P < 0.0001). Significantly fewer serum specimens had a microneutralization titer of 40 or above against 3391/2017 virus than that against 4801/2014 virus . Conversely, the geometric mean hemagglutination inhibition titer was slightly higher against 3391/2017 virus than that against the 4801/2014 virus . Moreover, 59.1% of specimens had a significantly lower microneutralization antibody titer (≥4-fold) against 3391/2017 virus than that against 4801/2014 virus, but none for hemagglutination titer (P < 0.0001). Similar results of microneutralization and hemagglutination titers were observed for day 21-post-vaccination sera. Hence, the 2017 A(H3N2) summer peak in Hong Kong was associated with a low-microneutralization titer against the circulating virus. Our results support the use of microneutralization assay with human serum in assessing population susceptibility and antigenic changes of A(H3N2) virus. Novel and available immunization approach, such as topical imiquimod followed by intradermal vaccination, to broaden the neutralizing antibody response of influenza vaccine should be considered.The 2017 Hong Kong influenza A(H3N2) summer season was unexpectedly severe. However, antigenic characterization of the 2017 circulating A(H3N2) viruses using ferret antisera did not show significant antigenic drift. We analyzed the hemagglutinin amino acid sequences of A(H3N2) virus circulating in Hong Kong in 2017, and found that viruses with hemagglutinin N121K substitution, which was rare before 2017, emerged rapidly and dominated in 2017 (52.4% of A[H3N2] virus in 2017 contains N121K substitution). Microneutralization assay using archived human sera collected from mid-2017 showed that the geometric mean microneutralization titer was 3.6-fold lower against a 2017 cell culture-grown circulating A(H3N2)-N121K virus (3391/2017 virus) than that against the cell culture-grown 2016–2017 A(H3N2) seasonal influenza vaccine-like vaccine virus (4801/2014 virus) (13.4 vs 41.8,  Humans are susceptible to seasonal influenza virus infections because of the continuous antigenic changes of influenza virus. These antigenic variants are caused by mutations in the viral surface glycoproteins hemagglutinin and neuraminidase, which allow the virus to escape from pre-existing humoral immunity induced by prior natural infection or vaccination2.Seasonal influenza virus poses a significant burden to human health4. A recent study has shown that microneutralization titer induced by vaccination correlates with protection, in which a microneutralization titer of 40 or above was associated with 60% protection5. Hence, the World Health Organization (WHO) determines antigenic drift of circulating influenza viruses by analyzing the hemagglutination inhibition titer using post-infection sera collected from ferrets infected with the vaccine virus strain, or with post-immunization sera from human vaccinees7. Virus neutralization assay is also performed on some strains to supplement the result from hemagglutination inhibition assay, especially for the recent A(H3N2) strains that are found to have poor agglutination with red blood cells7.Studies involving human volunteers experimentally infected with live influenza virus have shown that hemagglutination inhibition titer of 40 or above correlated with a protection of about 70%8. A similar severe summer influenza epidemic also occurred in Taiwan9. Unlike the 2014–2015 winter epidemic, which was associated with an antigenically drifted A(H3N2) virus10, there was no significant antigenic changes for the circulating A(H3N2) viruses in 2017. The WHO and the Center for Disease Control reported that circulating A(H3N2) viruses in 2017 were well-inhibited by ferret antisera raised against cell culture-propagated vaccine viruses (A/Hong Kong/4801/2014 [4801/2014] or similar viruses) recommended for the 2016–2017 northern hemisphere influenza season11.In 2017, an unexpectedly high-summer peak of influenza A(H3N2) occurred in Hong Kong. The number of hospitalizations and deaths due to A(H3N2) in 2017 summer influenza season was similar to those in 2014–2015 winter season14. The poor vaccine effectiveness for A(H3N2) has been attributed to mutations arising from egg-grown vaccine viruses16. Ferret antisera raised against egg-grown vaccine viruses have low-microneutralization titers against the circulating cell culture-grown A(H3N2) viruses16. Human serum panels from vaccinees showed a lower geometric mean hemagglutination inhibition and microneutralization titer against cell culture-grown circulating A(H3N2) viruses than those against the egg-grown vaccine virus7. Based on these data, the WHO has changed the recommendation for the vaccine to be used in 2018 southern hemisphere to A/Singapore/INFIMH-16-0019/2016 (H3N2)-like virus, in which even the egg-propagated virus can elicit good antibody response against the circulating strains.One possible explanation for the unusually severe A(H3N2) epidemic in 2017 was poor vaccine effectiveness conferred by the recommended influenza vaccine for the 2016/2017 northern hemisphere influenza seasonIn addition to poor vaccine effectiveness, other non-vaccine related factors may also affect the size of an epidemic. This study sought to assess the humoral immunity of the general population against the circulating A(H3N2) virus in 2017. We specifically used cell culture-grown viruses to avoid unwanted mutations that can be generated during virus passage in eggs. We have found that the geometric mean microneutralization titer was significantly lower against a representative 2017 circulating A(H3N2) virus than that against a pre-2017 A(H3N2) virus, suggesting that there is reduced protection conferred by the humoral immunity generated by prior natural wild-type virus infection or by immunization with egg-based influenza vaccine.The surveillance data of influenza A(H3N2) virus in Hong Kong from the Public Health Laboratory Services Branch showed that the number of influenza A(H3)-positive clinical specimens in 2017 summer influenza season was similar to that of 2014–2015 winter influenza season . Comparison of viruses collected in 2017 and in 2014–2016 showed that three substitutions at antigenic site A of the hemagglutinin  have rapidly emerged in Hong Kong  virus, 3391/2017 virus, which possesses N121K substitution for the subsequent microneutralization and hemagglutination inhibition assays.A(H3N2) viruses with N121K substitution were also found to be present in many A(H3N2) viruses from other countries17. The geometric mean microneutralization titer was 3.6-fold lower against 3391/2017 virus than that against 4801/2014 virus  (Table P < 0.0001). However, the geometric mean hemagglutination inhibition titer was 1.7-fold higher against 3391/2017 virus than 4801/2014 virus , and the proportion of individuals with hemagglutination inhibition titer ≥40 was significantly higher for 3391/2017 virus than that of 4801/2014 . The percentage of individuals with at least fourfold lower 3391/2017 virus MN titer than 4801/2014 virus MN titer was 59.1% (257/435), while none of the individuals have fourfold lower 3391/2017 than 4801/2014 virus HI titer (P < 0.0001)  virus (3391/2017 virus), and a pre-2017 A(H3N2) virus from Hong Kong (4801/2014 virus), which is antigenically similar to A(H3N2) viruses in the 2015–2016 northern hemisphere influenza seasonsP < 0.0001), while the geometric mean hemagglutination inhibition titer was significantly higher for 3391/2017 virus than that against 4801/2014 virus   and 4801/2014  viruses  and older (65 years or above) age groups. For the hospital patient cohort, the geometric mean microneutralization titers for younger individuals were significantly lower than those of the older individuals against both 3391/2017  virus sequences deposited into GISAID EpiFlu database showed that A(H3N2) viruses with mutations at antigenic site A, especially N121K, have rapidly emerged in 2017 in Hong Kong. Using archived human serum samples from hospital patients, the geometric mean microneutralization titer was significantly lower against a 2017 A(H3N2) virus with N121K substitution (3391/2017 virus) than that against the WHO recommended A(H3N2) virus for the 2016/2017 northern hemisphere vaccine (4801/2014 virus). The percentage of serum samples with microneutralization titer ≥40 was significantly less for 3391/2017 virus than that of 4801/2014 virus. Furthermore, the percentage of serum samples with at least fourfold lower titer against 3391/2017 virus than that against 4801/2014 virus was also significantly higher for MN titer than HI titer. Similar results were seen in the serum specimens from post-vaccination cohort. Our results support the hypothesis that antigenic changes of the 2017 circulating A(H3N2) virus was sufficient to escape the pre-existing immune response in the general population against the previous H3N2 virus, which was judged by standard ferret serum to be antigenically similar to presently circulating H3N2 virus.Hong Kong is located in the subtropical area with two influenza peaks per year. The winter influenza peak is usually more severe than the summer peak. However, the 2017 summer influenza season was at least as severe as the 2014–2015 winter influenza season in Hong Kong, despite the absence of a significant antigenic drift that can be detected by ferret antisera raised against the cell-grown A(H3N2) virus recommended for the 2016/2017 northern hemisphere vaccine19. In particular, amino acid position 145 was found to be one of the 7 important sites associated with major antigenic change. When we compared the A(H3N2) viruses from Hong Kong, we have identified three substitutions  at hemagglutinin antigenic site A which have rapidly emerged in 201720. N121K substitution was most frequently found, detected in over 50% of viruses collected in 2017. N121K is a major substitution emerging in 2017 A(H3N2) viruses worldwide13, and this substitution was found in both the clade 3C.2a and subclade 3C.2a19. T135K can result in loss-of-N-glycosylation site at position 133–13522. S144K is very close to the receptor binding site. However, T135K was not present in 3391/2017 virus which was used in our serological assays. Further studies are needed to determine whether these substitutions have led to the changes in microneutralization antibody titers.Substitutions at antigenic site A were previously found to be important for antigenic drift24. Previous studies have shown that the correlation between hemagglutination inhibition and microneutralization titer was poorer for A(H3N2) than that for A(H1N1) virus26. However, the reason for such discrepancy in hemagglutination inhibition and microneutralization titer is not clear. There are several possible reasons. First, a poor microneutralization titer may be related to the effect of internal genes on host humoral immune response. In our previous mice model with A(H7N9) virus infection, microneutralization antibodies were not detected despite the presence of hemagglutination inhibition antibody titer27. Experiments with reassortant viruses showed that the internal genes of the A(H7N9) virus was responsible for the lower microneutralization titer. Second, a high hemagglutination inhibition titer may be related to cross-reactive antibodies generated by prior infection by A(H1N1). It has been shown that natural A(H1N1) virus infection can induce cross-reactive hemagglutination inhibition antibodies against A(H3N2) virus in humans28. Nachbagauer R et al.29 has shown that patients infected with A(H1N1) generates antihemagglutinin antibodies against other influenza A virus subtypes. It is possible that prior A(H1N1) infection, especially during the 2016 A(H1N1) epidemic in Hong Kong, could have induced cross-reactive hemagglutinating inhibition antibodies against the current A(H3N2) virus.Unlike geometric mean microneutralization titer, geometric mean hemagglutination inhibition titer was significantly higher for the 3391/2017 virus than that of 4801/2014 virus, although the difference was <2-fold. Discrepancy between human serum hemagglutination inhibition and microneutralization titer has been well reported8. Another possibility is that more elderly have received the influenza vaccination as recommended by the Centre for Health Protection in Hong Kong30.It is interesting to note that the younger individuals (aged 18–64 years) have lower microneutralization titer against both 3391/2017 virus and 4801/2014 virus than the older individuals (aged 65 years or above) in the hospital patient cohort, but not among the vaccinees. One possibility is that more elderly were naturally infected than the younger population31. Several studies have postulated that the low-vaccine effectiveness against the presently circulating A(H3N2) may be related to the substitutions found in egg-propagated viruses. During egg passage, influenza virus acquires amino acid substitutions in the hemagglutinin protein, which improve the attachment of the virus to avian-type host sialic acid receptors32. Studies in the 20th century have already shown that viruses grown in eggs can be less immunogenic than those that are grown in MDCK cells33. Poor efficacy of the 2012–2013 egg-propagated influenza vaccine against A(H3N2) was attributed to three amino acid mutations in the hemagglutinin 34. Egg-propagated 4801/2014 virus has a hemagglutinin L194P substitution, which affects the structure of the receptor binding site15. Egg-propagated 3C.2a vaccine virus (A/Colorado/15/2014) has a hemagglutinin T160K substitution which leads to the loss of a glycosylation site16. In our study, we used mammalian MDCK cell-propagated viruses. The 3391/2017 virus used in this study has T at position 160 and L at position 194 of the hemagglutinin protein. Therefore, the significantly lower microneutralization titer induced against the circulating virus when compared with the vaccine virus cannot be explained by virus mutations previously shown to arise through passaging in chick embryo.The vaccine effectiveness for the 2016–2017 northern hemisphere influenza season was found to be low for A(H3N2), ranging from −52.1% in South Korea to 43% in the USAAlthough microneutralization titer may be a better assay for predicting population susceptibility to A(H3N2) infection, microneutralization assay is technically more demanding than hemagglutination inhibition assay. First, microneutralization assay requires live virus with an accurate virus titer, while hemagglutination inhibition assay can be performed using standard control virus antigens that are provided by the WHO kit. Second, since live virus is used, microneutralization assay must be performed inside biosafety cabinet. Third, microneutralization assay takes at least 2 days, while hemagglutination assay can be completed in a single day.Though a large number of serum samples have been tested, our study was limited by the collection of samples from one teaching hospital in Hong Kong. Due to the difficulty of getting sufficient volume of archived serum samples from children, no data is available for this age sector.35.Our result suggests that a reduction in microneutralization titer against A(H3N2) among the adult population may explain the severe summer influenza season in 2017, despite that there is no major antigenic changes detectable by WHO standard ferret serum. Microneutralization titer may be a better measure than hemagglutination inhibition titer for predicting the population susceptibility, severity of A(H3N2) influenza epidemic, and perhaps the effectiveness of the chosen vaccine. Until more data are available, we recommend that both microneutralization and hemagglutination inhibition assay should be performed. Broadening the antibody response of intradermal influenza vaccine by prior topical imiquimod, a Toll-like receptor 7 agonist, may enhance the vaccine effectiveness against these A(H3N2) viruses with antigenic changes35. Madin-Darby canine kidney (MDCK) cells were cultured at 37 °C with 5% CO2 in minimum essential medium (MEM) , supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. When MDCK monolayer culture reached about 85% confluency, the cells were trypsinized and seeded in 96-well plates at a density of 2 × 104 cells/well and cultured overnight for the microneutralization assay.All laboratory procedures were performed according to what we previously reported with minor modifications50) was determined in MDCK cells in the presence of 2 μg/ml l-1-tosylamide-2-phenylethyl-chloromethyl ketone-treated trypsin (TPCK-trypsin). Aliquots of virus stock were kept at −80 °C until use.The A(H3N2) virus A/Hong Kong/3391/2017 (3391/2017 virus) was collected from a patient on 10 July 2017. The A(H3N2) virus 4801/2014 was the recommended strain for the 2016–2017 and 2017–2018 northern hemisphere influenza season. Both 3391/2017 and 4801/2014 viruses were kindly provided by the Public Health Laboratory Service of Hong Kong. Influenza viruses were propagated in MDCK cells without any passaging in chick embryo. The hemagglutinin genes of each virus were sequenced to confirm that no mutations were introduced into the hemagglutinin sequence during virus propagation. The hemagglutination titer was determined using 0.5% fresh group O human red blood cells. The 50% tissue culture infective dose  epidemic, were randomly selected from the Clinical Biochemistry Division, Queen Mary hospital. In addition, sera from vaccinees of 2016/2017 northern hemisphere recommended quadrivalent influenza vaccine taken on day 21-post-vaccination from November 2016 to June 2017 were also used in this study. The collected sera were aliquoted and stored at −20 °C until testing to avoid repeated cycles of freeze and thaw. This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (HKU/HA HKW IRB).36. Serum samples were treated with receptor destroying enzyme (RDE)  overnight at 37 °C to remove non-specific inhibitors. Treated serum samples were heat inactivated at 56 °C for 30 min, and were twofold serially diluted in phosphate buffered saline (PBS) starting from 1:10 dilution. Diluted serum samples were mixed with 4 hemagglutinin units of A(H3N2) virus and incubated at room temperature for 1 h. After incubation, 0.5% human red blood cell suspension was added to the serum-virus mixture and incubated at room temperature for 1 h when the red blood cells were completely set in PBS control wells. The hemagglutination inhibition titer was expressed as the reciprocal of the highest serum dilution that completely inhibited haemagglutination. The hemagglutination inhibition assay was performed in duplicate for each sample, and the hemagglutination inhibition titer was taken as the average of the two titers.Hemagglutination inhibition assay was performed as we described previously with modifications37. Briefly, serum samples were heat inactivated at 56 °C for 30 min, and then serially diluted in twofolds with PBS starting at 1:10 dilution. Serial dilutions of heat-inactivated serum samples were mixed with 100 TCID50 of A(H3N2) virus and incubated at 37 °C for 1 h. The serum-virus mixture was then added to MDCK cells and incubated for 1 h. After incubation, the serum-virus mixture was removed, and serum-free MEM containing 2 µg/ml TPCK-trypsin was added to the cells, and was further incubated at 37 °C and 5% CO2. Cytopathic effect was determined by examination under inversion microscopy after 72 h of incubation. Culture supernatant was mixed with equal volume of 0.5% human red blood cell to confirm the absence of hemagglutination by virus. The microneutralization titer was defined as the highest dilution of serum that completely inhibited cytopathic effect and absence of hemagglutination in 50% of the wells as described previously38.Microneutralization assay was performed as we described previously39. Gene sequence of hemagglutinin gene, except 3391/2017 virus, was obtained from Global Initiative on Sharing All Influenza Data (GISAID) EpiFlu database (Supplementary Table 40.Laboratory surveillance data of influenza virus from 2014 to 2017 (up to week 49) from the Public Health Laboratory Services Branch was obtained from the Centre for Health Protection website42. Briefly, viral RNA was extracted using QIAamp Viral RNA Mini Kit , and cDNA was reverse transcribed with Uni12 primer (5′-AGCAAAAGCAGG-3′) and PrimeScript RT reagent Kit . The cDNA was amplified by influenza H3 specific primers. PCR products were gel purified with Wizard SV Gel and PCR Clean-Up System  before sequencing with an ABI Prism 3700 DNA Analyzer . Sequence fragments were assembled with Lasergene 7.1 (DNASTAR). The sequence has been deposited into NCBI GenBank (Accession number MG693001).Reverse transcription-polymerase chain reaction and sequencing of the hemagglutinin gene of 3391/2017 virus were performed as we described previouslyThe phylogenetic tree of the hemagglutinin gene was constructed using the Maximum Likelihood method based on the Hasegawa–Kishino–Yano model using MEGA software package version 7.0. The bootstrap values from 1000 replicates were calculated to evaluate the reliability of the phylogenetic trees.Amino acid sequences of all Hong Kong influenza A(H3N2) viruses with collection date between 1 January 2014 to 13 December 2017 and deposited into GISAID EpiFlu database.Bioedit version 7.0.9.0 was used for alignment and analysis of the amino acid residues.t-test. Fisher’s exact test was performed to compare categorical variables.Statistical analysis was performed using SPSS 21.0 and GraphPad PRISM version 6.0. Comparison of geometric mean antibody titer was performed using log-transformed antibody titer with pair-sampled Supplementary Table S1


Ticks are important vectors for the transmission of pathogens including viruses. The viruses carried by ticks also known as tick-borne viruses (TBVs), contain a large group of viruses with diverse genetic properties and are concluded in two orders, nine families, and at least 12 genera. Some members of the TBVs are notorious agents causing severe diseases with high mortality rates in humans and livestock, while some others may pose risks to public health that are still unclear to us. Herein, we review the current knowledge of TBVs with emphases on the history of virus isolation and identification, tick vectors, and potential pathogenicity to humans and animals, including assigned species as well as the recently discovered and unassigned species. All these will promote our understanding of the diversity of TBVs, and will facilitate the further investigation of TBVs in association with both ticks and vertebrate hosts. There are over 900 species of ticks in the world, and many of them are capable of transmitting pathogenic agents  , which causes an acute febrile illness accompanied with severe bleeding named Crimean–Congo hemorrhagic fever (CCHF) in humans. CCHF was first brought to modern medical attention in 1945 when over 200 Soviet military troops and local inhabitants contracted the disease and about 10% of the patients died  was first identified near Nairobi, Kenya in 1910, and Nairobi sheep disease virus (NSDV) was not found to be the causative agent until 1917 , Hughes virus (HUGV), Punta Salinas virus (PSV), Soldado virus (SOLV), and Zirqa virus (ZIRV). They are all TBVs vectored by soft ticks  in 1969 from Abu Dhabi (United Arab Emirates) . Most members of genus Orthobunyavirus are arboviruses transmitted by mosquitoes or culicoid flies except for a few that are vectored by ticks .The family et al.et al.Ixodes ricinus ticks collected in southern Finland (Saikku and Brummer-Korvenkontio http://wwwn.cdc.gov/arbocat/]). The UUKV group was not suggested to pose threat to public health, although antibodies to some members were detected in humans  is one of the novel pathogenic TBPVs and was first identified in China from patients with severe fever, thrombocytopenia, and leukocytopenia accompanied with gastrointestinal symptoms, chills, joint pain, and myalgia  in Kentucky in 1967  was first isolated from H. spinigera ticks in 1957  from Mononegavirales accommodates viruses with nonsegmented, linear, single-stranded negative-sense RNA genomes  , Nyamanini nyavirus (NYMV), and Sierra Nevada nyavirus (SNVV)  in northern California, USA. However, it was not the causative agent of EBA. SNVV has about 50% similarity to NYMV and MIDWV, and is phylogenetically related to them  and other unassigned rhabdoviruses. The virions of rhabdoviruses are characteristically bullet-shaped particles with the length of 100–430 nm and the diameter of 45–100 nm , Kolente virus (KOLEV), and Yongjia tick virus 2 (YTV-2)   collected from wild and domestic animals in Zhejiang Province, China, however, the virus was not isolated. Phylogenetic analysis showed that YTV-2 shares high amino acid similarity to all other ledanteviruses , and the Sawgrass virus group containing three TBVs.Am. americanum) collected in New York in a program for viral surveillance and discovered in ticks through high-throughput sequencing (HTS) , New Minto virus (NMV), and Sawgrass virus (SAWV), which were first isolated years ago in Asfarviridae has one genus, the genus Asfivirus, and comprises only single species, African swine fever virus (ASFV). It is the only known DNA arbovirus transmitted by ticks, and could infect and cause disease in swine. The African swine fever disease (ASF) caused by ASFV was first identified in Kenya in the 1920s  group, the mosquito-borne flavivirus (MBFV) group, and the no known vector group (NKV) . Terrible neurological symptoms were observed in patients with severe TBE, including muscle paralysis, disturbance of consciousness, and difficulties in swallowing, and verbal communication. Torturous sequelae were quite common among severe TBE patients  is the most notorious members of genus BEV Suss . Human iKyasanur Forest disease virus consists of two subtypes: Kyasanur Forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (AHFV). KFDV is the prototype of this species and is a zoonotic tick-borne viral disease that causes significant morbidity and mortality in human and monkey populations. It was known to exist in Kyasanur Forest of Karnataka State, India, since 1957 and has spread beyond into surrounding states  are distributed.Omsk hemorrhagic fever virus (OHFV) was identified to be the causative agent of Omsk hemorrhagic fever (OHF) which was first diagnosed in 1940s in Russia. OHFV was first isolated from a patient’s blood in 1947, and later from Ixodes spp. and can cause a fatal neuroinvasive disease in human. The virus was considered as a human pathogen when it was first isolated from the brain of a young boy who died of encephalitis in the town of Powassan (Canada)  in New England (USA) and was named deer tick virus (DTV)  is a tick-borne flavivirus transmitted by I. uriae) collected between 1975 and 1979 at Macquarie Island  in 1976  from forest rats caught near Kuala Lumpur in Malaya. LGTV can cause paralysis in adult mice and induce fever in macaque monkeys , Karshi virus (KSIV), Langat virus (LGTV) and Royal Farm virus (RFV) are not considered human pathogens at present. Knowledge about their ecology and epidemiology is limited. GGYV was first isolated from pools of seabird ticks  in 1826  was originally isolated from Orthomyxoviridae comprises viruses characterized with six to eight segments of linear, negative-sense RNA genomes  collected in 1964 from a Noddy Tern (Anous stolidus) nest, Sand Island, Johnston Atoll in the Central Pacific  and birds collected in Egypt, South Africa, Afghanistan, Nigeria, Kuwait, Iraq, Yemen, and Iran  and Quaranfil virus (QRFV) are included in genus Thogotovirus, and it was identified and isolated from Boophilus spp. and Rhipicephalus spp. ticks in Kenya (Africa)  is the type species of genus et al.et al.Interestingly, thogotovirus surface glycoproteins show no similarities to any influenza viral proteins but have striking sequence homology to a baculovirus surface glycoprotein  and nine belonging to the Spinareovirinae subfamily . Coltiviruses belonging to the Spinareovirinae subfamily and orbiviruses belonging to the Sedoreovirinae subfamily are arboviruses including TBVs.Coltivirus contains Colorado tick fever virus (CTFV), California hare coltivirus , and Eyach virus (EYAV)  in humans in northwestern America. It was initially confused with a mild form of Rocky Mountain spotted fever caused by Rickettsia rickettsii, until in 1946 when CTFV was isolated from human serum , but the natural cycle of the virus is still unclear. Serological tests have indicated that EYAV is associated with meningoencephalitis in humans  share low level identities with and are distantly related to other orbiviruses , Chenuda virus (CNUV), Essaouira virus (ESSV), Huacho virus (HUAV), Kala Iris virus (KIRV), Mono Lake virus (MLV), and Sixgun City virus (SCV). BAKUV was isolated in 1970 in the Soviet Union  was isolated in 1970 from Ornithodoros spp. ticks in Nepal. Serological evidence revealed the potential for infection by CGV to cattle, horses, sheep, buffalo, and humans , suggesting possible infection of GIV in seabirds   and Tribec virus (TRBV), which are quite closely related to KEMV, were isolated from adult I. ricinus ticks collected in Czechoslovakia in 1963  and Wad Medani virus (WMV). SELV was isolated from B. microplus ticks collected in the Seletar district, Singapore, in 1961. Serological evidence suggested the infection in cattle, camel, pigs, buffalo and rodents , and RA243 and RA257 (R. appendiculatus) (Alberdi et al.The species et al.et al.et al.et al.et al.et al.et al.et al.et al.Researchers are making efforts to identify, isolate, and characterize novel TBVs, and to determine whether these TBVs pose threats to public health, so that they can prepare for a rapid response to newly emerging diseases caused by novel TBVs. Taking advantage of NGS, a large number of novel virus-related sequences have been identified in different tick species; however, researchers failed to isolate these viruses (Li Since the discovery of first tick-borne pathogenic virus over 100 years ago, diversified TBVs with global distribution have been discovered and isolated belonging to at least 2 orders, 9 families, and 12 genera. In recent years, the rapid development of NGS has boosted the discovery of novel TBVs. An unexpected role of TBVs in the evolutions of RNA viruses has been revealed, which also suggests the significant role of ticks in circulating and transmitting TBVs. The known TBVs may just represent a tip of the iceberg, the most and rest part of which remains to be explored.Our current knowledge about the association of TBVs and tick species are still limited. Hopefully, ongoing researches from worldwide scientists on the immunomodulation mechanism of tick salivary, interactions between tick viruses and the transmitted-vector tick species, and anti-tick vaccines will have great significance in controlling of tick-borne viruses and protecting humans and livestock from pathogenic TBVs.


Hypnotics (sleeping pills) are prescribed widely, but the economic costs of the harm they have caused have been largely unrecognized. Randomized clinical trials have observed that hypnotics increase the incidence of infections. Likewise, hypnotics increase the incidence of major depression and cause emergency admissions for overdoses and deaths.  Epidemiologically, hypnotic use is associated with cancer, falls, automobile accidents, and markedly increased overall mortality.  This article considers the costs to hospitals and healthcare payers of hypnotic-induced infections and other severe consequences of hypnotic use. These are a probable cause of excessive hospital admissions, prolonged lengths of stay at increased costs, and increased readmissions. Accurate information is scanty, for in-hospital hypnotic benefits and risks have scarcely been studied -- certainly not the economic costs of inpatient adverse effects.  Healthcare costs of outpatient adverse effects likewise need evaluation. In one example, use of hypnotics among depressed patients was strongly associated with higher healthcare costs and more short-term disability. A best estimate is that U.S. costs of hypnotic harms to healthcare systems are on the order of $55 billion, but conceivably might be as low as $10 billion or as high as $100 billion. More research is needed to more accurately assess unnecessary and excessive hypnotics costs to providers and insurers, as well as financial and health damages to the patients themselves. Similarly, the American College of Physicians (ACP) recommended that cognitive behavioral therapy should be the first-choice treatment for insomnia, and the ACP guideline expressed doubt on whether hypnotics were worth the risks, even as secondary choices for short-term use3. A 2017 American Academy of Sleep Medicine (AASM) guideline reiterated that cognitive-behavioral therapy is the first-choice treatment for insomnia. For pharmacologic treatment of chronic insomnia, AASM gave “WEAK” positive recommendations for use of certain hypnotics, but qualified those recommendations by conceding inadequate evidence concerning the severe risks of hypnotic drugs discussed below, by conceding that their recommendations were limited to insomnia patients without much comorbidity (probably the minority), and by noting that their recommendations might not be applicable to payer perspectives5.Evidence of hypnotic harms is growing - the American Geriatrics Society has recommended that the popular hypnotic drugs be avoided for older patients, who are almost half of hospital patientsThe more severe risks of hypnotic drugs are rarely recognized.Randomized controlled trials show:6. Moreover, infections are associated with depression and suicide7.a) Hypnotics increase incidence of infections, with a mean 44% increase in controlled trials8.b) Hypnotics increase the incidence of major depression by more than two-fold7.Death certificates list hypnotics and other benzodiazepine agonists among causes of as much as 1 out of 3 U.S. opiate overdose deaths and may be present in about half of suicidesEpidemiologic studies demonstrate more risks associated with hypnotics:9. Outpatient falls are also increased.a) In-hospital falls, e.g. over 3 times as many falls have been observed among patients receiving zolpidem10.b) Hypnotic use is associated with up to double the motor vehicle crash rate11.c) Emergency room visits related to hypnotic ingestions have been increasing7.d) Rates of specific cancers, especially lung and esophagus, have multiplied among hypnotic users7.e) According to electronic records systems in 5 countries, overall mortality appeared increased 2-fold to 4-fold among patients receiving hypnotics, after adjustments for comorbid risk factors and confounders12. It is important to recognize that causality of some of these harms has been supported by randomized controlled trials or death certificates, and for other associated harms, demonstrated association may not be sufficient evidence for causality because of confounding comorbidities. Here the focus is on estimating financial harms to health systems even when lacking precise estimates of the hypnotics-caused components of these harms.Hypnotic harms to patients have been documented in more detail elsewhere, with critiques on the strengths and limitations of such evidenceWhat has been missing from current documentation is a detailed report on the cost of hypnotics to hospitals, insurers, and managed care, where the minimal benefits are weighed against the severe harms. Factual economic data have been so sparse that we must use fragmentary evidence and some speculation to estimate how much hypnotics cost the U.S. medical systems. Additional studies are needed before precise cost estimates can be made.7. Insufficient evidence of any benefit was found for the other benzodiazepine agonists3. Moreover, the authors stated that, “it is not known how many minutes’ change in SOL, TST, or WASO indicate clinically meaningful improvement3.” In other words, it is not known if the weak benefits reported were clinically meaningful even at high doses that are considered unsafe. An older definitive review of objective polysomnographic data that included data from unpublished trials concluded that hypnotics produced little or no objective improvements in total sleep in currently-recommended doses and no verified overall health benefits7.An authoritative systematic review biased towards hypnotics, limited to subjective outcomes of outpatient insomnia patients, restricted to published controlled trials, and including studies of greater-than-recommended doses, found low-strength evidence for weak benefits for two “Z” hypnotics used mainly in doses higher than recommended12. In a study of 55,000,000 managed care patients, only 31% of those receiving hypnotic medication had a diagnosis of insomnia13. The fraction of insomnia patients receiving hypnotics is quite variable depending on the patient samples and definitions of insomnia. Another complication is that using hypnotics may actually cause insomnia14, at least following hypnotic withdrawal. For example, in the long run, CBT-treated patients tapered off zolpidem slept better over time than a contrast group randomly provided with continuing zolpidem15. Consequences of insomnia such as absenteeism, automobile crashes, and increased medical costs were estimated to be costing the U.S. over $15 billion in 199316. Several more recent cost estimates have been far higher, as high as $216 billion in 2015 dollars17. These studies generally made little attempt to differentiate costs caused by insomnia itself from costs of confounding comorbidities and correlated hypnotic harms18. Some insomnia cost studies were sponsored by hypnotic manufacturers or others with interests in attributing the costs to insomnia.Up to now, medical literature has projected costs of insomnia harms but has hardly mentioned what the harms from treating with hypnotics may cost. The presence of insomnia is obviously confounded by association with prescription of hypnotics, though not as closely as one might expect19. Moloney et al. found that due to recent “medicalization” of sleeplessness, from 1993 to 2007, hypnotic prescribing grew much more rapidly than diagnoses of insomnia, so that in physician office visits, hypnotic prescribing grew to over 3 times the rate of sleeplessness-related complaints20. One study used a prescription claim for a hypnotic as an explicit marker for insomnia, in order to compare cohorts with and without insomnia among 87,461 depressed patients. Hypnotic use was associated with more comorbidity-adjusted hospitalization, more frequent ER visits, 12-month healthcare costs that were $3,918 higher, and more short-term disability21. For the authors to attribute these cost correlates of hypnotic prescription claims to insomnia (or underlying depression) and not to the hypnotics themselves seemed illogical. A similarly flawed study of a national sample of insured workers found yearly health costs were $936 higher among those with insomnia, but 2/3 of the insomnia cohort were defined by receiving hypnotics without having received a recorded diagnosis of insomnia22. Another nationwide study found that insomnia was correlated with prolonged hospital stay, but lacked data to determine whether length of stay was more closely correlated with hypnotic use rather than with insomnia diagnoses23. A study of insomnia patients both before and after treatment versus controls found an 85% increase in health costs of insomnia patients treated with sedatives/hypnotics as opposed to insomnia patients that were not treated with hypnotics24. The authors attributed this difference to more serious underlying conditions amongst those treated, without considering the possibility that the treatment itself was increasing costs. A study in Taiwan found that in contrast with a cohort without insomnia that did not use sedatives or hypnotics, a comorbidity-matched cohort with a diagnosis of insomnia suffered more acute myocardial infarction and stroke, but only among those taking hypnotics or other sedatives25. This may suggest that after control for insomnia, it was the sedative/hypnotics causing myocardial infarctions and stroke. Other studies relating insomnia to health care costs have explicitly found greater healthcare costs among those given prescription treatment for insomnia27.Several studies have attributed costs associated with hypnotic prescribing to insomnia, ignoring that less than half of the prescriptions are given to patients with diagnosed insomnia9. Although a 2% average increase in length of stay might appear small, such small mean increases would cost billions of dollars if pervasive throughout the United States. Another study found that in-hospital benzodiazepine prescriptions were associated with 23% higher readmissions28. We must recognize that without randomized placebo controls, none of these studies can offer definite proof on whether hypnotics or insomnia cause increased health costs.A Mayo Clinic study found that hospital patients who received zolpidem had a 2% longer length-of-stay , possibly due to their triple hazard of fallsIt is ironic that several of the studies that document hypnotic prescriptions as being associated with increased healthcare costs were sponsored by hypnotic manufacturers, when they had intended to attribute these costs to insomnia.29.” Personal communications indicate that routine hypnotic prescribing without evidence of benefit is still a common practice in many of the most renowned academic medical centers. The recent Mayo Clinic report is an example that listed only 32% of patients given zolpidem as having an insomnia diagnosis9.Fifty years ago, it was routine to prescribe an “as-needed” hypnotic with almost every hospital admission. In 1982, Perry and Wu reviewed 331 charts of a distinguished teaching hospital and reported that, “Most surgical patients (96%) and a large number of medical patients (46%) had hypnotic agents prescribed on admission without a recorded reason, without the patient’s request or knowledge, and without a statement in the medical chart indicating whether the therapeutic objectives were met30, even though several of the studies involved such intravenous drugs. Out of the 15 studies, 5 showed some evidence that oral benzodiazepines reduced sleep latency, but most of the treated patients still had abnormal sleep latencies exceeding 30 minutes. The review concluded with “insufficient evidence to suggest that pharmacotherapy improves the quality or quantity of sleep in hospitalized patients suffering from poor sleep30,” and no other health or cost benefits were documented.An up-to-date systematic review of 15 in-hospital controlled trials of hypnotic drugs going back to 1983 found that only one of the included trials (of intravenous dexmedetomidine) showed a convincing advantage for sleep efficiencyThe controlled hospital trials were not designed to assess the costs of hypnotic harms. Indeed, I know of no formal studies on the health cost of harms produced by in-hospital administration of hypnotics, whether randomized or not. It is hard to imagine how drugs that are known to increase the incidence of infection and depression and are strongly associated with in-hospital falls could fail to increase hospital costs. 32. Most outpatient hypnotic prescriptions have been renewals beyond recommended durations at above-recommended doses33. This lack of indication or documented benefit is characteristic of hypnotic prescribing, and it is hard to understand what could justify the risks and costs of supplying the benzodiazepine-agonist hypnotics.As previously mentioned, most prescriptions for hypnotics are given to patients without diagnosed insomnia, even though insomnia is the sole approved indication for most hypnotics. Zolpidem takes up over 70% of the contemporary U.S. hypnotics’ market. Most zolpidem prescriptions have been given to patients who had one or more hazardous contraindications, such as concomitant use of opiates or other sedatives, age over 60, alcoholism, history of depression or use of antidepressants34. Considering that about 27% of Medicare costs  are incurred in the last year of life, mainly shortly before death, the costs of hypnotics to Medicare in 2015 caused by increased mortality rates could be roughly $31 billion: 0.18 × 0.27 × $646.2 billion35. Current U.S. hypnotic prescriptions may be about as frequent as they were in 2006–2008, but current Medicare expenditures in 2017 would be a bit higher. Not all Medicare expenditures in the year before death would be related to the damage cause by hypnotics, but there would be counterbalancing hypnotic-related expenditures for patients before the year of death. Moreover, a substantial portion of the medical costs would have fallen on payers other than Medicare such as Medicaid, a government health provider for all-age indigent people costing $545.1 billion35. A somewhat different estimate of total population last-year-of-life care for 2011 was $205 billion36, of which 18% would be $36.9 billion. The number of deaths statistically associated with hypnotic use may greatly overestimate the deaths attributable causally to hypnotics, but likewise the attributable deaths may be underestimated12. A $31 billion yearly Medicare cost estimate is quite possibly inaccurate, but it represents, in my opinion, a reasonable estimate of the cost magnitude for hypnotic-caused mortality, based on current evidence.Excessive mortality may be the most expensive harm caused by hypnotics. It is possible to loosely estimate the related costs. The 2006–2008 estimate from the Geisinger Health Study supplement indicated that hypnotics cause roughly 18% of all adult deaths37. Between 5–10% of these costs came from readmissions. Hypnotics have been shown to cause infections6, e.g., benzodiazepines were associated with 54% higher rates of pneumonia38. Of course, not all infections treated in hospitals are caused by hypnotics or arose in-hospital. I might imagine that hypnotic-caused inpatient-treated infections could cost anywhere up to $20 billion per year. Also, hospital-acquired infections might not be reimbursable by Medicare payments but might fall on other funding sources.The costs of hypnotic-induced infections cannot be accurately estimated. We can gain a perspective on hospital infection costs from 2013 data on the U.S. hospital costs for treatment of septicemia and pneumonia alone, which together were estimated as reaching about $33 billion7. Estimating that there are about 14.8 million people in the U.S. each year suffering from major depressive disorders, and that 5.8% took hypnotics39, the total medical costs would add up to about $3.4 billion per year for depression attributable to hypnotics, if the added healthcare cost was around $3918 for each person39.Hypnotics increase the incidence of depression40. Of these costs, around 63% covered hospitalizations, 21% emergency department visits, and 16% outpatient visits. The average cost per fall was about $30,000. Unfortunately, there are no data available that estimate total numbers of outpatient or inpatient falls attributable to hypnotics in the U.S. However in 2010, among patients hospitalized at the Mayo Clinic, I infer from the number of falls among patients who received zolpidem, their adjusted hazard ratio, and the total falls, that 29% of total inpatient falls could be attributable to zolpidem, although there were only data on 11.8% of patients receiving any zolpidem9. Presumably, the costs of in-hospital falls were not reimbursed by Medicare charges.The medical costs of falls among U.S. adults aged 65 or older were estimated to be about $32 billion for 201541. It is known that people who take sedatives such as zolpidem have higher crash rates. Taken from Washington State health plan data10, sedative users had around twice the crash rate when compared to non-users, after controlling for comorbidities. Nationally, between 3–10% of adults take a hypnotic each year, so we might infer that 3–10% of crashes could be caused by hypnotics, costing roughly $0.6–2.0 billion per year for medical costs. I am inclined to think that the percentage of U.S. adults taking hypnotics in a given year is at the higher end of that estimate, since some studies give even higher estimates43, and press reports suggest current prescriptions are in a range of 40–50 million per year.Automobile crashes in 2010 were estimated to generate $23 billion in U.S. medical costs44. If we use the Geisinger Health Study34 as a model, $1 to $3 billion of cancer care costs could be associated with hypnotic use each year.National U.S. costs of cancer medical care are projected to reach $158 billion in 202022 and $391822, we can estimate the costs to fall in between $6.3 to $95.4 billion (probably close to the higher figures), consistent with the cumulative cost estimate taken from harm components. Wherever the true costs may fall, within that $10 to $100 billion likely range, these costs are great enough that studies are needed to assess the costs more reliably.Combining costs of excess mortality, infections, depression, falls, automobile crashes, and cancer, my best estimate is that hypnotics cost hospitals and medical payers somewhere around $55 billion per year, acknowledging an uncertainty range that likely falls between $10 billion and $100 billion. Similarly, assuming that from about 250 million adults in the U.S., 3% to 10% take hypnotics in a given year, and estimating yearly costs related to the hypnotics to range between $936With the recent expansion of electronic medical records, many hospital systems and insurance systems already have sufficient data in their existing electronic records to estimate the outcomes and costs associated with hypnotics prescribing, including hospital admissions, infections, falls, and incident delirium and dementia, lengths of stay, and readmissions. Such available data could give us a much clearer idea of costs associated with in-hospital hypnotic prescribing, but control for comorbidities and other confounders could not assure an accurate estimate of the causal component of associated costs.45. With the increasingly widespread development of personalized medicine, involving genotyping and whole-genome analyses, an increasing number of hospital systems will have accumulated sufficient genetic data to isolate the causal contribution of hypnotics to infection, hospitalization, depression, hospital readmissions, cancer and mortality.Fortunately, it is becoming increasingly possible to utilize genetic data and “Mendelian randomization” to effectively compare groups who received hypnotics due to random genetic propensities with those who did not46.Because of ethical and practical difficulties and manufacturer liability concerns, it appears unlikely that anyone will ever do large enough randomizing hypnotic vs placebo drug trials to demonstrate the costs of hypnotic harms accurately. Fortunately, an alternative randomizing strategy relying on patient choice after education and patient-empowerment has been suggested: such studies might be integrated into the wellness-promotion and cost-reduction programs of managed care organizationsIt might be years before more reliable data are assembled on the harm costs and cost-benefits of hypnotics. Meanwhile, hospital leaders and managed care and insurance administrators would be wise to infer from available evidence that the costs of hypnotic harms exceed any cost benefits of hypnotics. A decision to protect patients from hypnotics would also protect payer budgets. I have reviewed the revised version and believe that all issues and concerns have been adequately addressed.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have studied Version 2 of this article and read the authors comments and am satisfied that it should now be approved for publication.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The author has done considerable work in assembling data relating the use of sedatives and hypnotic drugs to harmful outcomes and increased healthcare costs. The main thrust of this article is, assuming that hypnotic use causes harmful health outcomes, what would be the economic costs of this harm? However, the question about hypnotic use by itself causing harm and health morbidity is still frustratingly not completely resolved. The author amasses a wealth of evidence associating the two, lending strength to the notion of causality but not confirming it. There is good experimental evidence showing negative effects of hypnotics including slowed reactions, cognitive and memory impairments, impaired balance arising from their sedating effects both before sleep and, for longer acting hypnotics, after sleep. However, the evidence for the direct effects of hypnotics on physical health measures is not as strong since these are largely correlation studies. The greater increase in health morbidity and mortality in those prescribed hypnotics may arise from the possibility that hypnotics are more likely to be prescribed by health professionals in cases with greater and more serious co-morbidity and those likely to have increased mortality risk. This self selection of hypnotic prescriptions to those likely to have higher morbidity and mortality may account for all or some of the subsequent elevated risks. The author, himself, admits that this may not be resolved without randomized control trials (with some diagnosed insomniacs not receiving hypnotics or receiving non-drug therapies instead). However, it may be difficult to obtain ethics approval for a study allocating insomnia sufferers to a non-treatment control over a long period of time . The only opportunity might be to compare pharmacotherapy alone with cognitive/behavior therapy with long term follow-ups of sleep and many health outcomes. This should be acceptable ethically. et al1. In their study they reported on an insomnia subset of 5,773 of which 75% were treated with a prescription medication and the other 25% were not and all were followed up after a 12 month period. The treated subset was more likely to have a mental health diagnosis and anti-depressant medications. These differences were significant between the groups at baseline which supports the notion that it is the higher morbidity patients more likely to receive hypnotic medication. These initial morbidity differences were then amplified at the follow-up and the medicated group showed greater increases in health costs ($4276 vs $2309) over that time. This is strong circumstantial evidence that the hypnotic medications were causing an increase in medical costs. At least one could say with greater certainty that the medications were not curing their insomnia and reducing health costs. The only lingering doubt, however, is not knowing the outcome had they not been medicated. Would they have been even worse off, no different, or, as Dr. Kripke is suggesting, better off? We simply cannot be certain until randomized controlled trials are conducted. The difficulty with drawing causal conclusions from epidemiological studies is nicely illustrated by one of the cited references with some of the most compelling evidence, the study by Anderson, A crude but good analogy to the point I am making is the effect of hospitalization. Should a 70 year old with a severe chest infection be hospitalized? Those admitted to hospital certainly have a higher morbidity and mortality risk. Does that mean someone with these symptoms will be better advised to stay out of the hospital? In the mean time, the weight of the evidence presented by the author is compelling. My only suggestion is for the author not to slip into the use of terminology that the causal connection is proven. By doing so he is in jeopardy of reducing the strength and consistency of the correlation data. Let the data be presented and just percolate in the reader's mind. It will thus be more effectively taken up than if the author takes the one step too far to insist upon the causal link. Already there is too much research being presented implying causal links between sleep variables  and health outcomes from epidemiological studies. Let's not contribute to this inappropriate trend.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Dr. Lack’s very thoughtful emphasis on the problem of causality highlights a crucial issue for readers of this manuscript about hypnotic costs.  His review contributes to the discussion already in the manuscript calling for more studies including studies of causality.  Likewise, the manuscript specifically acknowledged the problem of associational studies potentially being confounded by comorbidities.  The problem of comorbidities is emphasized a bit more in Version 2.  Until more reliable data become available, the manuscript argues that managed care and insurance administrators should weigh the best available evidence of whether the costs of hypnotic harms are likely to exceed the value of hypnotic benefits. As in most areas of medicine, timely decisions about hypnotic treatments and reimbursements must be made based on incomplete and imperfect evidence.  Decisions need to be made daily and cannot be deferred for years more study. We still do not have definitive randomized controlled trials about whether cigarettes cause cancer in humans.  After millions of people had died from cancers “associated with” cigarette smoking, it became time to advise against cigarette smoking based on available evidence that association probably indicates a large element of causation.  The reader may be sure that this manuscript on costs never implies causation without causal evidence, but uses the word “association” whenever there is insufficient evidence of an element of causation.  Some added emphasis on this distinction is included in the Version 2 revision.1,2  However, just as it would be an error to slip into language implying causation when only association has been demonstrated, it would be an error not to recognize existing human trials and meta-analysis confirming evidence of causation.  Moreover, in the case of hypnotics, as with cigarettes, there is much animal causal data andin vitro data supporting the evidence of association studies.  The sleep disorders community has been slow to recognize and replicate the existing findings as the causal evidence of serious hypnotic risks has accumulated.    This response to both reviews is a welcome opportunity to elaborate on the issues of causation versus association that were not presented at length in a manuscript about costs, since that would repeat much detail previously published.3 performed formal meta-analysis of 38 randomized controlled trials of 8828 participants randomized to 4 hypnotics and 4383 participants randomized to placebos, in whom the overall infection causation hazard ratio was 1.44 .  If any manufacturer could do a meta-analysis showing that this analysis was wrong, would they not have done so?  How much more evidence is needed?  As a matter of fact, Sanofi subsequently did an independent analysis of their Ambien data and informed the FDA that they could confirm that Ambien (zolpidem CR) causation of infections such as influenza was “probably real.”4 The FDA expert agreed. Would skeptics nevertheless question that hypnotic causation of infections such as influenza creates medical costs and sometimes deaths? Joya et al.5  If any manufacturer could do a larger, more formal meta-analysis showing that hypnotics do not cause depression, would one not think they would have published?  In an important study of 593 participants randomized to eszopiclone and 195 randomized to placebo, the rate of discontinuation for depression was 2.0% in the eszopiclone group and 0.0% in the placebo group, a result that appears statistically significant.  In their modesty, the authors did not even mention that the study had proven that eszopiclone causes infection.6  Note that these were new, incident depressions, not exacerbations, since the study excluded patients with any DSM-IV Axis I psychiatric diagnosis at baseline.6 Would skeptics nevertheless question that depression elevates medical costs and sometimes causes suicides? For an invited lecture, Kripke did an informal analysis of incident depressions in randomized trials of 4 hypnotics (including data available through the FDA that manufacturers did not publish). There were 5535 participants randomized to hypnotics with 2% incidence of depression and 2318 participants randomized to placebos with 0.9% incidence, yielding a depression causation risk ratio of 2.1 (P<0.002).1  For 2015, the U.S. CDC’s WONDER data base listed over 10,000 deaths with causes of death that included a barbiturate, benzodiazepine, or a Z-drug (the Z-drug category included anti-epileptics), but for many reasons, the 10,000 reported deaths is believed to be an underestimate.  Would skeptics nevertheless argue that death certificates listing a hypnotic as a cause of death are evidence for association, not causality? The previous detailed review also discussed at length the many death certificates that mention hypnotics as one of the causes of death and likewise discussed why these are probably underestimates. To summarize, there are certainly adequate data confirming that hypnotics cause some of the harms discussed in this manuscript and some of costs resulting. There are also controlled trials showing that hypnotics cause impaired balance and poor on-road driving performance, though controlled trials with automobile accidents or falls as endpoints have not yet reached adequate size. Further research is needed to better define the magnitude of costs from hypnotic harms, but some causal role of hypnotics in several of the harms resulting has already been confirmed. 7 Another alternative would be Mendelian randomization studies.  Randomizing studies of causality are feasible if those who advocate and provide hypnotics recognize their responsibilities to assess the risks and costs. Dr. Lack’s comments are very helpful in reiterating the need for expanded studies.  The problems of ethical approval are not insurmountable. If it is ethical to administer hypnotics to patients in short-term hospitalization, would it not be ethical to perform randomized trials focusing on whether hypnotic prescribing increases or decreases hospital costs?  Short-term hospital studies might also provide useful evidence regarding causality of serious infections and depression.  Even for early death as an endpoint, short-term randomizing clinical trials could be ethical and have adequate power.  Since the epidemiology suggests that the greatest risk-ratio of hypnotics may be within the first 30 administrations, mainly among frail and elderly patients, randomized placebo-controlled clinical trials might only need to last 30 days among frail and vulnerable participants (who currently receive much of the hypnotics prescribed). To be sure, large numbers of participants would be needed for adequate power in relatively brief randomization designs.  Another alternative would be the randomized education-patient-empowerment technique initiated by Dr. Tannenbaum.F1000Research. 2017;5: 918 ( http://dx.doi.org/10.12688/f1000research.8729.2 ). 1.  Kripke DF. Hypnotic drug risks of mortality, infection, depression, and cancer: but lack of benefit [version 2].Drug Saf. 2016;39: 93-107. 2.  Kripke DF. Mortality risk of hypnotics:  strengths and limits of evidence.J Clin Sleep Med. 2009;5(4): 377-83. 3.  Joya FL, Kripke DF, Loving RT, Dawson A, Kline LE. Meta-analyses of hypnotics and infections: eszopiclone, ramelteon, zaleplon, and zolpidem.https://www.accessdata.fda.gov/scripts/cder/daf/ under zolpidem. 4.  Farkas R. Center for Drug Evaluation and Research Approval Package for:  Application Number: 019908Orig1s032s034  021774Orig1s013s015.  Silver Spring, MD, FDA. Accessed 2013:  accessible throughBMC Psychiatry. 2007;7:42. 5.  Kripke DF. Greater incidence of depression with hypnotics than with placebo.Sleep. 2003;26(7): 793-9. 6.  Krystal AD, Walsh JK, Laska E, Caron J, Amato DA, Wessel TC, Roth T. Sustained efficacy of eszopiclone over 6 months of nightly treatment:  results of a randomized, double-blind, placebo-controlled study in adults with chronic insomnia.JAMA Intern Med. 2014;174(6): 890-8. 7.  Tannenbaum C, Martin P, Tamblyn R, Benedetti A, Ahmed S. Reduction of inappropriate benzodiazepine prescriptions among older adults through direct patient education: the EMPOWER cluster randomized trial. Overall this is an important contribution. A few relatively minor concerns: 1. The term "proven" is used quite a bit. It may be better to use the more qualified "shown" or "demonstrated." 2. The paper by Moloney and colleagues (2011) demonstrates quite clearly that rates of hypnotic use are high, not really tied to insomnia diagnosis, and often chronic. This may aid in making the case in this article. 3. In addition to mentioning the ACP guideline, even the recent AASM guidelines for prescription hypnotic use state that evidence for using hypnotics is overall low. 4. There is often causal language that may be more strong than the evidence warrants. For example, it is not clear that the role of hypnotics in suicides is causal. Certainly, anecdotal evidence suggests that people overdose on sleeping pills, but the cited report has a whole paragraph about suicides that makes mostly circumstantial connections. Clearly there is a relationship, but the causal role of hypnotics is not so clearly demonstrated. 5. If hypnotics don't produce benefits, then why do people keep taking them? For example, even if the medications do not directly impact on sleep, there may be evidence that the effects on suppressing memory may outweigh the effects on sleep. So they may not actually improve sleep much but reduce the memory or perception of sleeplessness. If this reduces the discomfort associated with insomnia  then it could potentially have some sort of reinforcing benefit in that regard. Just saying that there are no benefits may be a bit oversold. 6. The "Detailed estimates" section does not actually include detailed estimates. Rather, it provides several general estimates for a number of specific pathways by which hypnotics could induce harms. Perhaps this section could be better labeled.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Dr. Grandner’s has provided several very helpful suggestions which will be incorporated in a revision of the manuscript after the initial reviews.  He mentioned two points of interest deserving comment here.10.12688/f1000research.8729.2 .  There are several kinds of evidence.  Controlled trials and meta-analysis have shown that hypnotics cause incident depressions and cause incident infections.  Controlled trials are our best tests of causality.  There is much new evidence that infection causes depression and suicides, and depression itself is widely believed to be the main cause of suicide.  Second, in a narrower sense, when a medical examiner reports a hypnotic drug as a cause of intentional overdose death (and there are thousands of such examples), that is an expert finding of causality.  Since no specific costs of suicides and suicide attempts could be included in the hypnotic cost estimates offered, causality of suicide is not a key issue for the manuscript being reviewed.  More study is certainly needed.  Incidentally, my petition to the FDA specifically mentioned suicides as one endpoint which should be incorporated in expanded Phase IV randomized safety trials of hypnotics.         First, there is the question of whether there is evidence that hypnotics cause suicide, going beyond the extensive evidence for association.  This is discussed in more detail in my recent more detailed review of the evidence for hypnotic harms:  doi:            Second, there is the question of why people keep taking hypnotics.  This is a question for the ages, since throughout the history of magical beliefs and shamanism, people have consumed countless remedies including sleep remedies that are scientifically considered non-beneficial.  Moreover, benzodiazepine-agonist hypnotics are known to be addicting drugs that are sometimes sought for pleasure, but equally important, these hypnotics cause withdrawal insomnia.  There is much evidence that people persist in using hypnotics because of the withdrawal insomnia and anxiety that occur when consumers try to stop.  As the reviewer suggested, people may take hypnotics seeking the amnesia that hypnotics produce, just as alcoholics drown their sorrows.  That amnesia has not been recognized and advertised as a medical benefit.  I am not aware of evidence that hypnotic-induced amnesia is medically beneficial, though it does often elicit a favorable subjective report.  In hospitals, there might be an argument for helping patients to forget their anxieties, pains, and discomforts, but if the same amnesic effects contribute to falls, confusion, and dementia, it may be a costly strategy.


We report here the complete genome sequence of a West Nile virus (WNV) strain from the first laboratory-confirmed human case of neuroinvasive WNV infection in Cyprus. Phylogenetic analysis showed that this WNV strain grouped clearly into genetic lineage 1, clade 1a, cluster 2. Flavivirus genus within the Flaviviridae family. The virus is maintained in an enzootic cycle in birds, which act as amplifying hosts for ornithophilic mosquito vectors belonging to Culex spp., but it can be transmitted to other mammals, including humans serving as dead-end hosts (West Nile virus (WNV) is a neurotropic mosquito-borne pathogen belonging to the nd hosts , 2. WNV nd hosts . The aimPreviously, it was shown that whole-transcriptome amplification techniques, which are based on multiple-displacement amplification, can be successfully used for amplification of RNA virus genomes . BecauseSylvia nisoria) that was later sequenced and found to belong to lineage 2 (Culex are abundant in Cyprus (The first evidence of WNV in Cyprus stems from a serological and ectoparasite survey of migratory birds in the Eastern Mediterranean between 1966 and 1971 conducted by the U.S. Department of Defense. A WNV was isolated from a barred warbler, (ineage 2 . Since Cn Cyprus .In conclusion, we report the complete genome sequence of a West Nile virus strain obtained from the first human case of neuroinvasive WNV infection in Cyprus. Phylogenetic analysis revealed that the virus belongs to lineage 1a.MF797870.The nucleotide sequence of the WNV strain WNV_Cy2016 determined in this study has been deposited at GenBank under accession no.


Ten chikungunya virus isolates from human plasma collected in Haiti from May to August 2014, in the midst of a chikungunya fever outbreak, were fully sequenced. The resulting genomic sequences are nearly identical, and phylogenetic analyses indicate they belong to the Asian lineage of the virus. Alphavirus, family Togaviridae) is a mosquito-borne virus that is the cause of chikungunya fever (CF) in humans. Although infections by CHIKV may be asymptomatic, up to 97% of those infected develop CF, which is a fever-rash-arthralgia syndrome often associated with debilitating pain that may last up to several years (Chikungunya virus (CHIKV) -PCR; cDNA synthesis from extracted RNA was performed using a Moloney murine leukemia virus reverse transcriptase (MMLV-RT) and sequence-specific reverse primers, and PCR was performed as described by Cherabuddi et al. . For seqAedes aegyptii  and also have 99% identity with CHIKV strain WHCHK9, which was also collected from Haiti in May 2014 . Presently, three distinct CHIKV lineages have been identified: African, East/Central/South African (ECSA), and Asian –8. Phy strains , but thiKX702401, KX702402, and KY415978 to KY415985.The 10 complete CHIKV genome sequences of this study have been deposited in the GenBank database under the accession numbers


Penicillium digitatum and Penicillium italicum are the main destructive pathogens in the citrus industry, causing great losses during postharvest process. To our knowledge, only one mycovirus from P. digitatum has been reported, and the prevalence of such mycoviruses against citrus postharvest pathogenic fungi and their genotyping were still under investigation. In the present study, we showed that 39 of 152 Penicillium isolates from main citrus-growing areas in China were infected with various mycoviruses belonging to polymycoviruses, Narna-like viruses, and families Totiviridae, Partitivirdae and Chrysoviridae. The next generation sequencing (NGS) towards virus genome library and the following molecular analysis revealed two novel mycoviruses Penicillium digitatum polymycovirus 1 (PdPmV1) and Penicillium digitatum Narna-like virus 1 (PdNLV1), coexisting in P. digitatum strain HS-RH2. The fungicide-resistant P. digitatum strains HS-F6 and HS-E9 coinfected by PdPmV1 and PdNLV1 exhibited obvious reduction in triazole drug prochloraz resistance by mycelial growth analysis on both PDA plates and citrus fruit epidermis with given prochloraz concentration. This report at the first time characterized two novel mycoviruses from P. digitatum and revealed the mycovirus-induced reduction of fungicide resistance.Pathogenic fungi including  P. digitatum, P. italicum, P. crustosum, and other filamentous fungi during postharvest handling2. Currently, synthetic chemicals including triazole drugs have been predominantly applied to control these phytopathogens6. However, the application of such chemicals is becoming increasingly problematic due to the emergence of fungicide-resistant strains and the drug-induced toxicological risks to public health8. Thus, there is an urgent need to develop alternative method(s) like mycovirus-related biocontrol against these postharvest diseases.Citrus fruit is one of valuable crops in agricultural fruit markets. A quantity of harvested citrus fruits decay by Totiviridae, Partitiviridae and Chrysoviridae, and the ssRNA mycoviruses are classified into six families including Narnaviridae9.Mycoviruses have been described in all major group of fungi. The reported mycoviruses are mainly double-stranded (ds) RNA mycoviruses or single-stranded (ss) RNA mycoviruses. The dsRNA mycoviruses are classified into seven families including Beauveria bassiana virus screening but not accepted by current International Committee on Taxonomy of Viruses (ICTV)10. This newly proposed family mainly comprised non-conventionally encapsidated mycoviruses (polymycoviruses) with four to seven dsRNA segments (0.9–2.4 kbp in length) as genome structure11. According to the updated information on polymycovirus molecular biology, four largest dsRNAs (dsRNA1–4), encoding functional proteins, were found in almost all polymycoviruses as their essential genome components, while the remaining dsRNA segments were not uniformly identified concerning their function(s) in the mycovirus physiology. For details, dsRNA1 and dsRNA3 have been speculated to encode RNA-dependent RNA polymerase (RdRp) and methyltransferase respectively, and dsRNA2 and dsRNA4 encode proteins with specific motifs, cysteine-rich, zinc finger-like and arginine repeats  for the former and proline-alanine-serine (PAS) rich for the latter12. The arginine repeats in dsRNA2-encoding protein have been considered as endoplasmic reticulum (ER) retention signals, normally found in transmembrane proteins and functioning in virus replication10. The proline-alanine-serine (PAS) rich protein encoded by dsRNA4 was identified to coat viral dsRNA in an unconventional way10.Concerning RNA mycoviruses sharing characteristics between dsRNA and positive-strand ssRNA viruses, a novel family ‘Polymycoviridae’ has been proposed from Narnaviridae accommodates members each containing a single linear (+)ssRNA molecule, uncapsidated, 2.3–3.6 kb in length and encompassing a single ORF that encodes the RdRp13. According to subcellular location, the members of family Narnaviridae have been classified into two genera Narnavirus and Mitovirus, the former confined to cytosol and conventionally found in the yeast Saccharomyces cerevisiae and Phytophthora infestans, and the latter localized to mitochondria and only reported in filamentous fungi15. Up to date, the extensive evidence on dsRNA and/or ssRNA mycoviruses has been addressed in fungal phytopathogens including Cryphonectria parasitica, Sclerotinia sclerotiorum, Rosellinia necatrix, Botrytis species, and Fusarium species16. In contrast, apart from Penicillium digitatum virus 1 (PdV1)17, no other mycovirus has been reported from citrus pathogenic fungi (Penicillium species).As ssRNA mycoviruses with the simplest genomes, the family Cryphonectria hypovirus 1 have been identified to cause hypovirulence in their host fungi18. Additionally, the infection of dsRNA viruses to ascomycetous yeast Saccharomyce scerevisiae enables the host to secrete protein toxins to favor its growth19. The phenotypic response(s) to mycoviruses infection(s) are of interest to gain more knowledge on virus-host interactions.As previously reported, mycoviruses usually have little effects on their hosts with several exceptions. Mycoviruses such as Penicillium species) through a total of 152 isolates from main citrus-growing provinces in China. From one of all 39 mycovirus-infected Penicillium strains, we identified and characterized two novel mycoviruses belonging to Narna-like viruses and provisionally designated family ‘Polymycoviridae’, and further assessed host phenotypic response(s) to the infection of these identified mycoviruses under triazole drug prochloraz conditions.In this study, we evaluated the presence of mycovirus in citrus pathogenic fungi (Penicillium species), we isolated 152 strains from decayed citrus fruit epidermis collected from main citrus-growing provinces in China including Hubei, Sichuan, Jiangxi and Yunnan, and the results were summarized in Table P. digitatum isolates and 2) three P. crustosum and one P. italicum strains of the rest 63 Penicillium isolates. Based on the dsRNA banding pattern (Table Penicillium strains were classified into five groups (group1–5) with their representative electrophoretic profiles  and P. italicum isolate YN-1, consisted of 4 dsRNA segments between 2.9–4 kbp in length . Group2–5 represented P. digitatum strains with diverse dsRNA banding patterns. Group2 including 24 isolates harbored a monosegmented ~5.2 kbp dsRNA . Group4, corresponding to only one strain (HB-22), consisted of three dsRNAs with size of ~5.2 kbp, ~2.0 kbp and ~1.8 kbp , containing the two putatively novel mycoviruses, was prepared through curing the dsRNA1 virus (PdV1), and the lacking of target dsRNA segment was verified by northern blot  in the present study. For this reason, dsRNA2, 3, 4 and 6, labeled in order of increasing agrose-gel electrophoresis mobility  using Illumina platform. The obtained contigs were subjected to BLAST search in NCBI databases to identify viral sequences. The complete nucleotide sequences of these dsRNAs were finally obtained by contig assembling. BLAST analysis revealed that four dsRNAs  constituted the genome of a novel polymycovirus Fig. , tentatiity Fig. , were reity Fig. . A schemity Fig. , and werity Fig. . The fulAspergillus fumigatus tetramycovirus-1 (AfuPmV-1) by sharing 48% aa sequence identity , and also similar to the RdRp of Botryosphaeria dothidea virus 1  and putative RdRp of Cladosporium cladosporioides virus 1 . The deduced aa sequence of the Pm dsRNA1 ORF contained three conserved motifs (IV-VI) found in the picorna-like RdRp family of positive-strand, RNA eukaryotic viruses   Fig. . ConcernThe Pm dsRNA2 encoded a hypothetical protein, rich in arginine repeats , containing a cysteine-rich, zinc finger-like motif Fig. , and shaMelampsora lini  and BdRV1 . Additionally, a conserved protease domain of S-adenosylmethionine-dependent methyltransferases  was observed in the N-terminal region (positions 132–242) of the present Pm dsRNA-encoding protein, and a conserved catalytic motif of methyltransferase was also detected in its primary structure  and low similarity with unassigned proteins encoded by dsRNA of ure Fig. .Beauveria bassiana polymycovirus 1 .The Pm dsRNA4 encoded a Proline-Alanine-Serine rich protein (PASrp), containing 8.04% proline, 12.26% alanine and 6.90% serine residues out of total 261 aa and sharing 35% aa sequence identity with a hypothetical protein encoded by dsRNA4 of BdRV1 , a hypothetical protein of CcV1  and a PASrp of Beauveria bassiana small Narna-like virus  and contains four motifs (IV-VII) of RdRps, including the conserved GDD motif , and the full-length genomic cDNA sequence of its dsRNA was deposited in the GenBank database with accession number MF322872. Phylogenetic analysis based on the selected RdRps 17 . These evidences ensured the existence of PdPmV1 and PdNLV1 in the TEM sample and thus confirmed no regular virus particles associated with the viruses from HS-RH2. On the other hand, regarding viral protein components, SDS-PAGE showed a major protein band with molecular mass of ~27 kDa 17 Fig. . Thus, wkDa Fig. , just cokDa Fig. . Other tP. digitatum strain HS-RH2F was obtained from parental strain HS-RH2, using a combinational approach of protoplast regeneration and ribavirin treatment. However no colonies harboring just one of the viruses were recovered. The absence of PdPmV1 and PdNLV1 in HS-RH2F was confirmed by northern hybridization , indicating a virus-induced fungicide-conditioned hypovirulence. Such fungicide-conditioned hypovirulence was also reflected in the significantly lower EC50 value of prochloraz  and HS-E9 (relatively mild resistance with EC50~0.94 mg·L−1) as virus recipients to elucidate biological effects of PdPmV1 and PdNLV1 coinfection. These two virus-free strains were transfected by PdPmV1 and PdNLV1  to obtain virus-infected strains HS-F6I and HS-E9I, respectively. In the absence of prochloraz, there was also no significant difference in colony morphology  when coinfected with PdPmV1 and PdNLV1 . The resulting HS-F6RI and HS-E9RI, virus-infected isolates generated in the back-introduction virus transmission, exhibited highly similar phenotypes to their corresponding virus-donors, including colony morphology on PDA plates Fig. , prochloues Fig. , and virC. parasitica in North America, China and Japan22. Kondo et al. documented an extensive search for dsRNAs from field isolates of R. necatrix23. Zhong et al. reported 34 U. virens strains with dsRNA mycovirus infection in total 35 samples24. Recently, Kotta-Loizou and Coutts reported 16 B. bassiana isolates with such infection in total 75 samples from worldwide locations10. In this study, of all the 89 citrus postharvest pathogenic fungal strains from main citrus-growing areas in China, we have isolated 35 P. digitatum strains with mycovirus infection. The infection rates of 39.3% (35/89) suggested a well prevalence of dsRNA mycovirus infection in P. digitatum strains. According to partial sequencing analysis, 24 P. digitatum isolates are infected with mycoviruses, belonging to Totiviridae family and sharing great similarity with PdV117, and 9 P. digitatum isolates are infected with mycoviruses, belonging to Partitivirdae family and sharing great similarity with PsV-S24. Here, mycoviruses coinfection was observed in two P. digitatum strains. One strain harbors two mycoviruses belonging to Totiviridae and Partitivirdae families which members have been well documented in many fungal strains25. The other strain (HB-36) harbors three mycoviruses, the one (PdV1) previously reported as Totividae member17 and the other two characterized as polymycovirus and Narna-like virus. Here, the member of Narna-like viruses and the member of polymycovirus were at first time molecularly identified in Penicillium strains.Mycoviruses are widely distributed in almost all fungal groups. However, statistical investigation on incidence of mycovirus infection towards specific fungal host(s) was not extensively documented. The earlier extensive searches of dsRNA mycoviruses were carried out for the host 10. Some conserved motifs, previously identified to locate in virus RNAs terminal regions with presumably important functions27, have also been observed in the 5′- and 3′-terminal sequences of PdPmV1 dsRNAs, exhibiting highly structured as predicted by mfold program. These two lines of evidences obtained from sequence analysis tentatively classified PdPmV1 as a polymycovirus.An isolate HS-RH2 containing Pm and NL1 dsRNAs, i.e. dsRNA2-6 in order of electrophoresis mobility Fig. , was obtMonoegavirales28. Phylogenetic analysis has classified PdPmV1 as a polymycovirus, based on homolog alignment of selected RdRp sequences  that are functionally associated with endoplasmic reticulum (ER) retention signals, particularly involved in replication of positive-sense ssRNA viruses including caliciviruses and astroviruses30. These arginine-repeats-containing proteins have been implicated to favor dsRNA chaperones and/or replication machinery, achieving specific RNA virus life cycle11. In addition, a PAS-rich protein, initially described in the Phlebiopsis gigantean large virus 131, was also deduced to be translated from the ORF of Pm dsRNA4. The proline-rich domain, frequently found in some scaffold proteins, has been functionally associated with protein-protein interactions, interacting with membrane components to assemble virus replication complexes33. The PAS-rich proteins encoded by corresponding segments of reported polymycoviruses are associated with the dsRNA genome of the virus and functioned in viral coating in an unconventional manner11.Further, like all the reported polymycoviruses, the RdRp of PdPmV1, putatively encoded by Pm dsRNA1, contains a GDQN motif, a characteristic motif normally found in negative-strand ssRNA viruses of the order es Table . Actuall10 and AfuPmV-111. Zhai et al. reported “bacilliform virus-like particles” as presumable virions for polymycovirus BdRV1, but they did not correlate such viral particles with their designated cap proteins12. According to the methods described by Kotta-Loizou et al.11 and Zhai et al.12, we have also succeeded in purifying virus dsRNAs (Pm dsRNA1-4 and NL1 dsRNA) from HS-RH2. Such purified dsRNA mixture (naked PdPmV1 and PdNLV1 dsRNAs), verified by dsRNA electrophoresis profile, northern blotting, and further sequencing, could also be transfected as infectious entities to P. digitatum recipients including HS-RH2F, HS-F6 and HS-E9. However, we still failed to observe any viral or viral like particles under TEM scanning. Thus, all the polymycoviruses reported up to date including PdPmV1 (this study), BbPmV10, AfuTmV-111 and BdRV112 might exhibit a common feature that they have no or irregular encapsulating processes. For this reason, further studies are required to answer the questions how the protein conjugated with the viral dsRNAs and whether PdPmV1 presented in a form of unusual ribonucleoprotein.In this study, the PASrp encoded by Pm dsRNA4 was identified by PMF analysis, and accordingly, no conventional virion was detected by TEM, although the whole genomic dsRNAs of PdPmV1 could be recovered from pellets obtained after sucrose gradient differentiation combined with ultracentrifugation. The results revealed that PdPmV1 is not conventionally escapsidated in virions, a similar phenomenon occurred for BbPmV-1P. digitatum strain HS-RH2. The dsRNA5-encoding protein showed 72% aa sequence identity with the RdRp encoded by BbSNLV, one case of dsRNA mycovirus in Narna-like viruses (or Narnaviridae family)10. Regarding dsRNA structure, multiple stem-loops at the 3′- and 5′-terminial sequences, exclusively identified in members of Narna-like viruses and narnaviruses13, were also detected in the secondary structure of PdNLV1 genomic dsRNA. In the present study, using RdRp sequences from members of Narnaviridae family and Narna-like viruses, we constructed one phylogenetic tree, placing PdNLV1 and BbSNLV into a clade evolutionally close with typical members of Narnaviridae family  have been known as yeast nucleic acid elements and their fungal hosts been restrained to Saccharomyces yeasts36. However, unlike independent ability of narnavirus to infect yeasts, the transfection of PdNLV1 into P. digitatum strains, with no exception in this report, should cooperate with another virus PdPmV1, and more evidence are required to unravel the mechanism for such virus-coinfection.We have employed all available approaches to obtain an isolate solely infected with mycovirus PdPmV1 or PdNLV1, but failed. In the present study, using a specific fractionation of sucrose gradient ultracentrifugation or purified naked dsRNA mixtures, we only obtained the virus-coinfected fungal strains including HS-RH2FI, HS-F6I, HS-F6RI, HS-E9I and HS-E9RI, simultaneously containing Pm dsRNA1-4 and NL1 dsRNA . Meanwhile, through screening of virus-cured isolates, we only obtained fungal strains (HS-RH2F) simultaneously losing the mentioned mycoviruses (Pm dsRNA1-4 and NL1 dsRNA), i.e. also failing to obtain single cured progenies of HS-RH2. Our results that the naked viral dsRNAs possess infectious ability were in agreement with the previously reported polymycoviruses−1 prochloraz conditions, the virus-infected strains HS-RH2 and HS-RH2FI, both simultaneously containing PdPmV1 and PdNLV1, showed significantly lower fungicide EC50 values  to control citrus pathogenic fungi. According to our records on the prochloraz EC50 values of more virus-infected P. digitatum isolates (Table 17. This evidence associated the present prochloraz-conditioned hypovirulence with specific virus-host interaction(s). Nevertheless, as we have emphasized in the present study, it is hard to evaluate the contribution of single virus (independent PdPmV1 or PdNLV1) to the reduced prochloraz-resistance, and thus more field P. digitatum isolates including their virus-infected and virus-free progenies would be required to more definitely correlate prochloraz sensitivity to the mycoviruses.In the absence of prochloraz, compared to HS-RH2, the virus-cured strain HS-RH2F exhibited little difference in growth rate on PDA plates Fig.  and lesiues Fig.  and smalues Fig. , indicatues Fig.  and citrues Fig.  and 5, aes Table , interesPenicillium isolates collected from main citrus-growing areas in China. In total, 39 of 152 Penicillium strains were infected by various mycoviruses. Among them, we at first time molecularly characterized PdPmV1 and PdNLV1 as polymycovirus and Narna-like virus, respectively. A decrease in prochloraz resistance was further reported in some representative P. digitatum strains with PdPmV1 and PdNLV1 coinfection, and the fungicide-conditioned hypovirulence related was also recorded.In summary, the present work evaluated the prevalence of dsRNA elements in Penicillium strains used in this study were isolated from decayed citrus fruit epidermis collected from markets, packing houses and citrus orchards in Hubei, Sichuan, Jiangxi and Yunnan province of China from 2015 to 2016. The rDNA ITS  gene fragments of these strains were amplified and sequenced to confirm their identity. HS-RH2 was derived from HB-36 through curing the virus consisting of dsRNA1. HS-F6I and HS-E9I were derived from HS-F6 and HS-E9 respectively by transfection of PdPmV1 and PdNLV1. HS-RH2F was a virus-cured strain derived from HS-RH2, and the absence of virus dsRNA elements was assessed by agarose gel electrophoresis and northern blot. All the strains were cultured on potato dextrose agar (PDA) medium or in potato dextrose broth  on a rotary shaker (180 rpm) at 25 °C.The 38. The dsRNA preparation was further digested with S1 nuclease  and subsequently with RNase-free DNase I , electrophoresed on a 1% agarose gel, and then visualized by staining with ethidium bromide. The dsRNA was excised and purified with a gel extraction kit , dissolved in RNase-free water, and kept at −70 °C until use.The mycelia were harvested and homogenized in liquid nitrogen, and dsRNA was isolated by CF-11 cellulose  column chromatography following published procedure39 and then used for high-throughput deep-sequencing using the Illumina platform. Sequenced reads match with the host P. digitatum databases (isolate Pd1) were expurgated and the remaining reads were de novo assembled using Velvet (version 1.2.10) with default parameters40. All contigs were subjected to BLASTN and BLASTX alignments against the NCBI database to search for viral sequences. The 5′- and 3′-terminal sequences were further determined using RACE-PCR. Firstly, the 3′-terminus of each strand of dsRNA was ligated to the closed adaptor primer PC3-T7 loop with a phosphorylated (p) 5′ end and an NH2 3′ end (5′-p-GGATCCCGGGAATTCGGTAATACGACTCACTATATTTTTATAGTGAGTCGTATT A-OH-3′) by T4 RNA ligase  at 16 °C for 18 h. Then, the oligonucleotide-ligated dsRNA was reverse transcribed by a primer complementary to the oligonucleotide used for the RNA ligation (PC2 [5′-CCGAATTCCCGGGATCC-3′]) and sequence-specific primers corresponding to the 5′- and 3′-terminal sequences of the dsRNA in presence of M-MLV reverse transcriptase according to previously described protocol41. The amplified cDNA products were sequenced and subsequently assembled. The full-length sequences of the dsRNAs were further confirmed by RT-PCR with specific primers and sequencing. Northern blot hybridization analysis was performed as previously described42. The specific probes (~500 bp) were labeled with digoxigenin and used to probe the RNA blot. The positions of the probes were indicated in Figs A total of 5 μg purified dsRNA was subjected to library preparation following the a reported protocol43. Multiple sequence alignments of nucleotide and amino acid sequences were generated using the CLUSTAL_X program44. The phylogenetic trees for RdRps were constructed using the neighbor-joining method of MEGA version 6 software with bootstrapping analysis of 1000 replicates45.The obtained nucleotide sequences were subjected to BLAST program on the NCBI website to see the sequence similarity. Potential ORFs were deduced and translated using the DNAMAN software package with the default parameters and the ORF finder on the NCBI website−1 sodium phosphate buffer (pH 7.4), and were then ground into homogenate. The homogenate was subjected to differential centrifugation and ultracentrifugation in sucrose density gradients  and CsCl density gradients, respectively as described by the previously reported methods12. Aliquots about 1 mL were individually collected and subjected to UV absorbance assessment, and the fractions with a peak absorption were subjected to ultracentrifugation. The obtained VLPs were suspended in 100 μL of 0.01 M sodium phosphate buffer (pH 7.4) and then detected by SDS-PAGE analysis, transmission electron microscopy (TEM) and used for infectivity assays. The gel stripe with protein band on 12% (wt/vol) polyacrylamide gel was excised and subjected to peptide mass fingerprinting (PMF) analysis at ProtTech, Inc. . All the processes described above were performed as previously described with minor modifications47.To isolate the virus-like particles (VLPs), approximately 100 g mycelia (wet weight) were harvested and mixed with 400 mL of 50 mmol·L50. Based on the ribavirin protocol, we exploited HS-B36 as original strain to generate a curing library containing a large quantity of virus-cured and virus-partial-cured isolates, and finally got desirable isogenic strains HS-RH2 just lacking dsRNA1 element by library screening (not more than 10 target isolates per one round screening). Five biological replicates of HS-RH2 were set for further research. To cure mycoviruses PdPmV1 and PdNLV1 from their host HS-RH2, the conidia of HS-RH2 were incubated in PDB media containing 100 mmol·L−1 ribavirin for 10 h to generate virus-free progenies (HS-RH2F). For each parental strain HS-RH2, five biological replicates of HS-RH2F were selected for further experiments.To eliminate the mycoviruses from the host fungus, we treated the target strains with ribavirin as previously describedP. digitatum strains HS-F6 and HS-E9 were used as the recipients in the transfection experiments. Protoplasts of HS-F6 and HS-E9 strains, each containing five biological replicates, were prepared and mixed with the purified viral particles by Ultrafree-MC sterile centrifugal filters . In the PTC buffer , the purified virus particles and further purified naked dsRNA mixture containing PdPmV1 and PdNLV1 genome RNA elements were respectively introduced into the protoplasts of HS-F6 and HS-E9 in the presence of polyethylene glycol (PEG) according to the protocol as previously reported52. Transfected protoplasts were coated onto regeneration media  plates and incubated until single colony regeneration at 25 °C. The desirable transfectants HS-F6I and HS-E9I were selected and verified by dsRNA extraction and RT-PCR amplification with specific primer pairs. For each virus-infected strain (HS-F6I or HS-E9I), five biological replicates were prepared for further biological tests.The virus-free 6 spores mL−1) of the P. digitatum strains was coated onto a PDA plate and incubated at 25 °C for 24 h. Mycelia plugs (approximately 0.8 cm in diameter) were obtained from the plate using a punch. The mycelia plugs were placed on new PDA plates and cultivated for 4 d at 25 °C or according to the experimental need, and the diameters of different colonies were measured. EC50 values of prochloraz for P. digitatum strains were measured according to previous reports47. The diameters of the colonies cultivated on PDA with different concentrations of prochloraz were measured and the average of the colony diameters in each independent test was used for EC50 calculation using SPSS software (version 10.0). Virulence assays were performed directly on citrus fruits. The citrus fruits were washed with distilled water to remove the contamination. A 2 mm hole was made on the pericarp using a 1 mL pipette tip, and a 3 μL conidial suspension was injected (106 spores mL−1). The diameters of the disease spots were measured and compared after incubation for 5d at 25 °C or according to the experimental need.The conidial suspension (50 μL, 1 × 10Supplimentary Materials