Background: The first wave of the COVID-19 epidemic in China was brought under with 3 months—from mid-January 2020 to the end of March 2020. Less studies examined dynamic psychological effect and behaviors during COVID-19 pandemic. This study aims to examine perceived risk, anxiety, and behavioral response of the general public related to the outbreak of COVID-19 in four cross-sectional surveys conducted throughout China.Methods: In 2020, four cross-sectional, population-based online survey were conducted from January 28 to February 3, from February 10 to 12, from February 20 to 22, and from March 1 to 10, respectively. Convenience sampling was used for easy recruiting survey participants under the long-term impact of the COVID-19 epidemic. The four independent online questionnaires were sent from the same approach (WeChat and MicroBlog), and anyone who receives the questionnaire on the Internet or mobile phone and meets the inclusion criteria could fill in it. The same questionnaires repeatedly used in the four surveys. Socio-demographic information and individual protective practice were collected and the state-trait anxiety inventory (STAI) was used for measuring anxiety. Propensity score matching was used to adjust for differences in baseline characteristics among the four surveys. Wilcoxon signed ranks test was used to compare people's perceived risk, anxiety and protective behaviors changes in four stages. General linear model was used to identify associations between some demographic factors and perceived risk, anxiety scores, and protective behaviors.Results: The proportion of high perceived risk has dropped from 24.7 to 4.7%. The proportion of severe anxiety has declined from 12.2 to 1.2%. The proportion of people wore masks when they went out has increased from 97.0 to 98.3%. Women were more likely to develop anxiety  and more positively adopted recommended behaviors  than men. People at age 30–39 years, with high-degree education, with married status, and accompanied with poor self-rated health status were prone to have higher risk perception and anxiety. Perceived risk was significantly associated with anxiety over the entire periods. Anxiety levels had stronger associations with adoption of protective behaviors (wearing mask and avoiding crowed place) in the early epidemic periods than in the late epidemic periods.Conclusions: The levels of perceived risk and anxiety showed a trend of rising first and then falling. Gradually upward trend on initiative preventive behaviors including wearing mask and avoiding visiting crowded places also was observed through scanning data at four stages. People at age 30–39 years, with high-degree education, and accompanied with poor self-rated health status were prone to have higher risk perception and anxiety. Our findings showed that people simultaneously presented both high-level risk perception and anxiety across the four wave surveys, leading to their positive self-prevention and protective behavior. The COVID-19 pandemic has created an unprecedented crisis . COVID-1The COVID-19 pandemic has yielded a series of undesirable effects on all aspects of society, including physical health and mental health , 6. AccoAlthough previous studies revealed that anxiety was strongly associated with demographic factors and the perceived risk , 20, theIn this study we aimed to examine the risk perception, anxiety, and behavioral response related to the COVID-19 pandemic in the general Chinese population. The aims of this study were (1) to identify trends over time in perceived risk, anxiety, and behavior response and (2) to assess factors significantly associated with perceived risk, anxiety, and behavioral response .Four cross-sectional, population-based, online surveys were conducted, the first survey (S1) was from January 28 to February 3, 2020, the second survey (S2) was from February 10 to 12, 2020, the third survey (S3) was from February 20 to 22, 2020, and the fourth survey (S4) was from March 1 to 10, 2020. They were open online questionnaires for the people (1) aged ≥18 years, (2) resides in China, (3) willing to respond, (4) able to complete the questionnaire by mobile phone or computer. We use PASS  to calculate the necessary sample size on the basis of an expected minimal change of 5% in people's attention to the epidemic, psychological effect, and individual prevention practice with α: 0.05 and β: 0.20. In this study, 1,047 participants at most were required. Considering a possible dropout rate of 20%, at least 1,309 participants in total see . The stuWe designed a structured Chinese questionnaire and collected data on Wenjuanxing, an online platform that provides functions equivalent to Amazon Mechanical Turk. Convenience sampling was used for easy recruiting survey participants under the long-term impact of the COVID-19 epidemic. The four independent online questionnaires were sent from the same approach (WeChat and MicroBlog), and anyone who receives the questionnaire on the Internet or mobile phone and meets the inclusion criteria could fill in it. After a large number of questionnaires were collected, some samples were excluded according to the exclusion criteria. The same questionnaires repeatedly used in the four surveys, mainly including the following information: (1) socio-demographic information of respondents; (2) frequency of attention to COVID-19; (3) practices of preventive measures against COVID-19, including wearing masks, keeping physical distance, personal hygiene practices, and keeping the indoor and living environment clean; (4) anxiety toward COVID-19; and (5) perceived risks. The questionnaire consisted of 25 questions on average and could be completed in 3–5 min.Demographic information collected included age, sex, marriage, education, occupation, area, family members, and residence. Variables related to COVID-19 contact history included close contact with an individual with confirmed COVID-19, indirect contact with an individual with confirmed COVID-19 and contact with an individual with suspected COVID-19 or infected materials.Respondents expressed their degree of concern about the situation related to COVID-19 by one-item: “To what extent are you concerned about the current COVID-19-linked situation?” A five points Likert-type scale were used to ascertain the frequency of attention to COVID-19 .COVID-19 preventive measures practices included wearing masks, avoiding crowded places, personal hygiene practices, and keeping the indoor and living environment clean were measured with dichotomous variables. Questions were scored “1 point” (yes) or “0 points” (no).Participants' anxiety was measured via a five-item short form of the State Scale of the Spielberger state-trait anxiety inventory (STAIS-5)  and modiPerceived risk was assessed based on previous studies conducted among the general population , with onSubjective health status was measured via one item: “How would you define your health status?” Health status was divided into four categories: 1 = unhealthy, 2 = ordinary, 3 = good healthy, 4 = very healthy.p-values were two-sided and p < 0.05 was considered statistically significant.We used SPSS  and STATA  for data cleaning and statistical analysis. Categorical variables were expressed as absolute and relative frequencies. Perceived risk scores, anxiety scores, and measure practices scores were age-standardized using the China population in 2019 and the direct standardization method. Propensity score matching was used to adjust for differences in baseline characteristics among the four surveys. Matching was performed with the use of a 1:1 matching protocol without replacement , with a caliper width equal to 0.2 of the standard deviation of the logit of the propensity score. In the matched cohort, paired comparisons were performed with the use of McNemar's test for binary variables. Wilcoxon signed ranks test was used to compare perceived risk score, anxiety score, and preventive measures score changes in four stages. General linear model was used to analyze associations between socio-demographic factors and perceived risk, anxiety scores, and preventive measures. Odds ratio (OR) and their 95% confidence intervals (CI) were calculated as estimates of the correlations. All We monitored the progress of the survey daily. After the deadline, we checked the accuracy of data, and excluded the questionnaires if (1) the age range was below 18; (2) the answering time was <150 s; or if there were (3) logical contradictions between the answers to the questionnaire. All data were checked for consistency by two research members.This study was approved as ethically exempt by the Peking University Health Science Center Ethics Committee (IRB00001052). All subjects participated in the surveys voluntarily, and the information in the database was completely de-identified.Eleven thousand one hundred thirty-eight individuals participated in S1. Among these, 1,374 were excluded due to out of age range or incomplete questionnaire, and the effective rate was 87.7% . Three thousand five hundred ninety-seven individuals participated in S2. Among these, 315 were excluded due to out of age range or incomplete questionnaire, and the effective rate was 91.2% . Four thousand four hundred and fifty individuals participated in S3. Among these, 331 were excluded due to answering without serious consideration or out of age range or incomplete questionnaire, and the effective rate was 92.6% . One thousand nine hundred thirty-eight individuals in S4. Among these, 269 were excluded due to answering without consideration or out of age range or incomplete questionnaire, and the effective rate was 86.1% . The total effective rate was 89.2% .The participants covered 30 provincial administrative regions in Mainland China. Six thousand four hundred and ninety-one (34.5%) respondents were male; 5,648 (30.0%) respondents were younger than 30 years old; 15,459 (82.2%) respondents were with bachelor's degree or above; 5,617 (29.8%) were unmarried; and 2,223 (11.8%) were from Wuhan city .P < 0.0001). The proportion of high perceived risk has decreased significantly from 24.7% inS2 to 4.7% in S3 (P < 0.0001). Similarly, the proportion of severe anxiety has declined from 12.2% in S2 to 1.2% in S3 (P < 0.0001). The proportion of people wore masks when they went out and hand hygiene has increased from S1 to S4 , and the increase is statistically significant . The proportion of avoiding crowded places has declined from 98.5% in S1 to 94.4% in S4 (P < 0.0001)  paid daily attention to the epidemic. But in S4, the proportion of people who paid daily attention to it had dropped to 88.5% . The change was statistically significant ( 0.0001) .P < 0.001). Similarly, the level of anxiety from S1 to S2 mainly revealed an upward trend, and the level of the anxiety showed a downward trend from S2 to S3 (P < 0.001). The preventive measures score showed a trend of being relatively stable and then slightly increased (P < 0.001).In east region, with the change of time and epidemic, the level of perceived risk showed a first rising and then decline trend (P < 0.001). Similarly, the level of anxiety from S1 to S2 mainly revealed an upward trend, and the level of the anxiety showed a downward trend from S2 to S3 (P < 0.001). The preventive measures score showed a trend of being relatively stable and then slightly increased (P < 0.001).In central region, with the change of time and epidemic, the level of perceived risk showed a first rising and then decline trend (P < 0.001). Similarly, the level of anxiety from S1 to S2 mainly revealed an upward trend, and the level of the anxiety showed a downward trend from S2 to S3 (P < 0.001). The preventive measures score showed a trend of being relatively stable and then slightly increased (P < 0.001)  .Generalized linear models were performed to identify factors significantly associated with (1) perceived risk, (2) anxiety, (3) behavior in wearing mask, and (4) avoiding visiting crowded places. In this regression analysis variables of the survey in four periods  were included.The general linear model illustrated that women had a higher perceived risk than men for S1  . CompareOver the entire period, women, younger people, those with a bachelor's degree and above, and those with poor health status were more likely to experience anxiety. Women were more likely to experience anxiety compared to men  S1, . The mosIn S1, compared to men, women were more likely to wear masks   and avoiP < 0.001). P < 0.01).The COVID-19 pandemic presented health, economic, and social lost . We perfThis study found a decreasing trend over four study periods in the level of perceived risk and presence of anxiety and an increasing trend in preventive behaviors including wearing mask and avoiding visiting crowded places. Moreover, there is no difference among the East, Central, and West in China in these measures. The findings indicate that the level of perceived risk and presence of anxiety were gradually alleviated over time. These trends may be correlated with increased cognitive awareness toward the COVID-19 pandemic or with China's effective prevention and control measures and high-level trust between the public, government and scientists. As This study demonstrated that in the early stage of the outbreak, women had a higher level of risk perception than men as supported by the literatures , 31. OneConsidering age, the results showed that people younger than 30 years were more likely to have higher levels of risk perception compared to the other age groups in the early stage and peak of the epidemic. A similar study supported our findings and found that one in three U.S. young adults reported clinical cut-off symptoms of panic, anxiety as well as depression . As a woAmong factors influencing risk perception, psychological response, and preventive measures, people with higher levels of education were more likely to have higher level of risk perception and experience anxiety. Higher-level educated people were more likely to adopt preventive measures like wearing masks or avoiding crowded places. A study from Saudi Arabia reported similar results; higher-level educated participants were more likely to adopt protective practices . The resOur findings showed that people who had a poor self-rated health status had higher perceived risk about the outbreak as well as higher anxiety levels; a good self-rated health status was not associated with practicing protective behaviors. Similar findings were seen in other studies , 47. As Our findings showed that people simultaneously presented with both high-level risk perception and anxiety across the four surveys, leading to positive prevention behavior of avoiding visiting crowded places. With a consistent finding, a similar study found that higher perceived COVID-19 risk predicted greater mental problem . A signiSome limitations in the current study must be acknowledged. First, a convenience sample was adopted to collect data, which increased the potential for sampling bias. Second, the cross-sectional study cannot effectively and precisely judge the causal relationship between the variables. Moreover, four cross-sectional surveys were not equivalent to a longitudinal study. Third, the data were collected from self-reports from participants by means of an online survey, which is likely to introduce information bias from social desirability or negative affection. Fourth, several single-item tools in this study were used for collecting the data in order to abbreviate survey material and potentially increase response rates, but this may have reduced the validity and reliability of the measurements; therefore, a widely used measurement tool should be adopted in the future.The levels of perceived risk and anxiety showed a trend of rising first and then falling, indicating that psychological and mental issues caused by the COVID-19 pandemic gradually subsided over time. Additionally, the proportion of people practicing preventive behaviors such as wearing a mask and avoiding visiting crowded places also increased over the four survey periods, indicating that these behaviors gradually became a conscious habit. Women were more likely to experience anxiety, adopt recommended preventive behaviors, and practice social distancing than men. People aged <30 years, with high-degree education, or with poor self-rated health status were more likely to have higher risk perception and more likely to experience anxiety. Additionally, they also were more likely to practice positive preventive behaviors. Our findings showed that people simultaneously presented both high-level risk perception and anxiety across the four surveys, leading to their positive self-prevention and protective behavior. The findings contribute to the suggestion that health authorities and policy-makers should pay particular attention to those who are vulnerable and provide support and interventions related to psychological and mental health. The results in the current study can be used to guide the development of preventive strategies in vulnerable populations with a higher perceived risk of psychological and mental health.The original contributions presented in the study are included in the article/This study was approved as ethically exempt by the Peking University Health Science Center Ethics Committee (IRB00001052). All subjects participated in the surveys voluntarily, and the information in the database was completely de-identified.BL and FC: conceived and designed the study and approved the final manuscript for publication. BL, HL, and TS: performed the study. BL, BH, HL, and TZ: analyzed the data. BL, BH, HL, and XT: contributed reagents, materials, and analysis tools. BL, TS, and FC: wrote the paper. All authors have read and approved the manuscript.This work was supports by Fundamental Research Funds for the Central Universities and Peking University Health Science Center (grant numbers BMU20170607).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Southern tomato virus (STV) is a persistent virus that was, at the beginning, associated with some tomato fruit disorders. Subsequent studies showed that the virus did not induce apparent symptoms in single infections. Accordingly, the reported symptoms could be induced by the interaction of STV with other viruses, which frequently infect tomato. Here, we studied the effect of STV in co- and triple-infections with Cucumber mosaic virus (CMV) and Pepino mosaic virus (PepMV). Our results showed complex interactions among these viruses. Co-infections leaded to a synergism between STV and CMV or PepMV: STV increased CMV titer and plant symptoms at early infection stages, whereas PepMV only exacerbated the plant symptoms. CMV and PepMV co-infection showed an antagonistic interaction with a strong decrease of CMV titer and a modification of the plant symptoms with respect to the single infections. However, the presence of STV in a triple-infection abolished this antagonism, restoring the CMV titer and plant symptoms. The siRNAs analysis showed a total of 78 miRNAs, with 47 corresponding to novel miRNAs in tomato, which were expressed differentially in the plants that were infected with these viruses with respect to the control mock-inoculated plants. These miRNAs were involved in the regulation of important functions and their number and expression level varied, depending on the virus combination. The number of vsiRNAs in STV single-infected tomato plants was very small, but STV vsiRNAs increased with the presence of CMV and PepMV. Additionally, the rates of CMV and PepMV vsiRNAs varied depending on the virus combination. The frequencies of vsiRNAs in the viral genomes were not uniform, but they were not influenced by other viruses. CMV and PepMV are two pathogenic or acute viruses that are responsible for important economic losses [Amalgavirus , whose genome is 3.5 kb in length, which contains two overlapping open reading frames (ORFs): ORF 1 encodes for the 42 kDa putative coat protein (CP or p42) and ORF 2 encodes for the RNA-dependent RNA-polymerase (RdRp) by +1 ribosomal frameshifting [Southern tomato, Pepino mosaic, and Cucumber mosaic viruses  infect tomato c losses ,2. STV ishifting . High vishifting . Despiteshifting . Hence, shifting ,9.Potexvirus (family Flexiviridae) whose genome is 6.4 kb in length and contains five ORFs: ORF 1 encodes for the RdRp, ORF 2, 3, and 4 for the triple gene block proteins (TGB), involved in virus movement, and ORF 5 for the CP [PepMV is a (+) polarity single stranded RNA (ssRNA+) virus belonging to the genus r the CP ,11,12. Pr the CP ,14. No cr the CP ,16.Cucumovirus (family Bromoviridae): RNA 1 is 3.4 kb in length that contains the ORF 1a encoding a RdRp subunit; RNA 2 is 3.1 kb in length and contains the overlapping ORFs 2a y 2b, encoding the other RdRp subunit and the RNA silencing suppressor (VSR) 2b protein; RNA 3 is 2.2 kb in length and it contains the two separated ORFs 3a and 3b encoding for the cell-to-cell movement protein and CP, respectively [(Capsicum annuum) and the main way of virus transmission is by aphids in a semi-persistent manner. Symptoms that are induced by CMV depend on the host species and the presence of RNA satellite molecules: in tomato, the most common symptoms induced by CMV are plant stunting, mosaic, and leaf deformation, but the presence of the CARNA-5 satellite enhances the disease severity, inducing leaf and fruit necrosis and plant death [CMV is a tripartite ssRNA+ virus that belongs to the genus ectively . CMV infnt death ,19. TherWhen plants are infected by RNA viruses, viral dsRNAs (generated during virus replication) activate the post transcriptional gene silencing (PTGS), a plant defense mechanism that produces the degradation of invasive RNAs in small molecules of 21–24 nt . PTGS is also involved in the degradation of highly structured plant mRNA rendering micro RNAs (miRNAs), which are small RNA molecules that are equivalent in length to vsiRNAs. miRNAs are involved in the regulation of gene expression in many crucial plant processes, such as development, reproduction, and stress. The modification of the miRNA expression level could lead to disease development ,21. In aPotyvirus, family Potyviridae) as one of the viral partners. In this case, potyviral VSRs are involved in the increase of multiplication and plant symptom enhancing of other viral partner [Mixed-infections with two or more plant viruses are frequent in fields and they can interact in multiple and complicate ways . The int partner ,26. In t partner ,27 where partner ,29. The  partner .The number of studies on viruses in mixed infection has increased lately, providing valuable knowledge that may be useful in controlling complex diseases. However, information regarding interactions between persistent and acute viruses in is very scarce. In this work, we studied the interactions of the persistent STV and the acute PepMV and CMV in tomato. Plant symptoms, virus RNA accumulation, and miRNA and vsiRNA accumulation were assessed in single, double, and triple infections.Nicotiana benthamiana plants. To exclude possible mixed infections with other viruses, the plants that were infected with these CMV and PepMV isolates were tested by ELISA for the most common viruses infecting tomato in the collection region, such as CMV, ToMV, Tomato spotted wilt virus (TSWV), and Parietaria mottle virus (PMoV) [Tomato seedlings var. Roque were analyzed by RT-qPCR to determinate the presence and/or absence of STV . The abss (PMoV) ,35,36,37N. benthamiana plants in inoculation buffer  and rub-inoculation by using carborundum in the two first tomato (var. Roque) true leaf [w/w) of N. benthamiana plants infected with each virus. The assay consisted of a total of 71 tomato plants with the following virus combinations: five, eight, and 10 plants were single-infected with PepMV, CMV, and STV, respectively; 10 plants were co-infected with STV and PepMV, 10 were co-infected with STV and CMV, and eight were co-infected with CMV and PepMV, and, finally, 10 plants were triple-infected with STV, CMV, and PepMV. As control, 10 plants were mock-inoculated by using only the inoculation buffer. Because STV is not a mechanically transmitted virus, tomato plants that tested positive for STV by RT-qPCR were used as STV-single infected plants or were inoculated with CMV and/or PepMV to obtain the corresponding co- or triple-infections. Tomato plants were kept in a greenhouse with ventilation and the presence and accumulation of STV, CMV, and PepMV was evaluated by RT-qPCR at five, 10, 15, and 20 days post inoculation (dpi). Plant symptoms consisting in leaf deformation and mosaic were recorded in this period. A scale of symptom severity was established scoring from 0 to 3, where 0 corresponded to no symptomless, and 1, 2, and 3 to mild, moderate, and severe symptoms, respectively  with liquid nitrogen. The total RNA was extracted by using a phenol:chloroform:isoamyl alcohol standard protocol followed by ethanol precipitation .CMV and PepMV conventional RT-PCR was performed from the total RNA extracts. The RNA extracts were denatured in the presence of 0.8 µM of the corresponding reverse primer and cDNA was obtained with the SuperScript IV kit  at 55 °C for 20 min. and 80 °C for 10 min. PCR was done with 0.5 µM of the corresponding forward and reverse for each virus and the Taq polymerase kit  following the manufacturer’s instructions. The PCR conditions were cDNA denaturation at 94 °C for 5 min., 35 cycles of DNA amplification at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 40 s, and a final DNA chain extension of 72 °C for 5 min. The amplification products were separated by electrophoresis in 2% agarose gels and visualized by UV after staining with GelRed . Specific PCR products were purified with Qiagen minElute PCR purification kit  and sequenced by Sanger with an ABI 3130 XL capillary sequencer . CMV and PepMV nucleotide sequences were deposited in GenBank under the accession numbers MT785769 and MT785770, respectively.STV quantification was performed by RT-qPCR with primers and TaqMan probe set previously designed in the CP 1189–1257 nts) region 7 nts reg. For CMVN. benthamiana plants, as described in 23 molecules/mol) was used to calculate the number of RNA transcript copies. In order to generate external standard curves, 10-fold serial dilutions containing 1011–101 RNA copies of each transcript in total RNA extracts from mock-inoculated tomato plants were analyzed by RT-qPCR. For each dilution, three repeats  were done, and the Ct mean value was calculated. Quantitative optimal range were obtained from 1011 to 104 virus RNA copies/ng of total RNA for STV, from 1010 to 104 virus RNA copies/ng of total RNA for CMV and from 1011 to 103 copies/ng of total RNA for PepMV. For all of the viruses, standard curves showed a strong linear relationship with very high correlation coefficients of R2 = 0.99, low variation coefficient (<0.5%), and high amplification efficiencies (>99%).The templates for in vitro transcription were obtained by conventional RT-PCR from total nucleic acid extracts of STV-infected tomato and CMV- or PepMV-infected ® RNA assay Kit in a Qubit® 3.0 Fluorometer  and the NanoPhotometer® spectrophotometer , respectively. The RNA integrity was determined in the Agilent Bioanalyzer 2100 system with the RNA Nano 6000 assay Kit . cDNA was obtained from 1 µg of total RNA of each biological replicate by using the NEBNext® Multiplex Small RNA library Prep Set for Illumina®  and then sequenced by using the Illumina NextSeq550 platform . cDNA libraries were uploaded to the NCBI platform and published under the Bioproject PRJNA625104 and PRJNA574043. The reads were cleaned by trimming the sequencing adapters and low-quality reads were filtered using SeqTrimNext software applying the standard parameters for Illumina short reads [https://tools4mirs.org/software/precursor_prediction/oasis/, Access date, 7 March 2021): reads were aligned with the STAR program in the database RNAbase 2.1 , the known miRNAs were quantified with the FeatureCounts program , whereas the prediction and quantification of novel miRNA were done with the miRDeep2 program  [For the elaboration of the small RNA libraries, three independent biological replicates were used from tomato plants that were infected with STV, CMV, or PepMV, or the different virus combinations. Each biological replicate consisted of a mix of total RNA extracts that were obtained from two or three tomato plants at 15 dpi. As control, small RNA libraries from mock-inoculated plants were synthetized. RNA concentration and purity were determined using the Qubitch 2021) . For vsip-value < 0.05 corresponding to a log Fold-change > 0.56 was considered to be statistically significant.For plant symptoms, weight and height, and virus titer, the data were statistically analyzed using a mixed model PROC MIXED in the SAS software. Plant effect was included as a random effect, whereas time or inoculation was included as a fixed effect. Least Square Difference (LSD) was used for mean comparisons. The assumption of normal distribution of data was assessed using the normal probability plot of the residuals and the assumption of homoscedasticity using the Levene’s test. A 95% of confidence interval was considered in all cases. For miRNA differential expression analysis, a FDR adjusted The CMV and PepMV isolates showed nucleotide identities of 100% with the Japanese CM95 isolate (GenBank accession no. AB188236.1) in the 325 nt CP amplified region and with the European EU_CAHN8 isolate (GenBank accession no. JQ314457.1) in the 545 nt TGB3 amplified region, respectively. ELISA results analysis showed that PepMV and CMV isolates were no infected with other viruses, such as ToMV, TSWV, and PMoV: three replicates were used of each virus isolate and negative absorbance values were observed for ToMV (from 0.038 to 0.161), TSWV (from 0.047 to 0.075), and PMoV (from 0.039 to 0.059), whereas the positive control ranged from 0.903 to 2.076.STV-infected tomato plants were single inoculated with PepMV or CMV, and with a combination of both viruses, to study the effect of STV in mixed infection. Leaf deformation and mosaic  were observed in infected tomato plants at different times  . AdditioThe leaf symptoms severity values (mean of plants symptoms on each group) in tomato plants infected with STV, CMV, and PepMV in single and/or mixed infections varied depending on the time and the virus combination . As expeDifferences of height and weight  among groups of infected-tomato plants are shown in 4 − 2.29 × 105 virus RNA copies/ng total RNA) overtime for single-, co-, and triple-infections with CMV and PepMV, so the other viruses did not affect STV accumulation  to 15 dpi  and an increase at 20 dpi . CMV showed different accumulation patterns, depending on the virus combination , but it increased strongly at 20 dpi  in CMV single infected plants. However, co-infection with STV produced a high increase of CMV at 10 and 15 dpi , but, at 20 dpi, the CMV titer was similar in single- and STV + CMV co-infected plants . The pattern of CMV accumulation changed when co-infected with PepMV: the CMV titer decreased at 15 dpi  and increased slightly it at 20 dpi . Differences of CMV accumulation between CMV + PepMV co-infected and CMV-single infected plants were significant at 10, 15, and 20 dpi. STV infection increased strongly CMV titer in STV + CMV + PepMV triple- infection at 10, 15, and 20 dpi  to be similar to those of CMV single-infected plants. The differences of CMV accumulation were significant between STV + CMV + PepMV triple- and CMV + PepMV co-infection at 10, 15 and 20 dpi.Virus accumulation was studied at 5, 10, 15, and 20 dpi by RT-qPCR using specific primers and TaqMan probes for STV, PepMV and CMV . The spemulation , Panel Amulation , Panel Bbination , Panel CSolanum tuberosum, Oryza sativa, Glycine max, Prunus persica, or Arabidopsis thaliana. Three miRNAs with animal sequence homology, such as cow (Bos taurus) and mouse (Mus musculus), were also detected. It was found 5, 34, and 39 miRNAs with differential expression in STV, CMV, and PepMV single-infected plants, respectively. STV infection modified the number of miRNAs in STV + CMV and STV + PepMV co-infection with respect to CMV and PepMV single- infections   . Slight ctively) . Finallyctively) . In addiThe potential functions of 53 out of these 78 miRNAs were determined by searching on the bibliography or by analysis with the online psRNAtarget software and they were mainly related with fundamental plant process, such as cellular biotic and abiotic stress, metabolism, or plant development. For example, it was reported that sly-miR9470-5p was related to hydric and salt stress, and Potato virus Y (PVY) infection as well ,43. ThisTo obtain the vsiRNAs populations, the useful reads were aligned with the complete nucleotide sequence of STV, CMV, and PepMV . For each virus combination the percentage of vsiRNAs with respect the useful reads were calculated using the mean values of the biological replicates . It was With regard to CMV derived vsiRNAs, high quantities were detected in CMV single- and STV + CMV co- infected plants . However, in CMV + PepMV co-infected plants, CMV derived vsiRNAs were almost undetectable , but they increased markedly with the STV presence in STV + CMV + PepMV triple-infected plants . Contrarily, PepMV derived vsiRNAs decreased in STV + PepMV co- and CMV + PepMV co-infected plants  with respect to PepMV-single infected ones . However, in STV + CMV + PepMV triple infection , PepMV derived vsiRNAs increased with respect to PepMV single-infection.The polarity of the vsiRNA plus (+) or minus (−) was also determined by aligning the useful reads with the positive and negative genomic virus strands . For STVMoreover, the distribution of the plus and minus vsiRNAs  from each virus genome was determined by calculating the vsiRNAs frequency at each virus nucleotide position . STV andSTV is a persistent virus that is widespread, and high incidences have been reported in some Spanish tomato production areas, such as Valencian community and Canary Islands. Despite that STV was associated to some disorders, such as a lack of fruit maturation and coloration alteration , recent STV and CMV in co-infections established a synergistic interaction that produced the earlier apparition of leaf symptoms, increasing their severity, and increasing CMV titer in the first stages of infection. STV also produced an increase of plant symptoms in STV + PepMV co-infected plants, but it did not produce changes in PepMV titer. To our knowledge, this is the first report of a synergistic interaction between a persistent and two acute viruses. The best-known synergisms between acute viruses are those involving potyviruses. For example, infection with the potyvirus PVY and the potexvirus Potato virus X (PVX) increases the accumulation of PVX and the severity of symptoms ,48. It h(Beta vulgaris) infected plants [PepMV single-infected plants showed the maximum severity foliar symptoms (medium-severe) at 15 dpi and then decreases at 20 dpi. However, few changes of the viral titer were observed, with a slight decrease from 5 to 15 dpi and a recovery at 20 dpi. This accumulation pattern is not common in acute viruses, which normally increase the viral concentration at the beginning of the infection to reach a maximum that is followed by a stable or “plateau” stage, or sometimes with a slight decrease in the virus concentration ,39. Virud plants . The pred plants .(Cucurbita maxima) led to a reduction in ToLCNDV titer, decreasing the virus-induced symptoms [In CMV + PepMV co-infected plants, an antagonistic effect was observed with a decrease of CMV titer and symptoms were different to those that were induced by CMV or PepMV in single infections. Because CMV and PepMV are phylogenetically unrelated, this interaction cannot be explained as “cross protection” or “mutual exclusion”, which are produced between closely related viruses. To date, a few antagonistic interactions between phylogenetically distant viruses have been reported, but the mechanisms underlying these interactions have not been determined. For example, the simultaneous infection of Cucumber green mottle mosaic virus (CGMMV) and Tomato leaf curl New Delhi virus (ToLCNDV) in squash plants symptoms . STV preA. thaliana [In this research work the effect of the interaction between STV, CMV, and PepMV on the populations of both plant miRNAs and viral vsiRNAs was also studied. The differential expression of 78 miRNAs was determined in tomato plants in single and mixed infection conditions with respect to the control mock-inoculated plants. Of all these miRNAs, 47 corresponded to novel miRNAs that were described for the first time in tomato. It was previously reported that plant infection by viruses, such as PVY and Papaya ringspot virus (PRSV), stimulated the synthesis of novel miRNAs ,53. miRNthaliana .N. tabacum and by Tomato yellow leaf curl virus (TYLCV) in tomato target genes of these plants [STV, CMV, and PepMV plus and minus vsiRNAs were identified. The results obtained in this work showed that the vsiRNAs proportion varied, depending on the virus (single infection) and the combination whit other viruses (multiple infection). The amount of vsiRNAs generated in STV single-infected plants was very small, but it increased markedly in plants that were co-infected with PepMV or CMV (STV + PepMV and STV + CMV co-infections). These variations in the production of vsiRNAS from STV were not related to viral accumulation, since the concentration of STV did not change with the presence of other viruses. This is in concordance with the results reported by other authors that showed low vsiRNAs concentrations in STV single-infected tomato plants, but that increased in combination with other viruses in mixed-infections ,58,59. Ie plants ,23,60.The study of the frequency of STV, CMV, and PepMV derived vsiRNAs per nucleotide site, in the positive and negative strands of the viral genomes, showed that the distribution of plus and minus vsiRNAs was not uniform in these viruses. It detected regions of accumulation with peaks (hotspots) that were usually different for the plus and minus vsiRNAs. However, the distribution patterns of vsiRNAs for each virus were not influenced by the presence of the other viruses in mixed infection. Differences were only observed in the vsiRNAs accumulation level, which was correlated with the number of total useful reads. This agrees with the non-uniform patterns of STV and PepMV vsiRNAs frequencies found by other authors . AdditioTo date, the role played by STV in the development of some plant symptoms, such as disorders in tomato fruit coloration and maturation, was controversial. Despite recent studies showing that STV did not induce any plant symptoms in single-infections, the reported symptoms could be induced by the interaction of STV with other viruses. Here, we studied the effect of STV in co- and triple-infections with the widespread Cucumber mosaic virus (CMV) and Pepino mosaic virus (PepMV). The results showed that the persistent STV is relevant from a phytopathological point of view, since STV can interact with these viruses: (i) establishing a synergism with CMV or PepMV in which STV increased CMV titer and CMV induced symptoms at early infection stages, whereas PepMV titer did not change in spite that PepMV induced symptoms exacerbated, (ii) suppressing the antagonism between CMV and PepMV, restoring the CMV titer, and modifying the plant symptom severity with respect to CMV + PepMV co-infection, and (iii) modifying the accumulation of both plant miRNAs and viral vsiRNAs with respect to PepMV and CMV in single- or co-infections. Most of these miRNAs are involved in essential plant process, Additionally, vsiRNAs could mimic the action of miRNAs targeting plant genes. Thus, it is important to establish control measures to avoid STV spread by preventing the commercialization of STV-infected seeds, since the virus is only horizontally transmitted.


High hydrostatic pressure (HHP) treatment is a non-thermal processing technology, which is widely used in the food processing field at present. In this study, the effects of HHP treatment (100~500 MPa for 5 min) on the physicochemical properties, texture parameters, and volatile flavor compounds of oysters were investigated. The results showed that HHP treatment increased the water content while reducing the crude protein and ash content of the oyster. Texture parameters showed that HHP treatment improved the hardness, springiness, chewiness, and cohesiveness of oysters, compared with the control group. In addition, the saturated fatty acid (SFA) content was slightly increased after HHP treatment, while the difference in monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) content was not significant. Furthermore, HHP increased hexenoic aldehyde, 2,4-heptadienal, 1-octene-3-ol, and 2-octen-1-ol and decreased the contents of 3. 6-nadien-1-ol, 3-octanone, and 2-undecanone, suggesting that HHP might inhibit the fishiness of oyster and showed a positive effect on its flavor. Based on the above results, HHP improved the edible qualities such as texture properties and volatile flavor of oysters. This meets the requirements of consumers on the edible quality of seafood and provides new ideas for the development of seafood. Crassostrea gigas) is a kind of nutritious food, which constitutes a necessary ingredient for a healthy diet [Aquatic food is rich in nutritional value, widely consumed in the world, and has great consumption potential ,2. Oystethy diet ,4. The fthy diet . In addithy diet ,7. Howevthy diet . Every ythy diet ,10. Oystthy diet ,11.Vibrio vulnificus and other pathogenic microorganisms in oysters were eliminated [High hydrostatic pressure (HHP) treatment is a non-thermal processing technology that has attracted widespread attention in the field of food processing and preservation ,13,14,15iminated ,22. Moreiminated . HoweverBesides nutrition and freshness, texture characteristics and volatile flavor are also important factors for consumers’ acceptability of seafood products . The forThe proximate composition changes after different pressure treatments are shown in The pH value of aquatic products is an important indicator to evaluate the quality of food . As showp < 0.05). HHP treatment has been reported to cause degeneration and deformation of myofibrillar protein. With the increase of pressure, the morphology of myofibrillar protein usually changes from tight aggregation to dense uniformity [Hardness is an internal binding force for food to maintain its own shape . As showiformity . During iformity . Moreoveiformity .p < 0.05). Springiness has been reported to be closely related to sarcoplasmic proteins. Sarcoplasmic proteins form insoluble precipitates at a pressure above 140 MPa. In addition, when the mass concentration of sarcoplasmic protein was higher than 50 mg/mL, the protein formed a gel, and the strength of the gel increased with the increase of pressure, which directly led to the increase of springiness [Springiness reflects the degree of recovery of the oyster after being deformed by an external force, and the springiness of oyster closed muscles is positively related to the binding force between muscles . Table 2inginess . Furtheringiness .p > 0.05). However, as the pressure increased to 400 MPa, the chewiness of oysters increased significantly (p < 0.05). It was reported that the chewiness of chicken breast was improved after high-pressure treatment (300–600 MPa), which is similar to the present results [Chewiness represents the tenderness of food. The effect of HHP treatment on the chewiness of oysters is shown in  results . In this results .p < 0.05) when the pressure was greater than 300 MPa . Therefore, the ratio of n-3 PUFA to n-6 PUFA in the HHP treatment group was significantly higher than that in the control group (p < 0.05). The levels of long-chain n-3 and n-6 fatty acids  and their ratios (n-3/n-6) are considered important to human health [Marine lipids contain large amounts of long-chain triglycerides with high levels of unsaturated fatty acids, as well as high levels of unsaturated phospholipids, which are usually closely related to total lipid levels . In addin health .Principal component analysis (PCA) is a method used to determine the similarity between objects in a two-way data set (samples of sensors). The DI value indicates the degree of separation between samples. As shown in The GC–MS elution peak was searched through the database NIST0 8.L spectral library, and the composition and concentration of volatile components during different HHP treatments were further obtained through quantitative analysis. Twenty-nine volatile compounds were detected by HS–SPME/GC–MS, including seven aldehydes, five alcohols, four esters, two ketones, seven hydrocarbons, and four other classes of compounds . A totalThe amount and relative contents of volatile components of HHP-treated oysters and the control group are shown in Aldehydes play an important role in the flavor of oysters . In the Alcohol compounds have a higher odor threshold, so they contribute less to flavor unless the concentration is high or unsaturated alcohol . A totalCompared to alcohols, ketones have a lower threshold. Microbial, lipid oxidation, or amino acid degradation may produce volatile ketones . In thisThe highest content of volatile substances in the detected oyster samples are hydrocarbons. However, due to the higher odor threshold of hydrocarbons, they have little effect on the overall flavor of oysters .Crassostrea gigas) were purchased from the aquatic product market near Ocean University of China in December 2019. All oyster samples were processed experimentally within 24 h after harvest. Then, 48 oysters  with full gonads were selected for subsequent experiments. They were all washed with purified water, put into polyethylene plastic bags, and then vacuum-heat-sealed.Adult oysters , with water as the pressurized medium. Single-cycle treatments with different pressure, at 100, 200, 300, 400, and 500 MPa, were applied to oyster samples, the holding time was 5 min, and 0.1 MPa was used as the control group. The speed of pressure regulation was 100 MPa/min, and the time for maintaining the pressure was 5 min. Subsequently, one part was used for the determination of texture, and the other part was stored at −80 °C for the determination of biochemical and volatile components. Each HHP treatment condition was completed in three separate runs.Moisture was determined by drying (at 60 °C) to constant weight. The crude protein content was determined by using a fully automatic Kjeldahl analyzer . Crude fat was extracted through a Soxhlet extractor, according to previous reports . GlycogeThe oyster sample was homogenized with deionized water. The pH value of oysters after HHP treatment was measured with a pH meter .Fatty acids were determined according to previously reported methods, which were slightly modified . BrieflyThe adductor muscle part of the oyster was selected for measurement to ensure the uniformity of the sample. The texture meter , equipped with an aluminum cylindrical probe (diameter P/36–50 mm), was used to tested texture changes of the oyster. The oyster samples were compressed at a compression speed of 5 mm/s to 80% of the initial sample, and the test time was 5 s. Each oyster sample was evaluated by four texture parameters, including hardness, springiness, chewiness, and cohesiveness [Oyster flavor substances were determined according to previous methods . PEN 3 eThe headspace solid-phase microextraction (HS-SPME) was used to extract the volatile compound components of oysters based on the previous method with slight modification . Brieflyp < 0.05) of all data were obtained through the Turkey test. The iMPact of HHP treatment was evaluated by using ANOVA and SPSS14.0 software .The effect of different HHP treatments  on oyster samples was analyzed by one way of variance. To analyze the effect of HHP treatment on the physical and volatile substances of oyster samples, the mean differences (Overall, HHP treatment increased the water content of the oysters, which led to a decrease in the crude protein and ash. HHP also slightly changed the composition of fatty acids. Texture parameters showed that HHP significantly changed the texture of oysters by increasing hardness, springiness, chewiness, and cohesiveness. Moreover, HHP treatment increased the quantity and concentration of volatile components and improved the flavor of raw oysters. The present research results provide a reference for the influence of HHP on oyster flavor and also provide a new idea for the development of aquatic products.


Various viruses alter nuclear pore complex (NPC) integrity to access the nuclear content favoring their replication. Alteration of the nuclear pore complex has been observed not only in viruses that replicate in the nucleus but also in viruses with a cytoplasmic replicative cycle. In this last case, the alteration of the NPC can reduce the transport of transcription factors involved in the immune response or mRNA maturation, or inhibit the transport of mRNA from the nucleus to the cytoplasm, favoring the translation of viral mRNAs or allowing access to nuclear factors necessary for viral replication. In most cases, the alteration of the NPC is mediated by viral proteins, being the viral proteases, one of the most critical groups of viral proteins that regulate these nucleus–cytoplasmic transport changes. This review focuses on the description and discussion of the role of viral proteases in the modification of nucleus–cytoplasmic transport in viruses with cytoplasmic replicative cycles and its repercussions in viral replication. Most viruses with nuclear and cytoplasmic replication require nuclear proteins to facilitate their replication . Some viDifferent positive-strand RNA viruses modify the nucleus–cytoplasmic traffic, altering the Nuclear Pore Complex (NPC) through various mechanisms . These aEnterovirus, especially Poliovirus and Rhinovirus (Family Picornaviridae), and Flavivirus (Family Flaviviridae), affect the composition of the NPC and the consequences for nucleus–cytoplasmic traffic through the processing of Nups.Since it has been well established that disruption of the import–export mechanism is a checkpoint for virus replication, it is not surprising to find that viruses induce the degradation of some components of the NPC, such as nucleoporins (Nups), by the action of their own viral proteases. This review discusses how proteases of positive-strand RNA viruses from the genera The import and export of viral proteins requires interaction with the components of the nuclear envelope (NE), which is formed by a double membrane that harbor protein channels called nuclear pore complexes (NPCs). NPCs are formed by multiple copies of Nups that participate in the bi-directional nucleus–cytoplasmic transport of macromolecules, ribosomal subunits, viral proteins and RNAs  from both cellular and viral origin ,30,31.The NPC is made up of approximately 30 multi-copy of Nups, arranged from the cytoplasm to the nucleoplasm, which makes up the cytoplasmic filaments, cytoplasmic ring, internal pore ring, nuclear ring, and nuclear basket ,33. InsiMolecules smaller than approximately 40–50 kDa can pass freely through the nuclear envelope; however, higher molecular weight molecules such as proteins and RNAs from both cellular and viral origin are actively transported through the NPC, between the nucleoplasm and the cytoplasm. The nuclear import and export of molecules are regulated by NTR (nuclear transport receptors), such as importins, exportins, carriers, and small GTPases of the Ran family that regulate the activity of importins and exportins that transport cargo molecules  36,37,3,337,38.NTRs recognize specific sequences in cargo proteins that cross the nuclear membrane from the cytoplasm, such as nuclear location sequences (NLS) that contain repeated arginine (Arg or R) and lysine (Lys or K) amino acids. The classical NLS consists of five KKKRK amino acids. Moreover, some proteins possess bipartite NLS consisting of two groups of basic amino acids, separated by approximately ten amino acids ,40. On tOn the other hand, the nuclear export is given by the recognition of the nuclear export sequences (NES). Nuclear export begins with Ran-GTP binding to exportin , which causes an increased affinity for the export cargo. Then, the complex moves to the nuclear pore and Ran-GTP hydrolyze (activated by RCC1), which forms the export complex. The complex crosses the NPC, and in the cytoplasm, GAP deactivates RanGTP (hydrolyses the GTP in GDP), causing the export protein to be released from its cargo ,40,44.Flavivirus is a genus belonging to the family Flaviviridae, which contain viruses such as SPONV (Spondweni virus), YFV (Yellow fever virus), WNV (West Nile virus), JEV , DENV (Dengue virus), and ZIKV (Zika virus), widely distributed in tropical and subtropical areas. [Aedes spp. [l areas. ,47,48,49des spp. , causes Flaviviruses contain RNA genomes of positive sense that encode a polyprotein that gives rise to three structural proteins  present in the viral particles and seven non-structural proteins  essential for controlling several cell pathways for the replication of the viral genomes, assembly, and release of the viral particles from the infected cells A 50,51,,51,52,53Flavivirus has a cytoplasmic replicative cycle, translating and replicating its genome in the ER [n the ER ,55,56. Hn the ER ,58,59. On the ER ,61,62,63Flavivirus infections [The NS3 protein is the only protease encoded by these viruses and is required for the cleavage of the viral polyprotein and the unwinding of the viral RNA since it has the function of protease and helicase. Since this molecule is involved in the infection establishment, it has been used as a pharmacological target against the different fections ,65,66.Flavivirus is a trypsin-like serine protease. It requires a 40-residue hydrophilic segment of the NS2B transmembrane protein as a cofactor, and it is involved in the processing of the polyprotein that gives rise to the mature viral proteins [The NS3 protein of proteins ,68.NS3 protease has a catalytic triad conserved in the different members of the genus  and a total length of approximately 620 amino acids. NS3 protease cleaves between a basic residue (Arg-Lys) and another amino acid such as serine or threonine. In addition to the protease function, at its carboxyl terminus, NS3 has RNA triphosphatase, nucleoside triphosphatase, and helicase activities B. The coNS3 from ZIKV is mainly located in the perinuclear region of the infected cell, and alters the morphology of the nuclear lamina, a component of the nuclear envelope, forming extrusion sites; thus, it is suggested that it may affect the function of the centrosome . NS3 is In addition to the polyprotein processing, NS3pro from several Flaviviruses have other cellular proteolytic targets. Hill et al. have reported that NS2B-3 from ZIKV can process 31 different cellular proteins, such as JIP4, ATG16L1, eIF4G1, TAK1. Among them, 42% are involved in the processing of genetic information, 28% in transport and the cytoskeleton, 12% in metabolism, 10% in signal transduction, and 8% in immunity . On the Flavivirus NS3pro also cleaves components of the NPC [ the NPC , a subjeFlavivirus is still unknown [We have recently described the alteration of the NPC during the infection with ZIKV, DENV-2, and DENV-4. We found that NS2B-3 from DENV-2 and DENV-4 cleaves Nup153 (located in nuclear basket), Nup98 , and Nup62 (located in the inner pore ring). On the other hand, we found that NS2B-3 from ZIKV cleaves TPR (located in nuclear basket), Nup153, and Nup98 C. Beside unknown . However unknown .Flavivirus infections cause a spectrum of diseases, including fever, hepatitis, vascular shock syndrome, neurological and congenital abnormalities, and encephalitis [phalitis .Flavivirus such as JEV, WNV, and DENV have also been observed in the nucleus of infected cells [Although the Flaviviruses have a cytoplasmic replication cycle, it has been observed that some DENV and ZIKV proteins such as C, NS1, and NS5 are located in the nucleus of Vero cells. The transport to the nucleus of the viral proteins is mediated by the NLS present in its sequence ,58,59. Ied cells . Howevered cells ,79,80,81Flavivirus infections, some nuclear components are highjacked to avoid the antiviral immune response. The presence of the NS5 polymerase of WNV, DENV, and JEV in the nucleus mediates the IFN response pathway at different levels of the JAK/STAT signaling pathway and thereby prevents the entry of transcription factors into the nucleus to avoid the innate immune response [Besides internalization of viral proteins into the nucleus during response ,83,84,85On the other hand, nuclear proteins such as La (DENV), PTB (DENV), RNA Helicase A, TIA1/TIAR (WNV), and Tudor-DN/p100 (DENV) migrate to the cytoplasm to participate in the assembly of the viral replication machinery ,89,90,91Despite the knowledge of the presence of nuclear components in the viral replicative complexes, the mechanisms involved in this process are still unknown .Picornaviridae family is formed of at least 30 genera, including the Enterovirus genus, and more than 75 species [The  species .Picornaviridae family have a single-stranded RNA genome of positive polarity of 6.7 to 10.1 kb in length. They are small, non-enveloped viruses, which infect several vertebrates, such as mammals, birds, reptiles, amphibians, and fish. They are also human pathogens of medical relevance because they affect the central nervous system, heart, liver, skin, gastrointestinal tract, and upper respiratory tract [Viruses of the ry tract ,93,94.Picornaviridae is cytoplasmic, and the RNA genome is translated and replicated in the endoplasmic reticulum (ER). The genome of the Picornaviridae is composed of a single open reading frame (ORF), which codes for a polyprotein that, when cleaved by viral proteases, gives rise to three to four structural proteins that conform the capsid and seven non-structural proteins involved in viral replication and the modulation of the immune response. The genome at its 5′ end is covalently linked to a VPg protein involved in RNA replication and is polyadenylated at its 3′ end. Two untranslated regions (UTR) containing RNA stem-loop structures involved in the regulation of replication flanked the unique ORF; particularly, the 5′ UTR, of 1/10 of the total genome length promotes viral protein synthesis through an internal ribosome entry site (IRES). This polyprotein is processed by the viral proteases to produce P1–P3 regions that are further processed to produce precursor and mature proteins. The P1 region conformed by the structural proteins (VP1-VP4), and the P2 (2A–2C) and P3 (3A–3D) regions give rise to non-structural proteins. Intermediate protein precursors are also produced, having essential roles, such as the 3CD protease-polymerase protein  [The best-studied group of viruses altering the NPC by viral proteases belongs to the rus A–D) ,102.Although enteroviruses are cytopathic and mainly infect the gastrointestinal or the respiratory tracts, they can also infect the central nervous system like Poliovirus (PV). PV infects the gastrointestinal tract, where the infection is asymptomatic; however, it also has a tropism for motor neurons of the lower extremities, generating paralytic poliomyelitis in approximately 1% of the infected individuals ,104. On Enterovirus genus members, specifically PV and HRV.Particularly in the next section, we will discuss the alterations of the NPC by the viral proteases during the infection of the Unlike Flaviviruses, the polyprotein of both PV) and RV) is cleaved into functional proteins by two viral proteases: 2Apro and 3Cpro. While 2Apro cleaves P1 from P2 and P3, 3C pro is responsible for the cleavage of almost all precursors and mature proteins. Moreover, besides the polyprotein cleavage, both proteases are involved in establishing the infection .2Apro from PV and RV participates in the polyprotein processing at the P1 (VP1-2A) site B. InitiaIn addition to the polyprotein cleavage, 2Apro has other cleavage targets, such as the translation initiation factors (eIF4G-I and eIF4G-II), necessary to recognize the cap in the mRNAs by the ribosomes . TherefoAs 2Apro, 3Cpro is a chymotrypsin-like cysteine protease; however, it adopts a fold-like conformation of the serine proteases, responsible for the processing of different proteolytic targets . The strBesides its role in the polyprotein precursor cleavage, 3Cpro has different proteolytic targets that affect many cell pathways at various levels to promote its viral replication and the establishment of the infection. 3Cpro can locate in the nuclei of the PV infected cells and inhibits host RNA synthesis by processing cell transcription factors, such as factor 110 associated with TATA-binding protein (TAF110), TATA box-binding protein (TBP), binding protein to the response element to cAMP-1 (CREB-1), octamer-binding protein-1 (Oct-1), p53 and transcription factor IIIC (TFIIIC) ,118,119.The cleavage of RIG-I by 3Cpro from PV and RV can block the recognition of viral RNA by the host’s innate immune system ,126. On Furthermore, 3Cpro from PV and RV also alters the nucleus–cytoplasmic traffic by processing nucleoporins, such as Nup62, Nup153, Nup214, and Nup358, components of NPC ,17,19.As mentioned above, the viruses use these strategies to favor their replication by preventing the activation of genes related to the antiviral immune response and avoiding competition with cellular mRNA by the translational machinery.NPC alterations during viral infections due to specific cleavage of Nups by cysteine proteases with a chymotrypsin-like activity of cytoplasmic viruses such as PV and RV have been reported. The nucleoporins that are most commonly targets of these proteases are Nup358, Nup214, Nup153, Nup98, and Nup62, which results in the alteration of the nucleus–cytoplasmic transport of proteins and mRNAs , the redSome authors have reported that during infection with PV, the nuclear proteins La, Sam68, and nucleolin were delocalized towards the cytoplasm ,131,132.To analyze the classical nuclear import pathway (importin α/β), these authors used chimeric EGFP proteins coupled with the SV40 large T antigen NLS. Using confocal microscopy, they found that during infection with PV, the EGFP-NLS signal was located mainly in the cytoplasm, contrary to control uninfected conditions where the signal was in the cell nucleus. On the other hand, they analyzed the non-classical transport pathways, such as the one mediated by transportin 1 during infection. Transportin 1 participates in the bidirectional transport of proteins containing sequence M9-NLS , present in proteins such as the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), and some others involved in RNA binding, transcription, or processing . When thFurthermore, a battery of antibodies directed against proteins that use different transport routes was used to analyze their location. The hnRNP A1 (transportin 1 pathway ), hnRNP A necessary mediator for nucleus–cytoplasmic transport is the NPC, which was analyzed during infection with PV. Using the monoclonal antibody 414 (directed against FG-Nups) and specific antibodies against Nup153 and Nup62, a dramatic reduction in these proteins was observed in infected cells [In 2008, this same group described that 2Apro from PV is responsible for Nup153, Nup98, and Nup62 cleavage; therefore, for the nuclear–cytoplasmic traffic during infection with PV [Like PV, RV requires host nuclear proteins such as nucleolin, La, and Sam68 for its replication . Given tAs in PV infection, during the RV infection, Nup153 and Nup62 were also degraded, and consequently, the nuclear–cytoplasmic traffic was altered. Importin α/β (classic NLS), transportin 1 (M9-NLS), hnRNP A1 (transportin 1 pathway ), hnRNP Furthermore, it was found that 2Apro of HRV-2 is responsible for cleaving Nup62 and thus altering the nuclear–cytoplasmic traffic . In addiHRV 3Cpro and its precursor 3CD also participate in the cleavage of Nups and the alteration of the nuclear–cytoplasmic traffic. 3Cpro and 3CD degrade Nup153, Nup214, and Nup358 . FurtherFG-Nups (Phe/Gly repeats) are recognized by the nuclear–cytoplasmic transport machinery (carioferins) . Thus, tThe consequences of the inhibition of transport can occur at the antiviral level through the inhibition of transport via importin α/β of NF-KB , IRF-3 , and STAThe viral proteases of RNA-viruses are indispensable proteins for viral progeny production since they are the major components for the processing and maturation of viral proteins. Moreover, viral proteases also have essential roles in viral infection; for that, proteases alter the nucleus–cytoplasmic transport by targeting Nups in the NPC; thus, modifying different cellular pathways to favor viral replication and to evade the immune response. Thus, viral proteases constitute an attractive pharmacological target for the design of antiviral drugs.In this review and other related works, it becomes clear that there is a need to not only study the participation of the cell nucleus and its components but also the involvement of viral proteins in the nucleus during the promotion of viral replication and infection of RNA-viruses and, thus, to influence in the development of drug targets. Viral proteases also have essential roles in viral infection.


Caudovirales, with Siphoviridae and Podoviridae being the most abundant. Eukaryotic viruses were detected as early as two weeks after birth and remained present all along the first year of life. Thirty-four different eukaryotic virus families were found, where eight of these families accounted for 98% of all eukaryotic viral reads: Anelloviridae, Astroviridae, Caliciviridae, Genomoviridae, Parvoviridae, Picornaviridae, Reoviridae and the plant-infecting viruses of the Virgaviridae family. Some viruses in these families are known human pathogens, and it is surprising that they were found during the first year of life in infants without gastrointestinal symptoms. The eukaryotic virus species richness found in this work was higher than that observed in previous studies; on average between 7 and 24 virus species were identified per sample. The richness and abundance of the eukaryotic virome significantly increased during the second semester of life, probably because of an increased environmental exposure of infants with age. Our findings suggest an early and permanent contact of infants with a diverse array of bacteriophages and eukaryotic viruses, whose composition changes over time. The bacteriophages and eukaryotic viruses found in these children could represent a metastable virome, whose potential influence on the development of the infant’s immune system or on the health of the infants later in life, remains to be investigated.In this work, we determined the diversity and dynamics of the gut virome of infants during the first year of life. Fecal samples were collected monthly, from birth to one year of age, from three healthy children living in a semi-rural village in Mexico. Most of the viral reads were classified into six families of bacteriophages including five dsDNA virus families of the order  Humans are in constant close contact with a rich variety of bacteria, archaea, fungi, protists and viruses, which all together reside on, or within the human body, forming an ecosystem denominated microbiota. Distinct body sites are characterized by distinct microbial communities, with the gastrointestinal tract being the most populated site, playing an important role in human health and development. The bacterial component of the microbiota is the best characterized, having many functions in host physiology like metabolic processes, development and maturation of the immune system , 2. BactSiphoviridae, Inoviridae, Myoviridae, Podoviridae and Microviridae represent the most common families [Anelloviridae, Picobirnaviridae, and Circoviridae families being the most frequently reported [Adenoviridae, Astroviridae, Caliciviridae, Parvoviridae, Picornaviridae, and Polyomaviridae families have also been described [Alphaflexiviridae, Tombusviridae, Nanoviridae, Virgaviridae and Geminiviridae, have also been commonly reported.Contrary to bacterial composition, the virus assembly (known as virome) in the gut is much less understood. It has been observed that bacteriophages are the predominant viruses, where families –7. Eukarreported –11, althescribed , 9. In aAdenoviridae, Anelloviridae, Astroviridae, Parvoviridae, Picornaviridae, Picobirnaviridae and Reoviridae families, have been commonly identified in infant stool samples, even in the absence of clinical symptoms [Much less is known about the gut virome composition of healthy children during first years of life. Bacteriophages show high diversity and dynamic in the first months of life, decreasing over time and becoming stable until 2 years old. A number of phages have been identified to be shared between mothers and infants, suggesting their transmission through the mothers’ milk , 13 or tsymptoms , 22–24.In this work, the viral composition of the gastrointestinal tract of three healthy infants from a semi-rural town in Mexico was characterized in fecal samples collected monthly during their first year of life. The presence of eukaryotic viruses was detected as early as two weeks after birth and represented a diverse metastable and dynamic virome along the year of study formed by a rich and abundant mixture of viruses from plant and animal hosts.This study was carried out in the semi-rural community of Xoxocotla, in the state of Morelos, 130 km south of Mexico City. Healthy women who arrived for routine control at the pregnancy clinic of the Xoxocotla Health Center, during the last trimester of pregnancy, were invited to participate in the study. Written informed consent was obtained from all mothers after providing them with detailed information about the study and its characteristics. The protocol and the consent letter were approved by the Scientific and Ethics Committee of the Medical School of the National University of Mexico as well as by the Ministry of Health of the State of Morelos. Three mother-infant pairs were included in this study; the infants were healthy, full-term products, without any congenital condition and with normal weight at birth. A single fecal sample was obtained at the end of the last trimester of pregnancy from each mother. The infants were followed monthly during their first year of life between March 2015 and June 2016. The stool samples were collected immediately after defecation by the mothers, previously trained and under the supervision of a responsible nurse. Sterile plastic containers with screw caps were used to collect stool samples from the diapers; samples were not exposed to antiseptics or disinfectants. The samples were kept at 4°C and transported to the village laboratory where they were kept at -20°C for less than 1 week; The samples were transported to the Institute of Biotechnology where they were stored at -70°C, until use.9-3’5’–GTTTCCCAGTAGGTCTCN). The cDNA was generated by two consecutive rounds of synthesis with Sequenase 2.0 . The synthesized cDNA was then amplified with Phusion High fidelity polymerase (Finnzymes) using primer B (5’-GTTTCCCAGTAGGTCTC-3’) and 10 additional cycles of the program: 30 sec at 94°C, 1 min at 50°C and 1 min at 72°C. Then, the DNA was purified using ZYMO DNA Clean & Concentration-5 kit. Sequencing libraries were prepared using Nextera XT DNA library preparation kit (Illumina); samples were uniquely tagged, pooled and then deep sequenced on the Illumina NextSeq500 system, generating paired-end reads of 75 bases. The base calling was performed by Illumina Real Time Analysis (RTA) v1.18.54 software and the demultiplexing of reads by bcl2fastq v2.15.0.4.Nucleic acids were extracted from the stool samples as described before . BrieflyA viral metagenomics pipeline , which iThe taxa abundance differences were analyzed using Trimmed Mean of M-value (TMM) method . Unless Fecal samples from three apparently healthy infants, with no disease symptoms during the study, were collected monthly, starting two weeks after birth and until 12 months of age, obtaining 11 samples from infant 2, and 12 samples from infants 4 and 5. Mother samples were taken at a single time point around the eighth month of pregnancy. The characteristics of mothers and infants are described in Total nucleic acids were extracted from 38 fecal samples (35 from infants and 3 from mothers) to detect both DNA and RNA viruses and sequenced using the paired-end 2×75 bp Illumina HiSeq 500 system . Initially, 421.78 million paired-end reads were obtained for all libraries, with a mean of 12.05 ± 3.23 millions per sample . After qOn average, it was found that 36.1% (±27.6% s.d.) and 26.9% (±14.3% s.d.) of the valid reads in the infant samples had homology to at least one viral or bacterial reference sequence, respectively . In contVirgaviridae, the most abundant, was identified in all samples (35/35), representing an average of 26.6% of all eukaryotic viral reads. ii) Anelloviridae and Picornaviridae represented on average 24.8% and 18.7% of all eukaryotic viral reads, respectively, and were found in up to 80% of the samples. iii) Caliciviridae, Parvoviridae and Reoviridae were less abundant, on average between 5.8%-8.8%, and less prevalent, been found in around 60–71% of samples. Iv) Astroviridae and Genomoviridae were identified in between 43–49% of the samples, while Circoviridae in only 17%. As expected, the most abundant and prevalent viral families found have humans as hosts, except for the family Virgaviridae, whose natural hosts are plants. On the other hand, among the viral families identified in a single sample or in less than 10% of the samples, only 15% infect invertebrates. In the samples from the three mothers, 18.6% of the viral reads were eukaryotic, classified into 16 families, and the rest were bacteriophages, with Virgaviridae (90.6%) and Phycodnaviridae (8.3%) as the dominant virus families.In infants, 15.5% of the assigned viral reads correspond to eukaryotic viruses, which can be classified within 33 families , about hTobamovirus of the Virgaviridae family was the most prevalent genus, being found in all mother and infant samples, and was also the most abundant in infants and mothers, representing 28.5% and 91% of the eukaryotic viral reads per sample, respectively. A detailed characterization of the plant viruses found in the infants’ gut was recently reported by our group [At the genus level, 54 viral genera were identified in the infant samples and 18 were present in the samples obtained from their mothers . Tobamovur group .Virgaviridae family, were the most prevalent and abundant species in both, infant and mother samples. TSAMV was found in all samples, representing on average 15.9% of the eukaryotic viral reads per infant sample (range 0.001–89.4%) and 62.9% of reads in the mothers´ samples (range 0.7–95.9%). Whereas PMMoV represented, in average, 10.3% of the viral reads in the infant samples and 3.5% in the mothers’ samples; this virus was detected in 80% and 100% of infant and mother samples, respectively. Other abundant virus species found in more than half of the infant´s samples were Norwalk virus (NV), rotavirus A (RVA) and torque teno virus (TTV); there were also other 8 viral species identified in at least 30% of the samples and others that were identified sporadically  genomes were obtained from several viral species, by assembling them individually, such as, human bocavirus (4 genomes), enterovirus (3), human astrovirus (2), human rotavirus (3), norovirus (6), parechovirus (2), pepper mild mottle virus (7), rattail cactus necrosis associated virus (1), tomato mosaic virus (1), tropical soda apple mosaic virus (9) and sapovirus (2).Caudovirales were the most abundant: Siphoviridae , with an average of read abundance of 51.5% per sample; Podoviridae  with 20.2%; Myoviridae  with 12.7%; the provisional family crAss-like  with 10.4%; and Ackermannviridae, the new bacteriophage family proposed in 2017, with 2.9%. The ssDNA family Microviridae  represented, on average, 2.3% of children´s bacteriophage reads. Interestingly, in mothers, the crAss-like bacteriophages predominated in the gut, with an average of 79% of the total phage reads.As previously observed , 43–45, Pis4avirus from the Siphoviridae family and the genus G7cvirus from the Podoviridae family being the most common, since they were identified in all infant samples. Also, 8 other frequent and abundant genera were identified in more than 75% of samples, while more than 31 in a single sample or in less than 3 in the infants.Regarding phage genera, 97 were identified in infants and 31 in mothers , with thMyoviridae and Podoviridae families, 40 from the Siphoviridae and 1 from the Ackermannviridae, crAss-like and Microviridae.In contrast with previous reports that have shown that a stable community of bacteriophages exist in adults over a long period of time , 6, we oBacteroidales [Escherichia, Lactococcus, Salmonella, Streptococcus, Klebsiella, Staphylococcus, Clostridium and Enterococcus phages, with more than 50% of prevalence (The most abundant and frequent phage species in both infants and mothers has around 75% of homology to the Bacteroides phage crAss001 from the crAss(cross Assembly)-like family . This phroidales . We founevalence .E. coli virulent phages in the Myoviridae and Podoviridae families that were abundantly identified in some samples, persisting for prolonged periods of time  Positive correlation, where bacteria-phages abundances move in tandem, b) Negative correlation, where bacteria increase as the viruses decrease, and vice versa. For example, at 15 days of age, the crAssphages were more abundant (on average 35%) than the Bacteroidetes (12%), a trend that was positively correlated during the first semester, in a ratio that started to change during the second semester of life, becoming the inverse; thus, by month 10 the Bacteroidetes phages were more abundant than the CrAssphages . An addi of time . These pg blastp , to deteDiversity estimations were performed using the normalized matrixes of viral read and contig counts at the species level, and their association with factors of metadata . In infaThe beta diversity of the eukaryotic viruses and of bacteriophages was calculated among mothers and infants, to individually compare them and to discern if there were patterns in association with metadata. It is of upmost importance to understand the way the enteric virome develops during infancy and what impact on the development of the gastrointestinal tract and the human health it may have. Few studies have described the gut virome of infants during the first year of life –45, 49, Virgaviridae family, the most abundant and frequently found were viruses belonging to the families Anelloviridae, Astroviridae, Caliciviridae, Genomoviridae, Parvoviridae, Picornaviridae and Reoviridae. Some viruses in these families are common human pathogens, especially in children; it is thus surprising that they were commonly found in the absence of gastrointestinal symptoms all along the year of the study. The gut mucosa of infants is under a process of maturation and receptors to pathogens may still be absent; although other immunological or nutritional factors may also be involved.Regarding eukaryotic viruses, 33 families were identified in infants, but only nine were frequently found and made up to 97% of all eukaryotic viral reads identified. Aside for plant-infecting viruses in the Anelloviridae family were highly abundant, containing different species of torque teno mini virus and unclassified species in up to 80% of the samples. In previous studies, viruses from this family have been identified as the most abundant and frequent in healthy children [Picornaviridae family have also been frequently found in healthy children [In our study, members of the children , 50, 51,children , 22, 24,children , 53, AIDchildren , pulmonachildren , 56, canchildren , among ochildren . Our reschildren  and suggchildren –23, 50, children ; in thisCaliciviridae family have been found at a low frequency (<7%) in healthy infants in metropolitan areas of the USA [Viruses in the  the USA , 19, Sou the USA  and Bang the USA , while t the USA  and Ethi the USA . In case the USA , 51, 60.Parvoviridae and Genomoviridae, with most of them being insect and animal viruses. They were sporadically identified and in low abundance, possibly reflecting environmental contamination, except for human bocaviruses, which were identified in 11 of 35 infant samples. Such high prevalence was not surprising, as bocaviruses have been previously reported in feces of more than 40% of asymptomatic children [Reoviridae family, was identified in 66% of the infant samples. Interestingly, all rotavirus reads detected showed an identity of 100% with different genes of the RotaTeq vaccine strains. The rotavirus vaccine was administered to the three infants at around two, forth, and six months of age, except for infant 5 who did not receive the last dose. Surprisingly, we identified the rotavirus vaccine strain in 4 out of 5 samples just before their first vaccination, which suggests a frequent transmission of the vaccine strain by close contact with vaccinated people, as it has been suggested in a previous study of transmission from vaccinated infants to their unvaccinated co-twin [We identified a large set of 27 species of children . Rotavir co-twin . RotavirVirgaviridae family were frequently detected in the three children along the year of study. The Tobamovirus genus was the most frequent, with tropical soda apple mosaic virus, pepper mild mottle virus, and opuntia tobamovirus 2 being the most common species. Our results showed a large diversity of tobamoviruses circulating in the population, suggesting that infants are continuously exposed to an extensive and dynamic collection of these plant viruses, even before infants begin to ingest food other than mother’s breastmilk, including baby formula or other liquids, indicating a distinct source of origin for these viruses. We recently reported the genetic diversity and dynamics of tobamovirus infection in infants, as wells as the potential implications of these findings [Regarding plant-infecting viruses, those in the findings .Anelloviridae, Caliciviridae, Reoviridae and Virgaviridae families were found more abundantly in the second semester, as compared to the first six months of life. It is important to mention that we estimated diversity based on annotated taxa, not at contig level, since in our experimental procedure virus-like particles were not purified; and thus, our values cannot be compared with those of previous studies that use this method.The richness of eukaryotic virus species found in this work was higher than in previous studies in healthy children, in which less than 9 different virus species were found per individual samples , 50, 51.Siphoviridae, Myoviridae and Podoviridae families in the Caudovirales order. Although previous studies have reported that the majority of gut bacteriophages seem to engage in lysogenic interactions with their hosts [in vitro and in vivo [Most viral reads in this work were assigned to bacteriophages, both in infants (84.5 ∓ 24%) and mothers 61.4 ∓ 37%). Unlike eukaryotic viruses, and in contrast to previous findings , no diff1.4 ∓ 37%ir hosts , 63, 64,ir hosts . In this in vivo , althougCrAssphages have been reported to represent up to 95% of the total viral load in the adult´s gut, and to be present in 73% to 77% of samples analyzed in diverse human populations , 66. RecThe results and conclusions of this study are limited by a small sample size, although our primary goal was to have a first glimpse on the composition and dynamics of the eukaryotic and prokaryotic virome of healthy Mexican infants in a rural community during first year of life. Our findings suggest the existence of an early and constant contact of infants with a diverse array of eukaryotic viruses, whose composition changes over time. In addition to the phageome, that seems to be well established given the ubiquitous presence of their microbial hosts, the eukaryotic virus array could represent a metastable virome, whose potential influence on the development of the infant’s immune system or on the health of the infants during childhood, remains to be investigated.S1 Fig* Minimally non-redundant nucleotide database from NCBI . **Minim(TIF)Click here for additional data file.S2 FigSamples from infants are indicated according to their age in months and the single mother sample in each case, as M. The virus family name is indicated on the left side, while the host and type of virus genome are shown in the right side. The discontinuous yellow lines divide the years into two semesters.(TIF)Click here for additional data file.S3 FigSamples from infants are indicated according to their age in months and, from mothers, as M. The name of the viral genera is indicated on the left side while the family names are shown on the right side. The discontinuous yellow lines divide the year into semesters.(TIF)Click here for additional data file.S4 FigSamples from infants are indicated according to their age in months and from mothers as M. The type of genome of each viral family is indicated on the right side.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.