIDS in Nepal. A review reported that there were no sufficient research on economic issues of HIV/AIDS in Nepal . Only twThis is a cross-sectional study that employed a quantitative approach to collect information. A face-to-face structured survey obtained information from the respondents. In 2011, there were 23 HIV treatment and care centres in Nepal. Out of these, six treatment and care centres from six districts were selected purposively, based on their coverage, location and accessibility. These centres were: BP Koirala Institute for Health Sciences, Dharan, Sunsari; Sukraraj Tropical and Infectious Diseases Hospital, Teku, Kathmandu; Bharatpur Hospital, Bharatpur, Chitwan; Rapti Sub-Regional Hospital, Dang; Bheri Zonal Hospital, Nepalgunj, Banke; and District Hospital, Silgadi, Doti.A total of 446 respondents were approached and 415 respondents agreed to participate in the survey (93% response rate). The source of information for the survey was PLHIV aged 18 or over and who had been diagnosed HIV positive more than a month prior to the survey. The PLHIV who came to the treatment centres for check-ups, counselling and medicines were accessed for the study. A purposive sampling method was employed to select the participants for easy recruitment and the active participation of the respondents in the research .A survey questionnaire was used to collect data from the respondents  , the National Centres for AIDS and STD Control (NCASC), and the University of Aberdeen, UK. Informed consent was taken from the respondents before starting the survey. Anonymity and confidentiality were maintained throughout the research process from the data collection to the report dissemination phase. The collected information was solely used for the purpose of the study and kept confidential. No cash payment was made to the participants, in order to avoid ‘bias’ in the over-eager volunteering of information simply to please the researcher. This research has fully considered the ethical procedures at all stages of its research processes.Data obtained from the survey was entered into the SPSS version 20 spreadsheet, and after that coded and cleaned for analysis purposes. Data analysis was done using descriptive statistics - mainly means, standard deviation, frequencies, and percentages; and regression analyses. Measurement methods for direct costs, productivity costs and total costs are described below.Direct costs in this study was measured from the household’s perspective that means household was the payer. Average total direct costs for HIV/AIDS treatment were measured by combining all the average out-of-pocket medical and non-medical costs for HIV infected individuals, as well as costs for accompanying person/s. These included costs of doctors, diagnostic tests, clinic or hospital charges, travel, food, lodging and other items at the time of treatment. Direct costs incurred for accompanying person/s attending for diagnosis and/or treatment were included in the measure of average total direct costs. In Nepal, PLHIV under ART medicine need to visit a HIV/AIDS treatment and counselling centre every month for treatment, routine check-up and counselling services. In addition, while conducting the survey, it was found that PLHIV who were not under ART also visited clinics, hospitals or treatment centres every month to see doctors to check their general health, and treat non-HIV major and minor illnesses . Therefore, the direct cost for the last visit to the treatment centre was taken to be equivalent to the monthly direct costs due to HIV/AIDS in this study.In this study, productivity cost was defined as the inability to carryout normal daily activities (paid and or unpaid work), and their valuation. Normal daily activities were defined as formal and non-formal work carried out by individuals in rural and urban settings.To calculate the productivity losses, the inability of PLHIV was divided into absenteeism and presenteeism. Complete inability to carry out normal daily activities due to illness was defined as ‘absenteeism’, and ‘presenteeism’ was defined as reduced work efficiency due to ill health while still working. The sum of absenteeism and presenteeism was termed as ‘productivity losses’ and their monetary valuation was termed as ‘productivity cost’ in this study. To calculate the productivity losses due to presenteeism, two aspects were taken into account: the period during which losses were experienced and the extent to which work efficiencies were affected. To calculate the respondent’s days lost due to illness, respondents were asked whether they were completely unable to work in the last 2 months (60 days) or not. A visual analogue scale (VAS) ranging from blocks zero (0) to five (5) was used in asking individuals about work efficiency when they were ill but still working. Block zero meant ‘unable to work at all’, block one meant ‘one out of five efficiency’, and so on. Block five (5) meant illness did not affect work efficiency at all. Therefore, respondents would never answer block one or block five if they worked in a state of ill-health, because block zero means absenteeism and block five meant ‘not sick at all’. To calculate the days lost due to presenteeism, the total days worked in a state of poor health was multiplied by the inefficiency of the work during ill health.The days recovered were also calculated in the same way by using a VAS as presenteeism. Firstly, respondents were asked whether they got help or not from others when they were ill. If they said ‘no’, then the days recovered would be zero (0). If they said ‘yes’, then they were asked on how many days they got help. To operationalise this, they were asked on a VAS with blocks of zero to five, how much work was completed by others helping. To calculate the days recovered from other’s help, the days helped by others were multiplied by the amount of work done by other’s help. Although respondents were asked the total days lost due to HIV/AIDS (absenteeism and presenteeism) in the last 2-month period, the total days lost were divided into two, to get monthly days lost due to HIV/AIDS. Thus, in this study, total days lost due to HIV/AIDS before adjustment of coping strategies (sum of absenteeism and presenteeism), total days recovered by other’s help, and days lost after adjustment by coping strategies were calculated.Valuation of productivity losses in this study was done by using per capita gross domestic product (GDP) of Nepal . Valuation using per capita GDP was preferred, because this approach values time loss of rich or poor people by an average for the whole society: it is not appropriate to value rich people’s time at a high rate and poor people’s time at low rate. It is specifically true for HIV/AIDS which unlike other diseases like malaria is not a disease of rich or poor; it affects both rich and poor alike. Likewise, productivity costs ‘before coping strategies’, is preferred rather than ‘after coping strategies’, since coping strategies do not make productivity costs disappear. As the name implies, these strategies simply allow people to cope better. Therefore, productivity costs before coping strategies, were valued by using per capita GDP, and was preferred over productivity costs after coping strategies.The total costs in this study were calculated by summing the average total direct costs to the household, and the productivity costs in a monthly period.In this study, regression analyses were conducted to find the important predictor variables for treatment and access costs for PLHIV (as the direct costs), and productivity costs before coping strategies. Regression analyses for total direct costs to the household were not conducted because PLHIV who took an accompanying person obviously need to pay higher costs because of the additional costs required for the accompanying person/s. This might overshadow the impact of other important variables like the health status, and the income level of the PLHIV. Therefore, regression analyses for treatment and access costs in the place of total direct costs were used.To conduct the regression analyses for treatment and access costs and productivity costs, the predictor variables which were significant to the relevant outcome variables in the descriptive analyses, were taken into consideration. Occupation, ethnicity, CD4 level, self-reported health status, PLHIV accompanied or not, household income and study district were found to be statistically significant variables for the direct costs in the descriptive statistics. Likewise, education, occupation, ethnicity, CD4 level, self-reported health status, PLHIV accompanied or not, and study district were found significant variables for productivity costs in descriptive statistics. As the health status was measured in two forms: self-reported health status and CD4 level, one regression with self-reported health status and another regression with CD4 level were conducted separately along with other significantly contributing variables. Likewise, household income was also presented in two forms: as a continuous variable, and in income quintiles; one regression with income (as a continuous variable) and another regression with income quintiles were conducted separately along with other significantly contributing variables. Thus, four different types of regression analyses for treatment and access costs and three regression analyses for productivity costs were conducted before conducting the tests ; and then, final regression analyses. After that, a number of separate regression analyses were conducted to check if the other variables which did not contribute significantly in the descriptive analyses did emerge significantly as synergistic, or interactive variables. After checking all results, a final list of significantly contributing predictor variables was prepared and used for the final, reduced form of regression analysis.Before conducting the regression analyses, normality, linearity, multicolinearity, and outliers among the variables were checked. Normality and linearity were checked by using histogram and normal probability plot, multicolinearity was checked by using Pearson correlation, and outliers were checked by using Mahalanobis and Cook’s distance matrices. To correct the skewed data, log transformation (log10) was made for treatment and access costs, productivity costs (before coping strategies) and household income. All other variables which were either interval or nominal in nature, were rescored as binary variables. Examples of potential predictors which were changed into binary or dummy variables were: study districts, education of PLHIV, occupation of PLHIV, ethnicity of PLHIV, CD4 level of PLHIV, self-reported health status of PLHIV, and PLHIV accompanied with other.As our outcome variables were log transferred and some of the predictor variables were also log transferred, the regression equation was:Where,Log Y = outcome variable (log transferred)X1,X2,X3, andXk are predictor variablesα represents regression constant or interceptb1,b2,b3, and bk are the unstandardized regression coefficients, where k represents the number of predictor variables, and e is the error.X2 is log transferred.In the above regression equation, predictor variable Out of 415 respondents surveyed, 50.6% were male and 93.3% were aged 18 to 49 years (mean 36 years). Almost 64% of respondents were from rural areas and 66% were literate .3 to 400/mm3. Around 58% of respondents had self-reported good health status, and 29.2% of respondents were accompanied by a family member, friend or relative while visiting the treatment centre. Although the insurance system in Nepal does not cover HIV positive people, five out of 415 respondents reported that they had health insurance, but never used insurance support for their treatment  was NRs 1512 (US$ 20.4), which was 19.3% of the average household income (NRs 7837). Average treatment costs for HIV/AIDS on the last visit was NRs 922 (US$ 12.5), which was 61% of the average direct costs for the last visit. The highest treatment costs was accounted for by the cost for diagnostic tests  (32.2%) followed by cost of medicine (NRs 313 (US$ 4.2) (20.7%). Average access costs for PLHIV on the last visit were found to be NRs 445.4 (US$ 6), which is 29.4% of average direct costs for the treatment on the last visit. Among the access costs, transportation costs accounted 18.5% of the average direct costs. Thus, total treatment and access costs for PLHIV on the last visit was NRs 1367.5 (US$ 18.5), which is 90.4% of the average direct costs for the last visit. The costs for accompanying person/s accounted for 9.6% of the average direct costs (NRs 144.5) (Table 3 (NRs 2051.7) compared to the PLHIV who had CD4 counts more than 200/mm3 (NRs 1319.5) (p < 0.001).While looking at the total direct costs of treatment per visit based on CD4 counts, significantly higher costs were experienced by the PLHIV who had CD4 counts equal to or less than 200/mmThe study found that the average number of days on which a PLHIV was completely unable to carry out normal daily activities (absenteeism) in a month was 3.15, and average days lost while present at work in a state of poor health with reduced efficiency (presenteeism) in a monthly period were 1.9. Therefore, average productivity loss before adjustment by coping strategies in a monthly period was 5.05 days. Average total days recovered by others’ help was 1.45. After adjustment by coping strategies , average total days lost due to HIV/AIDS in a monthly period was 3.6. Here, coping strategies indicate the support from other people like family members, relatives, neighbours or friends who help the PLHIV for their care and any kind of work including household work.Average productivity costs were calculated by multiplying average days lost due to HIV/AIDS in a monthly period with the per capita GDP of the country in 2011. Through valuing productivity losses before adjustment of coping strategies, and after adjustment of coping strategies, two figures of productivity costs were obtained. Thus, average productivity costs due to HIV/AIDS in a monthly period before and after adjustment by coping strategies were NRs 721 (US$ 9.7) and NRs 514 (US$ 6.9) respectively.3 (NRs 1157.2/month) than the PLHIV who had CD4 counts more than 200/mm3 (NRs 565.7/month) (p < 0.001) , which was 28.5% of the average household monthly income. The proportion of average total direct costs was 67.7% of the total costs of the HIV/AIDS (NRs 1512 of NRs 2233), and the proportion of average productivity costs to the average total costs was 32.3% (NRs 721 of NRs 2233).p < 0.001), CD4 level 200–400/mm3 (p < 0.01), CD4 level <200/mm3 (p < 0.001), Kathmandu district (p <0.001) and PLHIV accompanied by others (p < 0.001) contributed positively to the treatment and access costs, but ‘household work’ as an occupation (p < 0.05) contributed negatively.The final reduced form of regression analysis Table  showed t3 to 200–400/mm3 was 20.3%, holding other variables constant. The expected increase in treatment and access costs from CD4 level >400/mm3 to <200/mm3 was 30.3%, holding other variables constant. The expected increase in treatment and access costs from the Doti district to the Kathmandu district was 24.7%, holding other variables constant. The expected increase in treatment and access costs from PLHIV unaccompanied to PLHIV accompanied by others was 16.6%, holding other variables constant. The expected decrease in treatment and access costs from agricultural occupation to household work was 17%, holding other variables constant  showed that the expected increase in treatment and access costs was 3%, with an increment of household income of 10%, holding other variables constant. The expected increase in treatment and access costs from CD4 level >400/mm3, PLHIV coming to Kathmandu district for treatment and PLHIV accompanied by others. The treatment and access costs were lowest among the PLHIV with household work occupation.The regression analyses confirm that household income, health status of PLHIV (CD4 level), occupation of PLHIV, PLHIV accompanied by others, and study district were the important predictor variables of the treatment and access costs. Thus, the treatment and access costs were highest for PLHIV having the highest household income, PLHIV having CD4 level <200/mmp < 0.001), self-reported poor health status (p < 0.001), Dalit (p < 0.01), LGBT  (p < 0.05), Sunsari district (p < 0.001), Chitwan district (p < 0.001), Dang district (p < 0.001), Banke district (p < 0.001), were positively contributing to the productivity costs.Final reduced form of regression analysis Table  showed tThe final regression analysis for the productivity costs (log) shows that the expected increase in productivity costs from the Brahmin/Chhetri to the Dalit caste was almost 29%, holding other variables constant. The expected increase in productivity costs from self-reported good health status to self-reported medium health status was 29.8%, holding other variables constant. The expected increase in productivity costs from self-reported good health status to self-reported poor health status was 128%, holding other variables constant. The expected increase in productivity costs from heterosexual PLHIV to LGBT  PLHIV was 24.2%, holding other variables constant. The expected increase in productivity costs from the Doti district to the Sunsari district was almost 50%, holding other variables constant. The expected increase in productivity costs from the Doti district to the Chitwan district was 70.4%, holding other variables constant. The expected increase in productivity costs from the Doti district to the Dang district was almost 43%, holding other variables constant. The expected increase in productivity costs from the Doti district to the Banke district was almost 38%, holding other variables constant  for HIV/AIDS treatment in our study were higher than other studies on HIV/AIDS conducted in Nepal , 29. HigOur study findings show that the highest proportion of direct costs was accounted for by diagnostic tests (32.2%). There is difference between costs of treatment  in government hospitals and private clinics or hospital. The treatment costs increase significantly if the PLHIV visits to the private clinics or hospitals. Although the PLHIV suspect that they might have HIV, they do not visit the hospital or clinic until they are very ill because of possible discrimination from families, relatives and society. This further helps to increase the costs of treatment. Access costs accounted for the second highest proportion (29.4%) of direct costs. There are no HIV/AIDS treatment and care services in every district of Nepal, meaning some PLHIV need to travel farther resulting into higher access costs. A previous study also mentioned the distance from health care facilities as a main problem in getting HIV/AIDS treatment services in Nepal . MoreoveIn our study, HIV-affected households spent more than a five times higher proportion of household income for HIV/AIDS (19.3%) than reported by the government of Nepal for general health care (3.3% of average household income) , even thWhile comparing our study findings with similar studies conducted in other countries, the average total direct costs of HIV/AIDS treatment in our study is similar to the total median costs in South India  and compAverage total productivity losses (absenteeism and presenteeism) due to HIV/AIDS before adjustment for coping strategies in Nepal was found to be very high in a monthly period (5.05 days). Per month absenteeism  was 3.15 days and presenteeism  was 1.9 days. Productivity costs before adjustment for coping strategies by using per capita GDP for valuation was NRs 721 (US$9.7) per month, which was 9.2% of the average monthly household income. The proportion of productivity costs to total costs (sum of direct costs and productivity costs) before adjustment of coping strategies was 32.3%, when doing a valuation using per capita GDP (NRs 721 of NRs 2233).There have been no studies conducted in Nepal, which measured productivity losses due to HIV/AIDS. The findings of the studies on kala-azar (VL) , 39, hepWe note that studies related to productivity losses due to HIV/AIDS in other countries, studies in India  and MalaAverage total costs  due to HIV/AIDS in Nepal was NRs 2233 (US$ 30.2), which was 28.5% of an average household monthly income. There were no studies in Nepal assessing the per month average total cost of HIV/AIDS to compare to our study. The average total costs due to HIV/AIDS in our study are considerably higher compared to other diseases in Nepal, because of its chronic nature. Other diseases like tuberculosis, kala-azar (VL), hepatitis E, and malaria can be healed through treatment; therefore, HIV/AIDS causes a higher and long-term economic burden on the HIV-affected households than is caused by the above-mentioned diseases.Looking for studies similar to our own, we found few that had developed a complex methodology of measuring economic costs that could be compared with our results. One study in Chad reported the total cost for AIDS care to be considerably higher than our study finding. However, they studied only AIDS patients (a more serious stage of HIV/AIDS), and included funeral costs in their study. A study in Spain reported total costs of care for asymptomatic HIV, symptomatic HIV and AIDS patients to be relatively higher than the total costs found in our study .3 – p < 0.01, CD4 level <200/mm3 – p < 0.001), household income (p < 0.001), occupation of respondents (p < 0.05), respondents accompanied by others (p < 0.01) and study district (p < 0.001).In our study, treatment and access costs (in terms of direct costs) were significantly determined by the health status of respondents  and the richest respondents (fifth income quintile) were almost the same (5.5 days vs 5 days). The poorest PLHIV may be sicker than the richest PLHIV due to the unaffordability of treatment in time and lack of a nutritious diet. However, they have to work every day for their own and their family’s livelihood, although they are sick . PerhapsThis is the first study of its kind in Nepal. The sample of the study is also representative of Nepal’s population. This is because it has a relatively large sample size (>400), and was conducted in six representative districts of Nepal, which cover five development regions, east to west, and hilly and mountainous to Terai (the plains) regions of the country. In addition, the study included both rural and urban areas unlike previous studies, which were focussed only in urban areas. While calculating costs of illness, previous studies had excluded some important cost components like ‘costs for accompanying person’ in attempting to calculate direct costs, and cost of presenteeism in calculating productivity costs. Our study has included all components of the direct costs and productivity costs.This study was conducted in government operated HIV/AIDS treatment centres. Therefore, it excluded those PLHIV who did not visit the treatment centre during the period of the study. Due to the limitations of time and resources, control groups  were not accessed. Cross-sectional studies only capture information from respondents at a certain point of time when data collection is carried out. Therefore, information collected in cross-sectional studies may not be as robust as information collected from longitudinal studies because variations in study subjects may be affected by weather, seasons and time of study. Most of the data in the study were derived from reporting of respondents who needed to recall their past. Therefore, there might be a possibility of recall bias by respondents about the given information and uncertainty about the robustness of the data. Moreover, the data in the study was collected in 2011. There might be changes in costs and burden to the HIV-affected households now due to changes in socioeconomic factors affecting the burden over the period.Although the doctor’s fees and medicine costs in the government hospitals in Nepal are minimal, the HIV-affected households are still paying a considerable amount of money (relative to their incomes) for diagnostic tests, transportation, food and lodging. Therefore, the Government of Nepal should make a policy for the affordable and accessible treatment of HIV/AIDS for everyone. It is recommended that the economic burden of HIV/AIDS could be reduced by decentralising HIV/AIDS related services to district level and putting in place more comprehensive service delivery for HIV/AIDS care, support and treatment.Subsidies should be provided from both private and governmental hospitals for the diagnostic tests for those PLHIV who come from remote rural areas, who have below poverty line income and who are marginalised (such as rejected PLHIV from family or community and being an impoverished widow/widower). Allowances should be provided to marginalised PLHIV, similar to the government current policy for pregnant women at the time of delivery. This policy would not only reduce the economic burden of PLHIV, but also encourage them to go for HIV testing, with adherence to the ART medicine. Early diagnosis means early treatment, lesser costs for treatment and loss of productivity.As the monthly direct costs and productivity losses are very high compared to other diseases, the HIV-affected household faces a considerable economic burden. PLHIV often have limited occupational skills and experience reducing their chances of economic independence. Therefore, we advocate that the Government of Nepal should establish a fuller policy to provide livelihood support (skill development and income generating programmes) for the PLHIV and their family members. The Government of Nepal could provide health insurance to HIV-infected people of as in Merauke, Indonesia.To implement all of these policies, there would need to be a larger budget for HIV/AIDS. This seems justified from the results of this study showing that HIV/AIDS exerts a greater economic burden on HIV-affected households than other diseases. More focus and support should also be given to those PLHIV who come from remote rural areas, who are below the poverty line income, and who are marginalised due to the disease, and their community reactions.In order to expand the knowledge of the economic burden experienced by PLHIV, further study of the economic impacts of HIV/AIDS upon the isolated, rejected, widow, widower and separated PLHIV (from family and society due to the disease) and the impact of HIV/AIDS affected households’ children’s education is recommended. In-depth knowledge of these issues would be beneficial in order to formulate a proper policy to enhance the quality of life for such individuals and their families. Likewise, we recommend conducting a longitudinal study for a 6-month period, including a control group to compare the different variables under investigation, involving all types of PLHIV from rural and urban areas.


Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the newly discovered viruses and the analysis of their impact at the biosecurity, commercial, regulatory, and scientific levels. This paper proposes a scaled and progressive scientific framework for efficient biological characterization and risk assessment when a previously known or a new plant virus is detected by next generation sequencing (NGS) technologies. Four case studies are also presented to illustrate the need for such a framework, and to discuss the scenarios. NGS technologies are progressively reaching the diagnostic field  has revolutionized the study of plant viruses by providing a powerful alternative for their detection and identification, without any Undoubtedly, these new scientific outcomes will have to be handled with extreme care in terms of dissemination from basic research to applied agronomy, and to plant health, agriculture and forestry policy makers and regulators. Indeed, the amount of information needed to assess the risk posed by a new virus species to a certain commodity or region is huge. Scientists may indeed have to work for years to provide the answers needed to conduct a thorough Pest Risk Analysis (PRA) according to international phytosanitary standards (ISPM 2 and ISPM 11) FAO, , 2007. TOverall, the advent of new NGS technologies wisely complemented by traditional or classical virus study methods is expected to globally promote plant health and plant protection, but may also cause mayhem in trade and agriculture if challenges and questions are not addressed properly.Quarantine and certification lists are regularly updated following new PRAs. They are based on 6 categories of information: (i) knowledge of the identity of the pest ; (ii) data on its distribution and (iii) host range; (iv) information on the modes and efficiency of spread and on the identity of any vector(s); (v) suitability of the local agro-environmental conditions for the pest [and vector(s)]; and (vi) the ability to cause a disease and impact the development, reproduction, or productivity of cultivated or wild host plants. Further, refinement of the risk assessment can be based on additional information such as the availability of efficient and easy-to-implement control methods.The PRA provides the risk assessor with a risk assessment tool whose details will depend both on data availability concerning the six points listed above and on the needs of the risk manager, whose role is to consider the available scientific information (risk assessment) as well as other factors  in reaching a decision on whether to regulate or address the pest in any specific way.When a known virus is detected by NGS technologies, the main questions are related to its legal status  or Grapevine pinot gris virus (Germany). In such a case, risk assessors cannot wait until enough data is available and first conclusions have to be reached based on whatever information is at hand. This must be done cautiously, while simultaneously taking into account the uncertainties associated to or caused by the lack of data and making them clear to the risk manager.In this context, the increasing identification of new viral sequences by NGS technologies may complicate the decision-making process for certification programs, quarantine processes, and more generally the trading of plant materials . The only information available may be the full or partial genome sequence with the additional complexity that it may have been detected in asymptomatic samples or in combination with other viruses. It is therefore increasingly important to understand the biology of any new viral sequence to provide a basis for assessing the risk they pose and take scientifically-based decisions. Scientists are therefore now challenged to provide biological data on these newly described viral sequences, in a short timeframe and with limited funding. The following chapters propose a framework of scaled actions to efficiently address the need for PRA of regulatory authorities.The detection of a new viral sequence should directly be followed by several immediate actions for its early biological characterization to reach a first decision level of notification to authorities -PCR using specific primers. If the concentration is very low, the sample can be enriched in viral particles through homogenization, filtration, and/or ultracentrifugation prior to nucleic acid extraction. Enriched viral particles can also be observed by electron microscopy, even if electron microscopy lacks sensitivity and viral particles can be missed. For DNA viruses, especially http://www.ictvonline.org). The detected viral sequences may also correspond to a bona fide virus infecting other organisms associated with the sample, including bacteria, fungi or arthropods  the frequent incompleteness of the genome sequence data obtained, (ii) the variable and irregular coverage of viral genomes, (iii) the incompleteness of viral taxonomy, and (iv) the variability of species, genus, or family molecular discrimination criteria. Specific guidelines can be found in the latest ICTV report  the putative host range for the tentative identification of alternative hosts, (ii) the prediction of the pathogenic potential in those hosts, (iii) the modes of horizontal and/or vertical transmission, including the identification of candidate vectors. These hypotheses have practical consequences such as the selection of appropriate hosts and experimental modes of inoculation, a first assessment of the possible epidemic potential of a new viral agent, or a tentative identification of possible alternative hosts in ecosystems. Altogether, this early step, which requires expertise in molecular biology, in plant virology, and basic bioinformatics skills, might lead to the elaboration of a draft epidemiological scenario  and time of appearance, to identify the plant species and cultivar, to describe the sampled plant part , the geographical origin of the sample, its intended use etc…Any missing information should be completed at this stage so as to be included in the first notification.de novo generated contigs, verify their exclusive viral origin and the absence of host-derived sequences. Taxonomic assignment can also be fine-tuned thanks to the whole genome sequence; but in a worst case situation, even the availability of a complete genomic sequence may be insufficient to settle such a question  based on NGS reads can result in three scenarios of genome coverage: (i) complete, (ii) incomplete but continuous, or (iii) a set of scaffolds and contigs. The reconstruction of the full genome sequence of a candidate new virus is mainly based on targeted (RT)-PCR amplification and sequencing to fill the sequence gaps between Identifying a new virus species, even from a partial genome sequence, triggers the immediate need to develop a diagnostic protocol. This is a fundamental step toward the management of viral diseases in cultivated crops or the unveiling of a new virus eco-epidemiological role in wild plants. The diagnostic of newly identified and confirmed viruses is usually done using PCR-based methods (the most popular ones for new viruses) or with the LAMP technology, as a recent alternative  and vector(s), their geographical spread and seasonality. Likewise, sampling methods enabling for the statistical linking of findings and field observations should be selected. Several scenarios are possible depending on the origin of the sample: field or trade/quarantine/certification.The presence of symptoms in the initial field host has often been perceived as a starting point for further investigations about the presence of a virus, but asymptomatic plants must also be included in the sampling because symptomatology reflects the complex interplay of infectious agent(s), the host plant metabolism, its defense systems and varietal specificities, as well as abiotic factors. The advent of wide metagenomic studies of environmental viromes  symptomatology for individual viruses and for mixture of viruses, (ii) host range studies and transmissibility, (iii) the assessment of natural or induced mutation/recombination rates in host plants, and (iv) the effects of targeted modifications on virus-host interactions and symptomatology. Mastering virus transmission will also be key to the development of research efforts to understand the potentially positive effects of viruses on plants. Inoculated plants from various species or cultivars can be submitted to biotic or abiotic stresses, and their reaction can be studied in laboratory and/or in greenhouse experiments to provide evidence of beneficial interactions.Large-scale surveys, organized on a national or international scale and based on the diagnostic technique(s) developed for the new agent, should be undertaken to evaluate its prevalence and distribution within a given ecosystem  and potentially prevent “conflicts” between import/export partners.All the authors significantly contributed to the writing and editing of the manuscript. SM created the figures. SM and TW coordinated the writing and editing of the manuscript.This article is based upon work from COST Action FA1407 (DIVAS), supported by COST (European Cooperation in Science and Technology).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.