To explore how being infected with the Zika virus during pregnancy was experienced by affected women, and how it influenced their family relationships and future family planning.We conducted a qualitative study, including 19 semistructured interviews with women of reproductive age and confirmed Zika infection during 2015–2018 in Medellin, Colombia. Purposeful sampling was applied, and participants were identified through National Public Health Surveillance System records. Interviews were recorded, transcribed verbatim, and analyzed using content analysis with inductive and deductive approaches.Of 19 women interviewed, eight women identified the pregnancy as unexpected and two women had undergone permanent sterilization. Women had mixed views about decision‐making related to family planning, and not having an abortion in a future pregnancy was influenced by religious beliefs. Women knew about vector‐borne transmission but were not well informed about sexual transmission of the virus. Women desired better support and guidance to ease concerns about Zika virus.All interviewed women expressed a need for more information about Zika virus and continuous support, specifically after delivery, from healthcare professionals. Communication strategies to enhance culturally sensitive messages and for accurate perception of information are recommended during Zika outbreaks. Women interviewed were unaware of sexual transmission of the Zika virus. Women desired continuous counselling and support during their pregnancy and after the birth. The susceptibility of becoming infected with Zika virus appears to be similar in pregnant compared with nonpregnant women, and up to 80% of infected individuals are asymptomatic, while the rest experience fever, arthralgia, conjunctivitis, and maculopapular rash, similar to chikungunya and dengue infection.The Zika outbreak in Colombia officially began in October 2015 when nine cases were confirmed by laboratory tests.Aedes aegypti mosquitoes, which also transmit dengue, chikungunya, and yellow fever.Zika virus is transmitted predominantly through the bite of infected The Zika virus outbreak highlighted barriers to sexual and reproductive health and rights (SRHR) in Latin American countries, such as Brazil and Colombia, including restrictive sexual and reproductive health (SRH) policies and reduced access to SRH services. In 2006, Colombia decriminalized abortion only under exceptional circumstances, including life‐threatening maternal conditions and severe fetal brain malformations, such as those seen in CZS.To improve prevention and management of Zika virus infection, the aim of the present study was to understand the perceptions among women of reproductive age who had been infected with Zika, the impact on their families, and their reproductive choices and plans.2A qualitative approach using content analysis was considered appropriate for our study to understand women's experience of being infected with Zika virus during pregnancy, concerns about CZS, and perceptions on family planning.The study was conducted in Medellin, Colombia—a city of over 2.8 million inhabitants in 2017, of whom an estimated 54.4% are women.Inclusion criteria for participants were: (1) age 18–45 years; (2) diagnosis of Zika virus during pregnancy; (3) permanent resident and residing in Medellin; (4) Zika virus infection reported to the National Institute of Health in Colombia and the infant considered a suspected case of CZS After establishing formal permission to conduct the study from the Secretariat of Health in Medellin, informed consent was provided over the telephone from women who were registered as a confirmed case of Zika infection and whose baby was suspected to have CZS. We informed the potential participants about the study objectives and goals, the voluntary aspects of participating in the interview, potential risks and measures taken to prevent those risks, as well as the mode of the participation for the face‐to‐face interview. After we had obtained written consent, the participants were still able to withdraw at any point during the face‐to‐face interview without any negative consequences for their healthcare services.Participants were asked if they preferred a male or a female interviewer and if there was a preference in the location of the interview, as we wanted them to feel comfortable. To facilitate attendance, we provided reimbursements for any transportation costs. Semistructured interview was chosen as the method of data collection. The research team constructed and piloted an interview guide with questions in Spanish, probing areas under topics related to awareness of Zika virus and means of information, experiences during the pregnancy and family planning in relation to abortion, as well as experiences surrounding healthcare staff and core support system , but when we were in school, they told us we were going to have a class about that, but I didn't remember all of it. I remembered the symptoms. I remember watching all that and saying, oh my God, this is what's going on. All 19 women had previously heard about Zika virus and knew where to find additional information. The women mentioned several sources of information about the virus, such as television or radio, from healthcare professionals or doctors, and attending workshops in their community or school seminars. One participant described:Participant 17, age 30Well, to tell you the truth, when I watched it on television, I didn't believe that could happen to me, there have been many types of mosquitoes, and that never happened. When they told me that I [had it], I didn't believe it because I thought it usually only happens on television, until I had to live it. I have this experience now. Participants mentioned that the information shown on television or heard on radio had a greater impact not only to become cautious about contracting Zika virus, but also learning about the different types of mosquitoes. A participant said:Participant 5, age 20I can't be with long sleeves and long pants every day. The mosquito can bite me here or even in the tip of my finger. That is very hard to control. Regarding prevention of Zika virus infection, all of the women were aware of different ways to prevent becoming infected with Zika virus by mosquito bites. For instance, preventing becoming bitten by mosquitoes was exemplified by the participants. One woman said:Participant 7, age 23We learned that it was a mosquito that bit pregnant women, and that made the baby born with malformation. Participants also reported means of preventing the spread of Zika virus by eliminating the breeding sites for the mosquito. The women knew about the possible consequences during pregnancy when Zika virus occurred; this was sometimes referred to as microcephaly, but mainly described as malformations to the baby's head. One of the participants stated:Participant 1, age 20I have heard about chikungunya and Zika, but I heard it was almost the same thing, the same results. When asked about the differences between Zika virus and other mosquito‐borne diseases, including chikungunya, one of the participants stated:The women indicated that they were familiar with the different mosquito‐borne diseases, and the true impact of dengue or chikungunya was better understood when people in their communities or close friends had experienced such disease.3.2Participant 19, age 33During the pregnancy, the father of my baby provided me with everything I needed and for my baby. Now, however, we are not living together. All 19 participants reflected on the type of support given at the time of diagnosis. Four subcategories emerged within this theme, including reliance on their partners and families, changes in their relationship with their partner, reliance on healthcare professionals, and health services. Most of the women received some form of financial support from her partner and family. Regarding care of the child, participants stated the possibility of leaving their child under the care of the extended family as an element that contributed to the quality of life of the family. When asked about the relationship with their partner after Zika virus diagnosis, many of the participants mentioned that there was significant change in the interaction between the father and their child. As described by one participant:Participant 11, age 36After seeing how the doctor behaves with my other daughter, I noticed a doctor discriminates her [the baby] a lot and wanted me to feel bad for my daughter's medical condition. A key aspect in terms of support provided to the women, with an influence on the quality of life of the child, is determined by the role of healthcare professionals. The participants positively assessed the possibility of sharing their doubts and concerns with healthcare professionals. However, their experiences concerning the type of healthcare support was contradictory; for instance, one of the participants expressed feeling unfairly judged when she had visited the doctor. The words of this mother illustrate this:Participant 18, age 38The last 15 days of my pregnancy the medical care was very good, but after she [the baby] was born, nothing is authorized, and I am sent to places where they tell me they don't have a healthcare specialist for her care. After they had been diagnosed, participants expressed the need for better support, guidance, information about Zika virus, and further advice from healthcare staff, particularly after giving birth.Participant 1, age 20The information on television was helpful, but I wanted to know more about the situation with Zika, so that you can take more preventive measures than what they say in the news. Even though most of the information about Zika virus was provided by the local radio news, several women mentioned that they would have preferred to be invited to an educational session with healthcare personnel during their pregnancy to talk more about microcephaly and other worries in relation to Zika virus. One participant mentioned:3.3Participant 4, age 31It was very difficult, very hard. Aside from the pregnancy, I would not worry about myself, but only for him [the baby]. The immediate emotional response mentioned by the participants was concern about the possible Zika‐related health complications during pregnancy, specifically for the fetus. Concerns about Zika virus were expressed in three subcategories: meeting other people with Zika virus, feeling worried about consequences at the time of diagnosis, and experiencing emotions about having a baby with microcephaly. All 19 women experienced feeling worried about their baby at the time of diagnosis, and other types of emotions were referred to as concerns and fear of the baby being born with microcephaly. When asked about their concerns surrounding babies born with microcephaly, one mother said:Participant 17, age 30I got scared when I was told my baby could come out with problems like microcephaly or brain paralysis or many other things – I imagined my baby was going to be born with all that. Another mother mentioned:The participants expressed that their concerns about Zika virus had not changed as the pregnancy progressed. All 19 participants summarized their fears of having a child diagnosed with microcephaly.Participant 5, age 20I tried not to read stuff about Zika, because it would have been more traumatizing to see what I saw after I had her. It was better not to see that, otherwise the pregnancy would have been more difficult. Women who had met someone infected with Zika virus also said that they became more worried about their prognosis at the time of pregnancy. The women were also disturbed by the images of newborns with microcephaly. One mother said:Participant 18, age 38I think no one is prepared to see children with microcephaly, because there are mothers who don't accept it and have difficulties to work – I think people are not prepared to work for the children affected with microcephaly. When asked about social interactions with other people who had had the Zika virus, participants mentioned potential social stigma. One mother said:3.4Participant 19, age 33[I don't want any more children]… because she [the baby] needs a lot of care and I have to pay a lot of attention to her. I could also have another baby with microcephaly, so No. Eight of the participants (42%) identified the pregnancy as unexpected/not planned, and two participants (10%) had undergone permanent sterilization to prevent a new pregnancy. When asked if the women were planning to have more children, many of the respondents expressed both desire and unwillingness to become pregnant again. One woman said:Participant 4, age 31I don't know if I want another baby, I would let God decide that. While another participant said:Participant 5, age 20I thought about it… if the doctors tell me that my baby was going to be born with malformations or with Down syndrome, and they gave me the opportunity to interrupt the pregnancy, I maybe would have done it. From a medical point of view, it's a disease and I would think about it, but from an emotional point of view, I can't consider deciding against my baby's life. Despite the mother's diagnosis of Zika virus at the time of pregnancy, not all children had microcephaly. Nevertheless, the participants had contemplated a decision about abortion in a future pregnancy, which was also influenced by healthcare professionals.Participant 17, age 30If the doctor tells me that the pregnancy might put me in danger, I would also think about it – I have three other girls and if the baby is coming sick, knowing that whatever God gives you is for a reason, I would… yes, it sounds difficult. Another mother explained that contemplating the decision would rely on the care of her other children, such as:Participant 18, age 38It would be very difficult for me… I would have him [the baby], eventually. It is still a life; he is not guilty of anything. When asked about the hypothetical situation of becoming pregnant again during another outbreak of Zika virus, all 19 mothers responded that they would continue the pregnancy based on personal or religious beliefs. One participant said:Participant 19, age 33I would take the risk, completely. He [the baby] is not guilty of anything and I would risk having him. If in case he's born with problems, that is God's will, but if God gives him to me and as long as I am able to have him, I would risk it no matter the difficulty. Most of the women sought prayer as support during pregnancy. The baby was not seen as responsible for the mother's decision to continue the pregnancy. One woman said:3.5Participant 4, age 31I had Zika and chikungunya tests, but at the time, it was also dengue… I did not know I had Zika, they said I probably had dengue and they would call, but they never called me. Information provided by different media sources and healthcare professionals about Zika virus was perceived as inaccurate by the participants. Despite being a registered case of Zika virus, many of the women were given another diagnosis other than Zika virus, which resulted in a late or unclear diagnosis. One woman said:The participants were knowledgeable and willing to be informed about prevention of Zika virus and mosquito‐borne transmission. The women were asked to share their understanding of how Zika virus is transmitted, and all 19 knew that it is transmitted by mosquitoes and it affects people, especially women who are pregnant. However, when the women were asked if they had heard that Zika virus can be transmitted through sexual contact, all 19 participants said they did not know.Participant 3, age 18I never heard that Zika could be transmitted through sexual contact, but it makes sense, because if that is in the blood then any kind of contact, it can also happen… then the media should also say that was [sexual transmission] if it is true about Zika. Hence, many women were surprised when asked what they had heard about Zika virus and its modes of transmission. One participant said:Participant 7, age 23God, in what moment did that mosquito bite me, I was always in places where it was cold. I was pregnant, and so the mosquito must have gone in my house where there was dirt. There were also misconceptions about Zika virus in relation to transmission only occurring in warmer climates. One of the participants said:4This qualitative study explored experiences and perceptions of Zika virus during pregnancy among 19 women in Medellin, Colombia. The participants were registered as cases of Zika virus in SIVIGILA that had been confirmed by healthcare staff on the basis of clinical symptoms or laboratory diagnosis made by reverse transcriptase PCR test. However, the majority of the women felt that their diagnosis was unclear. In addition, laboratory confirmation of Zika virus is challenging owing to the possible cross‐reactivity with dengue,All participants knew that the primary source of information was from the health authorities. The women independently sought more details about Zika virus from the local news coverage, such as the television or radio, and by accessing the internet. However, the women interviewed were not well informed about all transmission modes. Participants did not refer to condom use as a form of prevention against Zika virus, and this is explained by the participants’ low knowledge about sexual transmission. Other qualitative studies have indicated a high awareness of CZS among women of childbearing age at risk of Zika virus; however, little information was provided to them about sexual transmission.The interviews with the women revealed that cases of Zika virus were more common among young and unemployed women of low and middle socioeconomic status. Lack of proper infrastructure and poverty may lead to disparities in service provision and information. We found that all 19 participants had access to reproductive healthcare services. A recent study on Zika virus and microcephaly in Brazil found that almost all registered cases were from low‐income families, suggesting that the epidemic both reflects and may widen socioeconomic inequalities. For example, women in Brazil with a child born with Zika‐related health complications tend to be young, single mothers in relative poverty, living in remote parts of the country.All 19 women were the prominent caregivers to their babies. We found that the participants were reliant on either their partners and family or on healthcare services overall. Thus, individual quality of life and family dynamics are impacted in many ways; for instance, previous research highlighted several challenges families of children with CZS may face, such as complex medical care and economic burden, uncertainty for children about the unfolding consequences of CZS, limited professional knowledge, and potential social isolation and stigma.Women reported that support from healthcare providers for care of their children outweighed the immediate social and family support. Many of our participants were relieved to be able to share their doubts and concerns with healthcare professionals throughout their pregnancy. However, it was evident that the participants diagnosed with Zika virus desired further support from healthcare professionals, particularly after giving birth, to comprehend and gain more information on unexpected consequences related to Zika virus. Despite the existence of universal health coverage and an integrated social welfare support system in Colombia, no long‐term programs have been established to specifically support children born with Zika‐related health complications.The women did not report signs of mental health disorders. However, much of the necessary healthcare support described by the participants, such as providing accurate information and supportive communication, may also apply to other psychosocial support including management of mental disorders and strengthening social support and social networks. It is important to note that information for and referrals to specialized healthcare support services, such as psychological support or counseling, were provided to all of the women interviewed as part of the study protocol, and at least one of the participants sought counselling immediately after the interview.Colombia remains among the countries with the highest number of Zika virus cases, with 182 cases reported in 2019, as of May 31.More subtle but still significant is the impact of the disease during pregnancy and the unknown long‐term effects of the virus, which seem to influence decision‐making on family planning. SRHR regulations in Colombia aim to prevent unwanted pregnancies, and do not require parental consent for SRH counseling or access to contraception.Many of the women interviewed perceived the prevention of Zika virus transmission during their pregnancy as their sole responsibility, in part because the health authorities had failed to communicate clear information about sexual transmission of the virus. SRHR remains an essential component during the outbreak of Zika virus in Colombia and making an informed decision for family planning is crucial, both for couples who plan to get pregnant or to delay it, and for those who are already pregnant.In conclusion, this study explored the experiences and perceptions of Zika virus among women during their pregnancy. Further research is recommended to explore the impact of Zika virus on the uptake of family planning services, unmet needs from these services, and the role of male involvement in women's sexual and reproductive health. In addition, the sources of information, such as health authorities and governmental sources, should work to ensure provision of clear and unambiguous recommendations and advice that is culturally sensitive to stress the key messages in the context of Zika virus.All authors contributed to the planning and proposal of the research project. VT, SAMM, and BNRJ conducted the data collection, analysis, and writing of the original report. All authors contributed to reviewing drafts of the paper. All authors read and approved the final manuscript.The authors have no conflicts of interest.Data S1. Interview guide (Spanish/English).Figure S1. Mapping of the categories and subcategories developed using both inductive and deductive approaches (Spanish).Figure S2. Example of a trail analysis.Click here for additional data file.


Magnaporthe oryzae strains YC81-2, including one virus in the family Tombusviridae, one virus in the family Narnaviridae and four viruses in the family Botourmiaviridae. Since the RNA-dependent RNA polymerase (RdRp) of one botourmiavirus show the highest identity (79%) with Magnaporthe oryzae ourmia-like virus 1 (MOLV1), the virus that was grouped into the genus Magoulivirus was designated as Magnaporthe oryzae botourmiavirus 2 (MOBV2). The three other novel botourmiaviruses were selected for further study. The complete nucleotide sequences of the three botourmiaviruses were determined. Sequence analysis showed that virus 1, virus 2, and virus 3 were 2598, 2385, and 2326 nts in length, respectively. The variable 3′ untranslated region (3′-UTR) and 5′-UTR of each virus could be folded into a stable stem-loop secondary structure. Each virus consisted of a unique ORF encoding a putative RdRp. The putative proteins with a conserved GDD motif of RdRp showed the highest sequence similarity to RdRps of viruses in the family Botourmiaviridae. Phylogenetic analysis demonstrated that these viruses were three distinct novel botourmiaviruses, clustered into the Botourmiaviridae family but not belonging to any known genera of this family. Thus, virus 1, virus 2, and virus 3 were designated as Magnaporthe oryzae botourmiavirus 5, 6, and 7 , respectively. Our results suggest that four distinct botourmiaviruses, MOBV2, MOBV5, MOBV6, and MOBV7, co-infect a single strain of Magnaporthe oryzae, and MOBV5, MOBV6, and MOBV7 are members of three unclassified genera in the family Botourmiaviridae.Via virome sequencing, six viruses were detected from  Mycoviruses, viruses of fungi, are ubiquitous and diverse in all major phyla of fungi . They caAlphaflexiviridae, Barnaviridae, Gammaflexiviridae, Deltaflexiviridae, Narnaviridae, and Botourmiaviridae [Botourmiaviridae, a new family established by the International Committee on the Taxonomy of Viruses (ICTV) [Ourmiavirus, including plant ourmiaviruses, and three mycovirus genera, Scleroulivirus, Botoulivirus, and Magoulivirus, that include botourmiaviruses (previously named ourmia-like mycoviruses). However, several botourmiaviruses do not belong to the above three mycovirus genera, and have been clustered into an undefined clade.The genomes of most mycoviruses are double-stranded RNA (dsRNA) or linear positive-sense single-stranded RNA (+) ssRNA . Only a aviridae ,11. AmonMagnaporthe oryzae, the pathogen of rice blast, was discovered in 1971 [M. oryzae. Of these mycoviruses, dsRNA viruses are clustered into three families: Magnaporthe oryzae virus 1, 2, and 3  are classified into the family Totiviridae [Chrysoviridae [Partitiviridae. In addition, several (+) ssRNA mycoviruses have also been found to be associated with M. oryzae. Among them, Magnaporthe oryzae virus A is closely related to plant viruses of the family Tombusviridae. Magnaporthe oryzae ourmia-like virus 1 (MOLV1) and Magnaporthe oryzae ourmia-like virus 4 (MOLV4) are in the family Botourmiaviridae [P. oryzae isolate infecting wheat [M. oryzae, in the family Narnaviridae, Magnaporthe oryzae narnavirus virus 1 (MoNV1), was identified [The first mycovirus in  in 1971 . Since tiviridae ,14,15. Moviridae ,17,18. Moviridae , and 3 baviridae ,22,23. Ang wheat . Recentlentified .Chrysoviridae and Partitiviridae, have been reported to impact the the biological characteristics of their fungal host, M. oryzae. Among them, three chrysoviruses, MoCV1-A, MoCV1-B, and MoCV1-D, confer hypovirulence to M. oryzae. The infection of these hypovirulent viruses diminishes the pathogenicity of their fungal hosts by changing the biological characteristics of M. oryzae, such as by impairing hyphal growth, causing abnormal colony morphology, and reducing pigmentation [M. oryzae [M. oryzae sporulation and virulence to barley substantially decreased. However, the effects of the (+) ssRNA mycovirses from three families, Tombusviridae, Narnaviridae, and Botourmiaviridae, on M. oryzae have not been previously mentioned.To the best of our knowledge, only four dsRNA mycoviruses in two families, entation . In addi. oryzae . After iA single fungal strain can generally be infected by multiple mycoviruses. This kind of co-infection can be facilitated by chemical compound- or virus-mediated suppression of heterogenic recognition and RNA silencing ,27,28,29M. oryzae, we isolated and characterized four novel (+) ssRNA botourmiaviruses, designated as Magnaporthe oryzae botourmiavirus 2 (MOBV2), MOBV5, MOBV6, and MOBV7, from a single M. oryzae strain, YC81-2, that was isolated from a field in China. Sequence evaluations and phylogenetic analysis of their RdRps showed that MOBV2 belongs to the genus Magoulivirus, while MOBV5, MOBV6, and MOBV7 are clustered into three undefined clades.In this study, to expand the knowledge of mycoviral diversity in M. oryzae strain, P131, was used as a control [Diseased rice leaf samples with the typical symptoms of rice blast were collected from a rice field in Yichang, Hubei province, P. R. China. Single-spore  isolation was performed from lesions of the diseased rice leaf samples. Then, a single-spore isolate, YC81-2, was selected for further study. A virus-free  control . The colFor RNA extraction, a fresh mycelial sample of strain YC81-2 was ground into fine powder in liquid nitrogen. To detect RNA viruses in strain YC81-2, dsRNA extraction was performed as previously described . The dsRM. oryzae were filtered out from the raw data. Then, de novo assembly was performed with CLC Genomics Workbench (version: 6.0.4). Primary UniGenes were spliced to generate final UniGenes using CAP3 EST. The final UniGenes were then subjected to BLAST, using BLASTx to search for homology with viral sequences against non-redundant (NR). The contigs corresponding to the same viruses were assembled to generate viral fragments by DNAMAN.Virome sequencing was carried out on the Illumina HiSeq2000/2500 platform by GENEWIZ Technology Services . In brief, one microgram of total RNA was used to obtain ribosomal-depleted RNA and construct a cDNA library using a VAHTSTM Total RNA-seq (H/M/R) Library Prep Kit for Illumina . After sequencing, adapter-polluted, contaminated, paired-end reads shorter than 100 bp, low quality, high content of unknown base (N) reads, and the RNA and DNA sequences of 4 RNA ligase . The PC3-T7 loop-ligated ssRNA was purified, denatured in dimethyl sulfoxide , and then reversely transcribed into cDNA. The terminal sequences of the viral cDNA were amplified with the primer PC2 (5′-p-CCGAATTCCCGGGATCC-3′), a complementary primer to the PC3-T7 loop, and sequence-specific primer F1 or R1, corresponding to the 5′- or 3′-terminal sequences of all viral fragments. Then, using the first round PCR products as templates, primer pairs PC2/F2 and PC2/R2 were applied to conduct Nested PCR amplification of the 5′- and 3′-ends of the viruses, respectively. After purification with a gel extraction kit , the purified PCR amplicons were cloned into the pMD18-T vector  and sequenced. The experiment was repeated three times independently. Primers used in this study are listed in The ligase-mediated terminal amplification was performed to determine the terminal sequences of the viruses . The adaThe ORFs of the viruses were predicted based on standard genetic code, and their homologous amino acid sequences were searched in the NCBI database, using ORF finder and the BLASTp programs, respectively. Sequence alignment was performed using the CLUSTALX 1.8 program . AnnotatThe morphological characteristics of the strains YC81-2 and P131 on PDA were observed. The colony of strain YC81-2 was black and flat, with little mycelia. The colony of strain P131 was grayish white and flat, with dense mycelia A. There Tombusviridae, Magnaporthe oryzae virus A (MoVA), one novel virus in the family Narnaviridae, named as Magnaporthe oryzae narnavirus virus 2 (MoNV2), and four novel viruses in the family Botourmiaviridae. Based on the alignment with non-redundant protein sequence collection in the NCBI database using BLASTp, the RdRp of one novel botourmiavirus displayed the highest identity with the RdRp of MOLV1. Thus, this virus was classified in the genus Magoulivirus and designated as Magnaporthe oryzae botourmiavirus 2 (MOBV2). The other three novel botourmiaviruses were selected for further study. Since five botourmiaviruses (previously named ourmia-like mycoviruses), MOLV1, MOLV4, PoOLV1, PoOLV2, and PoOLV3, had been found in M. oryzae, the viruses found in this study were designated as MOBV5, MOBV6, and MOBV7, respectively. The 5′- and 3′-ends of each viral cDNA were determined using ligase-mediated terminal amplification , as shown in MOBV6 was also found to be a (+) ssRNA virus B. It was−42; query cover: 64%) in the genus Scleroulivirus  and the 3′-terminal sequence (nt positions 2576–2598) of MOBV5 could be folded into terminal stable stem-loop structures with ΔG values of −14.00 and −9.40 kcal/mol, respectively A. The MOBased on multiple alignments and a conserved domain database search with the previously identified Magnaporthe botourmiaviruses, typical conserved motifs of the RdRps of (+) ssRNA mycoviruses , includiBotourmiaviridae and Narnaviridae (Naranaviridae (mitovirus and narnavirus), while the third clade embraced viruses of the new proposed and accepted family Botourmiaviridae. Botourmiaviruses were clustered into four classified genera and two unclassified groups in the phylogenetic tree. The phylogenetic tree showed that MOBV5, MOBV6, and MOBV7 indeed belonged to the family Botourmiaviridae, but were not clustered into any classified genera. They were relatively distant from MOLV1, which belongs to the genus Magoulivirus. MOBV2 was grouped into the genus Magoulivirus. The results suggest that new genera should be supposed in the family Botourmiaviridae to embrace the new members.To define the evolutionary relationship of MOBV2, MOBV5, MOBV6, and MOBV7, a phylogenetic tree was constructed using the maximum likelihood (ML) method with 1000 bootstrap interactions for the complete RdRp amino acid sequences of viruses from the families aviridae . The ML M. oryzae were identified via virome sequencing. Of them, the RdRp of MOBV2 displayed 79% identity with that of MOLV1 and belongs to the genus Magoulivirus. Using ligase-mediated terminal amplification, the full-length nucleotide sequences of MOBV5, MOBV6, and MOBV7 cDNAs were determined as 2598, 2385, and 2326 nts, respectively. The genome of each virus consists of a single unique ORF. The proteins encoded by MOBV5, MOBV6, and MOBV7 show 52%, 34%, and 34% amino acid sequence identity with RdRps of three botourmiaviruses in the family Botourmiaviridae; PoOLV1, PoOLV2, and SLOV2, respectively. Multiple alignments with the RdRps of certain botourmiaviruses showed that these proteins contain conserved domains of the RdRp in (+) ssRNA viruses (motifs I-VIII) [Botourmiaviridae, but are significantly distant from known Botourmiaviruses, and are not clustered into any classified genera in this family.In this study, four novel (+) ssRNA mycoviruses  that co-infect an isolate of the plant pathogenic fungus  I-VIII) ,51, inclBotourmiaviridae was established by ICTV in 2018, and includes viruses that infect plants and filamentous fungi containing a (+) ssRNA genome that could be mono- or multi-segmented. It consists of four genera: Ourmiavirus (plant viruses), Botoulivirus, Magoulivirus, and Scleroulivirus . Members of the genera Botoulivirus, Magoulivirus, and Scleroulivirus that infect fungi have a genome with a single segment of 2000–3200 nts encoding an RdRp. The demarcation criteria for these three mycovirus genera are based on differences in the RdRp amino acid sequence. Members of different genera in the family Botourmiaviridae should be >70% different in their complete RdRp amino acid sequence. However, in the last two years, several ourmia-like viruses that could not be classified into any of these three established genera have also been reported, such as SsOLV4, PoOLV1, and MOLV4. The identities of amino acid sequences of these are all lower than 30% compared with representative mycoviruses  in three established genera [Botourmiaviridae. Our results also demonstrate that compared with BOLV, MOLV1, or SsOLV1, the identities of the amino acid sequences of MOBV5, MOLV6, and MOLV7 were all close to or lower than 30%. We also compared the identities of amino acid sequences between MOBV5 and MOBV6 (28%), MOBV5 and MOBV7 (27%), and MOBV6 and MOBV7 (31%). Taken together, these results indicate that MOVB5, MOBV6, and MOBV7 cannot be classified into any of the three established genera, and each of them may represent a new genus.The family d genera ,24, whicBotourmiaviridae might evolve quickly and have diverse functions. Their terminal sequences exhibit several different structural features. For example, MOLV1 and MOLV4 have 3′- and 5′-UTRs with variable lengths and sequences, which can be folded into a stable stem-loop secondary structure. BOLV contains stretches of 28 and 50 nt sequences at the 5′- and 3′- non coding regions (NCRs) that can form a slightly less stable stem-loop structure [Saccharomyces [Magoulivirus, has a poly (A) tail at its 3′-end. In mycoviruses, this structure was first reported in SsMV2/KL-1, a mitovirus infecting Sclerotinia sclerotiorum [Botourmiaviridae associated with M. oryzae.Due to the simple single strand structure, the botourmiaviruses in the family tructure . Furtheraromyces . The typrotiorum , and wasrotiorum ,56. Morerotiorum . Our preBotourmiaviridae have been found to be associated with fungal hypovirulence. However, mitoviruses that belong to the genus Narnaviridae and are closely related to botourmiavirus have been found to be relevant to the variable virulence of their fungal hosts. For example, Cryphonectria cubensis mitovirus 1b and Cryphonectria cubensis mitovirus 2a have no significant impact on their hosts [Botourmiaviridae is still unknown. It has been reported that certain mycoviruses infecting M. oryzae, such as MoCV1-C, MoV3, and MOPV2, have been successfully removed from their fungal hosts through single-spore isolation. However, in the present study, we failed to generate any virus-free strains of YC81-2 using the same strategy. This result indicates that MOBV2, MOBV5, MOBV6, and MOBV7 might vertically transfect via spores. In follow-up research, we will try to obtain virus-free strains using other methods, such as protoplast regeneration technology, to determine the impact of the four botourmiaviruses investigated here on host pathogenicity and the interactions among themselves.So far, none of the mycoviruses in the family ir hosts . On the ir hosts . The co-ir hosts . Whether


The estimation of evolutionary parameters provides essential information fordesigning public health policies. In short time intervals, however, nucleotidesubstitutions are ineffective to record all complexities of virus populationdynamics. In this sense, the current SARS-CoV-2 pandemic poses a challenge forevolutionary analysis. We used computer simulation to evolve populations inscenarios of varying temporal intervals to evaluate the impact of the age of anepidemic on estimates of time and geography. Before estimating virus timescales,the shape of tree topologies can be used as a proxy to assess the effectivenessof the virus phylogeny in providing accurate estimates of evolutionaryparameters. In short timescales, estimates have larger uncertainty. We comparedthe predictions from simulations with empirical data. The tree shape ofSARS-CoV-2 was closer to shorter timescales scenarios, which yielded parametricestimates with larger uncertainty, suggesting that estimates from these datasetsshould be evaluated cautiously. To increase the accuracy of the estimates ofvirus transmission times between populations, the uncertainties associated withthe age estimates of both the crown and stem nodes should be communicated. Weplace the age of the common ancestor of the current SARS-CoV-2 pandemic in lateSeptember 2019, corroborating an earlier emergence of the virus. The evolutionary analysis of virus genomes frequently relies on molecularphylogenies, which illustrate the ancestry of lineages in tree graphs . When trDifferent approaches were proposed to handle rate heterogeneity in order to carry outmolecular dating of sequence divergences. They can be roughly categorized intosmoothing methods and methods that employ explicit models of substitution rateevolution in a Bayesian framework .BayesiaOne of such factors is the age that the virus population is circulating in the hostspecies after the initial infection. Although frequently ignored, the age of thecirculating virus population will affect the sampling strategy required for accurateinference of evolutionary parameters. Depending on the mutation rate, viruspopulations that successfully infected a new host species may not accumulate enoughsubstitutions to allow for phylogenetic inference within a few generations after theinitial transmission, resulting in branch lengths close to zero, and an large numberof duplicated sequences . In a loAnother factor impacting the estimation of virus timescales is the mismatch betweenthe virus transmission history between populations and the genealogy of virusgenomes. When transmission events along geographic areas take place, the sampling ofthe genetic diversity of the donor virus population is often incomplete and, becauseof the stochasticity of the coalescent process, both the ages of common ancestors ofgenomes and the monophyly of the virus genetic diversity in geographic areas isaffected . This pr−4 substitutions/site/year (s/s/y)  of thepopulation from area A was 10 years. The transmission event toarea B took place 8 years ago, while transmissions fromB to areas C and Doccurred at 6 and one year ago, respectively. In the second scenario,2Y, the common ancestor of population Awas 2 years old, and the transmission to area B occurred 5months later. From population B, transmissions to areasB and C took place at 1 year and 6 monthsago, respectively. In the remaining two scenarios, we simulated the rapidgeographic spread of a virus within less than one year. In scenario6M, the age of population of A was 6months, the transmission to B occurred three months later, andtransmissions from B to C andD occurred at 3 and 1 month ago respectively. Finally, inscenario 1M, all transmissions took place within a single month(age of A): from A to B at 20days ago; from B to C and Dat 10 and 5 days ago respectively. For each scenario, we evolved 300 independentreplicates. In each replicate, sequences were sampled serially along timeintervals to yield approximately 28 sequences. This number was chosen to speedup computational time while ensuring the robustness of the results.This simple simulation allowed the investigation of the evolutionary parametersin different epidemiological timescales. Four scenarios were investigated byvarying the total temporal extent of the epidemy, which equaled the age of thepopulation from area dater function. The position of the root node was alsoinferred in TreeDater. We measured both the age of the stem and crown nodes forareas C and D  were retrieved from the TreeDater output. The frequency of themonophyly in areas C and D was measured usingthe built-in functions available in the ape R package.Analysis of the performance of evolutionary inference on the four scenarios wasimplemented by comparing features that are relevant for health policy evaluationand decision making: (1) the ages of the epidemic (the root node) and of thetransmission events, and (2) the frequency in which the genetic diversity inareas JSDtree functionin RPANDA. We used the spectral density metrics to arrange all the topologiessimulated into k groups (clusters) using thek-means clustering analysis. The number of clusters was chosenusing the gap statistic , as wellas long term circulating virus species .In order to investigate the effects of the timescale on evolutionary parametersinferred with empirical data, we compared our simulations with the 2019-2020outbreak of SARS-CoV-2, which consists of the main pandemic of the21We downloaded 358 SARS-CoV-2 genomes available in GenBank . Thesegdater function, and allowing the root node to be inferred.All tree topologies were inferred under the maximum likelihood (ML) frameworkimplemented in the IQ-TREE software  using th10Y scenario, in which the errors associated with theestimates were <5% of the total duration of the epidemic (10 years) (10Y) to 1157.2% (1M) of theroot age (6M and 1M), the best approximations ofthe ages of transmission events were inconsistent, because in four cases theages of the crown nodes were closer to the true value, whereas the remaining twocases were best inferred by the ages of the stem nodes. In most cases, the truetransmission ages lied between the estimated ages of the stem and crown nodes.The exception was the 1M scenario, in which the estimated stemand crown nodes did not bound the true value  . It is croot age . In shorue value .C and D, shorter timescales resulted inlower frequency of monophyly, indicating that inferred ML gene genealogies wouldsuggest incorrectly that these populations have multiple origins. While in the10Y scenario, allelic diversity from areasC and D were recovered as monophyletic in>90% of the replicates, in the 1M scenario this figuredropped to 78.3% (area 3) and 89.7% (area D).The frequency in which the reconstructed genealogy of alleles embedded inpopulations was monophyletic was also impacted by the timescale of the pandemic. In both1M scenario, this metric variedsignificantly between topologies (10Y and 1M scenarios. Thenumber of peaks (modes of evolution) was 1 in most topologies from the10Y and 2Y datasets. In both6M and 1M scenarios, the average number ofpeaks was 1.4, indicating incorrectly the existence of multiple modes ofdiversification in a single tree topology.The metrics calculated from the spectral densities of tree topologies were alsoaffected by the timescales of the epidemics. The asymmetries (skewness) of thedensity profiles were similar among the trees from the four scenariosinvestigated , whereaspologies . The twopologies . Similark-means algorithm was 4. When features measured from thespectral densities were used to cluster the topologies from the severalscenarios an interesting picture emerged. The 10Y and the2Y scenarios were distinguished from the shorter timescalescenarios . The inferred position of theroot node separated a Chinese sample from the remaining SARS-CoV-2 genomes. Thecomparison between the spectral densities of simulated trees with SARS-CoV-2tree topologies estimated for both the entire genome and each ORF independentlyindicated that the topologies from the 1M scenario exhibitedlower J-S distance for all SARS-CoV-2 genomic regions . Therefore, theshape of tree topologies contained information on the age of the epidemic.When our simulations were evaluated against other virus epidemics, it waspossible to differentiate long- and short-term circulating infections . The speOur simulations brought forth a number of shortcomings that arise when analyzing thedynamics of virus epidemics within a short timescale. If the age of the circulatinginfection is young, the inference of evolutionary parameters will suffer fromreduced accuracy and precision. For all parameters investigated , the scenarios of shorter timescales yielded estimates that deviatedmore from the true values.When inferring the ages of transmission events, a simple strategy that improves thesuccess rate of recovering the true age is to employ a bracketing approach, in whichboth the ages of the crown and stem nodes are communicated. However, when thegenetic diversity of the epidemic finds its common ancestor within few generationsago, the interval between the ages of the crown and stem nodes might fail toencompass the true ages. In fact, in such scenarios, the age of the common ancestorof the epidemic (root node) is also harder to estimate, as well as the dynamics ofviruses in space. In long-term epidemic scenarios, however, the age of the stem nodeis the best approximation of the true transmission times, although the age of crownnode is frequently used as an estimate of this parameter.We enumerate two major factors that explain the poor performance of molecular datingmethods in short-term epidemic scenarios. Firstly, the low genetic diversity ofrecent epidemics. If mutations accumulate randomly at a fixed rate, which was theassumption employed to simulate our sequences, then at short time intervals theprobability of observing new mutations is lower. The intervals between coalescenceevents, which define the internal branch lengths, will likely have zero length whenmeasured in units of substitutions/site. Therefore, the structure of the virusphylogeny will be largely random, because sequences lack information on theevolutionary history of virus populations. Such problem could be alleviated bysampling more sites of the virus genome. Assuming that mutation rate is homogeneousacross sites, sampling more sites will increase the probability of findingsubstitutions shared by more than two sequences . However,because virus genomes are limited in length, this may not be feasible.Another factor affecting the inference of evolutionary parameters is the variance ofthe coalescent process. Because the tree topology of the genealogy of genomes may bedifferent from the phylogeny of virus populations containing the transmissiondynamics - the well documented species tree/gene trees discordance  Figure  -parame1M trees. This suggests that the SARS-CoV-2 dataset maybe as challenging as the 1M simulation scenario to estimateevolutionary parameters. Our results demonstrated that the shape of serially-sampled virus phylogenies, asapproximated by the spectral density of the tree, provides information on thetemporal dimension - long- and short-term epidemics yielded distinct spectraldensity profiles. This observation, attained with simulated datasets, was replicatedwith empirical data. Samples from long-term circulating viruses were similar tolong-term simulated datasets as well. We showed that SARS-CoV-2 was most closelyrelated to -4 subst./site/year, is inagreement with the values calculated for RNA virus along the last decades  could bedistinguished from long-term circulating viruses .Metrics of SARS-CoV-2 phylogeny were similar to simulated scenarios of very recentpopulation dynamics, which resulted in parametric estimates with large uncertainty.Health policies drawn from SARS-CoV-2 evolutionary estimates should thus be designedcautiously. In this sense, simple strategies may be useful, as accounting for boththe ages of stem and crown nodes to approximate transmission times.


Of all cancer types, healthcare for lung cancer is the third most costly in Australia, but there is little detailed information about these costs. Our aim was to provide detailed population-based estimates of health system costs for lung cancer care, as a benchmark prior to wider availability of targeted therapies/immunotherapy and to inform cost-effectiveness analyses of lung cancer screening and other interventions in Australia.Health system costs were estimated for incident lung cancers in the Australian 45 and Up Study cohort, diagnosed between recruitment (2006–2009) and 2013. Costs to June 2016 included services reimbursed via the Medicare Benefits Schedule, medicines reimbursed via the Pharmaceutical Benefits Scheme, inpatient hospitalisations, and emergency department presentations. Costs for cases and matched, cancer-free controls were compared to derive excess costs of care. Costs were disaggregated by patient and tumour characteristics. Data for more recent cases identified in hospital records provided preliminary information on targeted therapy/immunotherapy.p<0.0001), smoking (p<0.0001) and unknown stage (p = 0.002). There was no evidence of differences by year of diagnosis or sex (both p>0.50). For 465 cases diagnosed 2014–2015, 29% had subsidised molecular testing for targeted therapy/immunotherapy and 4% had subsidised targeted therapies.994 eligible cases were diagnosed with lung cancer 2006–2013; 51% and 74% died within one and three years respectively. Excess costs from one-year pre-diagnosis to three years post-diagnosis averaged ~$51,900 per case. Observed costs were higher for cases diagnosed at age 45–59  or 60–69 , and lower for cases aged ≥80  and those with unspecified histology  or unknown stage . Factors associated with lower costs generally related to shorter survival: older age (Lung cancer healthcare costs are strongly associated with survival-related factors. Costs appeared stable over the period 2006–2013. This study provides a framework for evaluating the health/economic impact of introducing lung cancer screening and other interventions in Australia. Lung cancer is among the most common cancers worldwide . In AustLung cancer care is largely guided by tumour histology and disease stage at diagnosis , 6. LungIndividual patient care and the related costs vary based on patient and tumour characteristics. To date, little has been reported on population-based costs for patient subgroups in Australia, in part due to a lack of available data. The Australian healthcare system includes government-funded universal coverage of many medical costs, with other costs mainly covered by private health insurance or patients’ out-of-pocket costs. Previous Australian studies have assessed costs of treatment but did not include costs for other related aspects of cancer care, such as general practitioner or specialist consultations , 12, 13.To assess the potential impact of both existing and emerging lung cancer interventions on future healthcare costs, information is needed on the distribution of lung cancer care costs for key patient subgroups, referred to as the “usual care” scenario in cost-effectiveness analyses. For example, since 2013, a number of targeted therapies/immunotherapies  have been subsidised by the Australian government for the treatment of an expanding range of indications relating to lung cancer , and molOur aim, therefore, was to provide detailed population-based estimates of the health system costs for lung cancer care using linked patient-level data from New South Wales (NSW), Australia. We estimated lung cancer costs for several patient and tumour subgroups that are likely to be relevant to cost-effectiveness evaluations of lung cancer interventions prior to the wider introduction of new therapies and technologies. We analysed costs prior to diagnosis, for initial treatment, continuing care, and end-of-life care, providing a detailed resource for future healthcare planning. We also examined potential changes in practice related to use of targeted therapies for more recently diagnosed cases who were identified using hospital records.The study sample was selected from participants in the Sax Institute’s 45 and Up Study, a longitudinal study of 267,153 people in NSW aged ≥45 years. The study methods and cohort have been described previously . BrieflyThe linked health records included reimbursements for government-subsidised prescription medicines in the Pharmaceutical Benefits Scheme (PBS) and subsidised outpatient and medical services and some in-hospital procedures covered by the Medicare Benefits Schedule (MBS). The Sax Institute linked these records for study participants using a unique identifier. The Centre for Health Record Linkage  linked oPeople included for this analysis were 45 and Up Study participants with a NSWCR-registered lung cancer, diagnosed during 2006–2013 after study recruitment. We excluded participants with:probable linkage errors and those aged <45 years at baseline;invasive cancer diagnosed prior to study recruitment (NSWCR);any non-lung cancer diagnosed (except if first diagnosed with lung cancer) after recruitment;self-reported history of cancer (except keratinocyte cancers);healthcare subsidised by the Australian Government Department of Veterans’ Affairs (DVA), as their prescription medicine records were not available. DVA clients were identified via self-report or any mention of DVA coverage in hospital or ED records;unknown day/month of diagnosis, or first diagnosed after death.The remaining cases were matched to controls who had no record of cancer at any time and who were alive on the date their matched case was diagnosed. Up to three controls were matched to each case (without replacement) based on age (±5 years), sex , Local Government Area of residence (~150 areas in NSW) and smoking status  at recruitment. People with no information for the matching variables were excluded, as were cases with no matched controls . Lung caTo separately investigate the introduction of targeted therapies in real-world lung cancer care, we used hospital admission records to identify more recently diagnosed lung cancer cases in the 45 and Up Study cohort, with the sensitivity and positive predictive value of the method of diagnosis, compared to using NSWCR information, both estimated at 80–85% , 28. We For this study we focused on direct health system (government-funded) costs. Costs were estimated from records of individuals’ inpatient hospital episodes, ED presentations, and subsidised prescription medicines and medical services recorded in the PBS and MBS respectively. The period for which data on costs were available from all healthcare databases was 1 January 2005 to 30 June 2016 . This prCosts were estimated for time periods relative to the cases’ diagnosis and death dates where applicable. If the time period of interest extended beyond the end of the study period then the matched case-control group did not contribute to the estimates for that specific period (e.g. if the case was diagnosed in December 2013 then 3 years post-diagnosis is after 30 June 2016). Costs for all related items and procedures were included.The dates of supply of prescription medicines (PBS) and medical services (MBS) were used to assign costs to the relevant time period. Inpatient hospital costs were derived by linking the Australian Refined Diagnostic Related Group code for each hospitalisation to the average admission cost recorded in the 2010 National Hospital Cost Data Collection . As hospThe excess costs due to lung cancer were estimated for each case by taking their total healthcare costs and subtracting the average costs for their matched controls. If the case died then the included costs were censored at the date of death and the case-control group were excluded from calculations for subsequent time periods. If a control died, the matched case and their remaining control(s) were included in any subsequent estimates. Means of excess costs were calculated, along with the proportions of excess costs contributed by each source . Medians, standard deviations and inter-quartile ranges are reported in the Supporting Information ; non-small cell lung cancer ; other specified carcinoma; and other/unspecified . Cases wTo allow comparisons between patient subgroups defined by patients’ and tumour characteristics, we calculated an “average case” excess cost for each case-control group. This used the excess costs from 1 year before to 3 years after diagnosis as previous work showed little excess costs >1 year before diagnosis for lung cancer cases , and 92%Monthly costs around the case’s date of diagnosis and monthly costs at the end of life relative to the case’s death date were estimated. As described previously , costs wdied before July 2016, the final year up to and including the death date defined the terminal phase.survived ≤1 year after diagnosis, the terminal phase started at the diagnosis date and no costs were attributed to other phases.survived at least two years, the first year after diagnosis defined the initial phase.survived >1 year but ≤2 years, the initial phase was the period from diagnosis until the start of the 12-month terminal phase.survived >2 years after diagnosis, the period between the end of the initial phase and the start of the terminal phase, or 30 June 2016 if the case was still alive, defined the continuing phase. Costs for this phase were estimated as an annual rate, excluding any case-control group with the continuing phase <3 months (1.6% of groups).The University of New South Wales Human Research Ethics Committee approved the conduct of the 45 and Up Study. The NSW Population and Health Services Research Ethics Committee  approved this study.There were 997 eligible cases diagnosed with lung cancer between recruitment and December 2013 . At leasCompared with all lung cancers in NSW/Australia, the study cohort had a similar distribution for sex, age and disease stage at diagnosis, but a higher proportion of cases with adenocarcinoma and people from outside of major cities  or from more disadvantaged areas . Sixty-oFifty-one percent of cases died within one year of diagnosis (89% from lung cancer), 67% and 74% died within two and three years respectively; 5% of controls died within three years. The Kaplan-Meier estimate for 5-year lung cancer-specific survival was 27% and for overall 5-year survival was 21%, compared with 5-year relative survival of 18% for all lung cancer cases diagnosed in Australia 2011–2015 Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: YesReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: N/AReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: NoReviewer #2: NoReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: ReviewHealth services costs for lung cancer care in Australia: Estimates from the 45 and UpStudyOverview: In this descriptive study, the authors provided detailed population-based estimates of health system costs for lung cancer care, as a benchmark prior to wider availability of targeted therapies/immunotherapy.Overall, the study was done with academic rigor, using a unique dataset. However, I note below several concerns:Major comments:1. The overarching concern is that the study appears to be insufficiently motivated, in the strict sense that the stated purpose cannot logically be fulfilled by the results of the study. The reasons are that the study justifies its primary aim with the following sentence: “Given the large number of new cases every year, information about changing costs for lung cancer treatment is crucial to health system planning in Australia and in other developed countries.”a. First, there is no justification why Australia’s experience can guide health system planning in other developed countries. In fact, very little is given to highlight essential features of the Australian healthcare system, so that an international reader from another developed nation may not necessarily know how applicable lessons learned from this study would be to his or her national setting.b. The primary innovation from the study is stated to be the subgroup analyses in terms of how costs are generated for cancer care in Australia. This sentence (1 above) does not clearly speak to the need for subgroup analyses.c. Related to point (b) above, there was no explanation/justification as to why the specific subgroups were chosen for analyses. This justification should be tied into the motivation for the study.d. In fact, lines 75-77 actually provide the information that I was hoping to find earlier on in the introduction. I suggest that the authors move this critical sentence higher up: “It is important to establish methods for estimating overall costs of treatment over time, and to benchmark tumour-specific treatment costs prior to the wider introduction of new targeted therapies and the accompanying molecular testing.” But then see point 2 below:2. The second issue also relates to motivation. The manuscript states, in lines 75-77 (see 1d above), that we need to benchmark costs prior to wider introduction of new targeted therapies. However, the costs that are estimated and documented are actually costs prior to the new targeted therapies, so they have no direct relevance with the costs of the newer therapies. I find it difficult to understand the rationale for understanding the costs of “old technologies” before introducing “new technologies.” In fact, in line 83-84, the manuscript again notes “to understand future costs, we need to know the costs of key patient groups.” But I am not sure how knowing the costs of current technology for key patient groups will inform future costs, at least not without further explanation.3. The third issue again relates to motivation: If the rationale for the study is to understand the costs of care prior to adopting new technology, why is it important to understand the excess costs due to lung cancer (relative to patients with no lung cancer)? Should we need to know just how much it costs to treat lung cancer; not “how much more” lung cancer costs relative to someone who is similar, but has no lung cancer? In fact, the decision to define “excess” costs also leads to downstream issues, such as those identified in lines 227-229 (the need to shift costs to avoid negative excess costs). If we simply used actual costs (not excess costs), there would be no need to account for negative excess costs.4. There may be some confusion as to how “population-level” this study is. The abstract states that the study is “population-level,” but the methods section actually tells a different story – that information was gathered from a trial (that oversamples certain groups); and these patients were selected to participate in a study/survey. Lines 215-27 suggest that the study had to be re-weighted in order to match population-level data. So I would refrain to qualify this study as “population-level.”5. In the study sample section (starting on line 130), there are certain exceptions that are self-evident; but other exclusions are not so clear. For example, why exclude keratinocyte cancers?6. I am curious about the matching approach that includes matching based on smoking status, particularly matching based on current smoking status. If a case who has smoked for 20 years must be matched with a control who has smoked for 20 years without cancer, this would seem to introduce some odd selection issue.7. Finally, I would like to reiterate the importance of establishing the importance of identifying certain subgroups to compare costs. As currently written, the article reads like a collection of facts. I understand that not all manuscripts are hypothesis-driven and therefore will not necessarily progress in a linear fashion. However, a descriptive study that presents a lot of results by slicing the data in different ways actually ends up being difficult to read. Perhaps motivating the reasons for looking at costs for different subgroups may help lessen this sense? I.e., assessing costs by cancer stage seems quite appropriate. But what about all the statistics presented under “average costs”? Or facts and figures under “monthly costs”? Are not monthly costs highly correlated with the stage of cancer? Also, the exploratory analyses of new cases from 2014-2015 seem ad hoc – They seem to be an afterthought because they are not sufficiently developed relative to the other sections.8. Without ending on a morbid note, the final paragraph indirectly suggests that a cost-saving technique in cancer care is to diagnose later. I would suggest rewording the last paragraph, so that the paragraph does not begin with noting that “lower costs are associated with shorter survival time … and this is important information for health services planning.”Minor comments:1. What does it mean that ED costs were assigned based on triage category and discharge status? Why not just simply tally up all the costs during a specific ED visit?2. Line 303 seems to be contradicted by the table: Isn’t the cost for current smoker lower? The text says costs for current smokers are higher.Smoking status Not availableNever / Ex quit >15 years 461 46 51 53,737Ex-smoker quit <=15 years 249 25 47 54,046Current smoker 284 29 47 47,120Reviewer #2: The manuscript relies on descriptive stats and simple mean comparisons of matched samples to analyze the ‘costs’ of lung cancer treatment in Australia. The authors find that as expected the cost of lung cancer treatment is lower for older patients, varies across different types of tumor histology, and is strongly inflationary. That is all rather trivial.The authors also find an inverse u-shaped relation between socioeconomic status and treatment costs. Patients from most and least deprived socioeconomic strata are on average less costly. This is interesting, but the authors do explain these findings. One has to say that the manuscript remains unanalytical and simply describes differences without trying to explain them.From a methodological perspective I do not believe that patient characteristics are uncorrelated. Therefore, most of the reported mean differences will be biased if the treatment and control group are not perfectly matched in all relevant dimensions. What is clearly missing, to give an example, are the existence of diseases other than lung cancer.In sum, I find the manuscript borderline publishable. I also cannot see many implications. Yes, the costs of lung cancer treatments differ systematically, but so what?I also find it implausible that the data cannot be made publicly available. This should be a reason to reject the paper. If data is not shared and no replication policy in place, it is not science.Reviewer #3: This paper addresses an important issue of the Australian healthcare system. That of the lung cancer that was responsible for 9000 deaths in 2019 and accounts for a substantial proportion of Australian government healthcare costs. This shows us the seriousness of the problem in that country. The authors argued that It is important to establish methods for estimating overall costs of treatment over time and to benchmark tumor-specific treatment costs before the wider introduction of new targeted therapies and the accompanying molecular testing. However, there are little published Australian data quantifying these costs, and the authors proposed to provide detailed population-based estimates of the health system costs for lung cancer care using linked patient-level data from New South Wales (NSW), Australia.The authors give a good and detailed explanation of their data sources data collection as well as how the major variables such as cost were measured and the different assumptions that were made in their calculation.The authors’ findings indicate that excess costs from one-year pre-diagnosis to three years post-diagnosis averaged ~$51,900 per case. In addition, the costs were higher for cases diagnosed at age 45-59  or 60-69 , and lower for cases aged ≥80  and those with unspecified histology  or unknown stage . They argued this is the first study to provide detailed health system costs for lung cancer 391 care in Australia.The paper is well written, the data are well presented.The analysis is well done and the discussion to me looks fine.However, after knowing those different costs, one could ask the question of “so what?”They may be the need to related these costs to the standard of living of those patients. From the Australian standard is it something that an average individual can afford or some measure need to be put in place to support patients? This is important to better inform policymaking.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  25 Jun 2020The responses are in the attached file "Response to Reviewers.docx".AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 10 Aug 2020Health services costs for lung cancer care in Australia: Estimates from the 45 and Up StudyPONE-D-20-04316R1Dear Dr. Goldsbury,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Jason Chia-Hsun Hsieh, M.D. Ph.DAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. 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A method for the rapid detection of coronaviruses is presented on the example of the transmissible gastroenteritis virus (TGEV) directly in aqueous solutions with different conductivity. An acoustic sensor based on a slot wave in an acoustic delay line was used for the research. The addition of anti-TGEV antibodies (Abs) diluted in an aqueous solution led to a change in the depth and frequency of resonant peaks on the frequency dependence of the insertion loss of the sensor. The difference in the output parameters of the sensor before and after the biological interaction of the TGE virus in solutions with the specific antibodies allows drawing a conclusion about the presence/absence of the studied viruses in the analyzed solution. The possibility for virus detection in aqueous solutions with the conductivity of 1.9–900 μs/cm, as well as in the presence of the foreign viral particles, has been demonstrated. The analysis time did not exceed 10 min. Viral infections, including coronavirus infection, attract attention with enviable regularity and remain one of the global problems of our time. Coronavirus (Cov) is an acute viral infectious disease characterized by moderate intoxication, and predominantly, affection of the upper respiratory tract. Various types of coronaviruses are widespread in nature, causing various infectious pathologies, both in humans and in animals: pigs, chickens, dogs, cats, camels. Currently, the coronavirus family includes 2 types of viruses that cause severe respiratory infection in humans: SARS-Cov (severe acute respiratory syndrome coronavirus) or SARS coronavirus, which causes severe acute respiratory syndrome, and MERS-Cov (Middle East respiratory syndrome coronavirus) or MERS-coronavirus, causing Middle East respiratory syndrome. At the end of 2019, SARS-Cov-2 (Severe acute respiratory syndrome-related coronavirus 2), capable of causing viral pneumonia ,4. ThereQualitative analysis of viruses is determined by the reliability of methods for their determination and the ability to study a large number of samples in a short period of time. Various approaches are used to detect viruses (virions), such as microbiological and biochemical tests, genetic engineering and immunological methods ,6, polymA promising direction for the analysis of viruses is the use of acoustic biosensors. Previously, the possibility for viruses’ detection was shown on the example of bacteriophages using an acoustic sensor and microbial cells , and antTGEV). The general scheme of the experiments is shown in The aim of the current work was to develop an express method for detecting viruses using a sensor based on the slot mode in an acoustic delay line. As a model system, we used a representative of the Coronaviride family, transmissible gastroenteritisvirus, and antibodies specific for it  virus strain VN-96 was obtained from the Museum of Strains of the Virology Laboratory, Saratov Veterinary Research Institute . We used strain VN-96, which was deposited at the All-Russian State Collection of Microbial Strains Used in Veterinary Medicine and Animal Husbandry under accession number VN-96. The main properties of this virus were described earlier in Reference .We used also the bacteriophage M13K07, a kanamycin-resistant commercial preparation manufactured by Stratagene (Sweden), which was constructed on the basis of the wide-type phage M13 ,27.The concentration of the viral particles was measured on a Specord BS-250 device , as described in Reference .TGEV) was determined by the enzyme-linked immunosorbent assay (ELISA), as described in Reference [Antibodies were obtained by immunizing rabbits with the TGE viruses, and their sensitivity and specificity were determined by the method of dot-immunoassay . The titeference , and wasTransmission electron microscopy (TEM) analysis of the TGE viruses was carried out in accordance with Reference  by the L2PO4, 0.43; Na2HPO4, 1.68; NaCl, 7.2. Its conductivity varied from 4.1 to 900 μs/cm. The solutions conductivity was measured by the HI 8733 conductometer .We used a distilled water with conductivity 1.9 μs/cm and a standard phosphate buffered saline with pH 7.0 (g/L): KH3) with a propagating acoustic wave with shear horizontal polarization of zero order (SH0)  were significantly larger in comparison with the thickness, the plate was glued along the edges of a rectangular window with dimensions 38 × 18 mm2 of a plexiglass holder  . The plalder see . Thus, i3, was positioned above the delay line with the given gap between IDTs. The gap between the container and delay line was provided with 8 μm thick aluminum foil strips. This design of the sensor allowed us to use a removable liquid container, which in turn facilitated cleaning the container from the sample under study and accelerated the analysis of many samples. The constant air gap between the plates ensured repeatability of the results, and also prevented the damage to the delicate delay line during cleaning the container from the spent biological sample. The thickness of the bottom of the liquid container was equal 0.5 mm, the volume of the container was 1.5 mL. Such orientations of the LiNbO3 plate were also characterized by a significant value of electromechanical coupling coefficient for SH0 wave on the frequency 3 MHz. Thus, the sensor was a structure of two piezoelectric plates separated by an air gap. The sensor characteristics are described in detail in Reference [A removable liquid container, the bottom of which was made of a plate of Z-X+30° LiNbOeference .3 plate has a high value of the electromechanical coupling coefficient, the electric field accompanying this wave penetrates in the contacting air. Due to this property, there exists the slot wave propagating in two piezoelectric plates separated by the vacuum (air) gap. The excitation of the slot wave in such structure leads to the appearance of the clearly expressed resonant peaks on the frequency dependence of the insertion loss of the output signal of the delay line. The appearance of the peaks is determined by the fact that the bottom of the liquid sensor is limited in the direction of propagation of the acoustic wave. Each resonant peak corresponds to the case when the width of the second plate is equal to the whole number of the acoustic half-waves. The earlier studies [The analysis was performed using a network analyzer E5071C  to which an acoustic sensor was connected. The frequency dependence of the insertion loss of the sensor was measured in the frequency range of 2.6–3.8 MHz. The frequency dependence of the insertion loss of the sensor exhibits resonance absorption peaks associated with the excitation of a slot wave in the structure consisting of two piezoelectric plates with a gap between them . Since t studies  have sho studies .4 virus particles/mL), and for various amount of specific AbTGEV  added to the viral suspension. We used a distilled water with the conductivity of 1.9 μs/cm and the standard buffer solutions with the conductivity in the range 4.1–900 μs/cm. First, we filled a liquid container with a buffer solution with TGE viruses and measured the sensor readings. Then, anti-TGEV antibodies were added in the suspension and the sensor readings were recorded again. As analytical signal, we used the change in the depth and frequency of the resonant peaks on the frequency dependence of the insertion loss of the sensor after the interaction of viruses with antibodies. The scheme of experiments with an acoustic sensor is shown in We investigated the possibility of using an acoustic sensor based on the slot mode in an acoustic delay line for detecting viruses in aqueous solutions when they interact with the specific antibodies. Measurements were carried out for TGE viruses’ suspension (10p) ). Therefore, all the graphs stated below containing more than 1600 frequency points are given without the error bars, because in the presented scale, they will simply be invisible.All experiments were performed in 5 replicates, and the final results were calculated from the averaged values. The average error of 5 measurements at each point of the frequency range did not exceed ±2%. The results were statistically processed using standard procedures integrated into Excel 2007 . After the arithmetic mean and standard deviation (SD) were found for a given data sample, the standard error of the arithmetic mean and its confidence limits were determined taking into account the Student’s coefficients  Alphacoronaviruses, (2) Betacoronaviruses, (3) Gammacoronaviruses and (4) Deltacoronaviruses . Their nThe virus multiplies in the cytoplasm of mature epithelial cells located at the tips of the villi of the small intestine. Coronaviruses are infected by droplet–air and fecal–oral routes see . The virTGEV. Electron micrographs presented in TGEV. It can be seen that markers appear on the surface of the virus, i.e., it can be argued that anti-TGEV antibodies interact with TGEV and can potentially be used for its immunodetection.The morphology of viral particles, presence of the aggregates and individual subunits affects to the process of immunochemical interaction, which, in turn, changes the analytical signal. In this regard, at the beginning of the experiments, TGE viruses were investigated by electron microscopy (TEM). Then, experiments were carried out to detect the TGE viruses in aqueous solutions using an acoustic sensor. At the first stage, the interaction of viruses with different amounts of antibodies in water with a conductivity of 1.9 μs/cm was studied. First, virus-free distilled water was added to the sensor container and the sensor reading was measured.4 virus particles/mL) were placed in a liquid container and the sensor readings were taken again. This concentration of TGE viruses was selected based on the previous experiments. It was found that the addition of the virus to distilled water does not lead to a change in the depth and frequency of the resonant peaks in the frequency dependence of the insertion loss of the sensor (The frequency dependence of the insertion loss of the sensor showed peaks of resonant absorption (TGEV) were added to the container and the sensor readings were recorded for this case. The values of the concentration of antibodies were 5, 10, 15, 20, 25 and 30 μL/mL. It was found that the addition of AbTGEV to TGEV led to a significant decrease in the depth of resonance peaks and to a shift in resonance frequencies for any amounts of antibodies.Next, antibodies specific to this virus (AbTGEV (in the amount of 20μL/mL) to TGEV.Based on the obtained frequency dependences of the insertion loss of the sensor, the changes in the depth and frequency of the resonant peaks as functions of the concentration of antibodies added to the container with the virus for all resonance peaks were plotted. The dependences of changes in the depth and frequency of the resonance peak near the frequency of 2.82 MHz on the amounts of added antibodies are presented in One can see that the maximum change in the depth of the resonance peak is equal to 7.9 dB with the amount of antibodies being 20 μL/mL. At that, the minimum change turned out to be ~6 dB for antibodies in the amounts of 5 and 30 μL/mL. In this case, for all concentrations of antibodies, a shift in the frequency of the resonance peak by 0.01 MHz was observed. Approximately the same results were obtained for other resonance peaks. TGEV. It should be noted that the addition of only AbTGEV in liquid container in the virus’ absence did not lead to changes in the sensor parameters.Since the maximum change in the depth of the resonance peak was recorded when the amount of antibodies was 20 μL/mL, in subsequent experiments, we used exactly this amount of antibodies to register the interaction of TGE viruses with AbTGEV interaction during virus detection. The measurement time was ~10 min, including 4 min for the measurement process and the time for the introduction of the sample into the liquid container and cleaning the container after measurements.Thus, it has been shown that the addition of specific antibodies to the TGE virus under conditions of low conductivity of the medium (1.9 μs/cm) leads to a significant change in the depth of the resonance absorption peaks on the frequency dependence of the insertion loss of the acoustic sensor based on the slot mode. This indicates the possibility of using such acoustic sensor to register the TGE viruses–AbTGEV to buffer solutions with virus led to a decrease in the depth of resonance peaks on the frequency dependence of the insertion loss of the sensor and to a shift in the resonance frequency. In this case, the depth of the resonance peaks depended on the conductivity of the buffer solution. When developing a new method for virus detection, it is important to take into account the possibility of analyzing the viruses in solutions with increased conductivity. High conductivity creates significant interference in the analysis and most sensor devices are not adapted for measurements in real conditions . NeverthThis behavior of the dependence is in full agreement with the data obtained earlier in the study of the effect of liquid conductivity on the propagation of the slot mode in a structure of two piezoelectric plates separated by an air gap . It was Thus, the interaction of viruses with specific antibodies leads to an increase in the conductivity of the suspension. This suspension is in contact with one of the plates of the structure with a piezoactive slot mode accompanied by an electric field in the suspension. A change in the conductivity of a suspension alters the attenuation and phase velocity of the slot mode. This, in turn, changes the depth and frequency of the resonance peaks in the frequency dependence of the insertion loss of the sensor. The depth and frequency of resonance peaks are directly measurable quantities and are therefore used as an analytical signal, indicating the presence of viruses in suspension. Thus, the possibility of detecting the virus in aqueous solutions with increased conductivity, for example, in drinking and tap water, the conductivity of which is 500 and 900 μs/cm respectively, has been shown.For the development of virus method detection, an important point is to obtain a result in the presence of interfering factors and, above all, in the presence of other viral particles. Therefore, at the next stage, we measured the parameters of the sensor for a mixed suspension of viruses in the container. M13K07 bacteriophages were used as foreign viral particles. The measurements were carried out in the buffer solutions with the conductivity of 4.1, 50 and 100 μs/cm.TGEV. For this, the depth and frequency of the resonant peaks on the frequency dependence of the insertion loss of the sensor were measured before and after the addition of antibodies specific to TGE virus to the buffer solution with the M13K07. TGEV. It can be seen that nonspecific interaction “M13K07–AbTGEV” is absent and did not lead to a change in the sensor parameters.First, the control experiments were carried out to study the nonspecific interaction between M13K07 and AbTGEV. Then, the same measurements were carried out for buffer solutions with TGE virus and specific Ab4 particles/mL) and M13K07 (106 particles/mL), taken in the 1:1 ratio, was introduced into the sensor container. After that, the AbTGEV (20 μL/mL) was added. The sensor readings were measured before and after adding AbTGEV to the mixed virus suspension. TGEV. One can see that the addition of AbTGEV to a mixture of viruses leads to approximately the same change in the depth and frequency of resonant peaks, as in the case of a TGE virus suspension without M13K07.Finally, a mixed suspension of TGE viruses  in the buffer solutions with conductivity of 4.1, 50 and 100 μs/cm.For comparison, TGEV interaction with the TGE virus and a mixed viruses’ suspension (TGEV and M13K07) are comparable and are equal to 2–5 dB, depending on the buffer conductivity. The changes in this parameter after adding AbTGEV to the buffer solutions with the M13K07 are greatly smaller and are equal to 0.05–0.11 dB, which is within the sensor error. Thus, it can be argued that the presence of foreign viral particles does not interfere with the registration of the specific interaction of AbTGEV with TGE viruses by using an acoustic sensor.One can see that for various values of the conductivity of the buffer solution, changes in the depth of the resonant peaks in the cases of AbAs a result of the experiments, it has been found that an acoustic sensor based on the slot mode in an acoustic delay line successfully registers the interaction of the specific antibodies with the TGE virus and is promising for TGE virus detection in aqueous solutions with different conductivity, as well as in the presence of foreign viral particles. The interaction of viruses with specific antibodies leads to an increase in the conductivity of the suspension. A change in the conductivity of a suspension alters the attenuation and phase velocity of the slot mode. This, in turn, changes the depth and frequency of the resonance peaks in the frequency dependence of the insertion loss of the sensor. Based on the changes in the output parameters of the sensor for a virus suspension before and after biological interaction with specific antibodies, it is possible to draw conclusions about the presence (or absence) of the studied viruses in the analyzed suspension. So, the change in the depth and frequency of the resonant absorption peaks on the frequency dependence of the insertion loss of the sensor may be used as an analytical signal.-Detection of viral RNA,-Detection of antibodies produced upon contact with an infection .Let us compare the coronavirus detection method presented in the article with the known ones. In modern methods of detecting coronavirus infection, two main areas can be distinguished:The detection of viral RNA is usually performed by polymerase chain reaction (PCR) or nucleic acid hybridization.Viral antibodies or antigens can be detected by immunological and serological tests such as ELISA.-Sensors for the viral antigen analysis,-Sensors for the specific antibodies’ determination.Both directions of research are important, and they complement each other, since the determination of the RNA of the virus leads to the detection of the virus in its active stage, while serological tests help to identify the activity of the immune system in the production of antibodies to fight the infection. Exceptional growth in biosensor development for the analysis of viruses, including coronaviruses, is noted in 2019–2020 . BiosensBrief information about modern sensors developed for the coronavirus infection diagnosis is presented in As can be seen from the data presented here, with all the variety of sensor systems for diagnosing coronaviruses, acoustic biological sensors based on the slot mode in an acoustic delay line have not been used to solvethis problem. Acoustic sensors for detecting viral particles are known and described in the literature ,55, but The described method fundamentally differs from the known ones for detecting viruses with immobilized antibodies ,55,56,57The presented setup showed for the first time the ability to quickly detect viruses in the aquatic environment. Without a doubt, it can be modernized and made more convenient for analysis. It consists of a sensor and an expensive S-parameter meter E5071C  (frequency range 9 kHz–8.5 GHz). In the future, it is planned to make a cheaper S-parameter meter for the 1–10 MHz range, combined with a computer with software for fast processing of results.Thus, an acoustic sensor based on the slot mode in an acoustic delay line provides a unique opportunity for TGE virus detection under conditions of increased conductivity of the measurement medium and the presence of foreign viral particles. The analysis time did not exceed 10 min. The results obtained can serve as a basis for the development of a new direction in the use of acoustic sensors for detecting viruses.